Preparation and application of an iridium complex and its PLGA nanoparticles

Abstract

The present invention provides an iridium complex and its preparation and application of PLGA nanoparticles, belonging to the field of biomedicine technology. The iridium complex provided by the present invention can enter tumor cells, inhibit tumor cell proliferation and migration, and cause mitochondrial dysfunction leading to tumor cell death. The present invention also provides an iridium complex PLGA nanoparticle, which solves the problems of poor water solubility and high toxicity of the complex, while improving the bioavailability of the iridium complex, making the iridium complex drugable, and providing a new solution for the treatment of breast cancer. In addition, the iridium complex PLGA nanoparticles provided by the present invention can significantly inhibit cell growth, migration, etc., have the effect of reducing toxicity and increasing efficacy, do not change the mechanism of action of the complex, and promote cell apoptosis and cell ferroptosis caused by the complex.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the preparation and application of an iridium complex and PLGA nanoparticles thereof. Background Art

[0002] According to the World Health Organization (WHO), breast cancer has become one of the most common malignant tumors in women worldwide, posing a serious threat to women's health. Chemotherapy is a primary treatment strategy for cancer patients, but existing chemotherapy drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, hormonal agents, and targeted therapies, cannot distinguish between malignant and normal cells and have severe side effects. The development of new chemotherapy drugs or the reduction of their toxicity are needed.

[0003] Iridium complexes have demonstrated remarkable anticancer activity, making them promising candidates for antitumor drugs. Iridium(III) complexes possess unique physicochemical properties and are easily modifiable, offering the potential for designing more selective and effective therapeutic agents. Furthermore, their high luminescence efficiency and long lifetime have attracted widespread attention for their applications in bioimaging and cancer therapy. However, research on iridium complexes for tumor therapy remains in its early stages, with challenges such as weak antitumor activity, low bioavailability, and significant side effects.

[0004] Therefore, there is an urgent need for an iridium complex with strong anti-tumor activity, high bioavailability and small side effects to solve the above problems. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes the preparation and application of an iridium complex and PLGA nanoparticles thereof.

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides an iridium complex, which includes [Ir(piq)2(ZZIP)]PF6, [Ir(ppy)2(ZZIP)]PF6, and [Ir(bzq)2(ZZIP)]PF6;

[0008] The structural formula of the cation of [Ir(piq)2(ZZIP)]PF6 is:

[0009]

[0010] The structural formula of the cation of [Ir(ppy)2(ZZIP)]PF6 is:

[0011]

[0012] The structural formula of the cation of [Ir(bzq)2(ZZIP)]PF6 is:

[0013]

[0014] The iridium complex provided by the present invention can enter tumor cells, inhibit tumor cell proliferation and migration, and cause mitochondrial dysfunction, leading to tumor cell death. Its mechanism of action is as follows: (1) increased reactive oxygen species (ROS) levels cause mitochondrial dysfunction, inhibit the PAM (PI3K / AKT / m-TOR) and PARP pathways, activate the Bcl-2 family, and lead to apoptosis of tumor cells; (2) increased ROS leads to mitochondrial dysfunction, causing an increase in malondialdehyde (MDA) and a decrease in glutathione (GSH) and glutathione peroxidase 4 (GPX4), leading to ferroptosis of tumor cells.

[0015] The present invention provides a method for preparing the iridium complex described in the above technical solution, comprising the following steps:

[0016] (1) 4-isopropylbenzaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate are dissolved in glacial acetic acid and subjected to condensation reflux reaction, followed by cooling, pH adjustment, filtration, washing the precipitate and drying to obtain the ligand ZZIP;

[0017] (2) dissolving IrCl3·H2O and 3-phenylisoquinoline, benzo[h]quinoline or 2-phenylpyridine in a mixture of ethylene glycol monoethyl ether and water, condensing and refluxing under argon protection, cooling, filtering and vacuum drying to obtain iridium precursors Cis-[Ir(piq)2Cl]2, Cis-[Ir(bzq)2Cl]2 or Cis-[Ir(ppy)2Cl]2;

[0018] (3) dissolving the ligand ZZIP obtained in step (1) and the iridium precursor obtained in step (2) in a mixture of dichloromethane and methanol, refluxing the mixture under argon protection, and adding ammonium hexafluorophosphate after cooling to room temperature. After stirring, filtering and vacuum drying, the iridium complex is obtained.

[0019] Preferably, the preparation process of the iridium complex is:

[0020]

[0021] Preferably, in step (1), the molar ratio of 4-isopropylbenzaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate is (10-13):(10-13):(500-600).

[0022] Preferably, in step (1), the condensation reflux reaction time is 4-8 hours.

[0023] Preferably, in step (1), the reagent for adjusting pH is concentrated aqueous ammonia.

[0024] Preferably, in step (1), the drying temperature is 30-50°C.

[0025] Preferably, in step (2), the molar ratio of IrCl3·H2O to 3-phenylisoquinoline is (1:2)-(1:3), the molar ratio of IrCl3·H2O to benzo[h]quinoline is (1:2)-(1:3), and the molar ratio of IrCl3·H2O to 2-phenylpyridine is (1:2)-(1:3).

[0026] Preferably, in step (2), the condensation reflux time is 18-24 hours.

[0027] Preferably, in step (3), the molar ratio of the ligand ZZIP to the iridium precursor Cis-[Ir(piq)2Cl]2 is 2:1, the molar ratio of the ligand ZZIP to the iridium precursor Cis-[Ir(bzq)2Cl]2 is 2:1, and the molar ratio of the ligand ZZIP to the iridium precursor Cis-[Ir(ppy)2Cl]2 is (2:1)-(3:1).

[0028] Preferably, in step (3), the reflux reaction time is 4-8 hours.

[0029] Preferably, in step (3), the molar ratio of ammonium hexafluorophosphate to the ligand ZZIP is (8-20):0.5.

[0030] Preferably, in step (3), the stirring time is 20-50 min.

[0031] The present invention provides an iridium complex PLGA nanoparticle, comprising the iridium complex described in the above technical solution and poly (lactide-glycolide).

[0032] The iridium complex PLGA nanoparticles provided by the present invention can significantly inhibit the growth and migration of tumor cells, and have the effects of reducing toxicity and increasing efficacy. At the same time, the mechanism of action of the iridium complex is not changed, but rather the effect of the iridium complex is enhanced, and the problems of poor solubility and low bioavailability of the iridium complex are solved.

[0033] The present invention provides a method for preparing iridium complex PLGA nanoparticles according to the above technical solution, comprising the following steps: dissolving poly(lactide-glycolide) and the iridium complex according to the above technical solution in an organic solvent, then injecting them into a PVA solution, and performing ultrasonication and removing the organic solvent to obtain the iridium complex PLGA nanoparticles.

[0034] The invention first injects an organic solvent containing poly(lactide-glycol) and an iridium complex into a PVA solution, combines ultrasound with removal of the organic solvent, and prepares PLGA nanoparticles coated with the iridium complex.

[0035] Preferably, the mass ratio of the poly(lactide-glycol) to the iridium complex is 10:(0.1-5), more preferably 10:2.

[0036] The present invention controls the mass ratio of poly(lactide-glycol) and the iridium complex within the above range, thereby avoiding the precipitation of PLGA nanoparticles and further ensuring the anti-tumor effect of the iridium complex PLGA nanoparticles.

[0037] Preferably, the organic solvent is a mixed solvent of dichloromethane and ethanol.

[0038] The volume ratio of the dichloromethane to ethanol is (2:1)-(4:1).

[0039] The present invention adopts dichloromethane and ethanol as organic solvents, which is conducive to obtaining nanoparticles with moderate particle size and no precipitation, and can also reduce toxic solvents as much as possible.

[0040] Preferably, the mass concentration of the PVA solution is 1-4%.

[0041] Preferably, the power of the ultrasound is 200-300W, and the time is 10-30 minutes.

[0042] The present invention controls the power and time of ultrasound within the above range, which is conducive to obtaining PLGA nanoparticles with a particle size of about 150 nm. Increasing the ultrasound power and prolonging the ultrasound time will lead to a smaller particle size of the nanoparticles. If the particle size of the nanoparticles is too small, they will be cleared out of the body by the kidneys and liver. Reducing the ultrasound power and shortening the ultrasound time will lead to an excessively large particle size of the nanoparticles, making it impossible to achieve the EPR effect.

[0043] Preferably, the method of removing the organic solvent is vacuum rotary evaporation.

[0044] The invention adopts the reduced pressure rotary evaporation method to quickly solidify the nanoparticles, so that the iridium complex is fixed inside the PLGA and is not easy to leak.

[0045] Preferably, the present invention freezes the iridium complex PLGA nanoparticles at -20°C for 2-6 hours, then freezes at -80°C for 2-6 hours, and then freeze-dries at -60°C for 48-72 hours to obtain iridium complex PLGA nanoparticle freeze-dried powder.

[0046] The invention prepares iridium complex PLGA nanoparticle freeze-dried powder by freeze-drying the optimized prescription iridium complex PLGA nanoparticles, thereby solving the problem that nano drug aqueous solution is not conducive to long-term storage and transportation of nanoparticles.

[0047] The present invention also provides the use of the iridium complex or iridium complex PLGA nanoparticles described in the above technical solution in the preparation of anti-tumor cell drugs.

[0048] Preferably, the tumor cells include breast cancer cells.

[0049] Compared with the prior art, the present invention has the following advantages and technical effects:

[0050] (1) The three iridium complexes provided by the present invention can lead to increased ROS in breast cancer cells, resulting in mitochondrial dysfunction, thereby causing cell apoptosis and cell ferroptosis, broadening the research of anti-tumor drugs.

[0051] (2) The present invention solves the problems of poor water solubility and high toxicity of the iridium complex by preparing the iridium complex into PLGA nanoparticles. It also improves the bioavailability of the iridium complex, making the iridium complex druggable, and providing a new solution for the treatment of breast cancer. In addition, the iridium complex PLGA nanoparticles provided by the present invention can significantly inhibit cell growth and migration, etc., have the effect of reducing toxicity and increasing efficacy, do not change the mechanism of action of the complex, and promote cell apoptosis and cell ferroptosis caused by the complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of this application. The exemplary embodiments and descriptions of this application are intended to explain this application and do not constitute an improper limitation on this application. In the accompanying drawings:

[0053] Figure 1 The cytotoxicity graphs of PIQ in Example 1, PPY in Example 2, BZQ in Example 3, and paclitaxel (PTX) on MDA-MB-231, MCF-7, 4T1, MCF-10A, HepG-2, Hela, A549, U87, U251, and LO2 cells of the present invention are shown;

[0054] Figure 2 This is a graph showing the survival period of mice within 14 days after administration of PPY in Example 2 of the present invention;

[0055] Figure 3 This is the death of mice within 14 days after PPY administration in Example 2 of the present invention;

[0056] Figure 4 This is a graph showing the in vitro release curves of the iridium complex PLGA nanoparticles in Example 4 of the present invention after reconstitution under different pH conditions;

[0057] Figure 5 Figure 3 is a graph showing the cytotoxicity and safety of PPY-NPS in Example 4 of the present invention, PPY in Example 2, and blank group NPS (n=3);

[0058] Figure 6 The weight changes of mice in the normal group, negative group, PTX group, PPY group prepared in Example 3, and PPY-NPS group prepared in Example 4 within 14 days;

[0059] Figure 7 These are the tumor inhibition effect diagrams of the PTX group, the PPY group prepared in Example 3, and the PPY-NPS group prepared in Example 4;

[0060] Figure 8 The activity of heart, liver, spleen, lung, kidney, thymus and tumor in the normal group, negative group, PTX group, PPY group prepared in Example 3 and PPY-NPS group prepared in Example 4 are shown. DETAILED DESCRIPTION

[0061] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0062] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0063] The room temperature in the embodiments of the present invention refers to "25±2°C".

[0064] Unless otherwise specified, the raw materials in the examples of the present invention were purchased from commercial sources.

[0065] Example 1

[0066] An iridium complex [Ir(piq)2(ZZIP)]PF6, whose cationic structural formula is:

[0067]

[0068] The preparation method is:

[0069] (1) 1520 μL of 4-isopropylbenzaldehyde (12 mmol), 2.69 g of 1,10-phenanthroline-5,6-dione (12.50 mmol), and 38.80 g of ammonium acetate (500 mmol) were dissolved in 150 mL of glacial acetic acid. The mixture was refluxed under condensation for 5 h and then cooled to room temperature. The mixture was washed with water and transferred to a beaker. Concentrated ammonia was added dropwise to adjust the pH to neutral. A large amount of yellow precipitate was precipitated. The residue was collected by filtration, washed with water, and then dried in a vacuum drying oven at 40°C to obtain the ligand ZZIP.

[0070] (2) 0.97 g of IrCl3·H2O (2.76 mmol), 1.13 g of 3-phenylisoquinoline (5.52 mmol), 30 mL of ethylene glycol monoethyl ether, and 10 mL of water were placed in a three-necked flask and thoroughly mixed. The mixture was condensed and refluxed under argon for 24 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, and dried in vacuo to obtain a red solid product, which was the iridium precursor Cis-[Ir(piq)2Cl]2.

[0071] (3) 0.17 g of the ligand ZZIP (0.50 mmol) prepared in step (1), 0.38 g of the iridium precursor Cis-[Ir(piq)2Cl]2 (0.25 mmol) prepared in step (2), 28 mL of dichloromethane and 14 mL of methanol were placed in a three-necked flask and mixed evenly. Under argon protection, the mixture was refluxed for 6 h. After the reaction was completed, the mixture was cooled to room temperature. Then, 1.5 g of ammonium hexafluorophosphate was added and stirred for 30 min. The filtrate was filtered and dried in vacuo to obtain a wine-red solid crude product, which was the iridium complex [Ir(piq)2(ZZIP)]PF6, denoted as PIQ.

[0072] Example 2

[0073] An iridium complex [Ir(bzq)2(ZZIP)]PF6, whose cationic structural formula is:

[0074]

[0075] The preparation method is:

[0076] Step (1) is the same as in Example 1;

[0077] (2) 0.97 g of IrCl3·H2O (2.76 mmol), 0.99 g of benzo[h]quinoline (5.52 mmol), 30 mL of ethylene glycol monoethyl ether, and 10 mL of water were placed in a three-necked flask and mixed evenly. Under argon protection, the mixture was condensed and refluxed for 24 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, and dried in vacuo to obtain a yellow solid product, which was the iridium precursor Cis-[Ir(bzq)2Cl]2.

[0078] (3) 0.17 g of the ligand ZZIP (0.50 mmol) prepared in step (1), 0.30 g of the iridium precursor Cis-[Ir(bzq)2Cl]2 (0.25 mmol) prepared in step (2), 28 mL of dichloromethane and 14 mL of methanol were placed in a three-necked flask and mixed evenly. Under argon protection, the mixture was refluxed for 6 h. After the reaction was completed, the mixture was cooled to room temperature. Then, 1.5 g of ammonium hexafluorophosphate was added and stirred for 30 min. The filtrate was filtered and dried in vacuo to obtain a yellow solid crude product, which was the iridium complex [Ir(bzq)2(ZZIP)]PF6, denoted as BZQ.

[0079] Example 3

[0080] An iridium complex [Ir(ppy)2(ZZIP)]PF6, whose cationic structural formula is:

[0081]

[0082] The preparation method is:

[0083] Step (1) is the same as in Example 1;

[0084] (2) 2.43 g of IrCl3·H2O (6.90 mmol), 1972.5 μL of 2-phenylpyridine (13.80 mmol), 75 mL of ethylene glycol monoethyl ether, and 25 mL of water were placed in a three-necked flask and mixed evenly. Under argon protection, the mixture was condensed and refluxed for 24 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, and dried in vacuo to obtain an orange-yellow solid product, which was the iridium precursor Cis-[Ir(ppy)2Cl]2.

[0085] (3) 1.83 g of the ligand ZZIP (5.40 mmol) prepared in step (1), 2.71 g of the iridium precursor Cis-[Ir(ppy)2Cl]2 (2.7 mmol) prepared in step (2), 108 mL of dichloromethane and 54 mL of methanol were placed in a three-necked flask and mixed evenly. Under argon protection, the mixture was refluxed for 6 h. After the reaction was completed, the mixture was cooled to room temperature. Then, 1.5 g of ammonium hexafluorophosphate was added and stirred for 30 min. The filtrate was filtered and dried in vacuo to obtain an orange solid crude product, which was the iridium complex [Ir(ppy)2(ZZIP)]PF6, denoted as PPY.

[0086] Identification of iridium complexes by hydrogen and mass spectrometry: Weigh approximately 50 mg of PIQ, PPY, and BZQ, dissolve them in 1 mL of DMSO-d, and perform identification by hydrogen and carbon nuclear magnetic spectra.

[0087] PIQ's hydrogen spectrum and carbon spectrum: 11H NMR (400 MHz, DMSO-d6) δ 9.31 - 9.16 (m, 2H), 9.02 (d, J = 8.1 Hz, 2H), 8.40 (d, J = 8.1 Hz, 2H), 8.33 (d, J = 7.9 Hz, 2H), 8.03 - 7.97 (m, 2H), 7.95 - 7.80 (m, 8H), 7.45 (d, J = 6.6 Hz, 2H), 7.40 (d, J = 6.6 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.16 (t, J = 7.7 Hz, 2H), 6.96 (t, J = 7.4 Hz, 2H), 6.32 (d, J = 7.4 Hz, 2H), 2.98 - 2.88 (m, 1H), 1.23 (d, J = 6.9 Hz, 6H); 13 13C NMR (101 MHz, DMSO-d6) δ 168.34, 154.84, 149.62, 147.23, 145.83, 143.59, 141.09, 138.89, 136.91, 136.79, 133.34, 132.64, 132.40, 132.17, 131.03, 130.94, 129.77, 128.13, 127.48, 127.13, 127.01, 126.86, 126.78, 126.60, 126.01, 125.68, 122.65, 122.61, 33.80, 24.27.

[0088] 1H and 13C NMR spectra of PPY: 1 1H NMR (400 MHz, DMSO-d6) δ 9.27 (d, J = 7.3 Hz, 2H), 8.40 - 8.19 (m, 4H), 8.07 (d, J = 5.0 Hz, 2H), 7.95 (d, J = 6.7 Hz, 4H), 7.91 - 7.81 (m, 2H), 7.53 (d, J = 5.6 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.13 - 6.88 (m, 6H), 6.31 (d, J = 7.4 Hz, 2H), 3.02 - 2.87 (m, 1H), 1.24 (d, J = 6.8 Hz, 6H); 13C NMR (126MHz, DMSO-d6) δ167.41,155.16,155.04,151.22,150.27,149.59,148.00,147.88,144.52,144.06,142.20,139 .06,132.82,131.68,130.66,129.04,127.15,127.09,126.78,125.49,124.95,124.32,122.72,120.39,33.78,24.08.

[0089] BZQ's hydrogen spectrum and carbon spectrum: 1 H NMR(400MHz, DMSO-d6)δ9.16(s,2H),8.52(d,J=8.1Hz,2H),8.15(s,2H),8.09-7.93(m,6H),7.88(d,J=8.7Hz,2H),7.80( s,2H),7.58(d,J=7.9Hz,2H),7.47(s,2H),7.22(d,J=7.9Hz,4H),6.34(d,J=7.1Hz,2H),2.98-2.83(m,1H),1.19(s,6H); 13 C NMR(101MHz,DMSO-d6)δ156.98,149.79,149.19,148.11,148.07,144.31,143.22,141.98,140.89,140.73,137.92,137.87,137 .78,134.44,134.20,132.63,130.16,129.94,129.02,127.16,127.03,126.70,125.25,124.66,123.22,120.70,33.77,24.11.

[0090] Cytotoxicity assay of iridium complexes:

[0091] The CCK-8 method was used to determine the cytotoxicity of three complexes, PIQ, PPY, BZQ and paclitaxel PTX, against MDA-MB-231, MCF-7, 4T1, MCF-10A, HepG2, Hela, A549, U87, U251 and LO2 cells. The specific operation was as follows: cells in the logarithmic growth phase were taken and 5×10 3Cells were seeded at a density of 1000 cells / well in a 96-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours. Then, the culture medium containing PIQ, PPY, BZQ, or PTX was added as a replacement medium. After further culture for 48 hours, the culture medium was removed and a 10% (v / v) CCK-8 enhanced solution was added. The cells were incubated in the dark in an incubator for 30 minutes. The absorbance was measured at 450 nm using a microplate reader, and the cell inhibition rate was calculated according to the following formula. The results are shown in Table 1. Figure 1 and Table 1.

[0092] Cell inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0093] Where As is the absorbance of the experimental well, Ac is the absorbance of the control well, and Ab is the absorbance of the blank well.

[0094] Table 1 IC values ​​of PIQ, PPY, BZQ, and PTX 50 (n=3)

[0095] Type PIQ (μM) PPY (μM) BZQ (μM) PTX (μM) MDA-MB-231 39.06±6.15 4.55±0.49 32.96±5.78 38.35±6.37 MCF-7 11.56±0.07 1.81±0.12 16.51±2.13 21.47±4.95 MCF-10A 59.84±10.70 21.77±0.96 21.89±1.59 5.68±1.08 4T1 2.93±0.30 2.08±0.05 3.90±0.16 7.09±0.29 Hela 32.80±0.35 4.22±0.16 15.31±1.62 3.40±0.17 U87 5.32±0.27 1.28±0.22 15.68±1.39 2.77±0.45 HepG2 74.49±2.16 11.62±0.61 16.69±1.82 4.82±0.56 LO2 28.44±0.84 18.80±0.65 24.73±2.91 16.33±0.44 A549 7.89±0.11 1.22±0.08 3.49±0.61 0.53±0.04 U251 12.50±0.38 2.16±0.46 5.87±0.60 16.53±2.01

[0096] Depend on Figure 1 As can be seen from Table 1, the complex PPY has strong cytotoxicity to a variety of breast cancer cells and has a significant proliferation inhibitory effect compared with paclitaxel. 50 The value was 4.55 μM, and the IC value of MCF-7 cells was 50 The value is 1.81 μM, and the IC value for 4T1 cells is 50 The value is 2.08μM. In addition, the IC value of PPY for MCF-10A and LO2 cells is 50 The values ​​were 21.77 μM and 18.8 μM, respectively. The IC values ​​of paclitaxel for MCF-10A cells 50 The value is 5.68μM. The results show that PPY has a better inhibitory effect on breast cancer cells in vitro than paclitaxel, and its cytotoxicity to normal cells is lower than paclitaxel, indicating that PPY is more sensitive to breast cancer cells. At the same time, it was found that the three complexes have different inhibitory effects on proliferation of different cells. PPY not only has a strong inhibitory effect on breast cancer, but also has a strong inhibitory effect on U87. Its IC 50 The value is 1.28 μM.

[0097] Acute toxicity test on mice of PPY prepared in Example 3:

[0098] Experimental subjects: BALB / c female mice, weighing 18-20 g, 5 weeks old, purchased from Guangzhou Ruige Biotechnology Co., Ltd.

[0099] Experimental procedure: Weigh an appropriate amount of PPY, dissolve it in a mixture of PEG400 and ethanol (6:4), and dilute it with saline to an appropriate concentration for later use. 80 mice with good hair gloss and no abnormalities were selected for the experiment. All mice were randomly divided into 8 groups, with 10 mice in each group. The LD of mice was calculated using the Koch method. 50 . According to the results of the preliminary experiment, the groups were set as follows: concentration 1 (190 mg / kg), concentration 2 (168 mg / kg), concentration 3 (148 mg / kg), concentration 4 (131 mg / kg), concentration 5 (115 mg / kg), concentration 6 (102 mg / kg), concentration 7 (90 mg / kg), concentration 8 (0 mg / kg, i.e., solvent group). After the administration, the mice were observed for 14 consecutive days, and the death conditions and time of death of the mice were recorded. The mortality rate was calculated based on the number of deaths of mice, and the median lethal dose (LD) of the mice was calculated using the software Prism 8.0.2. 50 After 14 days, all mice were anesthetized, their eyeballs were removed and blood was collected. Serum was collected for biochemical analysis and liver and kidney function indicators (ALT, AST, CR, BUN) were tested.

[0100] Experimental results:

[0101] 1. Death of mice and time of death

[0102] Record the survival time of all mice within 14 days and draw a survival chart. The results are as follows Figure 2 and Figure 3 As shown, all mice in the 190 mg / kg group died on the same day after administration; 9 mice in the 168 mg / kg group died on the same day after administration; 5 mice in the 148 mg / kg group died on the same day, and 2 died the next day, for a total of 7 mice; 3 mice in the 131 mg / kg group died on the same day; 1 mouse in the 115 mg / kg group died on the same day; 1 mouse in the 102 mg / kg group died on the same day; no mice in the 90 mg / kg group died; and no mice in the vehicle group died.

[0103] 2. Mouse Blood Biochemical Indices

[0104] The surviving mice dosed with 131 mg / kg and 148 mg / kg were taken for blood biochemical analysis. The results are shown in Table 2.

[0105] Table 2 Blood biochemistry results of mice (n≥3)

[0106] ALT(U / L) AST(U / L) BUN (mM / L) CR (μM) 0mg / kg 31.66±4.51 81.33±6.11 8.48±0.34 9.97±1.29 131 mg / kg 25±4.24 80.75±13.58 7.51±0.98 10.48±0.38 148 mg / kg 27.5±3.41 92.25±5.96 8.42±0.64 10.85±1.05

[0107] As can be seen from Table 2, after 14 days, there was no significant difference in the liver and kidney function indicators of the mice in each group compared with the vehicle group, indicating that the body functions of the mice have recovered and no residue will remain in the body.

[0108] Example 4

[0109] A method for preparing iridium complex PLGA nanoparticles, comprising the following steps:

[0110] 100 mg of poly (lactide-co-glycolide) PLGA and 10 mg of PPY in Example 3 were dissolved in a mixed solvent consisting of 2 mL of dichloromethane and 1 mL of ethanol. The mixed solution was then rapidly injected into a 2% PVA solution using a syringe. Ultrasonication was performed for 15 min at an ultrasonic power of 260 W. After that, dichloromethane and ethanol were removed by vacuum rotary evaporation to obtain iridium complex PLGA nanoparticles, which were designated as PPY-NPS.

[0111] Performance test of the iridium complex PLGA nanoparticles prepared in Example 4:

[0112] 1. In vitro release behavior experiment: The in vitro release behavior of PPY-NPS was detected by dialysis under pH 5.2 and pH 7.4 conditions. The iridium complex PLGA nanoparticles prepared in Example 4 were redissolved with physiological saline to obtain PPY-NPS containing 1 mg of PPY. The PPY-NPS was transferred to a dialysis bag and placed in a phosphate buffer containing 40 mL of 2% sodium dodecyl sulfate (SDS). The mixture was shaken at a constant temperature (100 rpm / min) at 37°C and sampled at the following time points: 0.5h, 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, 72h, and 96h. 2 mL of sample was taken and 2 mL of release medium was added at the same time. The solution was taken out, diluted with the mobile phase, filtered through a 0.22 μm filter membrane, and detected by HPLC. The cumulative release amount was calculated according to the following formula, and the release curve was drawn. The experiment was repeated 3 times, and the results are as follows: Figure 4 As shown,

[0113]

[0114] Among them, Q n is the cumulative release rate of the complex, %; V is the total volume of the release medium, 40 mL; Cn is the concentration of the complex in the release medium at the nth sampling, μg·mL -1 ; m is the total amount of complex encapsulated by nanoparticles, μg; n is the number of sampling times.

[0115] Depend on Figure 4It can be seen that at 24 hours, the release of PPY-NPS at pH 7.4 reached 36%, while at pH 5.2 it reached 73%. At 48 hours, the release of PPY-NPS at pH 7.4 reached 51%, while at pH 5.2 it reached over 85%. This shows that PPY-NPS has a faster release rate and higher release rate in the acidic environment of the tumor, which indirectly indicates that PPY-NPS has pH-sensitive function.

[0116] 2. Cytotoxicity (using the aforementioned iridium complex cytotoxicity test method for detection, and using normal saline to redissolve the iridium complex PLGA nanoparticles)

[0117] The cytotoxicity and safety of the blank group (denoted as NPS), PPY-NPS, and PPY are shown in Table 3 and Figure 5 shown.

[0118] Table 3 Cytotoxicity and safety of NPS, PPY-NPS, and PPY (n=3)

[0119] NPS (μM) PPY (μM) PPY-NPS (μM) LO2 ＞200 18.80±0.65 ＞200*** 4T1 ＞200 2.08±0.05 1.45±0.10*** MCF-10A ＞200 21.77±0.96 ＞200***

[0120] Compared with the PPY group, ***P<0.001.

[0121] From Table 3 and Figure 5 It can be seen that NPS has almost no toxicity to LO2, MCF-10A and 4T1, indicating that the carrier is safe. 50 The IC values ​​of PPY-NPS on 4T1 cells were 18.8μM and 21.77μM respectively; PPY-NPS had almost no toxicity to LO2 and MCF-10A. 50 The IC value of PPY-NPS on 4T1 cells was 2.08 μM. 50 The concentration of PPY in PPY-NPS was 1.45 μM. In conclusion, PPY prepared into PPY-NPS can reduce toxicity and increase efficacy, and the difference is significant (P<0.001).

[0122] Comparative Example 1

[0123] The difference from Example 4 is that stirring and volatilization are used to remove dichloromethane and ethanol, and the remaining steps are the same as Example 4.

[0124] Comparative Example 2

[0125] The difference from Example 4 is that the ultrasonic power is 325W, and the remaining steps are the same as Example 4.

[0126] Comparative Example 3

[0127] The difference from Example 4 is that the ultrasonic power is 390W, and the remaining steps are the same as Example 4.

[0128] Comparative Example 4

[0129] The difference from Example 4 is that the ultrasonic treatment is performed for 10 minutes, and the remaining steps are the same as Example 4.

[0130] Comparative Example 5

[0131] The difference from Example 4 is that the ultrasonic treatment is performed for 20 minutes, and the remaining steps are the same as Example 4.

[0132] Comparative Example 6

[0133] The difference from Example 4 is that 100 mg of poly (lactide-co-glycolide) PLGA and 20 mg of PPY in Example 3 are dissolved in a mixed solvent consisting of 2 mL of dichloromethane and 1 mL of ethyl acetate, and the remaining steps are the same as in Example 4.

[0134] Comparative Example 7

[0135] The difference from Example 4 is that 100 mg of poly (lactide-co-glycolide) PLGA and 20 mg of PPY in Example 3 are dissolved in 3 mL of dichloromethane, and the remaining steps are the same as Example 4.

[0136] Comparative Example 8

[0137] The difference from Example 4 is that 100 mg of poly (lactide-co-glycolide) PLGA and 20 mg of PPY in Example 3 are dissolved in a mixed solvent consisting of 2 mL of dichloromethane and 1 mL of acetone, and the remaining steps are the same as in Example 4.

[0138] The particle size, polydispersity index (PDI), potential and stability of the iridium complex PLGA nanoparticles prepared in Example 4 and Comparative Examples 1-8 were tested. The results are shown in Tables 4-7.

[0139] Table 4 Particle size, PDI, potential and stability of iridium complex PLGA nanoparticles in Example 4 and Comparative Example 1

[0140] Particle size (nm) PDI Potential stability Comparative Example 1 --- --- --- Unstable Example 4 151.40±2.62 0.11±0.04 -3.95±0.15 No significant changes in 7 days

[0141] As can be seen from Table 4, the use of vacuum rotary evaporation (Example 4) to remove the organic phase can quickly solidify the nanoparticles, so that PPY is fixed inside the PLGA, is not easy to leak, and has good stability; while the stirring volatilization method (Comparative Example 1) cannot prepare nanoparticles.

[0142] Table 5 Particle size, PDI, potential and stability of iridium complex PLGA nanoparticles in Example 4 and Comparative Examples 2-3

[0143] Particle size (nm) PDI Potential (mV) stability Example 4 149.10±1.76 0.06±0.01 -4.56±0.05 No significant changes in 7 days Comparative Example 2 145.03±2.08 0.06±0.01 -2.21±0.05 No significant changes in 7 days Comparative Example 3 138.46±1.85 0.06±0.01 -2.29±0.15 No significant changes in 7 days

[0144] It can be seen from Table 5 that the particle size of the iridium complex PLGA nanoparticles decreases with the increase of ultrasonic power. This may be because the shear force increases with the increase of ultrasonic power, and the emulsion droplets formed are smaller, resulting in a smaller particle size of the nanoparticles.

[0145] Table 6 Particle size, PDI, potential and stability of iridium complex PLGA nanoparticles in Example 4 and Comparative Examples 4-5

[0146] Particle size (nm) PDI Potential (mV) stability Comparative Example 4 178.10±6.20 0.05±0.01 -5.16±0.07 No significant changes in 7 days Example 4 149.03±1.75 0.06±0.01 -2.21±0.15 No significant changes in 7 days Comparative Example 5 138.46±2.31 0.06±0.01 -5.24±0.05 No significant changes in 7 days

[0147] It can be seen from Table 6 that the particle size of the iridium complex PLGA nanoparticles decreases with the increase of ultrasonic time.

[0148] Table 7 Particle size, PDI, potential and stability of iridium complex PLGA nanoparticles in Example 4 and Comparative Examples 6-8

[0149] Particle size (nm) PDI Potential (mV) stability Comparative Example 6 125.17±1.55 0.10±0.03 -1.76±0.07 Precipitate within 2 days Comparative Example 7 168.96±3.35 0.11±0.02 -5.84±0.07 No significant changes in 7 days Example 4 149.10±1.76 0.06±0.01 -5.73±0.06 No significant changes in 7 days Comparative Example 8 124.23±0.72 0.11±0.01 -6.49±0.40 Next day precipitation

[0150] As can be seen from Table 7, the nanoparticles prepared in Comparative Example 6 using dichloromethane-ethyl acetate as the organic phase precipitated within 2 days and had a smaller particle size. The reason is that the hydrophilicity of ethyl acetate increases the solubility of the organic phase in the aqueous phase, reduces the surface tension of the oil-water interface, and forms smaller droplets under ultrasound, resulting in a smaller particle size. The precipitation within 2 days is due to the fact that during the solidification process of the nanoparticles, PPY penetrates the oil-water interface and the organic phase overflows from the aqueous phase, resulting in the precipitation of the nanoparticles. The particle size of the nanoparticles prepared in Comparative Example 7 using a single dichloromethane as the organic phase is too large, which may be due to the excessive removal of the organic phase during the rotary evaporation process, resulting in rapid solidification and formation of nanoparticles with a larger particle size. The nanoparticles prepared in Comparative Example 8 using dichloromethane-acetone as the organic phase precipitated the next day, which may be because water and acetone are miscible and cannot be completely removed, resulting in demulsification of the nanoparticles. The nanoparticles prepared in Example 4 using dichloromethane-ethanol as the organic phase have a moderate particle size and no significant change after 7 days, with good stability.

[0151] Antitumor effects of PPY prepared in Example 3 and PPY-NPS prepared in Example 4 in mice

[0152] Experimental subjects: BALB / c female mice, weighing 18-20 g, 5 weeks old, purchased from Guangzhou Ruige Biotechnology Co., Ltd.

[0153] Experimental process:

[0154] 1. Cell Culture

[0155] Mouse 4T1 breast cancer cells were selected and cultured in complete culture medium (10% fetal bovine serum, 1% double antibody, 89% 1640 culture medium). When the cell growth density reached 80%, the cells were passaged.

[0156] 2. Cell Seeding

[0157] 4T1 cells are homologous to BALB / c cells, so BALB / c female mice were selected as the model. Resuspend the cells in PBS and inject 100 μL of cell suspension subcutaneously into the axilla of each BALB / c mouse, approximately 5×10 5 cells.

[0158] 3. Palpation

[0159] Seven days after 4T1 cell inoculation, gently touch the inoculated area. If there is a movable hard lump the size of a mung bean and no inflammation at the inoculation site, it is determined that the tumor-bearing mouse model has been successfully established.

[0160] 4. Experimental Grouping and Dosage Regimen

[0161] When the tumor volume reaches 250±30mm 3 Forty-four mice with similar breast tumor volumes (i.e., model mice) and 11 normal mice were selected from the tumor-bearing model for the experiment. Paclitaxel (PTX), PPY, and PPY-NPS were dissolved in PEG400:anhydrous ethanol and then diluted with saline to the dosing concentration. The mice were then randomly divided into five groups of 11 mice each for a total of seven dosings, divided into the following groups:

[0162] ① Normal group: Normal mice were injected with normal saline via tail vein, with a volume of 0.1 mL·10 g -1 , administered every other day;

[0163] ② Negative group: Model mice were injected with normal saline via tail vein, with a volume of 0.1 mL·10 g -1 , administered every other day;

[0164] ③PTX group: PTX was injected into the tail vein of model mice, with a dosage of 0.1 mL·10 g -1 , the dose is 10 mg / kg, administered every other day;

[0165] ④PPY group: PPY was injected into the tail vein of model mice at a volume of 0.1 mL·10 g -1 , the dose is 10 mg / kg, administered every other day;

[0166] ⑤PPY-NPS group: PPY-NPS was injected into the tail vein of model mice at a volume of 0.1 mL·10 g -1 , the dose is 10 mg / kg, administered every other day.

[0167] Result determination:

[0168] During the experiment, mouse body weight and tumor volume were measured every other day. On day 14, six mice were selected from each group. After anesthesia, the eyeballs of the mice were removed and blood was collected. The mice were sacrificed by cervical dislocation and then dissected. The tumor, heart, liver, spleen, lung, kidney, and thymus were collected and weighed. Tumor volume, weight-based tumor inhibition rate, and volume-based tumor inhibition rate were calculated using the following formulas.

[0169]

[0170]

[0171]

[0172] 1. Mouse Weight Changes

[0173] The results of the weight changes of mice were as follows Figure 6 As shown, during the experiment, mice in the control group showed lethargy and rough, dull fur, while mice in the other groups had normal fur and no hair loss. At the end of the experiment, there was no significant difference in weight among the groups. The weight of mice in the PTX and PPY groups decreased over the first five days, then slowly increased. During the administration of PTX and PPY, the weight of mice in the PPY-NPS and normal groups increased. The weight of mice in the negative group remained largely unchanged during administration. This suggests that tail vein injection of PTX and PPY has certain toxic side effects, leading to slow weight gain and potentially affecting the mice's physical functions. PPY-NPS has good in vivo biosafety.

[0174] 2. Tumor inhibition effect

[0175] Tumor suppression effect Figure 7 As shown, a is the in vitro tumor image of mice, b is the change in tumor volume of mice within 14 days, c is the tumor mass image, d is the weight inhibition rate, and e is the volume inhibition rate (n=6, *P<0.05, ****P<0.0001). It can be seen that within 14 days of administration, the tumor volume of mice in the negative group increased significantly, while the tumor growth of mice receiving treatment was inhibited to varying degrees. Among them, PPY-NPS had the most obvious effect in inhibiting tumor growth. The in vitro tumor weight images of each group showed significant differences compared with the negative group (P<0.0001). Figure 7As shown in Figures d-7e, the weight-based tumor inhibition rates of PPY, PTX, and PPY-NPS were 62.63±13.83%, 63.14±9.28%, and 78.08±6.71%, respectively, with significant differences between the PPY, PTX, and PPY-NPS groups (P < 0.05). The volume-based tumor inhibition rates of PPY, PTX, and PPY-NPS were 69.26±9.82%, 67.90±5.84%, and 78.99±6.73%, respectively, with significant differences between the PPY and PPY-NPS groups (P < 0.05). PPY-NPS was more effective in inhibiting tumor growth than PPY and PTX, indicating that PPY-NPS can passively target tumors, allowing tumor cells to take up more PPY-NPS, thereby better exerting its anti-breast cancer effect and highlighting the advantages of nanoformulations in cancer treatment.

[0176] 3. H&E Staining

[0177] The internal organs and tumor sites of mice in each group were taken for HE staining and section analysis to examine the activity of each internal organ and tumor. The results are as follows Figure 8 As shown, cell nuclei appear blue. Compared with the normal group, the heart, kidney, and spleen of mice in all groups showed no significant morphological changes. Liver tissue sections from each group revealed that the negative group showed focal necrosis, hemorrhage, and inflammation; the PPY and PTX groups showed congestion; and the PPY-NPS group showed no significant differences from the normal group. Observation of lung tissue from each group revealed lesions and an abnormal proliferation of cells in the negative group, suggesting possible tumor metastasis. The remaining groups showed no significant changes compared to the normal group. Tumor cells in the negative group were abundant, densely packed, and plump, with large, darkly stained nuclei and virtually no necrosis. Tumor cell nuclei in all treatment groups showed significant cell shrinkage, with the order PPY-NPS > PPY > PTX > Negative. PPY-NPS nuclei were virtually absent, demonstrating the best therapeutic effect.

[0178] The above are merely preferred embodiments of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. An iridium complex, characterized in that The iridium complexes include [Ir(piq)2(ZZIP)]PF6, [Ir(ppy)2(ZZIP)]PF6, [Ir(bzq)2(ZZIP)]PF6; The structural formula of the cation of [Ir(piq)2(ZZIP)]PF6 is: The structural formula of the cation of [Ir(ppy)2(ZZIP)]PF6 is: The structural formula of the cation of [Ir(bzq)2(ZZIP)]PF6 is:

2. A method for preparing the iridium complex according to claim 1, characterized in that: The following steps are involved: (1) 4-isopropylbenzaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate are dissolved in glacial acetic acid and subjected to condensation reflux reaction, followed by cooling, pH adjustment, filtration, washing the precipitate and drying to obtain the ligand ZZIP; (2) IrCl3·H2O and 3-phenylisoquinoline, benzo[h]quinoline or 2-phenylpyridine are dissolved in a mixture of ethylene glycol monoethyl ether and water, and the mixture is condensed and refluxed under argon protection, cooled, filtered and vacuum dried to obtain iridium precursors Cis-[Ir(piq)2Cl]2, Cis-[Ir(bzq)2Cl]2, and Cis-[Ir(ppy)2Cl]2; (3) dissolving the ligand ZZIP obtained in step (1) and the iridium precursor obtained in step (2) in a mixture of dichloromethane and methanol, refluxing the mixture under argon protection, and adding ammonium hexafluorophosphate after cooling to room temperature. After stirring, filtering and vacuum drying, the iridium complex is obtained.

3. An iridium complex PLGA nanoparticle, characterized in that: The invention comprises the iridium complex according to claim 1 and poly(lactide-glycolide).

4. A method for preparing the iridium complex PLGA nanoparticles according to claim 3, characterized in that: The method comprises the following steps: dissolving poly(lactide-glycol) and the iridium complex according to claim 1 in an organic solvent, injecting the mixture into a PVA solution, performing ultrasonic treatment and removing the organic solvent to obtain iridium complex PLGA nanoparticles.

5. The method for preparing iridium complex PLGA nanoparticles according to claim 4, wherein The mass ratio of the poly(lactide-glycol) to the iridium complex is 10:(0.1-5).

6. The method for preparing iridium complex PLGA nanoparticles according to claim 4, wherein: The organic solvent is a mixed solvent of dichloromethane and ethanol.

7. The method for preparing iridium complex PLGA nanoparticles according to claim 4, wherein: The power of the ultrasound is 200-300W, and the time is 10-30 minutes.

8. The method for preparing iridium complex PLGA nanoparticles according to claim 4, characterized in that: The method of removing the organic solvent is vacuum rotary evaporation.

9. Use of the iridium complex according to claim 1 or the iridium complex PLGA nanoparticles according to claim 3 in the preparation of anti-tumor cell drugs.

10. The use according to claim 9, characterized in that The tumor cells include breast cancer cells.

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