Polyethylene glycol modified iridium coordination compounds, preparation and use thereof

Abstract

The application discloses a polyethylene glycol modified iridium coordination compound and a preparation and application thereof. The polyethylene glycol modified iridium coordination compound has the structure shown in the following formula I. The polyethylene glycol modified iridium coordination compound can self-assemble to form nanomicelles, and can be used for precise positioning of tumors after intravenous injection. The polyethylene glycol modified iridium coordination compound can also be combined with a polyethylene glycol modified indocyanine green to form a composition, the composition can self-assemble to form composite nanomicelles, and can be used for precise positioning of tumors after intravenous injection, and can be further used for photothermal treatment of tumors.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and pharmaceutics, specifically to a polyethylene glycol-modified iridium coordination compound and its preparation and application. Background Technology

[0002] Rapid and accurate tumor detection is crucial for subsequent cancer treatment. In recent years, in addition to upgrading and optimizing traditional imaging techniques such as positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI), significant progress has been made in the development of near-infrared dyes, such as cyanine dyes, phthalocyanines, porphyrin derivatives, and pyrroleboron compounds, which have had a profound impact on the field of tumor diagnosis. Among these, the cyanine dye indocyanine green has been approved by the U.S. Food and Drug Administration (FDA) and is used for imaging blood vessels, blood flow and tissue perfusion, lymph nodes, and lymphatic vessels. Although many studies have reported the use of indocyanine green and its derivatives for tumor tissue imaging, it has been observed that after intravenous injection, indocyanine green mainly accumulates in the liver, with some accumulation in the gastrointestinal tract, lymph nodes, spleen, or kidneys, leading to misdiagnosis of tumor sites.

[0003] As a significant characteristic of the tumor microenvironment and a marker of most solid tumors, hypoxia has been extensively studied in tumor diagnosis. To date, hypoxia probes can be broadly categorized into two types: one type utilizes specific enzymes (nitroreductases, quinone reductases, azobenzene reductases, etc.) found in the hypoxic microenvironment to catalyze chemical reactions in the functional groups (e.g., nitroaromatics, quinones, azobenzenes, etc.) of the probe structure, causing the probe to fluoresce under excitation wavelength illumination; the other type is designed based on the effect of oxygen concentration on probe fluorescence intensity. Specifically, at low oxygen concentrations, the probe fluoresces after excitation wavelength illumination, while fluorescence quenches when the oxygen concentration increases and reaches a certain threshold. Compared to normal human tissues, malignant tumor cells proliferate abnormally rapidly, constantly increasing their demand for oxygen and other energy substances. However, the inadequate and irregular development of new blood vessels in tumor tissues leads to… The blood supply balance of tumor tissue is disrupted, meaning the rate of blood oxygen supply cannot keep up with the rate of oxygen consumption by the tumor. This leads to a decrease in the oxygen content within the tumor, ultimately resulting in a hypoxic state. In this hypoxic state, the oxygen content can be less than 2%, and in extreme hypoxia, it can be below 0.1%. The degree of hypoxia is also commonly expressed in terms of oxygen partial pressure (pO2). When pO2 is below 14 mmHg, it is defined as a hypoxic state. When pO2 further decreases to below 2.5 mmHg, it enters a severe hypoxic state. Under normal physiological conditions, the average pO2 of tissues is 40 mmHg. Studies have shown that pO2 values ​​in tumor regions are often below 10 mmHg. One type of hypoxic probe is designed based on the effect of oxygen concentration on probe fluorescence intensity. It uses enzymes such as azobenzene reductase to catalyze chemical reactions in functional groups (e.g., nitroaromatics, quinones, azobenzene, etc.) within the probe structure, causing the probe to fluoresce under excitation wavelength light. Another type of hypoxic probe is designed based on the effect of oxygen concentration on probe fluorescence intensity. That is, when the oxygen concentration is low, the probe fluoresces after excitation wavelength irradiation, but the fluorescence quenches when the oxygen concentration increases and reaches a certain threshold. Compared to normal human tissues, malignant tumor cells proliferate abnormally rapidly, constantly increasing their demand for oxygen and other energy substances. However, the insufficient and irregular development of new blood vessels in tumor tissue disrupts the blood supply balance, meaning the rate of oxygen supply cannot keep up with the tumor's oxygen consumption. This leads to a decrease in oxygen content within the tumor, ultimately resulting in a hypoxic state. In this hypoxic state, the oxygen content can be less than 2%, and in extreme hypoxia, it can be below 0.1%. The degree of hypoxia is also commonly expressed using partial pressure of oxygen (pO2). When pO2 is below 14 mmHg, it is defined as a hypoxic state. When pO2 further decreases to below 2.5 mmHg, it enters a state of severe hypoxia. Under normal physiological conditions, the average pO2 of tissues is 40 mmHg.Studies have shown that pO2 values ​​in tumor regions are often below 10 mmHg.

[0004] Iridium coordination compounds (hereinafter referred to as complexes) have become a hot material for the preparation of hypoxia detection probes based on oxygen concentration due to their high phosphorescence yield, long lifetime, tunable excitation and emission wavelengths, and good biocompatibility. As the ligands of iridium complexes change, their application performance also changes accordingly. Currently reported host molecules of iridium complexes include benzothiophene pyridine, bis(2-(2'-benzothiophene)-pyridinecarboxylic acid-N,C3'), and 1-(9,9-dimethyl-9hfluorene-2-yl)isoquinoline, while co-ligand molecules generally include acetylacetone and 4,6-dioxolanic acid. Looking at the research reports on iridium complexes as hypoxia probes in recent years, most iridium complexes are hydrophobic molecules, making them unsuitable for intravenous administration. Xiao et al. designed and synthesized an iridium complex emitting red fluorescence, which was conjugated with bis(2-(2'-benzothiophene)-pyridinecarboxylic acid-N,C3') and 4,6-dioxovalerate to form a metal iridium complex probe, and encapsulated it in the hydrophobic cavity of cyanin-7 modified cyclodextrin to improve its water solubility (Xiao P, Liu C, Ma T, et al. A cyclodextrin-hosted Ir(III)complex for ratiometric mapping of tumor hypoxia in vivo. Advanced Science, 2021, 8(8): 2004044.); Ji et al. designed a small molecule probe based on an iridium complex, which was conjugated with iridium using 1-(9,9-dimethyl-9hfluorene-2-yl)isoquinoline as the main ligand to form a probe molecule, and encapsulated it in a composite hydrogel of carboxymethyl chitosan and sodium alginate for imaging of cell hypoxia (Ji S, Zhou S, Zhang X, et al. An oxygen-sensitive probe and a hydrogel for optical Imaging and photodynamic antimicrobial chemotherapy of chronic wounds. Biomaterials Science, 2022, 10(8): 2054-2061.); Both of these iridium complexes are hydrophobic molecules and can only be used as nano-hypoxic probes when loaded into nanocarriers formed by other materials. Summary of the Invention

[0005] This invention provides a polyethylene glycol-modified iridium coordination compound with a specific structure, using 6-benzo[b]thien-2-ylphenanthridine (BTPH) as the main ligand and polyethylene glycol-modified succinylacetone (SA-PEG) as the co-ligand. It exhibits hypoxia responsiveness, can be amphiphilic, and can self-assemble into nanomicelles. These micelles can be used directly without being loaded into nanocarriers formed by other materials, and have good water solubility, allowing for intravenous administration.

[0006] This invention discovers that the combined use of polyethylene glycol-modified iridium coordination compounds and polyethylene glycol-modified indocyanine green significantly enhances the photothermal effect of polyethylene glycol-modified indocyanine green. Therefore, it also provides a composition comprising polyethylene glycol-modified iridium coordination compounds and polyethylene glycol-modified indocyanine green, which can self-assemble into composite nanomicelles. These micelles can be used directly without being loaded into nanocarriers formed from other materials. This composition exhibits hypoxia responsiveness and photothermal effects, and can be used as or to prepare hypoxia fluorescent probes for tumor localization detection and imaging, or as or to prepare tumor photothermal therapy materials for photothermal treatment of tumors; it also has good water solubility and can be administered intravenously.

[0007] A polyethylene glycol-modified iridium coordination compound (i.e., polyethylene glycol-modified iridium complex, polyethylene glycol-modified iridium(III) complex, iridium(III) complex, (BTPH)2Ir(SA-PEG), Ir-PEG) has the structure shown in Formula I:

[0008]

[0009] Formula I.

[0010] The main ligand molecule of the Ir-PEG is 6-benzo[b]thiophene-2-phenanthridine, which has a large cyclic conjugated structure, resulting in a large Stockholm shift in the formed iridium(III) complex, which significantly enhances its photostability and imaging brightness; the emission wavelength has a significant redshift, and its penetration is stronger; the co-ligand molecule is polyethylene glycol-modified succinylacetone. The β-dicarbonyl structure of succinylacetone in this co-ligand molecule has the property of oxygen quenching after coordination with iridium(III), which makes Ir-PEG responsive to hypoxia. After succinylacetone is modified with polyethylene glycol of appropriate molecular weight, its hydrophobic properties are changed, and it becomes an amphiphilic molecule, so that the iridium(III) complex can self-assemble into nanomicelles.

[0011] In Formula I, n = 45-227. n is the number of repeating structural units (-CH2CH2O-) in Formula I. Within this range, Ir-PEG exhibits significant amphiphilicity (suitable hydrophilicity and lipophilicity) and readily self-assembles to form nanomicelles.

[0012] The polyethylene glycol-modified iridium coordination compound exhibits hypoxia responsiveness and can be used for precise localization of hypoxic tumors. It can be used directly as a hypoxia fluorescent probe or for preparing hypoxia fluorescent probes.

[0013] A polyethylene glycol-modified iridium coordination compound nanomicelle formed by the self-assembly of the aforementioned polyethylene glycol-modified iridium coordination compound. The self-assembly occurs in an aqueous environment.

[0014] The polyethylene glycol-modified iridium coordination compound nanomicelles exhibit hypoxia responsiveness and can be used for precise localization of hypoxic tumors. They can be used directly as hypoxia fluorescent probes or for preparing hypoxia fluorescent probes.

[0015] A composition comprising a polyethylene glycol-modified iridium coordination compound having the structure shown in Formula I and a polyethylene glycol-modified indocyanine green (ICG-PEG) having the structure shown in Formula II:

[0016]

[0017] Formula I;

[0018]

[0019] Formula II.

[0020] The composition exhibits hypoxia responsiveness and photothermal effect, and the combination of Ir-PEG and ICG-PEG can significantly enhance the photothermal effect of ICG-PEG.

[0021] In Formula II, m = 45-227. m is the number of repeating structural units (-CH2CH2O-) in Formula II. Within this range, ICG-PEG exhibits significant amphiphilicity (suitable hydrophilicity and lipophilicity) and readily self-assembles to form nanomicelles.

[0022] The values ​​of n and m can be the same or different, and have little effect on the performance of the composition.

[0023] There is no particular limitation on the amount of Ir-PEG and ICG-PEG in the composition. Since the hypoxia response of the composition is mainly attributed to Ir-PEG and the photothermal effect is mainly attributed to ICG-PEG, and the presence of Ir-PEG significantly enhances the photothermal effect of ICG-PEG, the amounts of both have little effect on the hypoxia response and photothermal effect exhibited by the composition. The amount of ICG-PEG in the composition can be designed according to the actual application scenario, such as therapeutic dosage. For example, the mass ratio of Ir-PEG to ICG-PEG in the composition can be designed to be 0.5-10:1.

[0024] The composition exhibits hypoxia responsiveness and photothermal effect, and can be used for precise localization and photothermal therapy of hypoxic tumors. It can be used directly as a hypoxia fluorescent probe for tumor localization detection and imaging, and can also be used to prepare hypoxia fluorescent probes and / or to prepare tumor photothermal therapy materials.

[0025] The composition self-assembles to form composite nanomicelles. This self-assembly occurs in an aqueous environment.

[0026] The composite nanomicelles exhibit hypoxia responsiveness and photothermal effect, and can be used for precise localization and photothermal therapy of hypoxic tumors. They can be used directly as hypoxia fluorescent probes for tumor localization detection and imaging, and can also be used to prepare hypoxia fluorescent probes and / or to prepare tumor photothermal therapy materials.

[0027] The hypoxia fluorescent probe includes hypoxia-induced tumor fluorescence localization material.

[0028] The preparation method of the polyethylene glycol-modified iridium complex includes the following steps:

[0029] (1) Synthesis of aminated polyethylene glycol (NH2-PEG): Polyethylene glycol was dissolved in a first organic solvent, and then phenyl p-nitrochloroformate, catalyst and triethylamine were added to the reaction system. The mixture was stirred overnight in an ice bath under inert gas protection. The filtrate was collected by filtration and precipitated with anhydrous diethyl ether. The precipitate was collected to obtain the intermediate product PEGylated phenyl p-nitrochloroformate. PEGylated phenyl p-nitrochloroformate and catalyst were dissolved in a first organic solvent to form a reaction system. Ethylenediamine dissolved in the first organic solvent was slowly added dropwise to the reaction system. After reacting at 20℃-30℃ for at least 12 h, the final reaction mixture was filtered. The filtrate was precipitated in diethyl ether and the precipitate was collected to obtain aminated polyethylene glycol (NH2-PEG).

[0030] (2) Synthesis of polyethylene glycol modified succinylacetone: Succinylacetone and NH2-PEG were taken, and 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl) were added. The mixture was stirred overnight under inert gas protection. After the reaction was completed, toluene was added, the filtrate was collected after filtration and dried to obtain the co-ligand polyethylene glycol modified succinylacetone.

[0031] (3) Synthesis of 6-benzo[b]thiophene-2-phenanthridine dichloride iridium(III): Tetra(triphenylphosphine)palladium, 6-chlorophenanthridine and benzo[b]thiophene-2-boronic acid were dispersed in a mixed solvent containing toluene, ethanol and saturated sodium carbonate aqueous solution. The mixture was refluxed under inert gas protection for at least 5 h. After the reaction was completed, water was added to the mixture after cooling, and then toluene was added for extraction. The organic phase was collected, washed with water, dried and purified to obtain 6-benzo[b]thiophene-2-phenanthridine. 6-benzo[b]thiophene-2-phenanthridine and iridium chloride trihydrate were added to a system of water and 2-ethoxyethanol. The mixture was refluxed under inert gas protection for at least 15 h. After the reaction was completed, the solid obtained by filtration was washed with water, methanol and petroleum ether in sequence, and dried to obtain 6-benzo[b]thiophene-2-phenanthridine dichloride iridium(III).

[0032] (4) Synthesis of polyethylene glycol modified iridium complex: 6-benzo[b]thiophene-2-phenanthrene dichloride iridium(III) and polyethylene glycol modified succinyl acetone were added to the system of 2-ethoxyethanol and triethylamine. The mixture was refluxed for at least 15 h under inert gas protection. After the reaction was completed, the reaction system was added dropwise to the first organic precipitant to precipitate. The precipitate was collected after filtration to obtain crude polyethylene glycol modified iridium complex. The crude product was dissolved in the second organic solvent, placed in a dialysis bag, and dialyzed in water for at least 5 h. The solution in the dialysis bag was collected and dried to obtain polyethylene glycol modified iridium complex.

[0033] In step (1), the amounts of polyethylene glycol and phenyl p-nitrochloroformate can generally be set according to the stoichiometric ratio of the chemical reaction, or polyethylene glycol or phenyl p-nitrochloroformate can be slightly or excessively added. The amounts of phenyl p-nitrochloroformate and ethylenediamine can generally be set according to the stoichiometric ratio of the chemical reaction, or phenyl p-nitrochloroformate or ethylenediamine can be slightly or excessively added to ensure complete reaction.

[0034] Optionally, the number of repeating structural units (-CH2CH2O-) in the polyethylene glycol is between 45 and 227, which is consistent with the number of repeating structural units (-CH2CH2O-) in the product.

[0035] Optionally, the first organic solvent is selected from organic solvents such as dichloromethane, acetone, and chloroform.

[0036] Optionally, the catalyst is 4-dimethylaminopyridine.

[0037] Maintaining the reaction temperature at 20℃-30℃ for more than 12 hours ensures the completion of the reaction.

[0038] In step (2), the amount of each reaction raw material can generally be set according to the stoichiometric ratio of the chemical reaction, or the amount of a certain raw material can be set to be slightly excessive or excessive.

[0039] The succinylacetone can be a commercially available product. Since commercially available succinylacetone is relatively expensive, it can be prepared using existing methods. For example, it can be prepared using the following method, which includes the following steps: dispersing sodium hydride and levulinic acid separately in a third organic solvent; slowly adding the system containing levulinic acid to the system containing sodium hydride at a uniform rate; stirring the reaction under ice bath conditions for at least 6 hours; after the reaction is completed, adjusting the pH of the reaction mixture to 1-2 with a pH adjuster; washing with saturated sodium chloride aqueous solution; separating the mixture; adding a back-extraction organic solvent to the aqueous phase for back-extraction; collecting all organic phases; drying and filtering to collect the filtrate; adding toluene to the filtrate and azeotropically boiling at 120℃-130℃ until the water is completely removed; and precipitating with ice-cold diethyl ether to obtain the brown solid product succinylacetone.

[0040] Optionally, the third organic solvent may be ethyl acetate or acetonitrile.

[0041] Optionally, the pH adjuster is an acidic pH adjuster, such as a commonly used acidic pH adjuster like a 10% (v / v) hydrochloric acid aqueous solution.

[0042] Optionally, the back-extraction organic solvent is selected from ethyl acetate, acetonitrile, N,N-dimethylformamide (DMF), etc.

[0043] In step (3), the amount of each reaction raw material can generally be set according to the stoichiometric ratio of the chemical reaction, or the amount of a certain raw material can be set to be slightly excessive or excessive.

[0044] The toluene, ethanol, and saturated sodium carbonate aqueous solution are used to disperse the reactants. There are no strict requirements on their amounts. Generally, the amount of saturated sodium carbonate aqueous solution used is less than that of toluene and ethanol. The volume ratio of toluene, ethanol, and saturated sodium carbonate aqueous solution can be 3-5:2-4:1.

[0045] Optionally, the purification is performed using a chromatography column, and the eluent is composed of ethyl acetate and petroleum ether in a volume ratio of 1:1.

[0046] The water, methanol, and petroleum ether are used as washing agents for the reactants. There are no strict requirements on the amount used; generally, the amount used is slightly more than the volume of the reactants to be washed.

[0047] In step (4), the amount of each reaction raw material can generally be set according to the stoichiometric ratio of the chemical reaction, or the amount of a certain raw material can be set to be slightly excessive or excessive.

[0048] Optionally, the first organic precipitant may be selected from diethyl ether, petroleum ether, cyclohexane, etc.

[0049] Optionally, the second organic solvent is selected from DMF, dimethyl sulfoxide (DMSO), acetonitrile, tetrahydrofuran, etc.

[0050] The polyethylene glycol-modified indocyanine green is obtained by esterification of carboxyl-terminated indocyanine green with aminated polyethylene glycol.

[0051] The preparation method of the polyethylene glycol-modified indocyanine green includes the following steps:

[0052] Carboxyl-terminated indocyanine green and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate were dissolved in a fourth organic solvent. N,N-diisopropylethylamine and amino-modified polyethylene glycol were added to the reaction system. The mixture was stirred overnight under inert gas protection. After the reaction was completed, some solvent was removed by concentration. The concentrated reaction system was added dropwise to a second organic precipitant to precipitate the precipitate. The precipitate was collected by filtration, dried and purified to obtain polyethylene glycol-modified indocyanine green (ICG-PEG).

[0053] The 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine are reaction catalysts, and their amounts are not strictly required. Generally, the molar amount of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate is equivalent to that of carboxyl-terminated indocyanine green, and N,N-diisopropylethylamine is used in excess of carboxyl-terminated indocyanine green (e.g., at least 6 times the amount). The amount of carboxyl-terminated indocyanine green can be used in excess according to the stoichiometric ratio of the chemical reaction, or it can be used in a molar amount twice that of the aminated polyethylene glycol.

[0054] Optionally, the fourth organic solvent, as the reaction solvent, can be one of organic solvents such as dichloromethane, trichloromethane, and tetrahydrofuran.

[0055] Optionally, the second organic precipitant can be one of the following: diethyl ether, petroleum ether, cyclohexane, etc.

[0056] Optionally, the purification is performed using a G-50 dextran gel chromatography column.

[0057] The inert gas used in this invention is selected from inert gases within the broad scope of the art, such as nitrogen, argon, and other inert gases.

[0058] The method for removing solvent and / or residual impurities from the solution can be an existing method such as rotary evaporation, dialysis, ultrafiltration, or neutral silica gel column chromatography.

[0059] The preparation method of the polyethylene glycol-modified iridium coordination compound nanomicelles includes the following steps:

[0060] Ir-PEG was dissolved in a fifth organic solvent and mixed evenly to obtain an Ir-PEG solution. The Ir-PEG solution was added dropwise to water and dialyzed to obtain polyethylene glycol-modified iridium coordination compound nanomicelles (M-Ir-PEG).

[0061] The fifth organic solvent is selected from DMF, DMSO, acetonitrile, tetrahydrofuran, etc. The fifth organic solvent is used to dissolve Ir-PEG, and there is no particular limitation on its amount; generally, 1 mg of Ir-PEG can be dissolved in 20 µL-30 µL of the fifth organic solvent.

[0062] The M-Ir-PEG has an average particle size of 122 nm ± 4.1 nm and a polydispersity index (PDI) of 0.187 ± 0.014.

[0063] The method for preparing the composite nanomicelles formed by the self-assembly of the composition includes the following steps:

[0064] Ir-PEG and ICG-PEG were dissolved in a sixth organic solvent and mixed evenly to obtain a mixed solution of Ir-PEG and ICG-PEG. The mixed solution of Ir-PEG and ICG-PEG was added dropwise to water and dialyzed to obtain Ir-PEG and ICG-PEG composite nanomicelles (M-Ir-ICG).

[0065] There is no particular limitation on the relationship between the amounts of Ir-PEG and ICG-PEG. Since the hypoxia responsiveness of the composition is mainly attributed to Ir-PEG, and the photothermal effect is mainly attributed to ICG-PEG, and the presence of Ir-PEG significantly enhances the photothermal effect of ICG-PEG, the amounts of both have little effect on the hypoxia responsiveness and photothermal effect exhibited by the composition. The amount of ICG-PEG in the composition can be designed according to the actual application scenario, such as therapeutic dosage. For example, the mass ratio of Ir-PEG to ICG-PEG in the composition can be designed to be 0.5-10:1. Different ratios of Ir-PEG to ICG-PEG will result in variations in the particle size of the composite nanomicelles. Studies show that the particle size of the composite nanomicelles decreases as the proportion of Ir-PEG increases. When the mass ratio of Ir-PEG to ICG-PEG is 10:1, the average particle size of the M-Ir-ICG is 172.6 nm ± 4.8 nm, and the PDI is 0.268 ± 0.028. When the mass ratio of Ir-PEG to ICG-PEG is 1:1, the average particle size of the M-Ir-ICG is 186.9 nm ± 5.4 nm, and the PDI is 0.279 ± 0.057. When the mass ratio of Ir-PEG to ICG-PEG is 1:2, the average particle size of the M-Ir-ICG is 330.2 nm ± 23.8 nm, and the PDI is 0.374 ± 0.062.

[0066] The sixth organic solvent is selected from DMF, DMSO, acetonitrile, tetrahydrofuran, etc. The sixth organic solvent is used to dissolve Ir-PEG and ICG-PEG, and there is no particular limitation on its amount; generally, 1 mg of Ir-PEG can be dissolved in 20 µL-30 µL of the sixth organic solvent.

[0067] The dialysis is performed in water using a dialysis bag (MW: 8000-14000) for at least 4 hours.

[0068] The polyethylene glycol-modified indocyanine green can be used to prepare single-compound nanomicelles. For example, the polyethylene glycol-modified indocyanine green self-assembles to form nanomicelles. The self-assembly occurs in a mixed environment of water and dichloromethane.

[0069] The preparation method of the polyethylene glycol-modified indocyanine green nanomicelles includes the following steps:

[0070] ICG-PEG was dissolved in dichloromethane, and deionized water was added to the dichloromethane. The mixture was then sonicated to completely emulsify the mixture, yielding polyethylene glycol-modified indocyanine green nanomicelles (M-ICG-PEG).

[0071] The ultrasound can be performed using a 600W ultrasonic probe with a 2-second working time and a 3-second interval.

[0072] The average particle size of the M-ICG-PEG is 305.0 nm ± 34.0 nm, and the PDI is 0.216 ± 0.035.

[0073] The tumors include those with a hypoxic environment, such as colon cancer.

[0074] The nanomicelles can be administered via intravenous injection, making them widely applicable in clinical settings.

[0075] The raw materials and reagents used in this invention can all be commercially available products.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] This invention utilizes 6-benzo[b]thiophene-2-phenanthridine and polyethylene glycol to modify the β-dicarbonyl group of succinylacetone and coordinate it with iridium(III) to obtain a polyethylene glycol-modified iridium(III) coordination compound. This compound contains a coordination structure of β-dicarbonyl group and iridium(III), which can generate phosphorescence after excitation. Since oxygen has a quenching effect on phosphorescence, and the oxygen partial pressure in the hypoxic region inside the tumor is very low, the phosphorescence generated by this compound and its nanomicelles in the hypoxic environment of the tumor can be used to achieve localization detection and imaging of the tumor.

[0078] This invention utilizes the absorption and scattering effects of indocyanine green molecules upon laser excitation, converting light energy into heat energy and raising the internal temperature of the molecules to produce a photothermal therapeutic effect on tumors. Polyethylene glycol-modified indocyanine green exhibits better photostability and water solubility than regular indocyanine green, resulting in greater accumulation at the tumor site and thus a better photothermal effect. Furthermore, this invention reveals that the combined use of polyethylene glycol-modified iridium coordination compounds and polyethylene glycol-modified indocyanine green significantly enhances the photothermal effect of polyethylene glycol-modified indocyanine green. The composite nanomicelles formed by Ir-PEG and ICG-PEG show a higher photothermal conversion efficiency than M-ICG-PEG, exhibiting a better photothermal effect. Therefore, the phosphorescence generated by the combination of polyethylene glycol-modified iridium coordination compounds and polyethylene glycol-modified indocyanine green, and the composite nanomicelles of the combination, in the hypoxic environment of tumors can be used for tumor localization, detection, and imaging. Moreover, the significant photothermal effect of the combination of these two compounds and the composite nanomicelles can be used for photothermal therapy of tumors. The composition of the present invention, and the composite nanomicelles of the composition, can not only accurately locate tumors in hypoxic environments, but also perform more efficient photothermal therapy on tumor tissues.

[0079] The polyethylene glycol-modified iridium coordination compounds and their nanomicelles, compositions, and composite nanomicelles of this invention are all applicable to various solid tumors with hypoxic environments. In hypoxic tumor environments, the excitation wavelength of the polyethylene glycol-modified iridium coordination compounds can be used to achieve direct and precise imaging and detection dependent on oxygen concentration. The compositions and composite nanomicelles can also utilize the excitation wavelength of polyethylene glycol-modified indocyanine green to provide highly efficient photothermal therapy to tumor tissue, achieving the goal of acting only on the tumor site without damaging other normal tissues. Furthermore, the detection-localization followed by photothermal therapy approach of the compositions and composite nanomicelles of this invention is a better integrated diagnostic and therapeutic method compared to existing technologies. It not only avoids the problem of inaccurate tumor localization caused by the tissue distribution of indocyanine green alone, but also utilizes and enhances the photothermal therapeutic effect of polyethylene glycol-modified indocyanine green on tumor tissue, giving the micelles both tumor localization and good photothermal therapeutic performance, making them highly clinically applicable.

[0080] The polyethylene glycol-modified iridium coordination compounds, their nanomicelles, compositions, and composite nanomicelles of this invention exhibit high sensitivity and good photochemical stability in hypoxia probe imaging analysis. Their large Stokes shift allows for excitation in the longer visible light wavelength region and provides good tissue penetration. Furthermore, they have good water solubility, making them suitable for intravenous administration.

[0081] The method for preparing nanomicelles in this invention is simple and suitable for large-scale industrial production and application. Attached Figure Description

[0082] Figure 1 This is a typical 1H NMR spectrum of the polyethylene glycol-modified iridium complex prepared in Example 1.

[0083] Figure 2 This is a typical 1H NMR spectrum of the polyethylene glycol-modified indocyanine green prepared in Example 2.

[0084] Figure 3A Transmission electron microscopy image of indocyanine green nanomicelles modified with polyethylene glycol alone; Figure 3B Transmission electron microscopy image of single polyethylene glycol-modified iridium complex nanomicelles; Figure 3C Transmission electron microscopy image of composite nanomicelles formed by self-assembly of polyethylene glycol-modified iridium complex and polyethylene glycol-modified indocyanine green in a mass ratio of 10:1. Figure 3D Transmission electron microscopy image of composite nanomicelles formed by self-assembly of polyethylene glycol-modified iridium complex and polyethylene glycol-modified indocyanine green in a 1:1 mass ratio; Figure 3E Transmission electron microscopy (TEM) image of composite micelles formed by self-assembly of polyethylene glycol-modified iridium complex and polyethylene glycol-modified indocyanine green in a mass ratio of 1:2.

[0085] Figure 4 Near-infrared thermograms of polyethylene glycol-modified indocyanine green nanomicelles (M-ICG-PEG), polyethylene glycol-modified iridium complex nanomicelles (M-Ir-PEG), composite nanomicelles (M-Ir-ICG), and PBS buffer under 808 nm laser illumination. In the figure, Time(s) represents time (seconds) and Temperature represents temperature.

[0086] Figure 5A Figure 1 shows the results of the cytotoxicity study of M-Ir-PEG before and after laser irradiation. Figure 5B Figure 1 shows the results of the cytotoxicity study of M-ICG-PEG before and after laser irradiation. Figure 5C The figures show the results of cytotoxicity studies of M-Ir-ICG before and after laser irradiation; where Concentration represents the concentration, Cell Viability represents cell viability, and NIR represents near-infrared laser irradiation (wavelength 808 nm).

[0087] Figure 6 This image shows the changes in fluorescence intensity of intracellular composite nanomicelles in CT-26 cells before and after laser irradiation; where NIR represents near-infrared laser irradiation (wavelength 808 nm), Concentration represents concentration, and FL intensity (10⁻¹⁰) represents the fluorescence intensity of the composite nanomicelles. 4 Counts / s) represents fluorescence intensity (10⁻⁶). 4 (counts / second), * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

[0088] Figure 7In vivo images of mice injected with M-ICG-PEG, M-Ir-PEG, and M-Ir-ICG via the tail vein under bright field and two laser channels; where Bright Field represents bright field; Ir channel represents Ir channel (excitation wavelength 540 nm) and ICG channel represents ICG channel (excitation wavelength 790 nm).

[0089] Figure 8 Infrared thermographs showing tumor temperature changes after laser irradiation with PBS buffer, M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG; where Time(s) represents time (seconds).

[0090] Figure 9 The images show H&E staining of tumors after different treatments with PBS buffer, M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG; where Laser+ indicates laser irradiation (wavelength 808nm) and Laser- indicates no laser irradiation.

[0091] Figure 10 The graphs show the tumor inhibition curves of PBS buffer, M-Ir-PEG, M-ICG-PEG and M-Ir-ICG treatments under no laser irradiation and laser irradiation; where Time (Day) represents time (days), Tumor Volume represents tumor volume, NIR represents near-infrared laser irradiation (wavelength 808nm), and * indicates p<0.05. Detailed Implementation

[0092] The principles and features of the present invention will be further described in detail below with reference to the specific embodiments shown, so that those skilled in the art can better understand the technical solution of the present invention. The specific embodiments listed are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0093] Example 1: Preparation of polyethylene glycol-modified iridium(III) complexes

[0094] (1) Preparation of aminated polyethylene glycol (NH2-PEG)

[0095] Polyethylene glycol 2000 (weight average molecular weight 2000, 10.0 g, 0.005 mol) was dissolved in dichloromethane (20 mL). Then, phenyl p-nitrochloroformate (6.0 g, 0.03 mol), 4-dimethylaminopyridine (61.0 mg), and triethylamine (2.0 mL) were added to the reaction system. The mixture was stirred overnight in an ice bath under an argon (Ar) protective atmosphere. The reaction mixture was filtered and precipitated in anhydrous diethyl ether. The precipitate was collected to obtain the intermediate product polyethylene glycolated phenyl p-nitrochloroformate. Polyethylene glycolated phenyl p-nitrochloroformate (974 mg, 0.45 mmol) and 4-dimethylaminopyridine (50 mg) were dissolved in dichloromethane (20 mL) to form a reaction system. Ethylenediamine (4.0 mL, 0.06 mol, with excess ethylenediamine to ensure complete reaction) was dissolved in an appropriate amount of dichloromethane and then slowly added dropwise to the reaction system. After reacting at room temperature for 12 h, the final reaction mixture was filtered and the resulting filtrate was precipitated twice in diethyl ether. The precipitate was collected to obtain aminated polyethylene glycol (NH2-PEG).

[0096] (2) Preparation of polyethylene glycol modified succinylacetone

[0097] Sodium hydride (12.0 g, 0.5 mol) was dispersed in 30 mL of ethyl acetate (0.307 mol) to obtain a system containing sodium hydride, and levulinic acid (12.0 g, 0.103 mol) was dispersed in 50 mL of ethyl acetate (0.512 mol) to obtain a system containing levulinic acid. The system containing levulinic acid was slowly added dropwise to the system containing sodium hydride through a constant pressure drop funnel. The reaction system was stirred for 6 h under ice bath conditions. After the reaction was completed, 10% (v / v) hydrochloric acid aqueous solution was added to the reaction mixture to adjust the pH to 1-2. The mixture was washed with saturated NaCl aqueous solution, separated, and back-extracted by adding ethyl acetate (100 mL × 3 times) to the aqueous phase. All organic phases were collected, and anhydrous sodium sulfate was added and allowed to stand overnight to remove water. After filtration, toluene (100 mL) was added to the filtrate and azeotropically boiled at 125 °C until water was completely removed. Finally, the product succinylacetone, a brown solid, was obtained by precipitation with ice-cold diethyl ether.

[0098] Take succinylacetone (0.339 g, 0.002 mol) and NH2-PEG (3.0 g, 1.44 mmol) obtained from the above reaction, add 4-dimethylaminopyridine (DMAP) (89 mg, 0.73 mmol) to the reaction system, stir for 30 min, then add EDC⋅HCl (356 mg, 1.86 mmol), and react and stir overnight under Ar atmosphere. After the reaction is complete, evaporate the solvent by rotary evaporation, add 50 mL of toluene, filter, and evaporate the filtrate to obtain the coligand polyethylene glycol modified succinylacetone.

[0099] (3) Preparation of 6-benzo[b]thiophene-2-phenanthridine iridium(III) dichloride

[0100] Tetra(triphenylphosphine)palladium (0.21 g, 0.18 mmol), 6-chlorophenanthridine (1.28 g, 6 mmol), and benzo[b]thiophene-2-boronic acid (1.28 g, 7.2 mmol) were dispersed in a mixed solvent consisting of toluene (10 mL), ethanol (5 mL), and saturated sodium carbonate aqueous solution (2 mL). The mixture was refluxed under an Ar atmosphere for 5 h. After the reaction was completed, 200 mL of water was added to the mixture, followed by extraction with toluene (50 mL × 3 times). The organic phase was collected and washed with deionized water (50 mL × 3 times). After drying with anhydrous sodium sulfate, the crude product 6-benzo[b]thiophene-2-phenanthridine was obtained by rotary evaporation. The crude product was purified by column chromatography using ethyl acetate:petroleum ether as eluent in a 1:1 (v / v) ratio to obtain pure 6-benzo[b]thiophene-2-phenanthridine as a pale yellow solid. The product obtained from the reaction, 6-benzo[b]thiophene-2-phenanthridine (685 mg, 2.2 mmol) and iridium chloride trihydrate (398.6 mg, 1.1 mmol), were added to a system of deionized water (10 mL) and 2-ethoxyethanol (30 mL). The mixture was refluxed for 15 h under an Ar atmosphere. After the reaction was completed, the solid obtained by cooling was filtered and washed successively with water, methanol and petroleum ether. The solid obtained was then dried in a vacuum drying oven. The final transition metal complex, 6-benzo[b]thiophene-2-phenanthridine dichloride iridium(III), was a purplish-brown solid powder.

[0101] (4) Preparation of polyethylene glycol-modified iridium(III) complexes

[0102] The 6-benzo[b]thiophene-2-phenanthridine iridium(III) dichloride (136 mg, 0.08 mmol) obtained in steps (2) and (3) and the co-ligand polyethylene glycol-modified succinylacetone (358.5 mg, 0.16 mmol) were added to a system of 2-ethoxyethanol (20 mL) and triethylamine (1.5 mL). The mixture was refluxed for 15 h under an Ar protective atmosphere. After the reaction was completed, the reaction system was added dropwise to diethyl ether to precipitate the precipitate. The precipitate was collected after filtration to obtain crude polyethylene glycol-modified iridium(III) complex. The crude polyethylene glycol-modified iridium(III) complex was dissolved in N,N-dimethylformamide (DMF) and placed in a dialysis bag (MWCO: 8000-14000). Deionized water was added for dialysis. After dialysis for 5 h, the product was lyophilized to obtain polyethylene glycol-modified iridium(III) complex (Ir-PEG).

[0103] A typical 1H NMR spectrum of Ir-PEG is shown below. Figure 1 This demonstrates the successful synthesis of Ir-PEG with the structure shown in Formula I.

[0104] Example 2: Preparation of polyethylene glycol-modified indocyanine green

[0105] Aminated polyethylene glycol (NH2-PEG) was prepared according to the method described in Example 1.

[0106] The synthetic route for carboxyl-terminated indocyanine green is as follows:

[0107]

[0108] 1,1,2-Trimethyl-1H-benzo[e]indole (compound 1; 4.3 g, 20.6 mmol) was weighed and dissolved in 20 mL of pre-dehydrated toluene. The reaction system was sealed after exhausting with high-purity (99.999% by volume) N2 and kept in the dark. Iodoethane (EtI, compound 2; 4.97 mL, 62.1 mmol) was slowly added dropwise with stirring. After the addition was complete, the mixture was refluxed at 120 °C (toluenereflux) for 12 h. The reaction was then allowed to cool naturally to room temperature. The mixture was filtered under reduced pressure, and the filter cake was washed successively with ice-cold diethyl ether and hexane. The cake was then dried under vacuum at room temperature to obtain a blue-purple powder, which is intermediate compound 3.

[0109] Compound 3 (4.0 g, 11.0 mmol) and pentadienal diphenylamine hydrochloride (compound 4; 4.0 g, 14.0 mmol) were weighed out, and 40 mL of acetic anhydride (Ac₂O) was added to the system. The reaction system was sealed after exhausting with high-purity N₂ and heated at 100 °C for 2 h in the dark. After the reaction was completed, the mixture was allowed to cool naturally to room temperature. The system was then slowly poured into 800 mL of deionized water, stirred in the dark for 20 min, and filtered under reduced pressure. The mixture was washed three times with deionized water. The product was dried under vacuum at room temperature to obtain a glossy black powder, which is intermediate compound 5.

[0110] 1,1,2-Trimethyl-1H-benzo[e]indole (4.2 g, 0.02 mol), 6-bromohexanoic acid (compound 6; 11.7 g, 0.06 mol), and potassium iodide (KI, compound 7; 9.96 g, 0.06 mol) were weighed sequentially. 20 mL of pre-dehydrated toluene was added to the system. The reaction mixture was vented with high-purity N2 and sealed, protected from light, and refluxed at 120 °C for 15 h. After the reaction was complete, the mixture was allowed to cool naturally to room temperature, filtered under reduced pressure, and washed successively with tetrahydrofuran, cold deionized water, and chloroform. The mixture was dried under vacuum at room temperature to obtain a pale yellow powder, which is intermediate compound 8.

[0111] Compound 8 (4.2 g, 9.3 mmol) and compound 5 (5.2 g, 9.3 mmol) were weighed, and 40 mL of anhydrous pyridine was added to the system. After purging with high-purity N2, the reaction system was sealed and kept in the dark. The reaction was heated at 40 °C for 2 h. After the reaction was completed, the pyridine was removed by rotary evaporation using an oil pump at 35 °C to obtain a dark green solid crude product. The product was purified by silica gel column chromatography with dichloromethane:methanol as the eluent at a ratio of 20:1 (v / v). The product was collected, and the eluent was removed by rotary evaporation using a water pump at 25 °C. The product was then dried under vacuum at room temperature to obtain a dark green solid compound 9, which is carboxyl-terminated indocyanine green (ICG-COOH).

[0112] Carboxyl-terminated indocyanine green (2.00 g, 3.20 mmol) and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (1.32 g, 3.20 mmol) were dissolved in dichloromethane (20 mL). N,N-diisopropylethylamine (18.25 mmol, 3.1 mL) and aminated polyethylene glycol (3.2 g, 1.60 mmol) were added to the reaction system. The mixture was stirred overnight under an argon atmosphere. After the reaction was completed, some solvent was removed by rotary evaporation. The concentrated reaction system was added dropwise to ice-cold diethyl ether to precipitate the product. The precipitate was collected by filtration and dried to obtain crude polyethylene glycol-modified indocyanine green (ICG-PEG). Purification was performed by G-50 dextran gel chromatography. The aqueous solution that passed through the gel chromatography column was lyophilized to obtain pure polyethylene glycol-modified indocyanine green (ICG-PEG).

[0113] A typical 1H NMR spectrum of ICG-PEG is shown below. Figure 2 This demonstrates the successful synthesis of ICG-PEG with the structure shown in Formula II.

[0114] Example 3

[0115] Preparation of M-Ir-PEG nanomicelles

[0116] 10 mg of polyethylene glycol-modified iridium(III) complex was dissolved in 200 µL of N,N-dimethylformamide and stirred until homogeneous to obtain an Ir-PEG solution. The Ir-PEG solution was then added dropwise to 1 mL of deionized water. The mixture was then transferred to a dialysis bag (MWCO: 8000-14000) and dialyzed in deionized water for 4 h. After dialysis, polyethylene glycol-modified iridium(III) complex nanomicelles (M-Ir-PEG) were obtained, which were formed solely from the polyethylene glycol-modified iridium(III) complex.

[0117] Preparation of M-ICG-PEG nanomicelles

[0118] Polyethylene glycol-modified indocyanine green (PEG) nanomicelles were prepared by an emulsification method. PEG-modified PEG (10 mg) was dissolved in dichloromethane (1.0 mL), followed by the addition of 2 mL of deionized water. The mixture was then completely emulsified using a 600 W ultrasonic probe (Ningbo Xinzhi JY92-2D) with a 2-second working interval followed by a 3-second interval, yielding PEG-modified PEG nanomicelles (M-ICG-PEG) formed solely from PEG-modified PEG.

[0119] Preparation of M-Ir-ICG nanomicelles

[0120] Polyethylene glycol-modified iridium(III) complexes and polyethylene glycol-modified indocyanine green were mixed at mass ratios of 10:1, 1:1, and 1:2, respectively, i.e., polyethylene glycol-modified iridium(III) complex (10 mg) and polyethylene glycol-modified indocyanine green (1 mg), polyethylene glycol-modified iridium(III) complex (10 mg) and polyethylene glycol-modified indocyanine green (10 mg), and polyethylene glycol-modified iridium(III) complex (10 mg) and polyethylene glycol-modified indocyanine green (20 mg), respectively, and dissolved in N,N-dimethylformamide (200 µL). Each of these three solutions was then added dropwise to 1 mL of deionized water. The mixtures were then transferred to dialysis bags (MWCO: 8000-14000) and dialyzed in deionized water for 4 h to obtain three different ratios of polyethylene glycol-modified iridium(III) complexes and polyethylene glycol-modified indocyanine green composite nanomicelles (M-Ir-ICG).

[0121] Example 4

[0122] Evaluation of in vitro photothermal performance

[0123] The M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG (Ir-PEG to ICG-PEG mass ratio 1:1) nanomicelle solutions prepared in Example 3, and PBS buffer solution (pH=7.4), were placed in quartz test tubes respectively. The solutions were then subjected to an 808 nm near-infrared laser (power 1W / cm²). 2 Under irradiation, the temperature changes of the solutions were recorded and photographed using a thermal imager (Fotric235, Shanghai, China). The temperature change curves of each solution over time are shown in the figure. Figure 4 The photothermal conversion efficiencies (i.e., photothermal conversion efficiencies) of M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG were calculated according to the formulas. The formulas are as follows: (1) and (2):

[0124]

[0125] Where h is the heat transfer coefficient, A is the container surface area (square meters), ΔTmax is the maximum temperature change of the M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG nanomicelle solutions (5.1℃, 14.9℃, and 22.5℃, respectively), ΔT is the temperature change at different time points, and Q is the temperature change at different time points. s The heat is related to the solvent's light absorption rate, I is the laser power, and A is the heat. λ The absorbance at 808 nm is A. 808 η is the photothermal conversion efficiency; Q s The value is 28mW, and I is 1.0W·cm. -2 A 808 The value is 2.4, m is 1g, and c is 4.2W / (g·℃).

[0126] According to formulas (1-2), the photothermal conversion efficiencies of M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG are 5.8%, 17.4%, and 20.2%, respectively. Compared with M-Ir-PEG, the photothermal conversion efficiencies of M-ICG-PEG and M-Ir-ICG are significantly improved (P < 0.001). The photothermal conversion efficiency of M-Ir-ICG is significantly higher than that of M-ICG-PEG (P < 0.05).

[0127] Figure 4 The results showed that the near-infrared heating temperature of pure M-Ir-PEG was not significant and was close to that of PBS buffer solution; the highest near-infrared heating temperature of pure M-ICG-PEG reached 39.9℃; the highest near-infrared heating temperature of M-Ir-ICG reached 46.9℃; under the same near-infrared laser irradiation time, the near-infrared heating temperature of M-Ir-ICG was significantly different from that of M-ICG-PEG (P<0.05), indicating that M-Ir-PEG in M-Ir-ICG can significantly enhance the near-infrared heating temperature of M-ICG-PEG, thus significantly increasing the near-infrared heating temperature of M-Ir-ICG.

[0128] Adjust to use 790nm-810nm near-infrared laser (power 1W / cm) 2 The materials were irradiated with 808nm near-infrared laser (power 1W / cm²), and the results were compared with those obtained by irradiating each group of materials with 808nm near-infrared laser (power 1W / cm²). 2 The results of the irradiation were quite similar.

[0129] Example 5

[0130] Cytotoxicity evaluation

[0131] Mouse colon cancer cells CT-26 were seeded in 96-well cell culture plates at a density of 2 × 10⁶ cells per well. 3Cells were cultured for 24 hours, and then different concentrations of M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG (Ir-PEG to ICG-PEG mass ratio 1:1) solutions (PBS buffer solution pH=7.4) were added. The concentrations of M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG ranged from 0-150 µg / mL. The concentrations of polyethylene glycol-modified iridium(III) complex and polyethylene glycol-modified indocyanine green in M-Ir-ICG were the same as those in M-Ir-PEG and M-ICG-PEG. Near-infrared illumination was performed using 808 nm near-infrared light (1 W / cm²). 2 Cells were irradiated (5 min). After 24 h of treatment, 32 μL of thiazolyl blue (MTT) solution (5 mg / mL) was added to each well. After 4 h of incubation, the liquid in the wells was aspirated and 200 μL of dimethyl sulfoxide was added. Cell viability was detected by MTT assay using a microplate reader (MK3, Thermo, America). Results are shown in the figure. Figure 5A , Figure 5B and Figure 5C .

[0132] Figure 5A , Figure 5B and Figure 5C The results showed that: there was no significant difference in cell viability between M-Ir-PEG without laser irradiation and after laser irradiation, indicating that M-Ir-PEG alone has no cytotoxicity against cancer cells such as colon cancer; however, there was a significant difference in cell viability between M-ICG-PEG alone without laser irradiation and after laser irradiation, with the cell viability after laser irradiation being significantly lower than that without laser irradiation, indicating that M-ICG-PEG alone has significant cytotoxicity against cancer cells such as colon cancer after laser irradiation; and there was a significant difference in cell viability between M-Ir-ICG composite micelles without laser irradiation and after laser irradiation, with the cell viability of M-Ir-ICG composite micelles after laser irradiation (17.7%-88.0%) being significantly lower than that of M-ICG-PEG alone after laser irradiation (22.4%-93.0%) (p<0.001), indicating that M-Ir-ICG composite micelles after laser irradiation has significant cytotoxicity against cancer cells such as colon cancer.

[0133] Example 6

[0134] Evaluation of the detection capability of probe M-Ir-ICG under hypoxic conditions

[0135] To investigate the sensitivity of M-Ir-ICG to hypoxia in mouse colon cancer cells CT-26, the fluorescence intensity of iridium in mouse colon cancer cells was detected. Cells were cultured at 1.0 × 10⁶ cells per well in 24-well plates. 5Mouse colon cancer cells CT-26 were cultured for 24 h and then treated with 100, 200, and 400 µg / mL M-Ir-ICG (Ir-PEG to ICG-PEG mass ratio 1:1) solution (PBS buffer solution at pH 7.4) as the solvent. After incubation with M-Ir-ICG for 24 h, the cells were then treated with an 808 nm laser (1 W / cm²). 2 Irradiation was performed for 5 minutes. Polyethylene glycol-modified indocyanine green, under laser irradiation, consumes oxygen molecules in the culture medium to generate reactive oxygen species (ROS), thus creating a hypoxic environment (simulating a hypoxic environment). The fluorescence intensity of the polyethylene glycol-modified iridium(III) complex in the cells was analyzed using flow cytometry (CytoFLEX S, Beckman, USA). The results are shown in [Figure number missing]. Figure 6 .

[0136] Figure 6 The results showed that without laser irradiation, the fluorescence intensity of M-Ir-ICG at all concentrations was very low. However, after laser irradiation, the fluorescence intensity increased with increasing M-Ir-ICG concentration. This indicates that M-Ir-ICG can enter CT-26 cells, and that the concentration of ICG-PEG in M-Ir-ICG also increases with increasing M-Ir-ICG concentration. Under laser irradiation, the hypoxia level of the environment increases, thereby enhancing the fluorescence intensity of Ir-PEG in M-Ir-ICG, indicating that M-Ir-ICG has a hypoxia-responsive characteristic.

[0137] Example 7

[0138] Evaluation of in vivo hypoxia sensitivity of M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG nanomicelles

[0139] BALB / c mice were injected with 3×10⁻⁶ saturates in the right axilla. 6 A subcutaneous tumor model was established using mouse colon cancer cells CT-26. When the tumor volume reached 400 mm², a tumor was successfully treated. 3 At approximately 2:00-7:00 h, micelles were prepared into solutions with a concentration of 0.5 mg / mL using PBS buffer at pH 7.4. These solutions were then injected via the tail vein with M-Ir-PEG (5 mg / kg), M-ICG-PEG (5 mg / kg), and M-Ir-ICG (Ir-PEG to ICG-PEG mass ratio 1:1) (10 mg / kg), respectively. In vivo imaging of mice was performed at each time point (2, 6, 12, 24, 48, and 72 h) using an IVISSpectrum (Perkin Elmer, USA) system. The excitation wavelength for the polyethylene glycol-modified iridium(III) complex was 540 nm and the emission wavelength was 710 nm, while the excitation wavelength for the polyethylene glycol-modified indocyanine green was 790 nm and the emission wavelength was 810 nm. Results are shown below. Figure 7 .

[0140] like Figure 7 As shown, the strong fluorescence of M-Ir-PEG in the Ir-PEG laser channel (i.e., the Ir channel, excitation wavelength 540 nm) accurately localized to the tumor site, while the strong fluorescence of M-ICG-PEG in the ICG-PEG laser channel (i.e., the ICG channel, excitation wavelength 790 nm) was mainly observed in tissues such as the liver. This indicates that M-Ir-PEG can be used as a hypoxia probe for localizing tumor sites or for preparing hypoxia probes for localizing tumor sites. Similarly, the strong fluorescence of M-Ir-ICG in the Ir-PEG laser channel (i.e., the Ir channel, excitation wavelength 540 nm) accurately localized to the tumor site, while the strong fluorescence in the ICG-PEG laser channel (i.e., the ICG channel, excitation wavelength 790 nm) was significantly deviated from the tumor site. The fluorescence intensity of the Ir channel reached its maximum value 12 h after administration and was still detectable after 72 h; this indicates that M-Ir-ICG can be used as a hypoxia probe for localizing tumor sites or for preparing hypoxia probes for localizing tumor sites. When used for precise tumor localization, the Ir channel, i.e., the excitation wavelength 540 nm, is preferred for more accurate localization. After accurately locating the tumor site, the tumor site is then irradiated with near-infrared light at a wavelength of 790-810nm (preferably 808nm) for photothermal therapy, achieving precise treatment and avoiding treatment errors.

[0141] Example 8

[0142] In vivo thermal performance evaluation of M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG

[0143] BALB / c mice were injected with 3×10⁻⁶ saturates in the right axilla. 6 A subcutaneous tumor model was established using mouse colon cancer cells CT-26. Each micelle was prepared into a solution with a concentration of 0.5 mg / mL using PBS buffer at pH 7.4. When the tumor volume reached approximately 100 mm², the tumor was... 3 At that time, PBS buffer solution (pH=7.4) (0.1 mL), M-Ir-PEG (5 mg / kg), M-ICG-PEG (5 mg / kg), and M-Ir-ICG (Ir-PEG to ICG-PEG mass ratio 1:1) (10 mg / kg) were injected via tail vein. 24 hours later, an 808 nm laser (1 W / cm²) was used. 2 The tumor site was irradiated for 5 minutes, and temperature changes at the tumor site were recorded and photographed using a thermal imager (Fotric235, Shanghai, China). Results are shown below. Figure 8 .

[0144] As shown in Figure 8, after irradiation with an 808 nm laser, the tumor site temperature in the M-ICG-PEG group and the M-Ir-ICG group was significantly higher than that in the PBS and M-Ir-PEG groups, with the highest local temperatures reaching 41.9℃ and 44.9℃, respectively. The results indicate that when M-ICG-PEG and M-Ir-PEG are co-assembled, the photothermal properties of M-Ir-ICG are significantly enhanced. Under the same near-infrared laser irradiation time, the near-infrared heating temperature of M-Ir-ICG at the tumor site was significantly different from that of M-ICG-PEG (P < 0.05), indicating that M-Ir-PEG in M-Ir-ICG can significantly enhance the near-infrared heating temperature of M-ICG-PEG at the tumor site, resulting in a significant increase in the near-infrared heating temperature of M-Ir-ICG at the tumor site.

[0145] Example 9

[0146] Evaluation of the in vivo antitumor effects of M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG

[0147] BALB / c mice were injected with 3×10⁻⁶ saturates in the right axilla. 6 A subcutaneous tumor model was established using mouse colon cancer cells CT-26. Each micelle was prepared into a solution with a concentration of 0.5 mg / mL using PBS buffer (pH 7.4). When the tumor volume reached 100 mm², the tumor was further investigated. 3 On days 1, 3, and 5, patients received intravenous injections via tail vein of PBS buffer solution (pH=7.4) (0.1 mL), M-Ir-PEG (5 mg / kg), M-ICG-PEG (5 mg / kg), and M-Ir-ICG (Ir-PEG to ICG-PEG mass ratio 1:1) (10 mg / kg). The near-infrared light irradiation group received 808 nm laser (1 W / cm²) on days 2, 4, and 6. 2 The tumor sites of mice were irradiated with laser light for 5 minutes. After 14 days, the mice were sacrificed, and major organs and tumors were collected for hematoxylin and eosin (H&E) staining. H&E staining images of tumors before and after laser irradiation are shown below. Figure 9 .

[0148] Figure 9The results showed that: in the PBS group, there were little change in tumor tissue before and after laser irradiation; tumor cell nuclei were clear and cell growth was good. In the M-Ir-PEG group, there were little change in tumor tissue before and after laser irradiation; tumor cell nuclei were clear and cell growth was good. In the M-ICG-PEG group, tumor cell nuclei were clear and cell growth was good before laser irradiation; after laser irradiation, tumor cell nuclei disappeared and cells died. In the M-Ir-ICG group, tumor cell nuclei were clear and cell growth was good before laser irradiation; after laser irradiation, tumor cell nuclei disappeared and cells died. Furthermore, the degree of cell nucleus disappearance and cell necrosis in the M-Ir-ICG group was significantly higher than that in the M-ICG-PEG group. Additionally, H&E staining results for other organs, including the heart, liver, spleen, lungs, and kidneys, showed no significant changes before and after laser irradiation, similar to those in the PBS group, indicating that other organs, including the heart, liver, spleen, lungs, and kidneys, were not damaged by the various treatments.

[0149] Example 10

[0150] Evaluation of the in vivo antitumor effects of M-Ir-PEG, M-ICG-PEG, and M-Ir-ICG

[0151] BALB / c mice were injected with 3×10⁻⁶ saturates in the right axilla. 6 A subcutaneous tumor model was established using mouse colon cancer cells CT-26. Each micelle was prepared into a solution with a concentration of 0.5 mg / mL using PBS buffer (pH 7.4). When the tumor volume reached 100 mm², the tumor was further investigated. 3 On days 1, 3, and 5, patients received intravenous injections via tail vein of PBS buffer solution (pH=7.4) (0.1 mL), M-Ir-PEG (5 mg / kg), M-ICG-PEG (5 mg / kg), and M-Ir-ICG (Ir-PEG to ICG-PEG mass ratio 1:1) (10 mg / kg). The near-infrared light irradiation group received 808 nm laser (1 W / cm²) on days 2, 4, and 6. 2 The tumor sites of mice were irradiated (5 min). Mouse weight and tumor volume were recorded every 2 days. The length and width of the mouse tumors were measured using calipers, and the tumor volume was calculated. The evaluation results of the antitumor effects of different formulations in vivo are shown below. Figure 10 .

[0152] like Figure 10 As shown, the tumors in the four groups (PBS, M-Ir-PEG, M-ICG-PEG, and M-Ir-PEG) that did not receive near-infrared irradiation grew rapidly, with no statistically significant difference; at the end of the experiment, the tumor volume of all groups was >1200 mmHg. 3In the groups receiving near-infrared irradiation: the PBS+NIR group and the M-Ir-PEG+NIR group showed some inhibition of tumor growth, but the final tumor volume in the PBS+NIR group was still greater than 1200 mm². 3 The final tumor volume in the M-Ir-PEG+NIR group was still greater than 700 mm. 3 In contrast, the M-ICG-PEG+NIR and M-Ir-ICG+NIR groups showed significant tumor inhibition effects, indicating that under near-infrared laser irradiation, both M-ICG-PEG and M-Ir-ICG exhibited significant tumor inhibition effects, with M-Ir-ICG showing the best effect, significantly better than M-ICG-PEG. At 12 days of treatment, the tumor inhibition rate (TIR) ​​of the M-Ir-ICG+NIR group was 89.1%, significantly higher than the 72.6% TIR of the M-ICG-PEG+NIR group, with a statistically significant difference (* indicates P < 0.05). This effect is attributed to the higher photothermal conversion efficiency of M-Ir-ICG. Therefore, compared to M-ICG-PEG, M-Ir-ICG has a better photothermal effect and thus a better tumor inhibition effect. Furthermore, the mice in each experimental group gradually gained weight normally, indicating that the 1W / cm² ratio selected in this invention... 2 The power is relatively mild for biological use. After treatment, the tumor tissues of each experimental group were separated and photographed. The tumor tissue volume of the M-Ir-ICG+NIR group was the smallest, less than 100 mm. 3 The results showed that M-Ir-ICG has significant anti-tumor effects and can be used as a tumor photothermal therapy material for treating tumors, and can also be used to prepare tumor photothermal therapy materials for treating tumors.

[0153] In this invention, polyethylene glycol with a weight-average molecular weight of 2000-10000 is suitable for modifying iridium coordination compounds and indocyanine green. It has no significant impact on the tumor localization and self-assembly of iridium coordination compounds modified with polyethylene glycol, the photothermal effect and self-assembly of indocyanine green modified with polyethylene glycol, etc.

[0154] In this invention, the reaction between aminated polyethylene glycol and carboxyl-terminated indocyanine green involves an amidation reaction between the amino group on the aminated polyethylene glycol and the carboxyl group on the carboxyl-terminated indocyanine green to form an amide bond; the reaction between polyethylene glycol-modified succinylacetone and dichloro-6-benzo[b]thiophene-2-phenanthrenediphenyl iridium(III) involves a coordination complexation reaction between the β-dicarbonyl group of polyethylene glycol-modified succinylacetone and iridium(III) to form a coordination compound. Therefore, any reaction conditions suitable for the amidation reaction between the amino group and the carboxyl group to form an amide bond, and any reaction conditions suitable for the coordination complexation reaction between the β-dicarbonyl group and iridium(III) to form a coordination compound, can ensure the progress of the reaction, thereby obtaining the polyethylene glycol-modified iridium(III) complex and polyethylene glycol-modified indocyanine green with the structure described in this invention.

[0155] Variations in the parameters of the micelle preparation method of this invention do not affect the preparation of single micelles or composite micelles. Therefore, any combination of parameters in the preparation method of this invention can achieve the preparation of single micelles or composite micelles. Further details will not be elaborated here.

Claims

1. A polyethylene glycol-modified iridium coordination compound, characterized in that, It has the structure shown in Equation I: In Equation I, n = 45 - 227.

2. The application of the polyethylene glycol-modified iridium coordination compound according to claim 1 as a hypoxia fluorescent probe or in the preparation of hypoxia fluorescent probes.

3. Polyethylene glycol-modified iridium coordination compound nanomicelles formed by self-assembly of the polyethylene glycol-modified iridium coordination compound according to claim 1.

4. The application of the polyethylene glycol-modified iridium coordination compound nanomicelles according to claim 3 as a hypoxia fluorescent probe or in the preparation of hypoxia fluorescent probes.

5. A composition, characterized in that, Including polyethylene glycol-modified iridium coordination compounds having the structure shown in Formula I and polyethylene glycol-modified indocyanine green having the structure shown in Formula II: In Equation I, n = 45 - 227; In Equation II, m = 45 - 227.

6. The composition according to claim 5, characterized in that, The mass ratio of the polyethylene glycol-modified iridium coordination compound to the polyethylene glycol-modified indocyanine green is 0.5-10:

1.

7. The use of the composition according to claim 5 or 6 in the preparation of hypoxia fluorescent probes and / or in the preparation of tumor photothermal therapy materials.

8. Composite nanomicelles formed by self-assembly of the composition according to claim 5 or 6.

9. The application of the composite nanomicelles according to claim 8 in the preparation of hypoxia fluorescent probes and / or in the preparation of tumor photothermal therapy materials.

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