CN114853758B

CN114853758B - Iridium complex for photo-thermal/photodynamic co-therapy, preparation method and application

Info

Publication number: CN114853758B
Application number: CN202210395873.1A
Authority: CN
Inventors: 胡黎文; 阳仁强; 肖标
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2022-04-14
Filing date: 2022-04-14
Publication date: 2023-03-28
Anticipated expiration: 2042-04-14
Also published as: CN114853758A

Abstract

The invention belongs to the technical field of antitumor drugs, and particularly discloses an iridium complex for photothermal/photodynamic synergistic treatment, and a preparation method and application thereof. The iridium complex of the invention has a chemical structure as shown in formula (I):

the iridium complex of the invention has long service life of triplet exciton, is easy to generate energy transfer with oxygen, and thus can be used for photodynamic therapy in hypoxic environment of tumor. The D-A-D unit chelated with the complex can be used for photothermal therapy, and the combination of the two can be used for photothermal/photodynamic synergistic therapy. The iridium complex Ir-M has a unique steric hindrance molecular configuration, so that the absorption response range of the iridium complex Ir-M is widened, and the near infrared region absorption is realized; the steric hindrance of the D-A-D unit is large, the accumulation of molecules in a high-concentration state of the nano particles can be effectively avoided, the interaction force among the molecules is weakened, and the photo-thermal effect is improved. The preparation method of the iridium complex has the advantages of easily available raw materials, mild synthesis conditions, simple preparation method, convenient and fast purification, easy realization and great application prospect.

Description

Iridium complex for photo-thermal/photodynamic cooperative therapy, preparation method and application

Technical Field

The invention belongs to the technical field of antitumor drugs, and particularly relates to an iridium complex for photothermal/photodynamic synergistic treatment, and a preparation method and application thereof.

Background

The existing cancer treatment method mainly takes traditional surgical excision as a main part and is assisted by chemical drug therapy and radiotherapy. The traditional treatment method has certain effect, but also has limitations, such as high recurrence possibility, high drug toxic side effect and the like. Photodynamic therapy is somewhat complementary to traditional methods. For example, photodynamic therapy has low toxic side effects, no drug resistance, can be used for repeated treatments, and can act synergistically with other treatments. As an emerging cancer treatment method, photothermal therapy has received wide attention due to its advantages of minimal or no trauma, short treatment practice, little effect on normal cells and tissues, and low side effects. One of the typical characteristics of tumors is hypoxia, and during the process of photodynamic therapy, intracellular oxygen is gradually depleted, so that the effect in the later period of the therapy is reduced and finally the expected therapeutic effect cannot be achieved. Therefore, it is an effective means to combine other therapeutic means (e.g. photothermal therapy, chemotherapy, etc.) to improve the therapeutic effect of cancer. Of these, the synergistic treatment combining photodynamic therapy with photothermal therapy has attracted great interest to scientists. The reason is that photothermal effect is generated during photothermal therapy, the temperature of cancer cells is increased, blood circulation is accelerated, more oxygen is promoted to be conveyed to focal tissues, and the condition of oxygen deficiency in the cancer cells is improved to a certain extent. In addition, the two treatment methods can use laser as a light source, have simple and convenient operation and can reduce the complexity and the treatment cost of cancer treatment. Therefore, it is of great importance to prepare photosensitizers capable of undergoing photodynamic/photothermal therapy.

At present, two or more components are mainly adopted to respectively play the functions of photodynamic therapy and photothermal therapy. For example, polydopamine (PDA) with good biocompatibility is first prepared into small-sized nanoparticles by loading hemoglobin (Hb provides oxygen) and photosensitizer (Ce 6) as a carrier, then the nanoparticles are wrapped in acid-sensitive PEG-PEI micelles, and then Hyaluronic Acid (HA) capable of tumor targeting is modified on the surface, so that the nanoparticles become a high-permeability and acid-sensitive release composite nanocarrier for photodynamic/photothermal synergistic therapy (ACS Nano 2020,14,12, 17046-17062). However, the environment in the organism is very complex, the drug stability of the blending system is poor, the preparation process is complicated, dissociation is very easy to occur in the organism, the expected treatment effect cannot be realized, side effects can be increased, and in addition, the possibility that two light sources with different wavelengths are needed for excitation is provided. The other is that the photosensitizer is combined with the photothermal material in a covalent bond mode, so that the stability of the photosensitizer can be improved, and the treatment effect can be enhanced. But this is difficult to synthesize. Therefore, photodynamic therapy and photothermal therapy are simultaneously realized through simple single molecules, the preparation process is simple, the single laser light source can be used for excitation, the treatment operation is simple, and the development of the photosensitizer has important practical significance.

Disclosure of Invention

To overcome the above-mentioned drawbacks and deficiencies of the prior art, it is a primary object of the present invention to provide an iridium complex for photothermal/photodynamic co-therapy.

Another object of the present invention is to provide a method for preparing the iridium complex for photothermal/photodynamic synergistic therapy.

The invention further aims to provide the application of the iridium complex in preparing a photosensitizer for tumor photothermal/photodynamic synergistic treatment.

The purpose of the invention is realized by the following technical scheme:

an iridium complex for photothermal/photodynamic synergistic therapy is named as Ir-M, and the chemical structure of the iridium complex is shown as the formula (I):

wherein R is a linear or branched alkyl group having 1 to 30 carbon atoms.

Preferably, R is a linear or branched alkyl group having 8 to 16 carbon atoms.

More preferably, R is 2-ethylhexane, corresponding to Ir-M having the formula:

more preferably, R is 2-hexyldecyl, and the formula corresponding to Ir-M is as follows:

the preparation method of the iridium complex for photothermal/photodynamic synergistic therapy comprises the following steps:

(1) Reacting 4, 7-dibromo-2, 1, 3-benzothiadiazole for 24 hours at 140 ℃ under the action of iron powder and glacial acetic acid to generate a ring-opening reaction to obtain a compound M1;

(2) Reacting the compound M1 for 12 hours at 25 ℃ under the action of glacial acetic acid and sodium nitrite, and performing a ring-closing reaction to obtain a compound M2;

(3) Dissolving a compound M2 in N, N-Dimethylformamide (DMF), carrying out alkylation reaction with alkyl bromide (R-Br) under the action of potassium carbonate, and reacting at the temperature of 110 ℃ for 12 hours to obtain a compound M3;

(4) Carrying out nitration reaction on the compound M3 in a concentrated nitric acid (the mass fraction is about 68%) and concentrated sulfuric acid (the mass fraction is about 98%), and reacting for 12 hours at 100 ℃ to obtain a compound M4;

(5) The compound M4 is subjected to a reduction reaction under the action of iron and glacial acetic acid, and the reaction is carried out for 20 hours at 140 ℃ to obtain a compound M5;

(6) Carrying out condensation reaction on the compound M5 and 1, 10-phenanthroline-5, 6-diketone under the action of glacial acetic acid, and reacting for 24 hours at 110 ℃ to obtain a compound M6;

(7) Carrying out Suzuki coupling on the compound M6 and 4-triphenylamine borate in an alkaline environment of palladium tetratriphenylphosphine and potassium carbonate by using absolute ethyl alcohol and tetrahydrofuran as solvents, and reacting at 80 ℃ for 12 hours to obtain a compound M7;

(8) Compound M7 is reacted with bis (1, 5-cyclooctadiene) iridium (I) chloride dimer in

Reacting the molecular sieve with o-xylene in a mixed system at 115 ℃ for 24 hours to obtain the iridium complex Ir-M.

Further, in the step (1), the molar ratio of the 4, 7-dibromo-2, 1, 3-benzothiadiazole to the iron powder is 1. The ratio of the molar weight (mmol) of the 4, 7-dibromo-2, 1, 3-benzothiadiazole to the volume (mL) of glacial acetic acid is 1.

In step (2), the molar ratio of compound M1 to sodium nitrite is 1. The ratio of the molar amount (mmol) of compound M1 to the volume of glacial acetic acid (mL) is 1.

Further, in the step (3), R-in the alkyl bromide (R-Br) is a straight-chain or branched-chain alkyl with 1-30 carbon atoms; preferably, R is a linear or branched alkyl group having 8 to 16 carbon atoms; more preferably, R is 2-ethylhexane or 2-hexyldecyl.

Further, the molar ratio of the compound M2, alkyl bromide (R — Br), and potassium carbonate in step (3) is 1 to 1.5, preferably 1; the ratio of the molar amount (mmol) of compound M2 to the volume (mL) of N, N-dimethylformamide is 1.

Further, in step (4), the ratio of the molar amount (mmol) of the compound M3 to the volume (mL) of concentrated nitric acid is 1. The volume ratio of the concentrated nitric acid to the concentrated sulfuric acid is 1.

In step (5), the molar ratio of the compound M4 to the iron powder is 1. The ratio of the molar amount (mmol) of compound M4 to the volume of glacial acetic acid (mL) is 1.

Further, in the step (6), the molar ratio of the compound M5 to the 1, 10-phenanthroline-5, 6-dione is 1. The ratio of the molar amount (mmol) of compound M5 to the volume of glacial acetic acid (mL) is 1.

Further, in step (7), the molar ratio of the compound M6, triphenylamine-4-borate, palladium tetratriphenylphosphine, potassium carbonate is 1. The ratio of the molar weight (mmol) of the compound M6 to the volume (mL) of absolute ethanol and the volume (mL) of tetrahydrofuran is 1.

Further, in the step (8), the molar ratio of the compound M7 to bis (1, 5-cyclooctadiene) iridium (I) chloride dimer is 1. Mass (g) of compound M7 and

the mass (g) of the molecular sieve and the volume (mL) ratio of o-xylene are 1.

The specific preparation route of the iridium complex Ir-M is as follows:

the synthesized iridium complex is applied to preparing a photosensitizer for tumor photothermal/photodynamic synergistic treatment.

When the iridium complex and the amphiphilic polymer are applied specifically, the iridium complex and the amphiphilic polymer are dissolved in an organic solvent, ultrapure water is added into a system, the system is dispersed uniformly by ultrasonic, then the organic solvent is removed, and water-soluble iridium complex nano-particles are obtained, wherein the apparent concentration of the water-soluble iridium complex nano-particles is more than or equal to 30 mu g/mL.

Further, the organic solvent is selected from one of tetrahydrofuran, acetone, N-dimethylformamide and dimethyl sulfoxide. Tetrahydrofuran is preferred.

Further, the amphiphilic polymer is F127 (amphiphilic triblock polymer formed by ethoxy-propoxy), DSPE-mPEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000), or DSPE-mPEG5000 (distearoylphosphatidylethanolamine-polyethylene glycol 5000).

Further, the tumor is a subcutaneous tumor.

The iridium complex for photo-thermal/photodynamic cooperative therapy can be used for photodynamic therapy in the hypoxic environment of tumors because the triplet exciton of the noble metal iridium has long service life and is easy to generate energy transfer with oxygen. The D-A-D unit chelated with the complex can be used for photothermal therapy, and the combination of the two can be used for photothermal/photodynamic synergistic therapy. The side chain contains a long alkyl chain, so that the solubility of the iridium complex Ir-M can be effectively improved, the preparation of nanoparticles is facilitated, the movement of the alkyl chain is facilitated, the non-radiative transition is improved, and the photo-thermal effect is enhanced. The iridium complex Ir-M has a unique steric hindrance molecular configuration, so that the absorption response range of the iridium complex Ir-M can be widened, and the near-infrared absorption can be realized; the steric hindrance of the D-A-D unit is large, the accumulation of molecules in a high-concentration state of the nano particles can be effectively avoided, the interaction force among the molecules is weakened, the intra-molecular motion is enhanced, and the photo-thermal effect is improved.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the iridium complex is a photosensitizer for tumor photothermal/photodynamic synergistic therapy, and can be used as a photothermal agent for photothermal therapy and a photosensitizer for photodynamic therapy. Can be excited by a single wavelength light source (wavelength range is 750-1700 nm) to be used for photodynamic/photothermal combination therapy, and has the advantages of multiple functions and simple and convenient operation.

2. The iridium complex has no toxicity to cancer cells under the action of laser and has good biocompatibility; has high damage effect on cancer cells under near infrared light irradiation (wavelength range of 750-1700 nm); the cells are cancer cells, particularly 4T1 cells.

3. Through reasonable structural design, the iridium complex Ir-M is beneficial to intramolecular movement, improves the nonradiative transition probability of the iridium complex Ir-M, further improves the photothermal conversion efficiency of the iridium complex Ir-M, and has better effect in near-infrared photothermal therapy. The iridium complex has excellent light stability and chemical properties, and also has high light-heat conversion efficiency and active oxygen yield; the absorption range is wide, the absorption response of a near infrared region I (750-1000 nm) or a near infrared region II (1000-1700 nm) can be realized, the photothermal therapy can be realized by penetrating into a focus region, the treatment efficiency is high, the side effect is less, and the application prospect is realized.

4. The preparation method of the iridium complex has the advantages of easily available raw materials, mild synthesis conditions, simple preparation method and convenient purification.

Drawings

FIG. 1 is a diagram of an ultraviolet-visible absorption spectrum of an iridium complex Ir-M1 in a tetrahydrofuran solution.

Fig. 2 is a graph of photothermal therapy test results of iridium complex Ir-M1 nanoparticles on 4T1 cells.

FIG. 3 is a photo-thermal performance test chart of the iridium complex Ir-M1.

Fig. 4 is an ultraviolet visible absorption spectrum of ABDA under Ir-M1 nanoparticles and light conditions.

Fig. 5 is a photo-thermal/photodynamic co-therapy test result diagram of iridium complex Ir-M1 nanoparticles on 4T1 cells.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

EXAMPLE one preparation of the Compound Ir-M1

(1) Preparation of 3, 6-dibromo-1, 2-phenylenediamine (M1)

4, 7-dibromo-2, 1, 3-benzothiadiazole (2.94g, 10mmol), iron powder (28mg, 0.5mmol) and glacial acetic acid (80 mL) were added to a 100mL two-necked flask under an argon atmosphere, and reacted at 140 ℃ for 24 hours. The reaction was stopped, quenched with water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, the solution was concentrated and recrystallized from tetrahydrofuran and ethanol to give compound M1 in 89% yield. The MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(2) Preparation of 4, 7-dibromo-1H-benzotriazole (M2)

Compound M1 (10.8g, 20mmol) and sodium nitrite (5.52g, 80mmol) were added to 100mL of glacial acetic acid under an argon atmosphere, and reacted at 25 ℃ for 12 hours. After the reaction was stopped, the reaction was quenched with water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, the solution was concentrated and recrystallized from tetrahydrofuran and ethanol to obtain compound M2 in 82% yield. MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(3) Preparation of 4, 7-dibromo-2- (2-hexyldecyl) -2H-benzo [ d ] [1,2,3] triazole (M3)

In a 250mL two-necked flask, compound M2 (1.38g, 5 mmol), 1-bromo-2-hexyldecane (1.68g, 5.5 mmol), potassium carbonate (4.14g, 30mmol), 50mLN, N-Dimethylformamide (DMF) were charged under an argon atmosphere, and reacted at 110 ℃ for 12 hours. After the reaction was stopped, the reaction was quenched with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solution was concentrated and purified by silica gel column chromatography, and the mixture of petroleum ether and dichloromethane (the volume ratio of petroleum ether to dichloromethane was 4) was used as an eluent, and dried to obtain compound M3 with a yield of 92%. The MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(4) Preparation of 4, 7-dibromo-2- (2-hexyldecyl) -5, 6-dinitro-2H-benzo [ d ] [1,2,3] triazole (M4)

Under an argon atmosphere, compound M3 (2.51g, 5 mmol) was added to a mixed system of 80mL of 98wt% concentrated sulfuric acid and 68wt% concentrated nitric acid (volume ratio 1. After the reaction was stopped, the reaction system was poured into ice water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, the solution was concentrated and purified by silica gel column chromatography with dichloromethane as eluent, and dried to obtain compound M4 with a yield of 75%. The MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(5) Preparation of 4, 7-dibromo-2- (2-hexyldecyl) -2H-benzo [ d ] [1,2,3] triazole-5, 6-diamine (M5)

Compound M4 (2.96g, 5 mmol) and iron powder (28mg, 0.5 mmol) were added to 80mL of glacial acetic acid under an argon atmosphere, and reacted at 140 ℃ for 20 hours. The reaction was stopped, quenched with water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, the solution was concentrated and recrystallized from tetrahydrofuran and ethanol to give compound M5 in 84% yield. The MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(6) Preparation of 10, 14-dibromo-12- (2-hexyldecyl) -12H-bipyridine [3,2-a:2',3' -c ] [1,2,3] triazolo [4,5-i ] phenazine (M6)

Compound M5 (2.66g, 5 mmol), 1, 10-phenanthroline-5, 6-dione (1.16g, 5.5 mmol) was added to 60mL of glacial acetic acid under an argon atmosphere, and reacted at 110 ℃ for 24 hours. The reaction was stopped, quenched with water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, the solution was concentrated and purified by silica gel column chromatography with a mixed solvent of petroleum ether and dichloromethane (volume ratio 1) as eluent, and dried to obtain compound M6 in 64% yield. The MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(7) Preparation of 4,4' - (12- (2-hexyldecyl) -12H-bipyridine [3,2-a:2',3' -c ] [1,2,3] triazolo [4,5-i ] phenazine-10, 14-diyl) bis (N, N-diphenylamine) (M7)

To a 150mL two-necked flask, under an argon atmosphere, compound M6 (2.17g, 3mmol), triphenylamine 4-borate (2.17g, 7.5 mmol), tetratriphenylphosphine palladium (0.17g, 0.15mmol), a 50% by mass aqueous solution of potassium carbonate (3.31g, 24mmol/3.31mL deionized water), 9mL anhydrous ethanol and 60mL tetrahydrofuran were added, and the mixture was heated to 80 ℃ and reacted for 12 hours. After the reaction was stopped, the reaction was quenched with water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, and the solution was concentrated and purified by silica gel column chromatography, and the mixture of petroleum ether and dichloromethane (the volume ratio of petroleum ether to dichloromethane was 2) was used as an eluent, and dried to obtain a compound with a yield of 72%. The MS and element analysis results show that the obtained compound is a target product M7, and the chemical reaction equation of the preparation process is as follows:

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(8) Preparation of Compound Ir-M1

To a 150mL two-necked flask, molecular sieves (A), (B), (C) and (C) were added under an argon atmosphere

4.615 g), bis (1, 5-cyclooctadiene) Iridium (I) chloride dimer ([ Ir (COD) Cl)] ₂ ) (0.60g, 0.89mmol), compound M7 (9.23g, 8.93mmol), 134mL of o-xylene, at 115 ℃ for 24 hours. After the reaction was stopped, the reaction was quenched with water, extracted with dichloromethane, and dried with anhydrous magnesium sulfate, and the solution was concentrated and purified by silica gel column chromatography, and the mixed solvent of petroleum ether and dichloromethane (the volume ratio of petroleum ether to dichloromethane was 2) was used as a eluent, and dried to obtain the compound Ir-M1 with a yield of 48%. MS and element analysis results show that the obtained compound is a target product Ir-M1, and the chemical reaction equation of the preparation process is as follows:

Ir-M1 (chemical structural formula is C) ₁₉₂ H ₁₈₇ IrN ₂₇ ) The theoretical molecular weight is 3063.51, and the test result of matrix assisted laser desorption tandem time of flight mass spectrometer (MALDI-TOF) is 3064.47[ deg. ] M + H ⁺ ]. Testing the contents of C, H and N elements in Ir-M1 by adopting a PerkinElmer element analyzer (the model is EA-2400 II), wherein the theoretical value is 75.24 percent of C; 6.15 percent of H; n is 12.34 percent. The measured value is C:76.36%; 6.21 percent of H; n is 12.56 percent. The mass spectrum and element analysis test results are close to the theoretical values, and the synthesized product is proved to be the target product. Testing the absorption spectrum of Ir-M1 in tetrahydrofuran solution by an ultraviolet visible absorption spectrometer, wherein the sample concentration is 5 multiplied by 10 ^-6 mol/L. From the test results (FIG. 1), it can be seen that Ir-M1 has an absorption spectrum ranging from 400 to 1200nm and can realize NAbsorption response in IRI and NIR II.

(9) Preparation of nanoparticles

In order to adapt to complex water environment in organisms, 10mg of oil-soluble photosensitizer Ir-M1 and 120mg of amphiphilic polymer F127 (amphiphilic triblock polymer formed by ethoxy-propoxy) are completely dissolved in 2.0mL of tetrahydrofuran solution, under the condition of ultrasonic treatment, the mixed system is rapidly added into 20mL of ultrapure water, the ultrasonic treatment is continued for 10min to ensure that the system is uniformly dispersed, then nitrogen is blown into a sample to remove tetrahydrofuran, and finally the sample is stored in a refrigerator at 4 ℃ for later use.

The apparent concentration of the prepared water-soluble Ir-M1 nanoparticles (Ir-M1 NPs) is 500 mug/mL. The size of Ir-M1 NPs was measured using a Malvern laser particle sizer (Mastersizer 3000), and the results indicated that the particle size was 124nm and the polydispersity PDI was 0.23.

EXAMPLE II preparation of the Compound Ir-M2

(1) Preparation of 4, 7-dibromo-2- (2-ethylhexyl) -2H-benzo [ d ] [1,2,3] triazole (M8)

In a 250mL two-necked flask, under an argon atmosphere, compound M2 (1.38g, 5 mmol), 1-bromo-2-ethylhexane (1.06g, 5.5 mmol), potassium carbonate (4.14g, 30mmol), 50mLN, N-Dimethylformamide (DMF) were charged and reacted at 110 ℃ for 12 hours. After the reaction was terminated, the reaction was quenched with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solution was concentrated and purified by silica gel column chromatography, and the mixture of petroleum ether and dichloromethane (the volume ratio of petroleum ether to dichloromethane was 4) was used as an eluent, and dried to obtain compound M8 with a yield of 89%. MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(2) Preparation of 4, 7-dibromo-2- (2-ethylhexyl) -5, 6-dinitro-2H-benzo [ d ] [1,2,3] triazole (M9)

Under an argon atmosphere, compound M8 (1.94g, 5 mmol) was added to a mixed system of 80mL of 98wt% concentrated sulfuric acid and 68wt% concentrated nitric acid (volume ratio 1. After the reaction was stopped, the reaction system was poured into ice water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, the solution was concentrated and purified by silica gel column chromatography with dichloromethane as eluent, and dried to obtain compound M9 with a yield of 70%. The MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(3) Preparation of 4, 7-dibromo-2- (2-ethylhexyl) -2H-benzo [ d ] [1,2,3] triazole-5, 6-diamine (M10)

Compound M9 (2.38g, 5 mmol) and iron powder (28mg, 0.5 mmol) were added to 80mL of glacial acetic acid under an argon atmosphere, and reacted at 140 ℃ for 20 hours. The reaction was stopped, quenched with water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, the solution was concentrated and recrystallized from tetrahydrofuran and ethanol to give compound M10 in 84% yield. MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(4) Preparation of 10, 14-dibromo-12- (2-ethylhexyl) -12H-bipyridine [3,2-a:2',3' -c ] [1,2,3] triazolo [4,5-i ] phenazine (M11)

Compound M5 (2.09g, 5 mmol), 1, 10-phenanthroline-5, 6-dione (1.16g, 5.5 mmol) was added to 60mL of glacial acetic acid under an argon atmosphere, and reacted at 110 ℃ for 24 hours. The reaction was stopped, quenched with water, extracted with dichloromethane and dried over anhydrous magnesium sulfate, the solution was concentrated and purified by silica gel column chromatography using a mixed solvent of petroleum ether and dichloromethane (petroleum ether to dichloromethane volume ratio of 2. The MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(5) Preparation of 4,4' - (12- (2-ethylhexyl) -12H-bipyridine [3,2-a:2',3' -c ] [1,2,3] triazolo [4,5-i ] phenazine-10, 14-diyl) bis (N, N-diphenylamine) (M12)

To a 150mL two-necked flask, under an argon atmosphere, compound M11 (1.77g, 3 mmol), triphenylamine-4-borate (2.17g, 7.5 mmol), palladium tetrakistriphenylphosphine (0.17g, 0.15mmol), a 50% by mass aqueous potassium carbonate solution (3.31g, 24mmol/3.31mL deionized water), 9mL anhydrous ethanol and 60mL tetrahydrofuran were added, and the mixture was heated to 80 ℃ and reacted for 12 hours. After the reaction was stopped, the reaction was quenched with water, extracted with dichloromethane and dried with anhydrous magnesium sulfate, and the solution was concentrated and purified by silica gel column chromatography using a mixed solvent of petroleum ether and dichloromethane (the volume ratio of petroleum ether to dichloromethane was 1) as an eluent, and dried to obtain compound M12 in a yield of 69%. The MS and element analysis results show that the obtained compound is a target product, and the chemical reaction equation of the preparation process is as follows:

(6) Preparation of the Compound Ir-M2

4.615 g), bis (1, 5-cyclooctadiene) Iridium (I) chloride dimer ([ Ir (COD) Cl)] ₂ ) (0.60g, 0.89mmol), compound M12 (8.23g, 8.93mmol), 134mL of o-xylene, at 115 ℃ for 24 hours. After the reaction is stopped, quenching the reaction by water, extracting by dichloromethane, drying by anhydrous magnesium sulfate, concentrating the solution, purifying by silica gel column chromatography,and (3) taking a mixed solvent of petroleum ether and dichloromethane (the volume ratio of the petroleum ether to the dichloromethane is 1). MS and element analysis results show that the obtained compound is a target product Ir-M2, and the chemical reaction equation of the preparation process is as follows:

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Ir-M2 (chemical structural formula is C) ₁₆₈ H ₁₃₉ IrN ₂₇ ) The theoretical molecular weight is 2727.13, and the test result of matrix assisted laser desorption tandem time of flight mass spectrometer (MALDI-TOF) is 2728.21[ 2 ] M + H ⁺ ]. Testing the contents of C, H and N elements in Ir-M2 by adopting a PerkinElmer element analyzer (the model is EA-2400 II), wherein the theoretical value is 73.96 percent; 5.14 percent of H; and N is 13.86 percent. The measured value is C:74.25%; 5.23 percent of H; n is 14.03 percent. The mass spectrum and element analysis test results are close to the theoretical values, and the synthesized product is proved to be the target product. Testing the absorption spectrum of Ir-M2 in tetrahydrofuran solution by an ultraviolet visible absorption spectrometer, wherein the sample concentration is 5 multiplied by 10 ^-6 mol/L. From the test results, the absorption spectrum range of Ir-M2 is 400-1200 nm, and the absorption response of an NIR I region and an NIR II region can be realized.

(7) Preparation of nanoparticles

In order to adapt to complex water environment in organisms, 10mg of oil-soluble photosensitizer Ir-M2 and 150mg of amphiphilic polymer F127 (amphiphilic triblock polymer formed by ethoxy-propoxy) are completely dissolved in 3.0mL of tetrahydrofuran solution, under the condition of ultrasonic treatment, the mixed system is rapidly added into 20mL of ultrapure water, the ultrasonic treatment is continued for 10min to ensure that the system is uniformly dispersed, then nitrogen is blown into a sample to remove tetrahydrofuran, and finally the sample is stored in a refrigerator at 4 ℃ for later use.

The apparent concentration of the prepared water-soluble Ir-M2 nanoparticles (Ir-M2 NPs) is 500 mug/mL. The size of Ir-M2NPs was measured using a Malvern laser particle sizer (Mastersizer 3000), and the results indicated that the particle size was 167nm and the polydispersity PDI was 0.33.

By comparing Ir-M1 and Ir-M2, the solubility of Ir-M1 and Ir-M2 is different due to different alkyl chains, and the amount of the solvent used in the process of synthesizing the nano-particles is different accordingly. In the process of preparing the nanoparticle, the longer the alkyl chain is, the better the solubility of the iridium complex is, and the smaller the particle size of the nanoparticle is, the more beneficial the nanoparticle is to be endocytosed by cells. Therefore, ir-M1 is more suitable for photothermal/photodynamic co-therapy.

The CCK-8 method is used for detecting the cancer cell toxicity of Ir-M1 and the photothermal treatment effect on 4T1 cells, and the specific experimental steps are as follows:

1) The nanoparticle stock solution at a concentration of 500. Mu.g/mL was diluted to a concentration of 30. Mu.g/mL, 60. Mu.g/mL, 90. Mu.g/mL, 120. Mu.g/mL, 150. Mu.g/mL with complete medium (DMEM containing 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin).

2) 4T1 cells (mouse breast cancer cells, ATCC) in logarithmic growth phase were digested with 0.25% trypsin and the cells were diluted uniformly to a concentration of 5X 10 ⁴ Individual cells/mL.

3) Adding the cell solution to a 96-well plate at a volume of 100. Mu.L/well, shaking gently and uniformly, and adding the cell solution at 37 ℃ and 5% CO ₂ The cultivation was carried out in the incubator of (1) for 24 hours.

4) Complete medium containing different concentrations of Ir-M1 nanoparticles was added to 96-well plates at 100 μ L per well, 10 wells per concentration were set, one set of 5 wells for 2 groups, i.e., illuminated and non-illuminated. Wherein 0. Mu.g/mL was set as a control group. And the 96-well plate was placed in an incubator for 12h.

5) The illuminated 96-well plate was removed and a 808nm laser (power 0.5W/cm) was used ² ) After 5.0min of irradiation, the mixture is put into an incubator for further incubation for 12h. The non-illuminated group 96-well plates did not need to be illuminated. Directly culturing for 24h.

6) The culture waste liquid in the 96-well plate was washed out in the light-irradiated group and the non-light-irradiated group, and 100. Mu.L of complete medium containing 10% of CCK-8 was added to each well, which was then returned to the incubator for 1 hour.

7) And (3) putting the 96-well plate of the illumination group and the non-illumination group into an enzyme-labeling instrument, testing the absorbance of an absorption peak of each well at 450nm, calculating the average value and the standard deviation of the absorbance of 5 wells of each group, and calculating the survival rate of the cancer cells. The CCK-8 test results are shown in FIG. 1.

As can be seen from FIG. 2, the survival rate of 4T1 cells was maintained at 90% or more in the absence of light at different concentrations of Ir-M1 NPs. Indicating that Ir-M1 NPs are not cytotoxic in the absence of light. Under the illumination condition, the survival rate of the cells is related to the concentration of Ir-M1 NPs, and the higher the concentration of Ir-M1 NPs is, the lower the survival rate of the cells is. At a concentration of 30. Mu.g/mL, ir-M1 NPs may kill 16% of 4T1 cells; at a concentration of 60. Mu.g/mL, ir-M1 NPs killed 28% of 4T1 cells; at a concentration of 90. Mu.g/mL, ir-M1 NPs killed 43% of 4T1 cells; at a concentration of 120. Mu.g/mL, ir-M1 NPs killed 60% of 4T1 cells; at a concentration of 150. Mu.g/mL, ir-M1 NPs killed 77% of 4T1 cells; thus, ir-M1 NPs have an excellent photothermal therapeutic effect on 4T1 cells.

The photo-thermal performance test of Ir-M1 NPs adopts 808nm laser. Placing an Ir-M1 NPs sample with the concentration of 60 mu g/mL under a laser light source with the wavelength of 808nm and the power of 1.0W/cm ² The temperature of the aqueous Ir-M1 NPs solution was initially recorded every 30 s. After 10min of irradiation, the light source was removed, allowed to cool naturally, and recorded every 30 s. The temperature rise and fall curve of Ir-M1 NPs is shown in FIG. 2.

As can be seen from FIG. 3, the temperature of the Ir-M1 nanoparticles is continuously increased under the 808nm laser irradiation, and the temperature rise rate is faster at the beginning of the irradiation and then becomes gentle. When not illuminated, the temperature of the Ir-M1 nanoparticle aqueous solution is 27 ℃, and after 30 seconds of illumination, the temperature rises to 34.2 ℃. After 10min of illumination, the temperature finally rose to 78.5 ℃. The Ir-M1 nanoparticles are rapidly cooled after the light source is removed. After removing the light source for 30s, the temperature was reduced to 75 ℃. The photothermal conversion efficiency of Ir-M1 was calculated to be 55% based on the cooling curve. This indicates that the iridium complex Ir-M1 has excellent photothermal conversion efficiency, and is expected to have excellent effects on photothermal therapy.

The photodynamic properties of the Ir-M1 nanoparticles are measured by the production of active oxygen. 9, 10-anthracenediyl-bis (methylene) dipropionic acid (ABDA) is a common singlet oxygen (one of active oxygen) probe, and Ir-M1 nano particles generate singlet oxygen under the illumination condition and can oxidize ABDA. Thus, in the systemThe amount of ABDA is consumed and the absorbance decreases as well. If Ir-M1 NPs cannot generate singlet oxygen under the illumination condition, the absorbance of ABDA remains unchanged. 3mL of test sample is prepared, wherein the concentration of Ir-M1 NPs is 60 mu g/mL, and the concentration of ABDA is 50 mu mol/L. FIG. 4 is an ultraviolet-visible absorption spectrum of ABDA of Ir-M1 NPs under illumination conditions (xenon lamp), wherein the illumination wavelength is 400-780 nm, and the illumination power is 50mW cm ^-2 . As can be seen from FIG. 3, the absorbance of the ABDA peak at 378nm gradually decreased with the change of the light irradiation time. After the irradiation of 300s, the absorbance of 0.746 is reduced to 0.195 when the light is not irradiated, and the reduction amplitude is 74 percent, which shows that the iridium complex Ir-M1 can efficiently generate active oxygen through the irradiation of light and can be used for photodynamic therapy.

The CCK-8 method is used for evaluating the photothermal/photodynamic synergistic treatment effect of Ir-M1 on cancer cells, and the specific experimental steps are as follows:

3) Adding the cell solution to a 96-well plate at a volume of 100. Mu.L/well, shaking gently and uniformly, and adding the cell solution at 37 ℃ and 5% CO ₂ The cultivation was carried out for 24 hours.

4) Complete media containing different concentrations of Ir-M1 NPs were added to 96-well plates at 100. Mu.L per well, 5 wells per concentration. Wherein 0. Mu.g/mL was set as a control group. And the 96-well plate was placed in an incubator for 12h.

5) The illuminated 96-well plate was removed and a 808nm laser (power 0.5W/cm) was used ² ) And xenon lamp (wavelength 400-780 nm, illumination power 50mW cm ^-2 ) After 5.0min of simultaneous irradiation, the mixture is put into an incubator for further incubation for 12h. The non-illuminated group 96-well plates did not need to be illuminated. Directly culturing for 24h.

6) Washing the waste culture medium in the 96-well plates of the light-irradiated group and the non-light-irradiated group, adding 100. Mu.L of complete medium containing 10% of CCK-8 per well, and returning to the incubator for 1 hour.

7) And (3) putting the 96-well plate into an enzyme labeling instrument, testing the absorbance of an absorption peak of each well at 450nm, calculating the average value and the standard deviation of the absorbance of 5 wells in each group, and calculating the survival rate of the cancer cells. The CCK-8 test results are shown in FIG. 4.

As shown in FIG. 5, the survival rate of 4T1 cells was maintained at 90% or higher for different concentrations of Ir-M1 NPs under no-light conditions. The result shows that Ir-M1 NPs have no cytotoxicity and good biocompatibility under the condition of no illumination. Under the combined illumination of the 808nm laser and the xenon lamp, the death rate of cancer cells is remarkably increased, and the concentration dependence is shown. At a concentration of 30. Mu.g/mL, ir-M1 NPs killed 24% of 4T1 cells; at a concentration of 60. Mu.g/mL, ir-M1 NPs killed 39% of 4T1 cells; at a concentration of 90. Mu.g/mL, ir-M1 NPs killed 52% of 4T1 cells; at a concentration of 120. Mu.g/mL, ir-M1 NPs killed 78% of 4T1 cells; at a concentration of 150. Mu.g/mL, ir-M1 NPs killed 89% of 4T1 cells; comparing fig. 2 and fig. 5, under the condition of the same concentration of Ir-M1 NPs, the lethality of 4T1 cells is further increased, which shows that after the photothermal therapy and photodynamic therapy are combined, the phototoxicity of Ir-M1 NPs to 4T1 cells is greater, i.e. Ir-M1 NPs have excellent photothermal/photodynamic synergistic therapeutic effect.

The photothermal/photodynamic co-therapy effect of Ir-M2NPs was evaluated in the same manner as described above, except that the complete medium containing Ir-M2NPs at different concentrations in step 4) was changed to a complete medium containing Ir-M2NPs at different concentrations. The result shows that the 4T1 cell survival rate of Ir-M2NPs with different concentrations can be maintained by more than 90% under the condition of no illumination. The result shows that Ir-M2NPs have no cytotoxicity and good biocompatibility under the condition of no illumination. Under the joint illumination of 808nm laser and a xenon lamp, the cancer cell death rate is obviously increased, and the concentration dependence is also shown. At a concentration of 30. Mu.g/mL, ir-M2NPs killed 21% of 4T1 cells; at a concentration of 60 μ g/mL, ir-M2NPs can kill 35% of 4T1 cells; at a concentration of 90 μ g/mL, ir-M2NPs can kill 50% of 4T1 cells; at a concentration of 120. Mu.g/mL, ir-M2NPs killed 74% of 4T1 cells; at a concentration of 150. Mu.g/mL, ir-M2NPs killed 86% of 4T1 cells; compared with the photo-thermal/photodynamic synergistic treatment effect of Ir-M1 NPs, the photo-thermal/photodynamic synergistic treatment of Ir-M1 NPs has larger lethality to cancer cells and better effect.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. An iridium complex for photothermal/photodynamic co-therapy, characterized in that: the iridium complex is named as Ir-M, and the chemical structure of the iridium complex is shown as the formula (I):

wherein R is a linear or branched alkyl group having 1 to 30 carbon atoms.

2. The iridium complex for photothermal/photodynamic synergistic therapy according to claim 1, wherein: in the formula (I), R is a straight chain or branched chain alkyl with 8-16 carbon atoms.

3. The iridium complex for photothermal/photodynamic synergistic therapy according to claim 1, wherein: in the formula (I), R is 2-ethylhexyl or 2-hexyldecyl.

4. The process for preparing an iridium complex for photo-thermal/photodynamic co-therapy according to any one of claims 1 to 3, wherein the iridium complex Ir-M is prepared by the following route:

5. the method for preparing an iridium complex for photothermal/photodynamic co-therapy according to claim 4, comprising the steps of:

(1) 4, 7-dibromo-2, 1, 3-benzothiadiazole reacts for 24 hours at 140 ℃ under the action of iron powder and glacial acetic acid to generate a ring opening reaction to obtain a compound M1;

(3) Dissolving a compound M2 in N, N-dimethylformamide, carrying out alkylation reaction with alkyl bromide R-Br under the action of potassium carbonate, and reacting at 110 ℃ for 12 hours to obtain a compound M3;

(4) Carrying out nitration reaction on the compound M3 in a concentrated nitric acid and concentrated sulfuric acid system, and reacting for 12 hours at 100 ℃ to obtain a compound M4;

(5) The compound M4 is subjected to a reduction reaction under the action of iron and glacial acetic acid to obtain a compound M5;

6. The method for preparing an iridium complex for photothermal/photodynamic synergistic therapy according to claim 5, wherein:

in the step (1), the molar ratio of the 4, 7-dibromo-2, 1, 3-benzothiadiazole to the iron powder is 1;

in the step (2), the molar ratio of the compound M1 to the sodium nitrite is 1;

in the step (3), the molar ratio of the compound M2, the alkyl bromide and the potassium carbonate is 1-1.5;

in the step (4), the molar weight of the compound M3 and the volume ratio of concentrated nitric acid are 1; the volume ratio of the concentrated nitric acid to the concentrated sulfuric acid is 1;

in the step (5), the molar ratio of the compound M4 to the iron powder is 1.05-0.15, the volume ratio of the molar amount of the compound M4 to the glacial acetic acid is 1;

in the step (6), the molar ratio of the compound M5 to the 1, 10-phenanthroline-5, 6-dione is 1;

in the step (7), the molar ratio of the compound M6, 4-triphenylamine borate, palladium tetratriphenylphosphine and potassium carbonate is (1-4);

in the step (8), the molar ratio of the compound M7 to bis (1, 5-cyclooctadiene) iridium chloride (I) dimer is 1

The volume ratio of the mass of the molecular sieve to the volume of the o-xylene is 1g (0.2-0.8) g (10-30) mL.

7. The method for preparing an iridium complex for photothermal/photodynamic synergistic therapy according to claim 6, wherein:

in the step (2), the molar ratio of the compound M1 to the sodium nitrite is 1;

in the step (3), the molar ratio of the compound M2, the alkyl bromide and the potassium carbonate is 1;

in the step (4), the molar quantity of the compound M3 and the volume ratio of the concentrated nitric acid are 1; the volume ratio of the concentrated nitric acid to the concentrated sulfuric acid is 1;

in the step (5), the molar ratio of the compound M4 to the iron powder is 1;

in step (7), the molar ratio of compound M6, triphenylamine 4-borate, palladium tetratriphenylphosphine, and potassium carbonate is 1.2.5;

The mass of the molecular sieve and the volume ratio of o-xylene are 1g.

8. Use of the iridium complex according to any one of claims 1 to 3 or the iridium complex obtained by the preparation method according to any one of claims 4 to 7 in the preparation of a photosensitizer for photothermal/photodynamic co-therapy of tumors.

9. The use of claim 8, wherein the tumor is a subcutaneous tumor.