CN114409711B - Aptamer-cyclometalated iridium conjugate and preparation method and application thereof - Google Patents

Aptamer-cyclometalated iridium conjugate and preparation method and application thereof Download PDF

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CN114409711B
CN114409711B CN202210099640.7A CN202210099640A CN114409711B CN 114409711 B CN114409711 B CN 114409711B CN 202210099640 A CN202210099640 A CN 202210099640A CN 114409711 B CN114409711 B CN 114409711B
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ligand
aptamer
conjugate
iridium
reacting
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CN114409711A (en
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陈锦灿
陈兰美
陈伟钢
郭欣华
黄鹤鸣
罗辉
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Guangdong Medical University
Southern Marine Science and Engineering Guangdong Laboratory Zhanjiang
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Guangdong Medical University
Southern Marine Science and Engineering Guangdong Laboratory Zhanjiang
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System compounds of the platinum group
    • C07F15/0033—Iridium compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention discloses an aptamer-cyclometalated iridium conjugate and a preparation method and application thereof. An aptamer-cyclometallated iridium conjugate comprising at least one of the following structural formulas: apIrC1, apIrC2, apIrC3, and ApIrC4; wherein the aptamer in the structural formula is AS1411 aptamer. The aptamer-cyclometalated iridium conjugate has an excellent inhibition effect on tumor cell lines such as lung cancer cells A549, cervical cancer cells HeLa, liver cancer cells HepG2, breast cancer cells MCF-7 and the like, has low toxicity on normal cells, can be specifically combined with cancer cells, and can be applied to fluorescence imaging and antitumor drugs.

Description

Aptamer-cyclometalated iridium conjugate and preparation method and application thereof
Technical Field
The invention belongs to the technical field of iridium conjugates, and particularly relates to an aptamer-cyclometaliridium conjugate, and a preparation method and application thereof.
Background
Cancer is the second leading cause of death worldwide, creating a heavy disease burden worldwide. At present, treatment strategies aiming at malignant tumors mainly comprise chemotherapy, radiotherapy, surgery and the like, but the treatment methods lack of selectivity on tumor cells, so that adverse reactions are generated, and the treatment effect of patients is obviously reduced. The use of highly cytotoxic chemotherapeutics, while killing tumor cells, inevitably causes severe non-specific damage to the normal tissues of the patient and induces the development of drug resistance to cancer cells.
Therefore, it is necessary to construct a targeted drug delivery system that increases the efficacy and bioavailability of non-targeted drugs and reduces the toxic side effects on normal cells. Among them, monoclonal antibodies (mAbs) have been 9 Antibody Drug Conjugates (ADCs) as one of the fastest growing classes of drugs in the targeted therapeutic area, and have been approved by the united states Food and Drug Administration (FDA). However, most monoclonal antibodies are too bulky to penetrate tumor cells effectively and are expensive to produce, and the process of binding to the drug is time consuming and cumbersome. Even though some monoclonal antibodies have been used clinically, the inherent cross-reactivity and immunogenicity of the antibodies themselves can lead to off-target effects and side effects, thereby affecting efficacy.
Disclosure of Invention
The first technical problem to be solved by the invention is as follows:
an aptamer-cyclometallated iridium conjugate is provided. The aptamer-cyclometallated iridium conjugate has a targeting effect on tumor cells.
The second technical problem to be solved by the invention is as follows:
a method of preparing the aptamer-cyclometallated iridium conjugate is provided.
The third technical problem to be solved by the invention is:
the use of said aptamer-cyclometallated iridium conjugate.
In order to solve the first technical problem, the invention adopts the following technical scheme:
an aptamer-cyclometallated iridium conjugate comprising at least one of the following structural formulas:
wherein,is an AS1411 aptamer.
Wherein, the aptamer refers to a single-stranded DNA or RNA sequence obtained by in vitro screening through an exponential enrichment ligand system evolution (SELEX) technology, can selectively bind to a specific target, and has high specificity and affinity. AS1411 is a guanine-rich aptamer that forms a G-quadruplex structure and specifically recognizes nucleolin proteins on the surface of cancer cells. Nucleolin is a highly conserved non-ribosomal phosphorylated protein in nucleolus, and is involved in a variety of important cellular processes such as cell adhesion, migration, rRNA transcriptional regulation, etc. Nucleolin is highly expressed on the surface of various cancer cell membranes compared with normal cells of human body, and is closely related to rapid proliferation of malignant tumors. Therefore, the development of effective nucleolin-targeted drugs and fluorescent probes is of great importance for the treatment and diagnosis of cancer. When an aptamer is combined with a drug to form an aptamer-drug conjugate (ApDC), apDC, as a low molecular weight molecule, hardly induces antibody production, and thus can be clinically used for a longer period of time. In addition, the aptamer has the advantages of no toxicity, wide target range, good thermodynamic stability, easiness in synthesis and modification and the like.
The cyclometallated iridium complexes have excellent photophysical and photochemical properties, strong anticancer activity and limited side effects. Compared with cisplatin (cispratin) and its analogues, which are widely used in clinic, the iridium complex has better ligand substitution kinetics, the peripheral ligand is easy to modify, the iridium complex has a molecular structure richer than a typical platinum complex square structure, the iridium complex has multifunctional anti-tumor activity, the unique spectral characteristics of the iridium complex, the iridium complex has longer luminous life and higher quantum yield than ruthenium complexes, and the iridium complex has adjustable emission wavelength and excitation wavelength. The combination of iridium complexes with natural active substances, polypeptides, drugs and the like has become an effective strategy for enhancing solubility, increasing selectivity and reducing toxicity.
In order to solve the second technical problem, the invention adopts the following technical scheme:
a method of preparing the aptamer-cyclometallated iridium conjugate comprising the steps of:
s1, mixing 4-formylbenzoic acid, 1, 10-phenanthroline-5, 6-diketone and ammonium salt, and reacting to obtain a ligand L1;
s2, mixing a ligand L1, N-hydroxysuccinimide, a condensing agent and a part of catalyst, and reacting to obtain a ligand L2;
s3, mixing ligand L2, 6-aminocaproic acid and the rest catalyst, and reacting to obtain ligand L3;
s4, mixing an iridium-containing compound, a fluorine-containing compound and a ligand L3, and reacting to obtain a ligand lr1;
s5, mixing the ligand lr1 with a chlorine-containing compound, and reacting to obtain a ligand lr2;
s6, mixing the ligand lr2 with the aptamer, and reacting to obtain the aptamer-cyclometalated iridium conjugate.
According to one embodiment of the invention, in step S6, the ligand lr2, sodium bicarbonate and the aptamer are mixed in a solvent, subjected to a room temperature vortex reaction, and purified by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the aptamer-cyclometalated iridium conjugate. Preferably, the solvent is selected from the group consisting of mixed solvents of water and N, N-Dimethylformamide (DMF), wherein the volume ratio of water to N, N-Dimethylformamide (DMF) is 2-3:1-2.
According to one embodiment of the invention, the molar ratio of the 4-formylbenzoic acid to the 1, 10-phenanthroline-5, 6-dione is 2-3:2-3, preferably 2-3:2.
according to one embodiment of the present invention, after the reaction of step S2, a step of concentrating under reduced pressure to obtain a viscous solid is further included.
According to an embodiment of the present invention, the condensing agent includes at least one of a carbodiimide-based condensing agent, an organophosphorus-based condensing agent, and an onium salt-based condensing agent.
According to one embodiment of the invention, the iridium-containing compound has the formula Ir 2 (R) 4 Cl 2 Wherein R comprises at least one of 7, 8-benzoquinoline, 2-phenylpyridine, 2- (2, 4-difluorophenyl) pyridine and 2- (2-thiophene) pyridine, and R is preferably 2- (2-thiophene) pyridine.
According to one embodiment of the invention, the ligand lr1 and the chlorine-containing compound are mixed under ice bath conditions and heated to reflux.
According to one embodiment of the invention, the molar ratio of ligand lr2 to aptamer is 100-200:4-6.
According to one embodiment of the invention, further comprising the steps of solid-liquid separation, drying and purification.
In a further aspect, the invention also provides application of the aptamer-cyclometallated iridium conjugate in preparing a fluorescent imaging agent or an anti-tumor drug.
One of the technical schemes has at least one of the following advantages or beneficial effects:
the aptamer-cyclometalated iridium conjugate has an excellent inhibition effect on tumor cell lines such as lung cancer cells A549, cervical cancer cells HeLa, liver cancer cells HepG2, breast cancer cells MCF-7 and the like, has low toxicity on normal cells, and can be applied to preparation of fluorescent imaging agents and antitumor drugs.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of L1 in example 1.
FIG. 2 is a hydrogen nuclear magnetic resonance spectrum of L2 in example 1.
FIG. 3 is a hydrogen nuclear magnetic resonance spectrum of L3 in example 1.
FIG. 4 shows a hydrogen nuclear magnetic resonance spectrum of Ir1 in example 1.
Fig. 5 is a spectrum test chart of AS1411, apIrC1 and Ir1 in example 1.
FIG. 6 is a chart showing the purity analysis of the ApIrC1 polyacrylamide gel electrophoresis of example 1.
FIG. 7 is a chart showing the stability analysis of the polyacrylamide gel electrophoresis of ApIrC1 in example 1.
FIG. 8 is a chromatographic test chart of ApIrC1 in example 1.
FIG. 9 is a chromatographic area percent report for ApIrC1 in example 1.
FIG. 10 is a confocal microscope image of ApIrC1 and Ir1 of example 1 after incubation with different cells.
FIG. 11 is a graph showing the cellular uptake levels of ApIrC1 and Ir1 after incubation with different cells in example 1.
FIG. 12 shows the average fluorescence intensity after incubation of ApIrC1 and Ir1 with different cells in flow cytometry analysis example 1.
FIG. 13 shows the detection of Ir (III) content by an inductively coupled plasma mass spectrometer after incubation of ApIrC1 and Ir1 with different cell lines for 6h in example 1.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
Example 1
A method of preparing the aptamer-cyclometallated iridium conjugate comprising the steps of:
s1 4-formyl-benzoic acid (0.3 g,2 mmol), 1, 10-phenanthroline-5, 6-dione (0.42 g,2 mmol) and ammonium acetate (0.77 g,10 mmol) were added to 10mL glacial acetic acid. Heating to 130 ℃ under argon, refluxing and stirring for 4 hours, cooling the reaction mixture to room temperature after the reaction is finished, diluting with 100mL of pure water, cooling overnight at 4 ℃ after precipitation, and collecting the obtained precipitate by suction filtration and washing with the pure water. The crude product was dried in vacuo and purified by silica gel column chromatography to give ligand L1. The nuclear magnetic resonance hydrogen spectrum of L1 is shown in FIG. 1.
S2 ligand L1 (0.17 g,0.5 mmol), N-hydroxysuccinimide (NHS) (0.1438 g,1.25 mmol) and N, N-Diisopropylethylamine (DIEA) (400. Mu.L) were dissolved in 10mL of N, N-dimethylformamide, and after stirring at room temperature for 2 hours, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (0.24 g,1.25 mmol) was added, and the reaction was continued at room temperature for 18 hours with stirring, and after completion of the reaction, the viscous solid was obtained by concentrating under reduced pressure. Pure water is added, ultrasonic treatment is carried out, a solid product is obtained through suction filtration and vacuum drying, and the crude product is purified through silica gel column chromatography, thus obtaining ligand L2. The nuclear magnetic resonance hydrogen spectrum of L2 is shown in FIG. 2.
S3 ligand L2 (0.1748 g,0.4 mmol), 6-aminocaproic acid (0.131 g,1 mmol), N-Diisopropylethylamine (DIEA) (0.9 mL) were dissolved in 10mL of N, N-dimethylformamide, and after stirring at room temperature for 18h, the reaction was stopped and concentrated under reduced pressure to give a viscous solid. Adding pure water, performing ultrasonic treatment, performing suction filtration to obtain a solid product, performing vacuum drying, and purifying the crude product by silica gel column chromatography to obtain a ligand L3, wherein the nuclear magnetic resonance hydrogen spectrum of the ligand L3 is shown in figure 3, and the ligand L3 has the chemical formula:
6-(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzamido)hexanoic acid(PICONH(CH 2 ) 5 COOH)。
s4 IrCl iridium trichloride hydrate IrCl in a three-necked flask 3 ·xH 2 O (0.298 g,1 mmol) is dissolved in 80mL of a mixed solution of 2-methoxyethanol/water (3:1, v/v), and excessive 2- (2-thiophene) pyridine (thpy) (0.483 g,3 mmol) is added, the mixture is refluxed and stirred for 24 hours at 120 ℃ under the protection of argon in a dark place, the solution is cooled to room temperature, and the produced precipitate is collected by suction filtration to obtain Ir 2 (thpy) 4 Cl 2 ；
Ir is used for 2 (thpy) 4 Cl 2 (0.1096 g,0.1 mmol) and ligand L3 (0.0906 g,0.2 mmol) were dissolved in a mixed solution (30 mL) of dichloromethane/methanol (2:1, v/v), refluxed under argon protection for 5h, cooled to room temperature after the reaction, suction filtered, the filtrate was concentrated under reduced pressure and transferred to a beaker, and potassium hexafluorophosphate (KPF) was added 6 ) Saturated aqueous solution. Standing, suction filtering, collecting precipitate, vacuum drying, and purifying by silica gel column chromatography with dichloromethane and methanol as eluent to obtain ligand lr1, wherein nuclear magnetic resonance hydrogen spectrum of lr1 is shown in figure 4, and ligand lr1 has chemical formula:
[Ir(thpy) 2 (PICONH(CH 2 ) 5 COOH)]PF 6 。
s5 slowly dropwise add 5mL SOCl to 30mg ligand lr1 under ice bath conditions 2 Stirring for 30min after the dripping is finished, transferring to room temperature, stirring for 30min, heating at 70 ℃ for reflux reaction for 3h, cooling to room temperature after the reaction is finished, and performing rotary evaporation under reduced pressure to obtain a ligand lr2, wherein the ligand lr2 has a chemical formula of:
[Ir(thpy) 2 (PICONH(CH 2 ) 5 COCl)]PF 6 。
s6 addition of NaHCO to aptamer AS1411 (5 nmol, 125. Mu.L) 3 The solution (0.1 m,10 μl), 60 μl of N, N-Dimethylformamide (DMF) and 30 μl of DMF mother liquor of ligand lr2 (200 nmol,10 mm) were placed on a vortex and reacted at room temperature for 36h, after the reaction was completed, the excess complex was removed by dialysis using a dialysis bag (mw=3000), the dialysate was a mixed solution of DMF/water=2/3 (v/v), and dialyzed 3 times in total, 15h each time, until no fluorescence was detected in the dialysate. And purifying by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the aptamer-cyclometallated iridium conjugate, namely ApIrC1.
ApIrC1 has the structural formula:
among them, aptamer AS1411 is:
5’-NH 2 (CH 2 ) 6 -GGTGGTGGTGGTTGTGGTGGTGGTGG-3’(SEQ ID NO.1)。
in example 1, the synthetic route for ligand L3 is:
in example 1, apIrC1 was synthesized by the following route:
example 2
A method of preparing the aptamer-cyclometallated iridium conjugate comprising the steps of:
s1 4-formyl-benzoic acid (0.3 g,2 mmol), 1, 10-phenanthroline-5, 6-dione (0.42 g,2 mmol) and ammonium acetate (0.77 g,10 mmol) were added to 10mL glacial acetic acid. Heating to 130 ℃ under argon, refluxing and stirring for 4 hours, cooling the reaction mixture to room temperature after the reaction is finished, diluting with 100mL of pure water, cooling overnight at 4 ℃ after precipitation, and collecting the obtained precipitate by suction filtration and washing with the pure water. The crude product was dried in vacuo and purified by silica gel column chromatography to give ligand L1.
S2 ligand L1 (0.17 g,0.5 mmol), N-hydroxysuccinimide (NHS) (0.1438 g,1.25 mmol) and N, N-Diisopropylethylamine (DIEA) (400. Mu.L) were dissolved in 10mL of N, N-dimethylformamide, and after stirring at room temperature for 2 hours, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (0.24 g,1.25 mmol) was added, and the reaction was continued at room temperature for 18 hours with stirring, and after completion of the reaction, the viscous solid was obtained by concentrating under reduced pressure. Pure water is added, ultrasonic treatment is carried out, a solid product is obtained through suction filtration and vacuum drying, and the crude product is purified through silica gel column chromatography, thus obtaining ligand L2.
S3 ligand L2 (0.1748 g,0.4 mmol), 6-aminocaproic acid (0.131 g,1 mmol), N-Diisopropylethylamine (DIEA) (0.9 mL) were dissolved in 10mL of N, N-dimethylformamide, and after stirring at room temperature for 18h, the reaction was stopped and concentrated under reduced pressure to give a viscous solid. Adding pure water, performing ultrasonic treatment, performing suction filtration to obtain a solid product, performing vacuum drying, and purifying the crude product through silica gel column chromatography to obtain a ligand L3, wherein the ligand L3 has a chemical formula of:
PICONH(CH 2 ) 5 COOH。
s4 IrCl iridium trichloride hydrate IrCl in a three-necked flask 3 ·xH 2 O (0.298 g,1 mmol) is dissolved in 80mL of a mixed solution of 2-methoxyethanol/water (3:1, v/v), and excessive 7, 8-benzoquinoline (bzq) (0.537 g,3 mmol) is added, the mixture is refluxed and stirred for 24 hours at 120 ℃ under the protection of argon, the solution is cooled to room temperature, and the produced precipitate is collected by suction filtration to obtain Ir 2 (bzq) 4 Cl 2 ；
Ir is used for 2 (bzq) 4 Cl 2 (0.1 mmol) and ligand L3 (0.2 mmol) were dissolved in a mixed solution (30 mL) of dichloromethane/methanol (2:1, v/v), refluxed for 5h under argon protection, cooled to room temperature after the reaction, suction filtered, the filtrate was concentrated under reduced pressure, transferred to a beaker, and potassium hexafluorophosphate (KPF) was added 6 ) Saturated aqueous solution. Standing, suction filtering, collecting precipitate, vacuum drying, and purifying by silica gel column chromatography with dichloromethane and methanol as eluent to obtain ligand lr3, wherein the ligand lr3 has the chemical formula:
[Ir(bzq) 2 (PICONH(CH 2 ) 5 COOH)]PF 6 。
s5 slowly dropwise add 5mL SOCl to 30mg ligand lr3 under ice bath conditions 2 Stirring for 30min after the dripping is finished, transferring to room temperature, stirring for 30min, heating at 70 ℃ for reflux reaction for 3h, cooling to room temperature after the reaction is finished, and performing rotary evaporation under reduced pressure to obtain a ligand lr4, wherein the ligand lr4 has a chemical formula of:
[Ir(bzq) 2 (PICONH(CH 2 ) 5 COCl)]PF 6 。
s6 addition of NaHCO to aptamer AS1411 (5 nmol, 125. Mu.L) 3 The solution (0.1 m,10 μl), 60 μl, N-Dimethylformamide (DMF) and 30 μl of DMF mother liquor of ligand lr4 (200 nmol,10 mm) were placed on a vortex and reacted at room temperature for 36h, after the reaction was completed, excess complex was removed by dialysis using a dialysis bag (mw=3000), the dialysate was a mixed solution of DMF/water=2/3 (v/v) and dialyzed 3 times in total, 15h each until no fluorescence was detected in the dialysate. And purifying by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the aptamer-cyclometallated iridium conjugate, namely ApIrC2.
ApIrC2 has the structural formula:
among them, aptamer AS1411 is:
5’-NH 2 (CH 2 ) 6 -GGTGGTGGTGGTTGTGGTGGTGGTGG-3’(SEQ ID NO.1)。
in example 2, apIrC2 was synthesized by the following route:
example 3
A method of preparing the aptamer-cyclometallated iridium conjugate comprising the steps of:
s1 4-formyl-benzoic acid (0.3 g,2 mmol), 1, 10-phenanthroline-5, 6-dione (0.42 g,2 mmol) and ammonium acetate (0.77 g,10 mmol) were added to 10mL glacial acetic acid. Heating to 130 ℃ under argon, refluxing and stirring for 4 hours, cooling the reaction mixture to room temperature after the reaction is finished, diluting with 100mL of pure water, cooling overnight at 4 ℃ after precipitation, and collecting the obtained precipitate by suction filtration and washing with the pure water. The crude product was dried in vacuo and purified by silica gel column chromatography to give ligand L1.
S2 ligand L1 (0.17 g,0.5 mmol), N-hydroxysuccinimide (NHS) (0.1438 g,1.25 mmol) and N, N-Diisopropylethylamine (DIEA) (400. Mu.L) were dissolved in 10mL of N, N-dimethylformamide, and after stirring at room temperature for 2 hours, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (0.24 g,1.25 mmol) was added, and the reaction was continued at room temperature for 18 hours with stirring, and after completion of the reaction, the viscous solid was obtained by concentrating under reduced pressure. Pure water is added, ultrasonic treatment is carried out, a solid product is obtained through suction filtration and vacuum drying, and the crude product is purified through silica gel column chromatography, thus obtaining ligand L2.
S3 ligand L2 (0.1748 g,0.4 mmol), 6-aminocaproic acid (0.131 g,1 mmol), N-Diisopropylethylamine (DIEA) (0.9 mL) were dissolved in 10mL of N, N-dimethylformamide, and after stirring at room temperature for 18h, the reaction was stopped and concentrated under reduced pressure to give a viscous solid. Adding pure water, performing ultrasonic treatment, performing suction filtration to obtain a solid product, performing vacuum drying, and purifying the crude product through silica gel column chromatography to obtain a ligand L3, wherein the ligand L3 has a chemical formula of:
PICONH(CH 2 ) 5 COOH。
s4 IrCl iridium trichloride hydrate IrCl in a three-necked flask 3 ·xH 2 O (0.298 g,1 mmol) was dissolved in 80mL of a 2-methoxyethanol/water (3:1, v/v) mixture and excess 2-phenyl was addedPyridine (ppy) (0.460 g,3 mmol), reflux stirring the mixture at 120deg.C under argon for 24 hr, cooling the solution to room temperature, and suction filtering to collect the precipitate to obtain Ir 2 (ppy) 4 Cl 2 ；
Ir is used for 2 (ppy) 4 Cl 2 (0.1 mmol) and ligand L3 (0.2 mmol) were dissolved in a mixed solution (30 mL) of dichloromethane/methanol (2:1, v/v), refluxed for 5h under argon protection, cooled to room temperature after the reaction, suction filtered, the filtrate was concentrated under reduced pressure, transferred to a beaker, and potassium hexafluorophosphate (KPF) was added 6 ) Saturated aqueous solution. Standing, suction filtering, collecting precipitate, vacuum drying, and purifying by silica gel column chromatography with dichloromethane and methanol as eluent to obtain ligand lr5, wherein the ligand lr5 has the chemical formula:
[Ir(ppy) 2 (PICONH(CH 2 ) 5 COOH)]PF 6 。
s5 slowly dropwise add 5mL SOCl to 30mg ligand lr5 under ice bath conditions 2 Stirring for 30min after the dripping is finished, transferring to room temperature, stirring for 30min, heating at 70 ℃ for reflux reaction for 3h, cooling to room temperature after the reaction is finished, and performing rotary evaporation under reduced pressure to obtain a ligand lr6, wherein the ligand lr6 has a chemical formula of:
[Ir(ppy) 2 (PICONH(CH 2 ) 5 COCl)]PF 6 。
s6 addition of NaHCO to aptamer AS1411 (5 nmol, 125. Mu.L) 3 The solution (0.1 m,10 μl), 60 μl, N-Dimethylformamide (DMF) and 30 μl of DMF mother liquor of ligand lr6 (200 nmol,10 mm) were placed on a vortex and reacted at room temperature for 36h, after the reaction was completed, excess complex was removed by dialysis using a dialysis bag (mw=3000), the dialysate was a mixed solution of DMF/water=2/3 (v/v) and dialyzed 3 times in total, 15h each until no fluorescence was detected in the dialysate. And purifying by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the aptamer-cyclometallated iridium conjugate, namely ApIrC3.
Wherein, the structural formula of ApIrC3 is:
in example 3, apIrC3 was synthesized by the following route:
example 4
A method of preparing the aptamer-cyclometallated iridium conjugate comprising the steps of:
s1 4-formyl-benzoic acid (0.3 g,2 mmol), 1, 10-phenanthroline-5, 6-dione (0.42 g,2 mmol) and ammonium acetate (0.77 g,10 mmol) were added to 10mL glacial acetic acid. Heating to 130 ℃ under argon, refluxing and stirring for 4 hours, cooling the reaction mixture to room temperature after the reaction is finished, diluting with 100mL of pure water, cooling overnight at 4 ℃ after precipitation, and collecting the obtained precipitate by suction filtration and washing with the pure water. The crude product was dried in vacuo and purified by silica gel column chromatography to give ligand L1.
S2 ligand L1 (0.17 g,0.5 mmol), N-hydroxysuccinimide (NHS) (0.1438 g,1.25 mmol) and N, N-Diisopropylethylamine (DIEA) (400. Mu.L) were dissolved in 10mL of N, N-dimethylformamide, and after stirring at room temperature for 2 hours, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (0.24 g,1.25 mmol) was added, and the reaction was continued at room temperature for 18 hours with stirring, and after completion of the reaction, the viscous solid was obtained by concentrating under reduced pressure. Pure water is added, ultrasonic treatment is carried out, a solid product is obtained through suction filtration and vacuum drying, and the crude product is purified through silica gel column chromatography, thus obtaining ligand L2.
S3 ligand L2 (0.1748 g,0.4 mmol), 6-aminocaproic acid (0.131 g,1 mmol), N-Diisopropylethylamine (DIEA) (0.9 mL) were dissolved in 10mL of N, N-dimethylformamide, and after stirring at room temperature for 18h, the reaction was stopped and concentrated under reduced pressure to give a viscous solid. Adding pure water, performing ultrasonic treatment, performing suction filtration to obtain a solid product, performing vacuum drying, and purifying the crude product through silica gel column chromatography to obtain a ligand L3, wherein the ligand L3 has a chemical formula of:
PICONH(CH 2 ) 5 COOH。
s4 IrCl iridium trichloride hydrate IrCl in a three-necked flask 3 ·xH 2 O (0.298 g,1 mmol) was dissolved in 80mL of a mixed solution of 2-methoxyethanol/water (3:1, v/v),adding excessive 2- (2, 4-difluorophenyl) pyridine (dfppy) (0.573 g,3 mmol), refluxing and stirring the mixture at 120deg.C under argon protection for 24 hr, cooling the solution to room temperature, and suction filtering to collect the precipitate 2 (dfppy) 4 Cl 2 。
Ir is used for 2 (dfppy) 4 Cl 2 (0.1096 g,0.1 mmol) and ligand L3 (0.0906 g,0.2 mmol) were dissolved in a mixed solution (30 mL) of dichloromethane/methanol (2:1, v/v), refluxed under argon protection for 5h, cooled to room temperature after the reaction, suction filtered, the filtrate was concentrated under reduced pressure and transferred to a beaker, and potassium hexafluorophosphate (KPF) was added 6 ) Saturated aqueous solution. Standing, suction filtering, collecting precipitate, vacuum drying, and purifying by silica gel column chromatography with dichloromethane and methanol as eluent to obtain ligand lr7, wherein the ligand lr7 has the chemical formula:
[Ir(dfppy) 2 (PICONH(CH 2 ) 5 COOH)]PF 6 。
s5 slowly dropwise add 5mL SOCl to 30mg ligand lr7 under ice bath conditions 2 Stirring for 30min after the dripping is finished, transferring to room temperature, stirring for 30min, heating at 70 ℃ for reflux reaction for 3h, cooling to room temperature after the reaction is finished, and performing rotary evaporation under reduced pressure to obtain a ligand lr8, wherein the ligand lr8 has a chemical formula of:
[Ir(dfppy) 2 (PICONH(CH 2 ) 5 COCl)]PF 6 。
s6 addition of NaHCO to aptamer AS1411 (5 nmol, 125. Mu.L) 3 The solution (0.1 m,10 μl), 60 μl, N-Dimethylformamide (DMF) and 30 μl of DMF mother liquor of ligand lr8 (200 nmol,10 mm) were placed on a vortex and reacted at room temperature for 36h, after the reaction was completed, excess complex was removed by dialysis using a dialysis bag (mw=3000), the dialysate was a mixed solution of DMF/water=2/3 (v/v) and dialyzed 3 times in total, 15h each until no fluorescence was detected in the dialysate. And purifying by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the aptamer-cyclometallated iridium conjugate, namely ApIrC4.
Wherein, the structural formula of ApIrC4 is:
in example 4, apIrC4 was synthesized by the following route:
performance test:
the aptamer-cyclometallated iridium conjugate of example 1, apIrC1, was characterized by dissolving AS1411, apIrC1 and Ir1 in acetonitrile and H at 298K 2 In the O mixture (1/1, v/v), an ultraviolet absorption spectrum (FIG. 5A) and a fluorescence emission spectrum (FIG. 5B) were measured, and as can be seen from FIG. 5 (A), apIrC1 has ultraviolet absorption peaks of both the complex and the aptamer, and as can be seen from FIG. 5 (B), apIrC1 exhibits strong fluorescence comparable to Ir1 as compared with the aptamer not bound to the complex.
The aptamer-cyclometallated iridium conjugate of example 1, apIrC1, was analyzed for purity using polyacrylamide gel electrophoresis (PAGE), and the results are shown in fig. 6. The polyacrylamide gel electrophoresis result shows that no impurity band appears in the first lane corresponding to ApIrC1. Furthermore, it can be seen that the synthesized ApIrC1 is slower than the pure aptamer, possibly due to the increase in molecular weight.
The stability of ApIrC1 in DMEM medium containing 10% Fetal Bovine Serum (FBS) was analyzed by PAGE and the image results (fig. 7) showed that bands of ApIrC1 were still observed after 36h incubation, demonstrating good serum stability of the conjugate.
The aptamer-cyclometallated iridium conjugate of example 1, apIrC1, was chromatographed and the results are shown in FIGS. 8-9. Wherein, fig. 8 is a chromatographic test chart, and fig. 9 is a chromatographic area percentage report. ApIrC1 was purified by RP-HPLC (reverse phase high performance liquid chromatography), and the purity of the synthesized ApIrC was calculated to be higher than 97% based on the chromatographic peak area ratio.
Toxicity of lr1, aptamer and ApIrC1 in ligand example 1 to normal human liver cell L-02 and human embryo kidney cell HEK-293T and inhibition effect of the ligand on tumor cell lines such as lung cancer cell A549, cervical cancer cell HeLa, liver cancer cell HepG2 and breast cancer cell MCF-7 are detected respectively by a thiazole blue (MTT) colorimetric method.
Cells in logarithmic growth phase (L-02, HEK293T, A549, heLa, hepG2 and MCF-7, commercially available) were taken, digested with 0.25% trypsin, and diluted to 4.0X10% in DMEM medium 4 Per mL of cell suspension, 100. Mu.L per well, at 37℃in 5% CO 2 After the culture was carried out under the condition for 24 hours,
the culture solution was aspirated, and 90. Mu.L of the fresh culture solution of the same formulation and Ir1, AS1411 and ApIrC1 liquid medicines of different concentrations were added. At the same time, a control group and zero-setting holes are arranged, the culture solution with the same volume is added, and 3 parallel holes are arranged at each concentration. After 48h incubation, 20. Mu.L of 5% MTT (5 mg/mL) was added to each well, incubation was continued for 4h, and then 150. Mu.L of DMSO (dimethyl sulfoxide) was added to each well. Oscillating for 20min in a micro-oscillator in a dark place, immediately colorizing after crystallization and dissolution, measuring an OD value by using an enzyme-labeling instrument, and colorizing with a blank Kong Diaoling. Experiments were repeated 3 times.
Calculating the cell proliferation rate according to the formula: cell proliferation inhibition (%) = (average OD value of 1-dosing well/average OD value of control well) ×100% and half Inhibition Concentration (IC) was calculated 50 ) The results are shown in Table 1.
TABLE 1 IC of ApIrC1 for different tumor cells and Normal cells 50 Value of
As can be seen from the results of Table 1, the inhibitory activity of ApIrC1 on 4 tumor cells after the combination of the complex and the aptamer was significantly improved, and IC 50 Values all reached nanomolar levels, where ApIrC1 IC for HeLa and MCF-7 cells 50 The values were 0.47.+ -. 0.3. Mu.M and 0.49.+ -. 0.2. Mu.M, respectively.
Furthermore, apIrC1 IC for Normal cells L-02 and HEK293T 50 IC's with values far higher than those of tumor cells 50 The values indicate that after the iridium complex is combined with the aptamer, the selective targeting effect on tumor cells is obtained.
The selective targeting effect and imaging capability of ApIrC1 on tumor cells are further studied. The total uptake of ligands lr1 and ApIrC1 in L-02, HEK-293T, A549, heLa, hepG2 and MCF-7 cells was analyzed using confocal microscopy, flow cytometry and inductively coupled plasma mass spectrometry (ICP-MS), respectively, as follows:
flow cytometry and confocal microscopy analysis:
1mL of cells (1X 10) 5 Per mL) was inoculated in 24-well plates and incubated for 24h prior to the experiment. After removal of the medium, the cells were washed twice with PBS. Then, the cells were incubated with 300nM Ir1, AS1411 and ApIrC1 for 3h at 37℃and washed once with PBS, and the cells were digested with 200. Mu.L trypsin. Cells were collected by centrifugation at 1000rpm for 3min and resuspended in PBS for flow cytometry analysis. Cells in the logarithmic growth phase were co-stained with ApIrC1 (300 nM,3 h), cell culture medium removed and cells were rinsed 3 times with PBS. Finally, the cells were imaged by a confocal microscope, and the uptake of the sample by the cells (excitation channel: 405nm; emission channel: 595.+ -. 20 nm) was observed, as shown in FIG. 10, wherein (a) in FIG. 10 is a confocal microscope image after incubation of ApIrC1 with different cells, and (b) in FIG. 10 is a confocal microscope image after incubation of Ir1 with different cells.
The results in FIG. 10 show that nucleolin-overexpressing cells (HeLa, MCF-7, A549, and HepG 2) exhibited significant cellular uptake of ApIrC1 after 3h incubation with ApIrC1, while the fluorescence intensity of ApIrC was negligible in normal cells (L-02, HEK 293T). Under the same incubation conditions, all cells treated with Ir1 observed significant fluorescence, with little difference in fluorescence intensity.
FIG. 11 is a flow cytometry analysis of uptake studies of ApIrC1 and Ir1 in various cells in example 1. FIG. 12 is a graph showing the average fluorescence intensity of ApIrC1 and Ir1 from example 1 and different cells.
From FIGS. 11 and 12, it can be seen that the fluorescence intensity was significantly increased in 4 cancer cells after ApIrC1 treatment, while only a weak fluorescence shift was detected in off-target cells (L-02 and HEK 293T). Whereas the uptake of Ir1 complexes without AS1411 shows little variability in normal and cancer cell lines.
These results all show that ApIrC1 can specifically bind to cancer cells, and can be used as a fluorescent molecular probe for tumor diagnosis.
Cell total uptake test of ApIrC 1:
taking L-02, HEK293T, A549, heLa, hepG2 and MCF-7 cell suspensions in logarithmic growth phase at 1×10 5 Inoculating into 6-well plate at 37deg.C with 5% CO 2 After 24h incubation in the incubator, ir1, AS1411 and ApIrC1 were added for various times.
After incubation is completed, cells are collected and lysed and the standard is prepared as a control.
The results in FIG. 13 show that the ApIrC1 content in tumor cells is significantly higher than that in normal cells, showing good cancer cell targeting ability.
The aptamer-cyclometallated iridium conjugates prepared in examples 2-4 have similar technical effects as the aptamer-cyclometallated iridium conjugate prepared in example 1.
The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in the relevant art are intended to be included in the scope of the present invention.
Sequence listing
<110> laboratory in Guangdong province of engineering in southern ocean science (Zhanjiang)
Guangdong Medical University
<120> an aptamer-cyclometalated iridium conjugate, and preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ggtggtggtg gttgtggtgg tggtgg 26

Claims (10)

1. An aptamer-cyclometallated iridium conjugate, characterized in that:
is at least one of the following structural formulas:
；
；
；
；
the synthetic route of the aptamer-cyclometallated iridium conjugate is at least one of the following routes:
；
；
；
；
the synthetic route of the L3 is as follows:
；
wherein,AS1411 AS aptamer;
the aptamer AS1411 is:
5’-NH 2 (CH 2 ) 6 -GGTGGTGGTGGTTGTGGTGGTGGTGG-3’。
2. a method of preparing an aptamer-cyclometallated iridium conjugate according to claim 1, wherein: the method comprises the following steps:
s1, mixing 4-formylbenzoic acid, 1, 10-phenanthroline-5, 6-diketone and ammonium salt, and reacting to obtain a ligand L1;
s2, mixing a ligand L1, N-hydroxysuccinimide, a condensing agent and a part of catalyst, and reacting to obtain a ligand L2;
s3, mixing ligand L2, 6-aminocaproic acid and the rest catalyst, and reacting to obtain ligand L3;
s4 mixing iridium-containing compound and KPF 6 Reacting the saturated aqueous solution with a ligand L3 to obtain a ligand lr1;
s5 Mixed ligands lr1 and SOCl 2 Reacting to obtain ligand lr2;
s6, mixing the ligand lr2 with the aptamer, and reacting to obtain the aptamer-cyclometalated iridium conjugate.
3. The method according to claim 2, characterized in that: the molar ratio of the 4-formylbenzoic acid to the 1, 10-phenanthroline-5, 6-dione is 2-3:2-3.
4. The method according to claim 2, characterized in that: after the reaction in the step S2, the method also comprises the step of decompressing and concentrating to obtain a viscous solid.
5. The method according to claim 2, characterized in that: the condensing agent includes at least one of a carbodiimide condensing agent, an organophosphorus condensing agent, and an onium salt condensing agent.
6. The method according to claim 2, characterized in that: the chemical formula of the iridium-containing compound is Ir 2 (R) 4 Cl 2 Wherein R is one of 7, 8-benzoquinoline, 2-phenylpyridine, 2- (2, 4-difluorophenyl) pyridine and 2- (2-thiophene) pyridine.
7. The method according to claim 2, characterized in that: mixing the ligand lr1 and SOCl under ice bath conditions 2 And (5) heating and refluxing.
8. The method according to claim 2, characterized in that: the molar ratio of the ligand lr2 to the aptamer is 100-200:4-6.
9. The method according to claim 2, characterized in that: further comprises the steps of solid-liquid separation, drying and purification.
10. The use of an aptamer-cyclometallated iridium conjugate according to claim 1 in the preparation of a fluorescent imaging agent or an anti-tumor drug.
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