A near-infrared second-zone emitting iridium phthalocyanine complex and its application

Abstract

The invention discloses an iridium phthalocyanine complex, wherein the iridium complex has tetra-β-alkylphthalocyanine as a main ligand and pyridine as an auxiliary ligand. The research results of the invention show that the iridium phthalocyanine complex and its nanoprobe have near-infrared zone II emission, and under hypoxic conditions, have higher near-infrared zone II fluorescence imaging resolution, higher tissue detection depth, and lower background interference, which is of great significance for exploring oxygen transport and distribution, diagnosis and surgical navigation of tumors.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probes for bio-optical imaging, and specifically relates to a novel near-infrared second-zone emitting iridium phthalocyanine complex and application thereof. Technical Background

[0002] The oxygen partial pressure in hypoxic tumor tissue is about 0-3% O2, and the oxygen partial pressure in normal oxygen supply environment under physiological conditions is about >5% O2. There is a significant difference between the two. Iridium complex is a reversible oxygen probe that changes with the oxygen concentration and is a good choice for amplifying the difference in oxygen content signals. Therefore, using iridium complex fluorescence imaging, a non-invasive, high-resolution, and high-sensitivity technology for tumor imaging has become a hot topic for scientists. Compared with near-infrared zone 1 fluorescence imaging (detection of fluorescence signals in the range of 750-900nm), near-infrared zone 2 fluorescence imaging (detection of signals in the range of 900-1700nm) has higher imaging resolution, higher tissue detection depth, and lower background interference. Therefore, the use of near-infrared zone 2 hypoxia imaging is more significant for exploring the oxygen transport and distribution, diagnosis, and surgical navigation of tumors. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides a near-infrared second-zone emitting iridium phthalocyanine complex and application thereof.

[0004] The specific scheme of the present invention is as follows:

[0005] A phthalocyanine iridium complex has tetra-β-alkylphthalocyanine as a main ligand and pyridine as an auxiliary ligand.

[0006] The iridium complex has the following structure: Wherein R1 to R4 are the same or different and represent a C1-C6 alkyl group substituted at the β position.

[0007] Preferably, the tetra-β-alkylphthalocyanine has the following structure:

[0008] R1 to R4 are the same or different and represent C1-C6 alkyl. Preferably, R1 to R4 are the same and represent tert-butyl.

[0009] The present invention also provides a method for preparing the iridium phthalocyanine complex, comprising the following steps:

[0010] (1) adding 4-alkylphthalonitrile to an octanol solution under nitrogen protection and lithium catalysis to react and generate tetra-β-alkylphthalocyanine, wherein the alkyl group is a C1-C6 alkyl group, and the 4-alkylphthalonitrile can be a single alkyl-substituted compound or a mixture of different alkyl-substituted compounds;

[0011] (2) Tetra-β-alkylphthalocyanine, methoxy(cyclooctadiene)iridium dimer and pyridine are heated to react under nitrogen protection to obtain a phthalocyanine iridium complex.

[0012] The present invention also provides a nano probe of iridium phthalocyanine complex, which is the iridium phthalocyanine complex of the present invention wrapped by amphiphilic block copolymer polyethylene glycol-polycaprolactone to form nano micelles.

[0013] The present invention also provides the use of the iridium phthalocyanine complex or nanoprobe of the present invention in preparing a fluorescent probe for near-infrared second-zone imaging, wherein the fluorescent probe is a fluorescent probe that responds to hypoxia. A specific application is a tumor imaging probe.

[0014] Advantages of the present invention:

[0015] The iridium phthalocyanine complex and nanoprobe described in the present invention elevate near-infrared hypoxia imaging to near-infrared zone II for the first time, broadening the application scope of near-infrared in vivo imaging. Near-infrared zone II fluorescence imaging (detection signal in the range of 900-1700nm) has higher imaging resolution, higher tissue detection depth, and lower background interference. Therefore, near-infrared zone II hypoxia imaging can better present tumor oxygen-related details, which is of greater significance for exploring tumor oxygen transport and distribution, diagnosis, and surgical navigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The ultraviolet absorption spectrum (A) and phosphorescence absorption emission spectrum (B) of the iridium phthalocyanine complex PcIrpy2 of the present invention.

[0017] Figure 2 This is a schematic diagram of the preparation of the iridium phthalocyanine complex nanoprobe of the present invention.

[0018] Figure 3 The ultraviolet absorption spectrum (A) and phosphorescence absorption emission spectrum (B) of the iridium phthalocyanine complex nanoprobe of the present invention.

[0019] Figure 4 This is a transmission electron microscope photo of the nanoprobe of the iridium phthalocyanine complex of the present invention (Bar: 50nm).

[0020] Figure 5 This is the dynamic light scattering characterization result of the iridium phthalocyanine complex nanoprobe described in the present invention.

[0021] Figure 6 This is the emission spectrum of the iridium phthalocyanine complex nanoprobe of the present invention under different oxygen environments.

[0022] Figure 7 This is the in vivo imaging result of the iridium phthalocyanine complex nanoprobe of the present invention on tumor-bearing mice.

[0023] Figure 8 This is the result of using the iridium phthalocyanine complex nanoprobe of the present invention for imaging blood vessels around tumors. DETAILED DESCRIPTION

[0024] The specific steps of the present invention are described below by way of examples, but are not limited to the examples.

[0025] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0026] The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0027] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0028] Example 1 Preparation of iridium phthalocyanine complex PcIrpy2

[0029]

[0030] 1. Synthesis of Tetra-tert-butylphthalocyanine H2Pc(β-C4H9)4

[0031] Under nitrogen protection, add 100mL of n-octanol and 16mg of lithium to a round-bottom flask. After the lithium is completely dissolved, add 1.6g of 4-tert-butylphthalonitrile. After the solution turns green, heat it to 100°C and react for 2 hours. Remove the solvent by vacuum distillation. Dissolve the reactant in chloroform and precipitate it in ethanol. Collect the precipitate and separate the target product by column chromatography. The mobile phase is petroleum ether. Collect the product: 980mg.

[0032] 2. Synthesis of iridium phthalocyanine complex PcIrpy2

[0033] Add 100 mg H2Pc(β-C4H9)4, 21.4 mg methoxy(cyclooctadiene)iridium dimer [Ir(cod)OMe]2 and a small amount of pyridine to a round-bottom flask, and then heat the mixture at 100°C for 4 h under nitrogen protection. After the reaction, remove the solvent by distillation under reduced pressure, and separate the target product by column chromatography with a mobile phase of CH2Cl2 / MeOH=2:1. Collect product: 35 mg.

[0034] Structural characterization:

[0035] H2Pc(β-C4H9)4: 1H NMR (400MHz, Chloroform-d) δ9.68 (s, 1H), 9.22 (dd, J = 7.0, 2.0Hz,

[0036] 1H),9.13(d,J=8.4Hz,1H),7.96–7.84(m,3H),7.79(d,J=8.5Hz,1H),7.72(s,1H),1.83(s,36H).ESI-MS: 738.47.

[0037] PcIrpy2: 1 H NMR (400MHz, Chloroform-d) δ9.50 (d, J=13.7Hz, 3H), 9.41 (ddd, J=21.6,

[0038] 8.1,3.7Hz,3H),9.33(s,1H),9.31–9.26(m,1H),9.21–9.09(m,2H),8.32(dd,J=8.1,3.6H z,3H),8.11–8.02(m,2H),6.51(s,2H),5.55(s,3H),1.86–1.71(m,36H).ESI-MS: 1087.45.

[0039] The UV absorption spectrum and phosphorescence absorption emission spectrum of PcIrpy2 are shown in Figure 1 In DMSO solution, the maximum absorption peak of PcIrpy2 is 655nm, and the maximum emission peak is 975nm.

[0040] Example 2 Preparation of phthalocyanine iridium complex PcIrpy2 nanoprobe

[0041] PEG-PCL (10 mg) and PcIrpy2 (1 mg) with a weight ratio of 10:1 were dissolved in 1 ml of tetrahydrofuran solution. The tetrahydrofuran stock solution of the preparation was added to 10 ml of water under ultrasound. The initial aqueous solution of the nanoprobe was then dialyzed to remove tetrahydrofuran and other small molecule impurities. After 24 hours of dialysis, the dialyzed solution was filtered with a 0.22 μm filter membrane to remove large insoluble matter. Finally, the nanoprobe was obtained by ultrafiltration and concentration. The concentration of the iridium complex photosensitizer in the preparation was quantified by ultraviolet absorption. The schematic diagram of the preparation route is shown in the figure. Figure 2 The UV absorption spectrum and fluorescence emission spectrum of the nanoprobe are shown in Figure 3 In aqueous solution, the maximum absorption peak of PcIrpy2 nanoprobe is 655nm, and the maximum emission peak is 989nm.

[0042] Transmission electron microscope photos of iridium phthalocyanine complex nanoprobes Figure 4 The dynamic light scattering characterization results are shown in Figure 5 The results show that the size of the nanoprobe is 15.4 nm as characterized by transmission electron microscopy, and the size of the probe in aqueous solution is 38.4 nm as characterized by dynamic light scattering.

[0043] Example 3 Investigation of the hypoxic response of the iridium phthalocyanine complex PcIrpy2 nanoprobe

[0044] An aqueous solution of the nanoprobe of Example 2 was prepared, nitrogen was passed into the solution for 10 minutes, and the fluorescence spectra of the PcIrpy2 nanoprobe were detected before ventilation (normoxia) and after ventilation (hypoxia).

[0045] The results are as follows Figure 6 The results showed that in a hypoxic (0% O2) environment, the emission intensity of the PcIrpy2 nanoprobe was twice that in a normoxic (21% O2) environment, suggesting that the PcIrpy2 nanoprobe can be used for near-infrared second-zone hypoxia imaging.

[0046] Example 4 In vivo imaging of phthalocyanine iridium complex PcIrpy2 nanoprobe in tumor-bearing mice

[0047] Phthalocyanine iridium complex PcIrpy2 nanoprobe was used for in vivo imaging experiments in 4T1 tumor-bearing mice. The mouse tumor model was inoculated into Babl / c mice by mammary gland orthotopic injection (4T1 mouse breast cancer cells were injected into the second to last mammary gland on the left side, 10 7 cells), and when the tumor volume reaches 100 mm 3 The nanomicelle probe was dissolved in pH = 7.4 PBS (0.01M) to prepare a 1 mg / mL solution (the concentration was determined by the ultraviolet absorption of the iridium complex), and 100 μL was injected into the mouse through the tail vein. The tumor-bearing mice were imaged using a near-infrared in vivo imaging system. Figure 7 The results show that the optical signal of the probe detected at the tumor site is much stronger than the signal of the surrounding tissues from 1 hour to 48 hours after the probe is injected. The results of in vivo imaging tests show that the iridium phthalocyanine complex nanoprobe of the present invention can achieve high signal-to-noise ratio tumor detection. The imaging results of the iridium phthalocyanine complex nanoprobe surrounding the tumor are shown in Figure 8 As shown in Figure A, the densely distributed blood vessels in the tumor are selected, and two blood vessels (the dotted circle is the tumor area) ① and ② are selected inside and outside the tumor for statistical analysis after imaging. A straight line is drawn perpendicular to the direction of the blood vessel using ImageJ software to obtain the cross-sectional fluorescence intensity distribution curve along the straight line, and the fluorescence intensity curve is Gaussian fitted to calculate the half-peak width of the curve. The results are shown in Figure 1. Figure 8As shown in B, the widths of the two selected tumor blood vessels ① and ② are 476.5 μm and 240.3 μm, respectively. The experimental results show that the use of the iridium phthalocyanine complex nanoprobe of the present invention has the advantage of near-infrared zone II hypoxia imaging. When performing near-infrared zone II in vivo imaging, tumor blood vessels can be clearly identified and the width of the blood vessels can be calculated. Imaging of blood vessels around tumors can be achieved.

Claims

1. A phthalocyanine iridium complex, characterized in that The iridium complex has tetra-β-alkylphthalocyanine as the main ligand and pyridine as the auxiliary ligand, and the iridium complex has the following structure: Wherein R1 to R4 are the same or different and represent a C1-C6 alkyl group substituted at the β position.

2. The iridium phthalocyanine complex according to claim 1, characterized in that The tetra-β-alkylphthalocyanine has the following structure: R1 to R4 are the same or different and represent a C1-C6 alkyl group.

3. The iridium phthalocyanine complex according to claim 1 or 2, characterized in that The R1 to R4 are the same and represent tert-butyl.

4. The method for preparing the iridium phthalocyanine complex according to any one of claims 1 to 3, characterized in that The steps include: (1) adding 4-alkylphthalonitrile to an octanol solution under nitrogen protection and lithium catalysis to react and generate tetra-β-alkylphthalocyanine, wherein the alkyl group is a C1-C6 alkyl group; (2) Tetra-β-alkylphthalocyanine, methoxy(cyclooctadiene)iridium dimer and pyridine are heated to react under nitrogen protection to obtain a phthalocyanine iridium complex.

5. The preparation method according to claim 4, characterized in that The 4-alkylphthalonitrile is a compound substituted with a single alkyl group or a mixture substituted with different alkyl groups.

6. A nanoprobe of iridium phthalocyanine complex, characterized in that The iridium phthalocyanine complex described in any one of claims 1 to 3 is wrapped by the amphiphilic block copolymer polyethylene glycol-polycaprolactone to form nano micelles.

7. Use of the iridium phthalocyanine complex according to any one of claims 1 to 3 or the nanoprobe according to claim 6 in preparing a fluorescent probe for near-infrared second-zone hypoxia imaging.

8. The use according to claim 7, characterized in that The fluorescent probe is a hypoxia-responsive fluorescent probe.

9. The use according to claim 8, characterized in that The fluorescent probe is a tumor imaging probe.

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