A near-infrared heptamethine cyanine photosensitizing dye conjugate and its preparation method and application

Abstract

The present invention relates to the technical field of biochemical materials, and specifically to a near-infrared heptamethine cyanine photosensitizer dye conjugate, and a preparation method and application thereof. The near-infrared heptamethine cyanine photosensitizer dye conjugate of the present invention has a long-wavelength near-infrared absorption function. The preparation method of the near-infrared heptamethine cyanine photosensitizer dye conjugate of the present invention is simple and easy to adjust. The near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles of the present invention have specific tumor targeting, water solubility and biocompatibility, can actively target tumor sites, do not aggregate in normal tissues or clear them out in a short time, thereby not affecting their clinical application. The near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles of the present invention can successfully target tumor sites and stay for a long time, and can achieve the effects of in vivo diagnosis and treatment under 808nm near-infrared laser irradiation, have certain clinical application prospects, and are applied to clinical intraoperative navigation.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical materials, and in particular to a near-infrared heptamethine cyanine photosensitizing dye conjugate and a preparation method and application thereof. Background Art

[0002] At present, cancer is one of the main culprits that seriously endanger the health of Chinese residents besides cardiovascular disease, and it seriously endangers human health. Clinical treatments for tumors mainly include surgical resection, chemotherapy drugs, radiotherapy drugs, etc. Traditional treatments, including radiotherapy, chemotherapy, and surgical local resection, are associated with high recurrence rates, low specificity, serious side effects, and drug resistance. In order to obtain precise treatment and less side effects, phototherapy technology is a promising cancer treatment method because it has specific light-induced diagnosis and targeted cytotoxicity to cancer cells, high spatiotemporal accuracy, and non-invasiveness. In particular, fluorescence imaging (FLI) in the near-infrared II region (NIR-II, 1000-1700 nm) is able to penetrate deep into tissues with minimal interference and photodamage to tissues. On the other hand, photodynamic therapy (PDT) and photothermal therapy (PTT) can be applied as cancer treatment strategies at the same time by utilizing light-induced reactive oxygen species (ROS) generation and heat production.

[0003] Albumin is the main component of serum proteins. It can bind and deliver various small molecule drugs and has become the most utilized protein in nanomedicine. Due to its complete biocompatibility, good biodegradability and non-immunogenicity, albumin is widely used as a nanocarrier for clinical drug delivery. Therefore, more and more protein-related nanoplatforms have been constructed for treatment, including albumin-bound paclitaxel (PTX), methotrexate-albumin complexes, and albumin-bound doxorubicin (DOX) precursors. Therefore, it is of great significance to construct albumin nanocomposites for phototherapy in small molecule drug delivery.

[0004] So far, some highly selective small molecule tyrosine kinase inhibitors have been clinically studied as anticancer drugs. Among them, we found that crizotinib is an FDA-approved oral ATP competitive inhibitor. Crizotinib can specifically target tumor tissues, activate pathways to further lead to tumor cell apoptosis, and play a role in tumor treatment. In the process of developing tumor-specific fluorescent probes, anti-tumor drugs with tumor-specific killing effects have certain research value and can be used for the development of specific fluorescent probes to further expand the application value of drugs and be used for surgical navigation to remove tumors. Summary of the invention

[0005] One of the purposes of the present invention is to provide a near-infrared heptamethine cyanine photosensitizing dye conjugate having long-wavelength near-infrared absorption.

[0006] The second object of the present invention is to provide a method for preparing a near-infrared heptamethine cyanine photosensitizer dye conjugate, which is simple and easy to adjust.

[0007] The third object of the present invention is to provide a method for preparing composite nanoparticles, which are coated with bovine serum albumin (BSA) to improve their water solubility and biocompatibility.

[0008] A fourth object of the present invention is to provide an application of composite nanoparticles.

[0009] The scheme adopted by the present invention to achieve one of the purposes is: a near-infrared heptamethine cyanine photosensitizer dye conjugate, the structural formula of which is as follows:

[0010] .

[0011] The scheme adopted by the present invention to achieve the second purpose is: a method for preparing the near-infrared heptamethine cyanine photosensitizer dye conjugate, comprising the following steps: reacting compound 2 and compound 3 at a certain temperature under an inert atmosphere, and purifying the product after the reaction is complete to obtain the near-infrared heptamethine cyanine photosensitizer dye conjugate; the compound 2 is ;

[0012] The compound 3 is .

[0013] Under an inert atmosphere, compound 2 and compound 3 are dissolved in a solvent and reacted at a certain temperature. A certain amount of alkali may be added during the reaction or may not be added. When the alkali is added, the alkali may be triethylamine (TEA) or N-ethyldiisopropylamine (DIPEA); the solvent is N,N-dimethylformamide (DMF) or N,N-dimethylacetamide (DMAc).

[0014] Preferably, the molar ratio of compound 2 to compound 3 is 1: (1-2), and the reaction temperature is 60-80°C.

[0015] Preferably, the purification process is to add ether to the reaction product for slurry filtration, and then chromatographically separate the filter cake.

[0016] Preferably, the chromatographic column is octadecylsilane bonded silica gel with a particle size of 75-150 μm.

[0017] Preferably, the eluent of the column chromatography is a mixture of methanol and water in a volume ratio of 1:1 to 4:1.

[0018] The solution adopted by the present invention to achieve the third purpose is: a method for preparing composite nanoparticles, comprising the following steps: under ultrasonic conditions, adding an ethanol or methanol solution of a near-infrared heptamethine cyanine photosensitizer conjugate to an aqueous solution of bovine serum albumin, and then stirring at 50-70°C to obtain the composite nanoparticles.

[0019] The solution adopted by the present invention to achieve the fourth purpose is: an application of the composite nanoparticles, wherein the composite nanoparticles are used to prepare tumor diagnostic agents and therapeutic agents.

[0020] Another solution adopted by the present invention to achieve the fourth objective is: an application of the composite nanoparticles, wherein the composite nanoparticles are used to prepare NIR-II fluorescent imaging contrast agents.

[0021] Another solution adopted by the present invention to achieve the fourth objective is: an application of the composite nanoparticles, wherein the composite nanoparticles are used to prepare a PDT / PTT combined therapeutic agent guided by fluorescence imaging.

[0022] The reaction route is as follows:

[0023] ;

[0024] The present invention has the following advantages and beneficial effects:

[0025] (1) The near-infrared heptamethine cyanine photosensitizing dye conjugate of the present invention has a long-wavelength near-infrared absorption function.

[0026] (2) The preparation method of the near-infrared heptamethine cyanine photosensitizing dye conjugate of the present invention is simple and easy to adjust.

[0027] (3) The near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles of the present invention have specific tumor targeting, water solubility and biocompatibility, can actively target the tumor site, do not accumulate in normal tissues or be cleared away in a short time, and thus do not affect its clinical application. The near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles of the present invention can successfully target the tumor site and stay for a long time. Under the irradiation of 808 nm near-infrared laser, it can achieve the effect of in vivo diagnosis and treatment, has a certain clinical application prospect, and is used in clinical intraoperative navigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the preparation of near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles of the present invention;

[0029] Figure 2 This is the characterization of the near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles of the present invention, wherein Figure a is a transmission electron microscope, and Figure b is a DLS particle size distribution;

[0030] Figure 3 is the absorption spectrum of the near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles of the present invention;

[0031] Figure 4 It is the fluorescence spectrum of the near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles of the present invention;

[0032] Figure 5 This is a thermal imaging image of the near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles under laser light;

[0033] Figure 6 The in vitro singlet oxygen production of the near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles of the present invention;

[0034] Figure 7 The present invention is the in vivo imaging of the near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles in mice bearing colorectal cancer CT26;

[0035] Figure 8 The near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticle tumor phototherapy animal experiment verification of the present invention;

[0036] Fig. 9 These are the hematoxylin-eosin stained sections of the main organs and tissues of mice in different experimental groups. DETAILED DESCRIPTION

[0037] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0038] The experimental methods in the examples described below are conventional methods unless otherwise specified; the materials, reagents, instruments, etc. used are all available from commercial sources unless otherwise specified.

[0039] Example 1 Preparation of near-infrared heptamethine cyanine photosensitizing dye coupling

[0040] 0.15 mmol of compound 2 was added to a 25 mL reaction bottle, and 3 mL of anhydrous N,N-dimethylformamide (DMF) was added and stirred to dissolve. Under nitrogen protection, 0.3 mmol of compound 3 and triethylamine were added, and the reaction was completed by stirring at 80°C for 2 h. The reaction solution was poured into 25 mL of ether, and a blue solid was precipitated. The solid material on the upper layer was collected by filter paper. Octadecylsilane bonded silica gel, particle size: 75-150 μm, was separated and purified by silica gel column chromatography, and the eluent was a mixture of methanol and water in a volume ratio of 1:1. 50 mg of blue metallic crystals were obtained as heptamethine cyanine photosensitizer dye conjugates. The structure was confirmed by nuclear magnetic hydrogen spectrum and mass spectrometry. 1HNMR (600 MHz, MeOD) δ 7.90 (d, J = 4.8 Hz, 1H), 7.78 (d, J = 12.8 Hz, 2H), 7.65 (d, J = 2.8 Hz, 1H), 7.38 (s, 2H), 7.33 (m, 4H), 7.13 (dt, J = 7.8, 4.3Hz, 4H), 7.00 – 6.95 (m, 1H), 6.90 (dd, J = 10.3, 3.5 Hz, 1H), 6.05 – 5.91(m, 2H), 4.64 (s, 1H), 3.99 (t, J = 7.4 Hz, 4H), 3.82 – 3.56 (m, 4H), 3.50(d, J = 12.7 Hz, 1H), 3.22 – 3.18 (m, 2H), 2.67 – 2.46 (m, 6H), 2.37 (m, 4H), 2.23 (t, J = 7.4 Hz, 8H), 1.85 (d, J = 6.3 Hz, 4H), 1.79 (s, 3H), 1.65 (s,12H), 1.48 – 1.46 (m, 2H). HRMS-ESI (m / z) Calcd for (C 63 H 73 Cl 2 FN 7 O 5 + ) ([M-Br] + ):1096.5029, found: 1096.5034.

[0041] Example 2 Preparation of Near-Infrared Heptamethine Cyanine Photosensitizing Dye Coupling

[0042] 0.15 mmol of compound 2 was added to a 25 mL reaction bottle, and 3 mL of anhydrous N,N-dimethylformamide (DMF) was added and stirred to dissolve. Under nitrogen protection, 0.3 mmol of compound 3 was added and stirred at 80°C for 2 h to terminate the reaction. The reaction solution was poured into 25 mL of ether, and a blue solid was precipitated. The solid material on the upper layer was collected by filter paper. Octadecylsilane bonded silica gel, particle size: 75-150 μm, was separated and purified by silica gel column chromatography, and the eluent was a mixture of methanol and water in a volume ratio of 1:1. 50 mg of blue metallic crystals were obtained as heptamethine cyanine photosensitizer dye conjugates. The structure was confirmed by nuclear magnetic hydrogen spectrum and mass spectrometry. 1H NMR (600MHz, MeOD) δ 7.90 (d, J = 4.8 Hz, 1H), 7.78 (d, J = 12.8 Hz, 2H), 7.65 (d, J= 2.8 Hz, 1H), 7.38 (s, 2H), 7.33 (m, 4H), 7.13 (dt, J = 7.8, 4.3 Hz, 4H),7.00 – 6.95 (m, 1H), 6.90 (dd, J = 10.3, 3.5 Hz, 1H), 6.05 – 5.91 (m, 2H),4.64 (s, 1H), 3.99 (t, J = 7.4 Hz, 4H), 3.82 – 3.56 (m, 4H), 3.50 (d, J =12.7 Hz, 1H), 3.22 – 3.18 (m, 2H), 2.67 – 2.46 (m, 6H), 2.37 (m, 4H), 2.23(t, J = 7.4 Hz, 8H), 1.85 (d, J = 6.3 Hz, 4H), 1.79 (s, 3H), 1.65 (s, 12H),1.48 – 1.46 (m, 2H). HRMS-ESI (m / z) Calcd for (C 63 H 73 Cl 2 FN 7 O 5 + ) ([M-Br] + ):1096.5029, found: 1096.5034.

[0043] The difference between Example 2 and Example 1 is that no alkali (triethylamine) was added in the reaction. The structure was confirmed by H NMR and MS, and the near-infrared heptamethine cyanine photosensitizer conjugate of compound 1 was successfully synthesized.

[0044] Example 3 Preparation of near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles

[0045] Use 1 mL 1×PBS (pH=7.2-7.4) to dissolve 66 mg (BSA). Then, dissolve 1 mM of compound 1 in methanol. Dilute 1 mM BSA and compound 1 to 10 μM with PBS. Then, add 10 μM of compound 1 dropwise to the BSA solution. The combined solution was vortexed for 10 seconds and heated to 70°C for two hours. Then, add double distilled water and purify using an ultrafiltration centrifuge tube (30 kDa). The near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles were stored at 4°C.

[0046] Example 4 Characterization of Near-Infrared Heptamethine Cyanine Photosensitizing Dye Conjugate Composite Nanoparticles

[0047] The hydrodynamic diameter of the near-infrared heptamethine cyanine photosensitizer conjugate composite nanoparticles was measured using a Malvern Zetasizer Nano series ZS-90. The morphology of the samples was observed using a transmission electron microscope (TEM) Hitachi TEM (HT7700, Japan). Figure 2 As shown in a, it can be seen that the nanoparticles are evenly distributed in the water. Figure 2 b It can be seen that the average particle size of the nanoparticles is 156.3 nm.

[0048] Example 5 Spectral measurement of near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles.

[0049] Use a 1 / 10,000 analytical balance to accurately weigh the heptamethine cyanine photosensitizer dye conjugate composite nanoparticles and prepare a 10mM solution for use. Dilute it to a 4 μM dye solution for testing, and use a Shimadzu UV-3600 UV fluorescence spectrophotometer and Lumina Fluorescence Spectrometer to measure the ultraviolet absorption spectrum and fluorescence emission spectrum of the compound, respectively. The absorption spectrum of the near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles is shown in Figure 2. Figure 2 As shown in the figure, it can be seen that the conjugate composite nanoparticles in water show a clear absorption peak at 778 nm similar to the photosensitizer dye in DMSO, which is significantly clearer than the photosensitizer dye in water. The fluorescence spectrum of the near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles is shown in Figure 3 As shown in the figure, it can be seen that the fluorescence intensity of the photosensitizer dye in water is very weak, while the fluorescence intensity of the conjugate composite nanoparticles in water and the photosensitizer dye in DMSO are both relatively strong, indicating that the composite nanoparticles in water protect the photosensitizer dye from quenching caused by aggregation.

[0050] Example 6 Photothermal effect of near-infrared heptamethine cyanine photosensitizing dye conjugate composite nanoparticles

[0051] Use PBS to prepare a 10 mM stock solution of the heptamethine cyanine photosensitizer dye conjugate composite nanoparticles for later use. Take a certain amount of the mother solution from each of the above storage solutions and add it to PBS to prepare a 10 μM sample working solution, which is added to 1.5 mL centrifuge tubes. Place the centrifuge tubes containing the detection solution at 808 nm, 1.0 W / cm 2 The near-infrared laser was irradiated for 5 minutes, and the temperature change was recorded every 10 seconds. The thermal imaging image of the near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles under the laser is shown in the figure. Figure 4 As shown in the figure, it can be seen that the drug can produce a photothermal effect under laser irradiation, and with the increase of time and laser intensity, the temperature generated gradually accumulates. 20 μM drug can heat water to about 50°C under 5 min irradiation.

[0052] Example 7 In vitro singlet oxygen production of near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles

[0053] DPBF probe (prepared with 100% methanol solution) was added to 2 mL of 10 μM near-infrared heptamethine cyanine photosensitizer conjugate composite nanoparticle solution. The DPBF concentration was 1.5 μM and the methanol content was 2%. 2 The samples were irradiated with 808 nm laser for 5 min, and the absorption spectrum intensity at 415 nm was immediately measured to quantify the generation of singlet oxygen. Figure 5 As shown in the figure, it can be seen that the drug can produce active oxygen under laser irradiation, and the active oxygen produced gradually increases with the increase of time and laser intensity.

[0054] Example 8 Tumor Targeting Ability of Near-Infrared Heptamethine Cyanine Photosensitizing Dye Conjugate Composite Nanoparticles

[0055] A subcutaneous tumor-bearing model of Balb / c mice (male) was established using CT26 cells. Near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles were administered via the tail vein. Near-infrared fluorescence real-time imaging was performed using a small animal in vivo imaging system to observe the metabolic distribution of the compound in the mouse at different time points. The in vivo imaging of near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles in colorectal cancer CT26 tumor-bearing mice is shown in Figure 2. Figure 6 As shown in the figure, it can be seen that the drug shows stronger fluorescence in the tumor site than in other normal tissue sites, indicating that the drug accumulates more in the tumor site, proving that the drug has good tumor targeting ability.

[0056] Example 9 Application of near-infrared heptamethine cyanine photosensitizer dye conjugate composite nanoparticles in tumor phototherapy in tumor-bearing mice

[0057] A subcutaneous tumor-bearing model of Balb / c male mice was established using CT26 cells. The mice were divided into (1) PBS group, (2) group treated with heptamethine cyanine photosensitizer dye conjugate composite nanoparticles alone, (3) group treated with PBS plus laser irradiation, and (4) group treated with heptamethine cyanine photosensitizer dye conjugate composite nanoparticles plus laser irradiation, with 5 mice in each group. The mice were given drugs through the tail vein, and 24 hours later, the tumors of groups (2) and (4) were treated with 1 W / cm 2The mice in the four groups were irradiated with 808 nm laser for 5 minutes, and the weight and tumor volume changes were continuously observed and measured. Figure 7 As shown in the figure, it can be seen that the tumor growth rate of mice in the drug group slowed down, while the combined drug + light treatment group significantly inhibited tumor growth, with a good tumor inhibition effect.

[0058] Example 10 Hematoxylin-eosin stained sections of major organ tissues of mice in different experimental groups.

[0059] The mice in the PBS group and the other three experimental groups were killed. The heart, liver, spleen, lung and kidney were collected and fixed in 4% paraformaldehyde. Then paraffin-embedded sections and hematoxylin-eosin (H&E) staining were performed according to standard procedures. The hematoxylin-eosin staining sections of the main organs and tissues of mice in different experimental groups are shown in the figure. Figure 8 As shown in the figure, it can be seen that there was no obvious damage to the main organs of the mice (heart, liver, spleen, lungs, and kidneys), indicating that the drug is safe.

[0060] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A near-infrared heptamethine cyanine photosensitizer dye conjugate, Features: Its structural formula is as follows: The preparation method of the near-infrared heptamethine cyanine photosensitizing dye conjugate comprises the following steps: reacting compound 2 and compound 3 at a certain temperature under an inert atmosphere, and purifying the product after the reaction is complete to obtain the near-infrared heptamethine cyanine photosensitizing dye conjugate; the compound 2 is The compound 3 is 2. The near-infrared heptamethine cyanine photosensitizer dye conjugate according to claim 1, Features: The molar ratio of compound 2 to compound 3 is 1:(1-2), and the reaction temperature is 60-80°C.

3. The near-infrared heptamethine cyanine photosensitizer dye conjugate according to claim 1, Features: The purification process is to add ether to the reaction product for slurry filtering, and then chromatographically separate the filter cake.

4. The near-infrared heptamethine cyanine photosensitizing dye conjugate according to claim 3, Features: The chromatographic column is octadecylsilane bonded silica gel with a particle size of 75-150 μm.

5. The near-infrared heptamethine cyanine photosensitizing dye conjugate according to claim 3, Features: The eluent of the column chromatography is a mixture of methanol and water in a volume ratio of 1:1 to 4:

1.

6. A method for preparing composite nanoparticles, comprising the following steps: under ultrasonic conditions, dropping an ethanol or methanol solution of a near-infrared heptamethine cyanine photosensitizer dye conjugate into an aqueous solution of bovine serum albumin, and then stirring at 50-70°C to obtain the composite nanoparticles, wherein the near-infrared heptamethine cyanine photosensitizer dye conjugate is the near-infrared heptamethine cyanine photosensitizer dye conjugate described in any one of claims 1 to 5.

7. Use of the composite nanoparticles as claimed in claim 6, Features: The composite nanoparticles are used in preparing tumor diagnostic and therapeutic agents.

8. Use of the composite nanoparticles as claimed in claim 6, Features: The composite nanoparticles are used to prepare NIR-II fluorescence imaging contrast agents.

9. Use of the composite nanoparticles according to claim 6, Features: The composite nanoparticles are used to prepare PDT / PTT combined therapeutic agents guided by fluorescence imaging.

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