Preparation of mitochondrial targeting photosensitizer with I-type photodynamic effect and tumor phototherapy

Abstract

The invention discloses preparation of a mitochondrial targeting photosensitizer with a type I photodynamic effect and tumor phototherapy. The invention discloses preparation of a series of cyanine photosensitizers with a cell mitochondrial targeting effect and type I ROS (reactive oxygen species) generation capability and tumor phototherapy combining photothermal and photodynamic effects. The photosensitizer modifies an alkyl chain connected with a nitrogen atom on an indole ring of a heptamethine cyanine molecule, a water-soluble carboxyl group is introduced to one side of the indole ring, the length of the carboxyl chain is changed, the hydrophilicity of the photosensitizer is improved, the mitochondrial targeting ability of the photosensitizer is improved, and meanwhile the length (n = 1-10) of an alkyl chain on the other side of the indole ring is changed, so that the mitochondrial targeting ability of the heptamethine cyanine molecule is improved. And respectively synthesizing asymmetric cyanine molecules. Tetraphenylethylene (TPE) groups are introduced into asymmetric cyanine molecules, the asymmetric cyanine molecules (n = 1-10) coupled with the TPE are synthesized respectively, and the mitochondrial targeting ability, the photo-thermal heating ability and the ROS generation efficiency of the photosensitizer are enhanced. Therefore, a series of photosensitizers disclosed by the invention are expected to be applied to clinical tumor tracing and enhancement of anti-cancer phototherapy in a tumor hypoxia environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent biological diagnosis and treatment, and specifically relates to the preparation of a series of cyanine photosensitizers with cell mitochondria targeting effects and phototherapy combining photothermal and photodynamic therapy. Background Art

[0002] Phototherapy is mainly divided into photothermal therapy (PTT) and photodynamic therapy (PDT). It is a treatment method that converts light energy into heat energy or reactive oxygen species (ROS) to cause cancer cell death in a non-invasive way. Photodynamic therapy includes type II PDT that strongly depends on oxygen and type I PDT that does not depend on oxygen. Due to the hypoxic characteristics of the tumor microenvironment, the conventional type II PDT treatment efficiency is low. However, most photosensitive molecules currently have type II photoreactions as the main reaction, which greatly limits the efficiency of phototherapy.

[0003] PDT drugs targeting the cell nucleus can effectively kill tumor cells, but due to the risk of genetic mutation, they are not conducive to tumor treatment. Mitochondria are organelles involved in energy production, metabolism and signal transduction, providing ATP for cell growth, and therefore play an important role in the development and metastasis of cancer; in addition, mitochondria are the main site of cell respiration, are highly sensitive to temperature and reactive oxygen species, and are involved in cell apoptosis. PDT drugs targeting mitochondria can cause cellular oxidative stress under light activation and affect the biological functions of cells. Therefore, mitochondrial-targeted photosensitizers are expected to improve the efficacy of phototherapy. Some photosensitive molecules easily form aggregates in water, which reduces the effective concentration of the compound, thereby weakening the interaction with the mitochondrial membrane and limiting the ability of photosensitive molecules to enter the mitochondria. Therefore, introducing a water-soluble group carboxyl into the structure is expected to enhance the mitochondrial targeting ability of the molecule.

[0004] The photodynamic therapy process is divided into type I PDT and type II PDT. The triplet photosensitizer absorbs hydrogen atoms or electrons to form free radicals, which then interact with oxygen to produce reactive oxygen species (ROS), such as O 2 •− , OH•, i.e., type I reactive oxygen species, or triplet photosensitizers transfer energy directly to tissue oxygen, generating 1 O 2 , that is, type II reactive oxygen species. Unlike type II PDT, type I PDT can make full use of disproportionation reaction, Haber-Weiss reaction or Fenton reaction to compensate for O 2The depletion of oxygen does not rely on oxygen, and can achieve the treatment of hypoxic tumors and improve the treatment efficiency. Structural modification is the main strategy to enhance the type I PDT effect, which is mainly reflected in: (1) enhancing the efficiency of ROS generation by strengthening the electron donor-acceptor (DA) interaction and reducing the singlet-triplet energy gap. The usual practice is to introduce groups with DA effects into the molecule, such as tetraphenylethylene, to improve the intramolecular mobility; (2) introduce heavy atoms such as halogen atoms to enhance the intersystem crossing (ISC) process to improve the efficiency of reactive oxygen generation. Summary of the invention

[0005] The purpose of the present invention is to provide a series of cyanine photosensitizer molecules with cell mitochondrial targeting and type I PDT. Among them, the photosensitizer T780CAR-I has good photostability and ROS generation ability, as well as good cell mitochondrial targeting ability; it shows effective tumor targeting and retention ability in mice, and can be used for fluorescence imaging-guided phototherapy.

[0006] The present invention provides a series of cyanine photosensitizer molecules with cell mitochondrial targeting and type I PDT, and its structural formula (1) is as follows: Figure 1 As shown:

[0007] The synthesis steps of the photosensitizer provided by the present invention are shown in the attached Figure 2

[0008] The present invention provides a method for preparing a photosensitizer, comprising the following steps: Asymmetric cyanine molecules: Under nitrogen protection, acetic anhydride as solvent, 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde, sodium acetate, indole iodide with different alkyl chain lengths and indole iodide with different carboxyl chain lengths were stirred and refluxed at 130 °C for 1 h. The solvent was evaporated and the crude product was purified by silica gel chromatography to obtain an asymmetric monocyanine molecule (R=1-10). Asymmetric cyanine molecules coupled with TPE: Under nitrogen protection, anhydrous DMF as solvent, 1,2-di(4-hydroxyphenyl)-1,2-diphenylethylene, potassium carbonate and asymmetric monocyanine molecules were stirred and refluxed at 70 °C for 8 h. The solvent was evaporated and the crude product was purified by silica gel chromatography to prepare an asymmetric biscyanine molecule (R=1-10).

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The cyanine photosensitizer for cell mitochondrial targeting and type I PDT of the present invention has the following characteristics: (1) The photosensitizer of the present invention modifies the alkyl chain connected to the nitrogen atom on the indole ring of the heptamethine cyanine molecule, and introduces a water-soluble group carboxyl group on one side of the indole ring, thereby reducing the aggregation of the molecule and improving the mitochondrial targeting ability; (2) The photosensitizer of the present invention changes the length of the alkyl chain and carboxyl chain on the indole ring of the heptamethine cyanine molecule to synthesize an asymmetric cyanine molecule 780CAR-I; (3) A tetraphenylethylene (TPE) group is introduced into the photosensitizer 780CAR-I of the present invention to enhance its photostability, photothermal conversion efficiency and ROS generation efficiency; (4) The photosensitizer T780CAR-I of the present invention can target the tumor site and accumulate for a long time; (5) The photosensitizer T780CAR-I of the present invention has low dosage, one-time injection, operability of multiple irradiations and effective inhibition of tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The invention discloses a synthetic route for the photosensitizer.

[0012] Figure 2 The invention discloses a synthetic route for the photosensitizer.

[0013] Figure 3 This is the synthetic route of the photosensitizer intermediate 780CAR-I of the present invention.

[0014] Figure 4 This is the synthetic route of the photosensitizer final product T780CAR-I of the present invention.

[0015] Figure 5 The UV absorption graph and fluorescence emission graph of the photosensitizer (10 µM) of the present invention in DMSO and aqueous solution, respectively.

[0016] Figure 6 Schematic diagram of thermal cycle and photothermal influencing factors of the photosensitizer of the present invention.

[0017] Figure 7 The figure is a comparison chart of the ROS generating ability and type of the photosensitizer of the present invention.

[0018] Figure 8 This is a confocal microscopic image of the co-localization of the photosensitizer T780CAR-I of the present invention and MitoTracker Green in A549 cells.

[0019] Fig. 9 Graph showing the photoactivated cytotoxicity of the photosensitizer T780CAR-I of the present invention under normoxic and hypoxic conditions.

[0020] Fig.10The fluorescence imaging of the photosensitizer T780CAR-I of the present invention after intravenous injection into 4T1 tumor-bearing BALB / c mice, and the distribution of fluorescence in major organs 24 hours and 120 hours after injection. Ex=745 nm, Em=800 nm.

[0021] Fig.11 After intravenous injection of the photosensitizer T780CAR-I into 4T1 tumor-bearing BALB / c mice (24 h, 48 h, 72 h), 808 nm laser (0.8 W cm -2 ) Temperature changes of mouse tumor sites at different irradiation times. Diagram of the inhibitory effect on tumors.

[0022] Fig.12 These are the tumor change graph, body weight change graph and safety evaluation graph of the photosensitizer T780CAR-I of the present invention during the treatment period. DETAILED DESCRIPTION

[0023] The photosensitizer of the present invention and the specific implementation of its preparation method and application are further described below in conjunction with the accompanying drawings and examples.

[0024] Embodiment 1:

[0025] Synthesis of probe 780CAR-I:

[0026] The synthesis method is as follows Figure 3 shown.

[0027] The synthesis method is as follows:

[0028] Specific steps: iodinated 2,3,3-trimethyl-3H-indole (500 mg, 1.755 mmol) and 6-bromohexanoic acid (513 mg, 2.63 mmol) were placed in a 10 mL two-necked flask, a reflux device was assembled, vacuum-nitrogen replacement was performed 3 times, 3 mL of anhydrous acetonitrile was added with a syringe, and the mixture was heated to 80°C and refluxed for 48 hours. The reaction solution was concentrated under reduced pressure to obtain a brown solid crude product. The crude product was placed in dichloromethane to obtain an insoluble white solid iodinated carboxyindole. Place the magnet, iodo-2,3,3-trimethyl-1-propyl-3H-indole-iodide (250 mg, 0.549 mmol), 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (189.06 mg, 1.099 mmol), iodocarboxyindole (526.42 mg, 1.099 mmol) and sodium acetate (45.06 mg, 0.549 mmol) in a 10 mL two-necked flask, assemble a reflux device, replace the vacuum-nitrogen atmosphere three times, and then add 3 mL of acetic anhydride with a syringe. Under nitrogen protection, the reaction system was heated to 130°C and stirred at reflux for 1 h. TLC detection (developing solvent CH2 Cl 2 :CH 3 OH=15:1) After the reaction is complete, the solvent is evaporated with an oil pump, and the crude product is separated by silica gel chromatography. 2 Cl 2 :CH 3 The mixture was eluted with OH=15:1 eluent to obtain 207 mg of green solid compound 780CAR-I with a yield of about 40%. 1H NMR (400 MHz, Methanol-d4) δ 8.42 (dd, J = 14.5, 9.4 Hz, 2H), 7.88 (s, 2H), 7.76 (t, J =7.6 Hz, 2H), 7.16 (dd, J = 13.0, 8.5 Hz, 2H), 6.30 (t, J = 14.0 Hz, 2H), 4.13 (dd, J = 17.2, 8.7 Hz, 4H), 2.73 (s, 4H), 2.22 (t, J = 7.4 Hz, 2H), 1.95 (d,J = 8.7 Hz, 3H), 1.88-1.81 (m, 4H), 1.71 (d, J = 7.9 Hz, 12H), 1.47 (t, J =7.8 Hz, 3H), 1.04 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, Methanol-d4) δ137.60, 131.41, 127.46, 112.94, 112.81, 53.41, 48.26, 48.04, 47.83, 47.62,47.41, 47.19, 46.98, 45.42, 43.80, 33.33, 31.68, 30.45, 29.39, 29.37, 29.08,26.83, 26.63, 25.95, 24.30, 22.34, 20.43, 13.07, 10.24.

[0029] Embodiment 2:

[0030] Synthesis of probe T780CAR-I:

[0031] The synthesis method is as follows Figure 4 shown.

[0032] The synthesis method is as follows:

[0033] Specific steps: Place the magneton, 780CAR-I (120 mg, 0.127 mmol), 1,2-di(4-hydroxyphenyl)-1,2-diphenylethylene (46.28 mg, 0.127 mmol) and potassium carbonate (23.36 mg, 0.169 mmol) in a 10 mL two-necked flask, assemble a reflux device, replace the vacuum-nitrogen atmosphere three times, and then add 3 mL of DMF with a syringe. Under nitrogen protection, reflux at 70°C in the dark for 8 h. TLC monitoring (developing solvent CH 2 Cl 2 :CH 3 OH=10:1) after the reaction, the reaction solution was concentrated under reduced pressure. The obtained solid was extracted three times with dichloromethane and saturated brine (2: 1, v: v), and then the organic phases were combined and washed with anhydrous Na 2 SO 4The crude product was purified by silica gel column using DCM: MeOH (15: 1, v: v) as eluent to obtain 15 mg of green solid T780CAR-I with a yield of about 15%. 1H NMR (400 MHz, Methanol-d4) δ 7.82 (dt,J = 14.4, 4.7 Hz, 2H), 7.69 (t, J = 2.2 Hz, 2H), 7.63 (dt, J = 8.4, 2.0 Hz,2H), 7.00 (dd, J = 8.7, 5.1 Hz, 2H), 6.96-6.86 (m, 10H), 6.86-6.81 (m, 2H), 6.78 (d, J = 7.1 Hz, 2H), 6.72 (t, J = 8.1 Hz, 2H), 6.36 (dd, J = 10.4, 7.7Hz, 2H), 6.04 (dt, J = 14.1, 3.3 Hz, 2H), 3.96 (dt, J = 14.7, 7.5 Hz, 4H), 2.59 (s, 4H), 2.13 (t, J = 7.3 Hz, 2H), 1.88 (s, 3H), 1.69 (q, J = 7.3 Hz,4H), 1.56 (t, J = 7.6 Hz, 3H), 1.27 (d, J = 6.6 Hz, 12H), 0.91 (t, J = 7.4Hz, 3H). 13C NMR (101 MHz, Methanol-d4) δ 142.12, 137.54, 133.04, 132.12,131.27, 130.92, 127.50, 127.34, 126.06, 122.89, 114.40, 113.76, 48.04, 47.83,47.72, 47.66, 47.62, 47.40, 47.19, 46.98, 35.42, 27.06, 27.02, 26.61, 26.17,25.06, 23.72, 20.97, 20.29, 10.26. HRMS (ESI): m / z: 1191.3029 ([M]+, calcd,1191.3030).

[0034] Embodiment 2:

[0035] UV-visible absorption spectrum and fluorescence absorption spectrum: 10 µM 780CAR-I and T780CAR-I were prepared in DMSO and aqueous solution, respectively, and the fluorescence spectrum and UV absorption spectrum were measured. Figure 5 .

[0036] Embodiment 3:

[0037] Photothermal stability: Prepare 2 mL of DMSO solution of 780CAR-I and T780CAR-I at 10 µM, respectively, and place the solution under 808 nm laser (0.3 W / cm 2 ) for 10 min, and then naturally cooled to the initial temperature. Four heating-cooling photostability cycles were performed. The temperature of the solution was recorded every 30 s, and the color change of the solution was recorded after each cycle. Photothermal factors: 780CAR-I and T780CAR-I were prepared into 2.5 μM, 5 μM, 10 μM, and 20 μM working solutions. 2 mL of each solution was placed in a 24-well plate and exposed to 808 nm laser (0.3 W / cm 2 ) for 10 min, and the temperature of the solution was recorded every 30 s. The photothermal effects of the solutions with different molecular concentrations were compared. 10 μM 780CAR-I and T780CAR-I working solutions were prepared, 2 mL of each was placed in a 24-well plate, and 808 nm lasers of different intensities (0.3 W / cm2, 0.5 W / cm 2 , 0.8 W / cm 2 , 1.0 W / cm 2 ) for 10 min, and the temperature change of the solution was recorded every 30 s to compare the photothermal effect of the photosensitive molecular solution under different laser powers. Figure 6 .

[0038] Embodiment 4:

[0039] ROS generation: Weigh an appropriate amount of ABDA and dissolve it in DMSO to prepare a 10 mM stock solution; prepare 780CAR-I and T780CAR-I into a 10 μM working solution. Take 10 μL of ABDA stock solution and add it to 1990 μL of the molecule solution to be tested (50 μM) and evenly illuminate it under an 808 nm laser (0.3 W / cm 2) for 10 min, scan the full wavelength of UV (200 nm~1100 nm) every 1 min, and record the UV absorption intensity at 378 nm. Weigh an appropriate amount of DHR123 powder and dissolve it in DMSO to prepare a 10 mM mother solution; prepare 780CAR-I and T780CAR-I into a 10 μM working solution. Take 1 μL of DHR123 mother solution and add it to 999 μL of the molecule solution to be tested (10 μM) and mix well. 2 ) for 8 min, and the fluorescence emission spectrum (500 nm~650 nm) was scanned every 1 min, and the fluorescence emission intensity at 525 nm was recorded. 2 •− Characterization of production capacity: T780CAR-I was prepared into a 10 μM methanol solution, 100 μL was taken into a 96-well plate, DMPO (100 μM) was added, mixed, and then irradiated under 808 nm laser (0.5 W / cm 2 ) for 3 min, and test its electron paramagnetic resonance spectrum. Characterization of OH• production ability: 2 •− is different from OH•, which is tested in an aqueous environment. T780CAR-I was prepared into a 10 μM aqueous solution, 100 μL was taken into a 96-well plate, DMPO (100 μM) was added, mixed, and laser irradiated as above to test its electron paramagnetic resonance spectrum. The results are shown in Figure 7 .

[0040] Embodiment 5:

[0041] Fluorescence confocal laser scanning microscopy imaging: A549 cells were plated at 3×10 5 The cells were inoculated at a density of 100 / dish in a confocal glass-bottomed cell culture dish and cultured for 24 h. Take 1 μL of T780CAR-I mother solution (10 mM) and add 999 μL of 1640 complete culture medium to dilute it into a 10 μM working solution. Replace the original culture medium in the culture dish and incubate the cells with T780CAR-I for 4 h. Discard 500 μL of culture medium, add 500 μL of 100 nM Mito-Tracker Green solution, and incubate for 30 min. Discard the culture medium, wash twice with PBS, and then add 1 mL of PBS for laser confocal imaging. The results are shown in Figure 2. Figure 8 .

[0042] Embodiment 6:

[0043] Cell phototoxicity evaluation: A549 cells and 4T1 cells were cultured at 2×10 4The cells were inoculated at a density of 100 μM / well in a 96-well plate and cultured for 24 h. 780CAR-I and T780CAR-I were diluted to different concentrations (10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM), and three replicate wells were set for each concentration. They were co-incubated with cells. One group was cultured normally for 24 h, and the other group was induced to hypoxia for 24 h using a hypoxic chamber. The cells were then irradiated with an 808 nm laser (0.3 W / cm2) for 5 min, and continued to be cultured under hypoxic conditions for 24 h. 20 μL of MTT solution was added to each well, incubated for 4 h, and repeated three times. As above, the cell viability was tested and calculated using the MTT method. The results are shown in Fig. 9 .

[0044] Embodiment 7:

[0045] In vivo fluorescence imaging: The mother solutions of 780CAR-I and T780CAR-I (10 mM) were diluted to 100 μM in PBS and injected into the mice through the tail vein at a dose of 100 μL / mouse. The mice were anesthetized with isoflurane at 1 h, 5 h, 9 h, 24 h, 48 h, 72 h, 96 h, and 120 h, and in vivo imaging was performed using the small animal three-dimensional optical in vivo quantitative analysis system (IVIS Spectrum). The fluorescence emission wavelength was 745 nm and the receiving wavelength was 800 nm. At 24 h and 120 h after injection, one mouse was randomly selected from each group and killed. The heart, liver, spleen, lung, kidney, and tumor were dissected for organ imaging to observe the distribution of fluorescence signals of photosensitive molecules. The results are shown in Fig.10 .

[0046] Embodiment 8:

[0047] In vivo photothermal evaluation: When the tumor volume reaches about 100 mm 3 At 1:499, v / v, a 100 μM T780CAR-I solution was prepared. The mice in the T780CAR-I group and T780CAR-I+laser group were intravenously injected with 100 μL of T780CAR-I solution. The mice in the control group and control group+laser group were intravenously injected with 100 μL of PBS. At 24 h, 48 h, and 72 h after intravenous injection, the mice were illuminated with an 808 nm laser (1.0 W cm -2) irradiated the tumor sites of mice in the control group + laser group and T780T-TPP + laser group for 10 min each time. Then, 100 μL of T780T-TPP solution was intravenously injected into the mice in the T780T-TPP group and T780T-TPP + laser group again, and the above laser operation was repeated. During each laser irradiation, the temperature changes of the tumor sites of mice in the PBS group and T780T-TPP + laser group were recorded with a thermal imager. The results are shown in Fig.11 .

[0048] Embodiment 9:

[0049] Tumor inhibition and safety evaluation: The body weight and tumor volume of mice were measured every day during the 14-day treatment period. The tumor volume was calculated as follows: Volume = (tumor length) × (tumor width) 2 / 2. After the 14-day treatment cycle, the heart, liver, spleen, lung, kidney and other major organs and tumors of the mice were removed and stained with hematoxylin and eosin (H&E). The tumors of mice in each group were stained with H&E and immunohistochemistry (Bcl-2, IDO, PD-L1). The results are as follows Fig.12 .

Claims

1. A series of cyanine photosensitizer molecules with cell mitochondrial targeting and PTT / PDT synergistic phototherapy effects, whose structural formula (1) is shown in Figure 2.

2. The cyanine photosensitizer according to claim 1, characterized in that: One side of the indole ring of the heptamethine cyanine molecule is a water-soluble group carboxyl connected to alkyl chains of different lengths, and the other side of the indole ring of the heptamethine cyanine molecule is an alkyl chain of different lengths (n=1-10).

3. The photosensitizer as claimed in claim 1, wherein a tetraphenylethylene (TPE) group is introduced into the asymmetric cyanine molecule to synthesize a series of asymmetric cyanine molecules (n=1-10) to enhance the mitochondrial targeting ability, photothermal heating ability and type I ROS generation efficiency of the photosensitizer.

4. The photosensitizer according to claim 1, wherein the series of photosensitizers have cell mitochondrial targeting and accumulation effects and PTT / PDT synergistic phototherapy effects.

5. The photosensitizer as claimed in claim 1, wherein the asymmetric cyanine molecule has different targeting effects on tumors when the length of the alkyl chain on one side of the indole ring is different.

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