Near-infrared selenium rhodamine photodynamic photosensitizer as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a near-infrared selenium rhodamine photodynamic photosensitizer as well as a preparation method and application thereof. In order to provide a selenium rhodamine photosensitizer with exciting light wavelength in a near-infrared region, a photodynamic photosensitizer AN-SeR without heavy atoms is designed and synthesized by taking a selenium rhodamine dye as a parent chromophore and an electron acceptor and anthracene as an electron donor, the photosensitizer exists in two forms of a monomer and a J-aggregate in an aqueous solution, and the photodynamic photosensitizer AN-SeR has the characteristics of high sensitivity, high sensitivity, high sensitivity and the like. According to the J-aggregate, the singlet oxygen quantum yield can be improved by reducing the energy level gap between the singlet state and the triplet state, and more importantly, the excitation light wavelength of AN-SeR is expanded from a visible region to a near-infrared region more suitable for biological application. Under the irradiation of near-infrared light (650 nm), the median lethal concentration of AN-SeR on cancer cells is 0.052 [mu] M, and the AN-SeR has potential biological application value.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a near-infrared selenorhodamine photodynamic photosensitizer and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a minimally invasive, low-side effect, and highly effective phototherapy method that plays an increasingly important role in cancer treatment. Photodynamic therapy is a combination of photosensitizers (PSs), light sources, and oxygen (O2) that cause local oxidative stress in cancer cells, thereby achieving the purpose of treating tumors. Specifically, photosensitizers absorb light of a specific wavelength and change from the ground state ( 0 PS) to the singlet excited state ( 1 PS*), and then decays to the triplet excited state ( 3 PS*), the photosensitizer in the triplet excited state interacts with biological molecules to generate superoxide anions (O2 ·- ), hydroxyl radicals (·OH) and other reactive oxygen species (ROS), which is the Type I photodynamic reaction. The photosensitizer in the triplet excited state can also transfer energy to convert oxygen molecules into singlet oxygen ( 1 O2), which is a Type II photodynamic reaction.

[0003] In photodynamic therapy, the tissue penetration depth of the excitation light is a key factor affecting photodynamic therapy. Selenorhodamine and its derivatives are excellent photodynamic photosensitizers, which is mainly attributed to the ISC process promoted by selenium atoms in their molecular structure. However, this type of photosensitizer usually has a shorter excitation wavelength, which results in the inability of the excitation light to penetrate deep tissues, thereby limiting its therapeutic effect in treating deep tumors below the skin. Therefore, the development of selenorhodamine-type photosensitizers with excitation wavelengths in the near-infrared region has certain practical significance for the treatment of deep tumors. Summary of the invention

[0004] In view of the above problems, the present invention provides a near-infrared selenorhodamine photodynamic photosensitizer and a preparation method and application thereof. Selenorhodamine dye is used as a parent chromophore and an electron acceptor, anthracene is used as an electron donor, and a heavy atom-free photodynamic photosensitizer AN-SeR is designed and synthesized based on the SOCT-ISC mechanism. The photosensitizer exists in the form of monomers and J-aggregates in aqueous solution. The J-aggregates can not only reduce the energy level gap (ΔE) between the singlet state (S1) and the triplet state (T1), but also can effectively inhibit the photodynamic activity of the photosensitizer. ST) to increase the singlet oxygen quantum yield, and more importantly, to extend the excitation light wavelength of selenorhodamine-based photosensitizers to the near-infrared region which is more suitable for biological applications.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a near-infrared selenorhodamine photodynamic photosensitizer, the structural formula of which is:

[0007]

[0008] The present invention also provides a method for preparing a near-infrared selenorhodamine photodynamic photosensitizer, comprising the following steps:

[0009] Step 1, under N2 protection, 9-bromoanthracene is dissolved in ultra-dry THF and cooled, and n-butyl lithium is added dropwise, and after the addition is completed, the reaction is continued at the same temperature to obtain a mixed solution;

[0010] Step 2, dissolving selenopyrrolidone in ultra-dry THF, and then adding it dropwise to the above mixed solution. After the addition is completed, the temperature is raised to room temperature to continue the reaction. After the reaction is completed, HCl solution is added to quench the reaction. After extraction, drying and column chromatography purification, the photosensitizer, i.e., AN-SeR, is obtained.

[0011] Furthermore, the molar ratio of 9-bromoanthracene, n-butyl lithium and selenopyrrolidone is 3:3:1.

[0012] Furthermore, in step 1, the mixture is cooled to -78°C and the reaction is continued for 30 minutes.

[0013] Furthermore, in step 2, the temperature is raised to room temperature and the reaction time is continued for 4 hours.

[0014] Furthermore, the concentration of the HCl solution in step 2 is 1 mol / L.

[0015] Furthermore, in step 2, dichloromethane is used for extraction, and the developing solvents of the column chromatography are CH2Cl2 and CH3OH, and the volume ratio thereof is 10:1.

[0016] The present invention also provides an application of a near-infrared selenorhodamine photodynamic photosensitizer, which is used to kill cancer cells under near-infrared light irradiation.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The absorption wavelength of selenorhodamine photosensitizers reported so far is in the visible region. During photodynamic therapy, visible light can cause certain damage to surrounding normal tissues. The photosensitizer AN-SeR provided by the present invention can exist in two forms in aqueous solution: monomer (600nm) and J-aggregate (642nm). Under near-infrared light (650nm), AN-SeR shows strong 1 O2 and O2 ·- Its ROS generation ability in vivo and in vitro is stronger than that of the commercial photosensitizer MB, and it has potential biological application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 AN-SeR photosensitizer 13 CNMR image.

[0020] Figure 2 AN-SeR photosensitizer 1 H NMR spectra.

[0021] Figure 3 This is the HRMS graph of the photosensitizer AN-SeR.

[0022] Figure 4 This is the UV-visible absorption spectrum of the photosensitizer AN-SeR as it changes with concentration.

[0023] Figure 5 For DCFH, laser (650nm, 10mW / cm 2 ) fluorescence emission spectra of ROS generated by capturing photosensitizers under irradiation, where A is DCFH and AN-SeR, B is DCFH and MB, and C is DCFH, AN-SeR and Triton X-100.

[0024] Figure 6 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) was irradiated with laser (650nm, 10mW / cm 2 ) shows the change of the UV-visible absorption spectrum of the singlet oxygen produced by the photosensitizer under irradiation, where A is ABDA and AN-SeR, B is ABDA and MB, and C is ABDA, AN-SeR and Triton X-100. D is the change of the absorbance value of ABDA at 380nm in A~C over time.

[0025] Figure 7 For DHR 123, laser (650nm, 10mW / cm 2 ) captures the photosensitive O2 under irradiation ·-Fluorescence emission spectra of , where A is DHR 123 and AN-SeR, B is DHR 123 and MB; C is DHR 123, AN-SeR and Triton X-100.

[0026] Figure 8 The cytotoxicity test results of photosensitizers AN-SeR and MB, wherein A is the dark toxicity and phototoxicity test results of photosensitizer AN-SeR, and B is the dark toxicity and phototoxicity test results of MB.

[0027] Fig. 9 These are the experimental results of the death mode of A549 cells induced by the photosensitizer AN-SeR, where A is the phototoxicity and dark toxicity of A549 cells when AN-SeR was co-incubated with different death inhibitors; B is the live / dead cell imaging of A549 cells treated with AN-SeR, AN-SeR+hν, and AN-SeR / Fer-1+hν, respectively. DETAILED DESCRIPTION

[0028] In order to further illustrate the technical solution of the present invention, the present invention is further described below through embodiments.

[0029] A near-infrared selenorhodamine photodynamic photosensitizer in this embodiment has the structural formula:

[0030]

[0031] The preparation method of the near-infrared selenorhodamine photodynamic photosensitizer comprises the following steps:

[0032] (1) Under N2 protection, 9-bromoanthracene (0.2 g, 0.9 mmol) was dissolved in ultra-dry THF (10.0 mL) and cooled to -78°C. n-Butyl lithium (2.5 M, 348 μL, 0.9 mmol) was added dropwise to the above solution. After the addition was completed, the mixture was reacted at this temperature for 30 minutes;

[0033] (2) Selenopyrrolidone (100 mg, 0.3 mmol) was dissolved in ultra-dry THF and added dropwise to the above mixture. After the addition was completed, the reaction solution was warmed to room temperature and continued to react for 4 hours. After the reaction was completed, 1M HCl (10.0 mL) was added to quench the reaction. The reaction solution was extracted with dichloromethane, dried and purified by column chromatography (DCM / MeOH=10 / 1) to obtain photosensitizer AN-SeR (blue-purple solid). 13 CNMR images, 1 H NMR and HRMS Figures 1 to 3 shown.

[0034] 1H NMR (600MHz, DMSO-d6) δ8.95(s,1H),8.29(d,J=8.2Hz,2H),7.83(s,2H),7.61-7.54(m,2H),7.47-7.40(m,2H),7.30(d,J=8.3Hz,2H),6.83(d,J=8.9

[0035] Hz, 2H), 6.72 (d, J = 9.7Hz, 2H), 3.18 (s, 12H); 13 C NMR (150 MHz, DMSO-d6)δ

[0036] 158.27,153.44,145.19,137.29,131.09,130.60,130.03,129.41,129.32,12 8.05,126.33,125.55,120.22,116.60,110.18,55.36,40.75,40.59; HR-MS[M] + :calculated for 507.1344,Found 507.1340.

[0037] Example 2

[0038] 1. Solution preparation

[0039] The stock solutions (2 mM) of AN-SeR and methylene blue (MB) were prepared with chromatographically pure DMSO, and the stock solutions (2 mM) of 2,7-dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123 (DHR 123) were prepared with chromatographically pure DMF. When testing, they were diluted to the corresponding concentrations with PBS (10 mM, pH = 7.4).

[0040] 2. Titration study

[0041] First, the absorption spectrum of AN-SeR in PBS (10 mM, pH = 7.4) was tested using a UV-visible absorption spectrometer. Figure 4 As shown in the figure, at low concentrations (<1μM), AN-SeR exists in monomeric form (600nm), and at high concentrations (>1μM), AN-SeR exists in both monomeric and aggregated forms (642nm). The maximum absorption peak of the aggregated state is 42nm red-shifted compared to the monomer, which is within the therapeutic window more suitable for biological applications and has practical application value. When the concentration of AN-SeR is 8μM, AN-SeR shows an obvious aggregation peak in PBS, and the maximum absorbance value is about 0.2. Therefore, in Subsequent implementation In the experiment, 650 nm was selected as the excitation wavelength and 8 μM was the test concentration of AN-SeR.

[0042] 3. Study on the in vitro performance of photosensitizers

[0043] 1) Photosensitizer AN-SeR under near-infrared light irradiation (650nm, 10mW / cm 2 ) Ability to generate active oxygen

[0044] The ability of AN-SeR to generate ROS in PBS was tested using a commercial reactive oxygen species fluorescent probe DCFH and compared with that of MB. Figure 5 As shown, when the DCFH / AN-SeR or DCFH / MB solution was continuously irradiated with a 650nm laser, DCFH was oxidized by the generated ROS, and the fluorescence intensity at 526nm gradually increased. Compared with MB, AN-SeR generated more and faster ROS under laser irradiation; when the depolymerization reagent Triton X-100 was added to the DCFH / AN-SeR solution, the fluorescence intensity at 526nm hardly changed after irradiation. The above results show that under 650nm laser irradiation, AN-SeR has a stronger ROS generation ability than the commercial photosensitizer MB.

[0045] 2) Reactive oxygen species generated by photosensitizer AN-SeR under light

[0046] First, take ABDA as 1 O2 capture agent, methylene blue (MB) as reference, AN-SeR (8 μM) and AN-SeR (8 μM) / Triton X-100 (disaggregating agent) were tested in PBS solution 1 O2's ability. Figure 6 As shown in Figure 2, when the ABDA / AN-SeR solution (or ABDA / MB solution) is continuously irradiated with a 650nm laser, the absorbance value of ABDA at 380nm decreases rapidly. It can be calculated that the singlet oxygen generation efficiency of AN-SeR (Φ Δ ) is 0.56, which is similar to MB 1 The O2 production capabilities were comparable; however, when the ABDA / AN-SeR / TritonX-100 solution was irradiated with light, the absorption peak of ABDA at 380 nm remained almost unchanged due to the disappearance of the aggregation peak.

[0047] Next, use O2 ·- The fluorescent probe DHR 123 was used to test the O2 production of AN-SeR and AN-SeR / Triton X-100 in PBS. ·- Such as Figure 7 As shown in Figure 2, when the DHR 123 / AN-SeR solution (or DHR 123 / MB solution) was continuously irradiated with a 650 nm laser, the fluorescence intensity at 530 nm gradually increased, and AN-SeR produced more O2 than MB.·- ; After adding Triton X-100 to the DHR 123 / AN-SeR solution, the fluorescence intensity at 530nm hardly changed. The above results show that the J-aggregates formed by AN-SeR in water can effectively reduce ΔE ST Under near-infrared light irradiation, a large amount of 1 O2 and O2 ·- .

[0048] 4. Study on the effect of photosensitizer photodynamic therapy

[0049] 1) Biological application effects of AN-SeR

[0050] The biological toxicity and photodynamic therapy effect of AN-SeR were tested by CCK8 experiment and compared with MB. A549 cells were incubated with different concentrations (0-0.5μM) of AN-SeR and MB for 1 hour, and the non-light-exposed (dark toxicity) group was placed in a cell culture incubator for 24 hours, and the light-exposed (phototoxicity) group was treated with laser (650nm, 30mW / cm 2 ) and then placed in a cell culture incubator for 24 hours. Figure 8 As shown in Figure 2, AN-SeR has acceptable dark toxicity in the range of 0-0.5 μM, and its phototoxicity gradually increases with the increase of concentration. The half lethal concentration (EC 50 ) was 0.052 μM. Under the same conditions, the survival rate of A549 cells treated with 0.5 μM MB was as high as 69.7%.

[0051] 2) AN-SeR-induced death of A549 cells

[0052] The cell viability was determined when AN-SeR was co-incubated with different death inhibitors. Fig. 9 As shown in (A), when A549 cells were co-incubated with apoptosis inhibitor (Z-VAD-FMK), necrosis inhibitor (Nec-1) and autophagy inhibitor (3-MA), A549 cells died rapidly under light, while ferroptosis inhibitor Fer-1 could significantly increase the survival rate of A549 cells.

[0053] Calcein-AM / PI staining experiments further confirmed this result. Fig. 9As shown in (B), A549 cells loaded with AN-SeR showed bright green fluorescence, indicating that AN-SeR had almost no dark toxicity; A549 cells loaded with AN-SeR showed bright red fluorescence after illumination, indicating that AN-SeR had strong phototoxicity; and A549 cells loaded with AN-SeR / Fer-1 showed similar green fluorescence to that of cells loaded with AN-SeR alone after illumination, indicating that Fer-1 effectively inhibited the death of A549 cells. The above results show that the mode of death of A549 cells induced by AN-SeR is ferroptosis.

[0054] In summary, the present invention constructs a heavy atom-free photodynamic photosensitizer AN-SeR by introducing a hydrophobic anthracene structure at the center of selenorhodamine. The J-aggregate formed by the photosensitizer in water can not only effectively reduce ΔE ST , which makes AN-SeR have a high efficiency in generating reactive oxygen species, and the half-lethal concentration for cancer cells under light is 0.052μM; importantly, the maximum absorption wavelength of the J-aggregate is 42nm red-shifted compared to the monomer, thus extending the excitation wavelength of selenorhodamine photosensitizers from the visible region to the near-infrared region, which is more suitable for biological applications, and has potential biological application value.

[0055] The above shows and describes the main features and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0056] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A near-infrared selenorhodamine photodynamic photosensitizer, characterized in that: The structural formula of the photosensitizer is:

2. The method for preparing a near-infrared selenorhodamine photodynamic photosensitizer according to claim 1, characterized in that: The following steps are involved: Step 1, under N2 protection, 9-bromoanthracene is dissolved in ultra-dry THF and cooled, and n-butyl lithium is added dropwise, and after the addition is completed, the reaction is continued at the same temperature to obtain a mixed solution; Step 2, dissolving selenopyrrolidone in ultra-dry THF, and then adding it dropwise to the above mixed solution. After the addition is completed, the temperature is raised to room temperature to continue the reaction. After the reaction is completed, HCl solution is added to quench the reaction. After extraction, drying and column chromatography purification, the photosensitizer, i.e., AN-SeR, is obtained.

3. The method for preparing a near-infrared selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: The molar ratio of the 9-bromoanthracene, n-butyl lithium and selenopyrrolidone is 3:3:

1.

4. The method for preparing a near-infrared selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: In the step 1, the mixture is cooled to -78°C and the reaction is continued for 30 minutes.

5. The method for preparing a near-infrared selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: In step 2, the temperature is raised to room temperature and the reaction is continued for 4 hours.

6. The method for preparing a near-infrared selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: The concentration of the HCl solution in step 2 is 1 mol / L.

7. The method for preparing a near-infrared selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: In step 2, dichloromethane is used for extraction, and the developing solvents of column chromatography are CH2Cl2 and CH3OH, and the volume ratio thereof is 10:

1.

8. The use of a near-infrared selenorhodamine photodynamic photosensitizer according to claim 1, characterized in that: Used to prepare products that kill cancer cells.

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