A near-infrared selenorhodamine photodynamic photosensitizer and its preparation method and application
By designing the near-infrared selenium rhodamine photodynamic photosensitizer AN-SeR, the problem of short excitation wavelength of selenium rhodamine photosensitizers was solved, and efficient ROS generation in the near-infrared region was achieved, which has a strong ability to kill cancer cells.
Patent Information
- Application Number
- CN202411373127.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2025-10-14
- Estimated Expiration
- 2044-09-29
AI Technical Summary
Existing selenorhodamine photosensitizers have a short excitation wavelength and cannot effectively penetrate deep tissues, which limits their application in treating deep tumors.
A near-infrared selenorhodamine photodynamic photosensitizer AN-SeR was designed. Selenorhodamine dye was used as the parent chromophore and anthracene was combined as the electron donor. Through the SOCT-ISC mechanism, a heavy atom-free photosensitizer was synthesized. It can exist in aqueous solution in the form of monomers and J-aggregates, reducing the gap between singlet and triplet energy levels and extending the excitation light wavelength to the near-infrared region.
Under near-infrared light irradiation, AN-SeR showed strong 1O2 and O2·- generation capabilities, and its ROS generation ability was stronger than that of the commercial photosensitizer MB. It has potential biological application value and can effectively kill cancer cells.
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Figure CN119241495B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fluorescent probes, and particularly relates to a near-infrared selenorhodamine photodynamic photosensitizer, a preparation method thereof, and an 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 three-way interaction of photosensitizers (PSs), light sources, and oxygen (O2) that induces local oxidative stress within cancer cells, thereby achieving the purpose of treating tumors. Specifically, photosensitizers absorb light of a specific wavelength and transform from a ground state ( 0 PS) transitions 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 make oxygen molecules form 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 order to solve the above problems, the present invention provides a near-infrared selenorhodamine photodynamic photosensitizer and its preparation method and application. Selenorhodamine dye is used as the parent chromophore and electron acceptor, anthracene is used as the 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 reaction 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] In order to achieve the above-mentioned purpose, 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. After the addition is complete, 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 complete, 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 reaction time after the temperature is raised to room temperature is 4 hours.
[0014] Furthermore, the concentration of the HCl solution in step 2 is 1 mol / L.
[0015] Furthermore, dichloromethane is used for extraction in step 2, and the developing solvents for column chromatography are CH2Cl2 and CH3OH, with a volume ratio of 10:1.
[0016] The present invention also provides an application of a near-infrared selenium rhodamine 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 spectrum.
[0021] Figure 3 This is the HRMS diagram of the photosensitizer AN-SeR.
[0022] Figure 4 The UV-visible absorption spectrum of the photosensitizer AN-SeR changes with concentration.
[0023] Figure 5 For DCFH, laser (650nm, 10mW / cm 2 ) fluorescence emission spectra of ROS generated by the captured photosensitizer under irradiation, where A represents DCFH and AN-SeR, B represents DCFH and MB, and C represents 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 ) UV-visible absorption spectra of singlet oxygen generated by photosensitizers captured under irradiation. A shows ABDA and AN-SeR, B shows ABDA and MB, and C shows ABDA, AN-SeR, and Triton X-100. D shows the absorbance variation of ABDA at 380 nm over time in A–C.
[0025] Figure 7 For DHR 123, laser (650nm, 10mW / cm 2 ) captures 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 These are the cytotoxicity test results of the photosensitizers AN-SeR and MB, where A is the dark toxicity and phototoxicity test results of the photosensitizer AN-SeR, and B is the dark toxicity and phototoxicity test results of MB.
[0027] Figure 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 is 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 examples.
[0029] The structural formula of a near-infrared selenorhodamine photodynamic photosensitizer in this embodiment is:
[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 complete, 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 complete, the reaction solution was warmed to room temperature and the reaction was continued 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 spectra 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. Preparation of solution
[0039] AN-SeR and methylene blue (MB) stock solutions (2 mM) were prepared with chromatographically pure DMSO, and 2,7-dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123 (DHR 123) stock solutions (2 mM) were prepared with chromatographically pure DMF. During testing, the solutions 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 monomeric state, 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 a clear aggregation peak in PBS, and the maximum absorbance value is about 0.2. Therefore, in In the subsequent experiment, 650 nm was selected as the excitation wavelength, and 8 μΜ was selected as the test concentration of AN-SeR. Figure 5
[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 produce reactive oxygen species
[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 MB. Figure 6 As shown, when a 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 ROS and faster under laser irradiation. When the disaggregation reagent Triton X-100 was added to the DCFH / AN-SeR solution, the fluorescence intensity at 526nm remained almost unchanged after irradiation. These results indicate that under 650nm laser irradiation, AN-SeR has a stronger ROS-generating 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 to generate 1 O2's capabilities. Figure 7 As shown in the figure, 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 consistent with 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 8 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 indicate 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 biotoxicity and photodynamic therapy effects of AN-SeR were tested by CCK8 assay and compared with MB. A549 cells were incubated with AN-SeR and MB at different concentrations (0-0.5 μM) for 1 hour. The non-illumination (dark toxicity) group was cultured in a cell culture incubator for 24 hours. The illumination group (phototoxicity) was treated with laser (650 nm, 30 mW / cm 2 ) and then placed in a cell culture incubator for 20 minutes and continued to culture for 24 hours. Figure 9 As shown in the figure, AN-SeR has acceptable dark toxicity in the range of 0-0.5 μM, and its phototoxicity gradually increases with the increase of concentration. 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. Figure 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 improve the survival rate of A549 cells.
[0053] Calcein-AM / PI staining experiments further confirmed this result. As shown in (B), A549 cells loaded with AN-SeR exhibited bright green fluorescence, indicating that AN-SeR has almost no dark toxicity; A549 cells loaded with AN-SeR exhibited bright red fluorescence after illumination, indicating that AN-SeR has strong phototoxicity; and A549 cells loaded with AN-SeR / Fer-1 exhibited similar green fluorescence after illumination as those loaded with AN-SeR alone, indicating that Fer-1 effectively inhibited A549 cell death. These results indicate that the mode of death of A549 cells induced by AN-SeR is ferroptosis.
[0054] In summary, the present invention introduces a hydrophobic anthracene structure into the center of selenorhodamine to construct a heavy atom-free photodynamic photosensitizer AN-SeR. 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 expanding the excitation wavelength of selenium rhodamine 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 foregoing shows and describes the principal features and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims, not the foregoing description, and all variations that come within the meaning and range of equivalents of the claims are intended to be embraced therein.
[0056] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method 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 can also be appropriately combined to form other implementation methods 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. After the addition is complete, 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 complete, 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 was cooled to -78°C and the reaction was 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 for column chromatography are CH2Cl2 and CH3OH, with a volume ratio of 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.