Preparation method and application of BODIPY oligomer NIR-II nano fluorescent probe

Abstract

The invention relates to a preparation method and application of a BODIPY oligomer NIR-II nano fluorescent probe, a BODIPY tetramer capable of being used for NIR-II fluorescence imaging is prepared through a Stile coupling reaction, then the water solubility and biocompatibility are improved through covalent connection of polyethylene glycol (PEG) with the molecular weight of 5000, the PEGylated fluorescent probe has excellent photothermal conversion capacity under 980 nm laser excitation, and the fluorescence probe can be used for fluorescence imaging of NIR-II. The material has ablation and killing effects on tumors, and shows a good photo-thermal treatment effect. Moreover, the probe has no obvious dark toxicity, can clearly display the blood vessels of the whole body of the mouse and the blood vessels at the tumor position in a living body, and can be enriched at the tumor position through high permeability and retention (EPR) effect. The probe prepared by the preparation method disclosed by the invention realizes NIR-II fluorescence imaging guided tumor photothermal therapy and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of nano-biomedical materials, and in particular to the design of a NIR-II nano-fluorescent probe prepared by PEGylating a BODIPY oligomer, and the application of the nano-fluorescent probe at the cellular and animal levels. Background Art

[0002] Deep tissue fluorescence imaging, with its high signal-to-noise ratio and high fluorescence intensity, has profound implications for in vivo biological research and disease diagnosis. Current research indicates that compared to conventional near-infrared imaging techniques in the first region (NIR-I, 650-900nm), near-infrared fluorescence imaging in the second region (NIR-II, 1000-1700nm) has longer excitation and emission wavelengths, resulting in lower tissue autofluorescence, higher spatiotemporal resolution, and high-penetration depth imaging. This allows for the precise location of microscopic lesions, offering significant advantages and potential for application in biomedical fields.

[0003] Compared to the inherent shortcomings of cyanine fluorescent probes, such as poor stability and low yield, boron dipyrrole (BODIPY) fluorophores are a commonly used NIR-II fluorophore due to their low toxicity, excellent photostability, and structural tunability. They have been a research hotspot in recent years. Furthermore, preparing BODIPY oligomers by increasing the number of repeating units and extending the conjugation is an effective way to achieve red-shifted absorption and emission wavelengths. BODIPYs are also excellent photothermal agents, effectively converting light energy into heat energy, thereby achieving cancer treatment.

[0004] In order to meet the requirements of biocompatibility, PEGylation of fluorophores to prepare nanoparticles can not only improve water solubility and biosafety, but also effectively increase the residence time of nanoparticles in the blood and increase the passive targeting ability of tumors. Based on the existing research status, the inventors of this application have developed a BODIPY tetramer with NIR-II imaging capabilities and excellent photothermal conversion capabilities. In order to better meet the conditions for in vivo use, the imaging agent is covalently linked to PEG to prepare a nanofluorescent probe. With the guidance of fluorescence imaging and the high penetration depth of lasers in the NIR-II region, NIR-II fluorescence imaging is combined with photothermal therapy, which can effectively improve the therapeutic effect of nanofluorescent probes on malignant tumors. Summary of the Invention

[0005] The present invention aims to provide a preparation method and application of a BODIPY tetramer NIR-II fluorescent probe based on the existing research status and technical deficiencies.

[0006] The technical solution adopted by the present invention is as follows: In order to achieve the above technical objectives, the present invention provides a nano fluorescent probe, which specifically comprises a BODIPY tetramer and a PEG chain, wherein the BODIPY tetramer has the following structure:

[0007]

[0008] The present invention provides a method for preparing a PEGylated nano fluorescent probe, comprising:

[0009] A certain proportion of BODIPY tetramer and PEG were mixed in a certain amount of acetonitrile, and the mixture was stirred under reflux at room temperature for 4-8 hours. The acetonitrile and unreacted raw materials were removed by dialysis, and then the solution was passed through a 0.22 μm aqueous membrane to obtain a uniform nanofluorescent probe.

[0010] Wherein, the molar ratio of the BODIPY tetramer to PEG is 1:1-2 (preferably 1:1.2).

[0011] Wherein, the dialysis process uses a dialysis bag with a molecular weight cut-off of 4500-7000 (preferably 6000).

[0012] The present invention provides a study on the photothermal conversion performance of a nanofluorescent probe. The experimental results show that the nanofluorescent probe has sufficient photothermal conversion ability under 980nm laser irradiation.

[0013] The present invention provides a study on the in vivo distribution of nano-fluorescent probes. The experimental results show that the nano-fluorescent probes can effectively realize blood vessel and tumor imaging and outline the contours of tumors with a high signal-to-background ratio.

[0014] The present invention provides an in vitro cytotoxicity study of the nano-fluorescent probe. The experimental results show that the nano-fluorescent probe has no obvious toxicity under no light conditions, but can effectively kill cancer cells after 980nm laser irradiation.

[0015] The present invention provides a method of injecting nanofluorescent probes through the tail vein to monitor the accumulation effect at the tumor and conduct photothermal therapy experiments. The results show that the nanofluorescent probes can effectively accumulate at the tumor through high permeability and retention (EPR) and have the ability to ablate tumors under laser irradiation.

[0016] The beneficial effects achieved by the present invention using the above structure are as follows: The preparation method and application of the BODIPY tetramer NIR-II fluorescent probe of the present invention have the following advantages:

[0017] (1) The BODIPY tetramer with NIR-II absorption and emission provided by the present invention has an absorption wavelength of 963 nm and an emission wavelength of 1009 nm in tetrahydrofuran, which expands the application range of BODIPY-based NIR-II fluorophores;

[0018] (2) The nanofluorescent probe prepared by PEGylation in the present invention has extremely high photostability and does not require strict light protection;

[0019] (3) The nanofluorescent probe has excellent NIR-II imaging performance, which can achieve high-penetration depth and high-contrast imaging of blood vessels and tumors;

[0020] (4) Nanofluorescent probes have the ability to perform photothermal therapy guided by NIR-II fluorescence imaging, which can effectively achieve tumor ablation. Therefore, they have application potential in the fields of fluorescence imaging and cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0022] Figure 1 The UV-visible absorption spectrum (a) and emission spectrum (b) of the nano fluorescent probe of the present invention are shown;

[0023] Figure 2 The photostability of the nano fluorescent probe of the present invention;

[0024] Figure 3 The in vitro photothermal conversion capability of the nano fluorescent probe of the present invention;

[0025] Figure 4 The survival rate of 4T1 cells under the irradiation of the nano fluorescent probe of the present invention without 980nm laser and with 980nm laser;

[0026] Figure 5 Whole-body vascular imaging and half-peak width intensity analysis of mice using the nano-fluorescent probe of the present invention under 980nm laser;

[0027] Figure 6 The nano fluorescent probe of the present invention is used to image blood vessels in the tumor of 4T1 tumor-bearing mice under 980nm laser;

[0028] Figure 7 The fluorescence intensity of the nano fluorescent probe of the present invention at the tumor of 4T1 tumor-bearing mice is monitored over time under 980nm laser;

[0029] Figure 8 The tissue distribution of the nanofluorescent probe in 4T1 tumor-bearing mice 48 hours after tail vein injection of the nanofluorescent probe;

[0030] Figure 9 This is the temperature rise curve of the tumor under 980nm laser irradiation after the nano fluorescent probe is injected into the tail vein of the 4T1 tumor-bearing mouse of the present invention;

[0031] Figure 10 The graph shows the tumor growth curves of 4T1 tumor-bearing mice under different treatment conditions after the tail vein injection of PBS or the nano-fluorescent probe of the present invention. DETAILED DESCRIPTION

[0032] The following describes preferred embodiments of the present invention with reference to the accompanying drawings to demonstrate that the present invention can be implemented. These embodiments will fully explain the present invention to those skilled in the art, making the technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments described herein.

[0033] like Figure 1 — Figure 10 As shown, the technical solution adopted by the present invention is as follows:

[0034] Example 1. Preparation method of nano fluorescent probe:

[0035] In this example, a nanofluorescent probe was prepared using a BODIPY tetramer and PEG as raw materials. The BODIPY tetramer and PEG were mixed in a predetermined amount of acetonitrile at a ratio of 1:1.2. The mixture was stirred under reflux at room temperature for 6 hours. The mixture was then dialyzed against ultrapure water using a dialysis bag (MWCO = 6000) to remove the acetonitrile and unreacted raw materials. Following dialysis, the solution was passed through a 0.22 μm aqueous membrane to obtain a uniform nanofluorescent probe. Figure 1 The UV-visible absorption spectrum (a) and emission spectrum (b) of the nanofluorescent probe are shown, demonstrating its applicability to NIR-II fluorescence imaging. The UV-visible absorption spectrum was acquired using a SHIMADZU UV-2600 (version 2.70) instrument with a spectral scan range of 500–1200 nm. The emission spectrum was acquired using an iHR320 imaging spectrometer with a spectral scan range of 850–1400 nm.

[0036] Example 2. Photostability of nanofluorescent probes:

[0037] In this example, the photostability of the nanofluorescent probe was tested, with indocyanine green (ICG) as a control. A fixed concentration of the nanofluorescent probe aqueous solution and an ICG aqueous solution were added to a 1.5 mL centrifuge tube and placed under a NIR-II fluorescence imager. The tube was continuously irradiated with an 808 nm laser for 1 hour, and the fluorescence intensity changes were recorded at a certain time. Figure 2 In the experiment, ICG itself has poor photostability, while the nanofluorescent probe does not show a significant decrease in fluorescence intensity, showing excellent photostability.

[0038] Example 3. Verification of the photothermal conversion capability of the nanofluorescent probe:

[0039] This example demonstrates the excellent photothermal conversion capability of the nanofluorescent probe under 980nm laser irradiation. -2 Under the experimental conditions, the nano fluorescent probe aqueous solution with a concentration of 100μM was irradiated and the temperature change was recorded in real time. Figure 3 As shown in the figure, the temperature of the nanofluorescent probe increased by 49.63°C compared to the initial temperature after 5 minutes of irradiation, and its heating ability was significantly better than that of the control ultrapure water. It is worth noting that after 5 heating-cooling cycles, the nanofluorescent probe did not show any significant weakening of its photothermal conversion ability, indicating that the nanofluorescent probe has photothermal stability.

[0040] Example 4. In vitro photothermal therapy capability of nanofluorescent probes:

[0041] In order to study the application of the nanofluorescent probe of the present invention in photothermal therapy, the inventors first conducted an in vitro cell experiment. Taking 4T1 cells as the research object, the CCK-8 method was used to test the cytotoxicity, and the absorbance at 450nm was measured by a microplate reader. In this embodiment, the cytotoxicity of the nanofluorescent probe was first tested without laser irradiation. After the nanofluorescent probes with concentrations of 0, 25, 50, 75 and 100 μM were co-incubated with 4T1 cells for 24 hours, the cell survival rate was higher than 90%, indicating that the nanofluorescent probe has good biocompatibility. After the nanofluorescent probes with concentrations of 0, 12.5, 25, 35, 50, 75 and 100 μM were co-incubated with 4T1 cells for 24 hours and irradiated with 980nm laser for 5 minutes, as shown in FIG. Figure 4 As shown, the cell survival rate decreased significantly and exhibited concentration dependence, indicating that the nanofluorescent probe has photothermal therapeutic ability.

[0042] Example 5. In vivo vascular imaging using nanofluorescent probes:

[0043] In this example, 1 mM of the nanofluorescent probe was injected into BALB / c mice via the tail vein, and whole-body vascular imaging was performed. Figure 5 As shown, the nanofluorescent probe can clearly display the capillary contours in the abdomen and legs of mice with high temporal and spatial resolution and low tissue autofluorescence.

[0044] Example 6. In situ 4T1 tumor vascular imaging using nanofluorescent probes:

[0045] In this example, the tumor model is a 4T1 orthotopic tumor model. 4T1 cells were cultured in DMEM medium containing 5% fetal bovine serum and 1% penicillin-streptomycin in a cell culture incubator at 37°C and 5% CO2. The 4T1 cells were cultured at a density of 5×10 5 PBS solution was injected into the mammary pad of the mouse abdomen. 3 Blood vessel imaging at the tumor site was performed at around 2 p.m.

[0046] 1mM nanofluorescent probe was injected into the tail vein of mice with orthotopic 4T1 tumors, and the blood vessels in the tumor were monitored in real time. Figure 6 As shown, due to the tumor's demand for energy and nutrition during growth, the blood vessels in the tumor are more abundant than those in healthy mice.

[0047] Example 7.4 Monitoring of fluorescence intensity of T1 subcutaneous tumors:

[0048] In this example, the tumor model is a 4T1 subcutaneous tumor model. 4T1 cells were cultured in DMEM medium containing 5% fetal bovine serum and 1% penicillin-streptomycin in a cell culture incubator at 37°C and 5% CO2. The 4T1 cells were cultured at a density of 5×10 5 PBS solution was subcutaneously injected into the lower limbs of the mouse back. When the tumor volume grew to 80-100 mm 3 Tumor imaging was performed around 9 p.m.

[0049] 1mM of the nanofluorescent probe was injected into the subcutaneous 4T1 tumor mice via tail vein injection. The mice were anesthetized and placed under the NIR-II imaging instrument for tumor imaging at 0.1, 2, 8, 12, 24 and 48 hours after injection. Figure 7 In the experiment, over time, the nanofluorescent probes can be passively targeted to the tumor through the EPR effect and light up the tumor.

[0050] Example 8. Organ distribution of nanofluorescent probes in 4T1 tumor-bearing mice:

[0051] 48 hours after the tail vein injection of the nanofluorescent probe, mice bearing 4T1 subcutaneous tumors were sacrificed (n=3) and the heart, liver, spleen, lung, kidney and tumor tissues were removed by dissection. The stains were washed with saline and the tissues were imaged under a NIR-II imager to observe the distribution of the probe in the above tissues. Figure 8 As shown, the nanofluorescent probes mainly accumulated in the liver and spleen, and tumor imaging under dissection also proved that the probes effectively entered the tumor.

[0052] Example 9. Temperature rise curve of laser irradiation at the tumor site after tail vein injection of nanofluorescent probe:

[0053] In this example, the tumor model used was a 4T1 mouse subcutaneous tumor model. The tumor volume was 80-100 mm. 3 The experiment was conducted on mice of about 1000 nm. 980 nm laser irradiation was performed 24 hours after the injection of the nanofluorescent probe. During this period, a thermal infrared imager was used to monitor the temperature rise in the tumor in real time. Figure 9 As shown, the tumor temperature of mice in the nanofluorescent probe group (Probe+Laser) increased significantly, and the temperature was higher than the 55°C group to kill cancer cells, while the control group PBS group (PBS+Laser) only increased slightly in temperature, and the increased temperature could not ablate the tumor.

[0054] Example 10. Photothermal therapy of 4T1 tumor mice using nanofluorescent probes:

[0055] In this example, the tumor model used was a 4T1 mouse subcutaneous tumor model. The tumor volume was 80-100 mm. 3 About 100 mice were subjected to photothermal therapy experiments.

[0056] BALB / c tumor mice were divided into the following four treatment groups:

[0057] a) Five animals received tail vein injection of PBS without illumination (PBS-Laser);

[0058] b) Five animals received tail vein injection of PBS and 980 nm laser irradiation for 5 min (PBS+Laser);

[0059] c) 5 animals received tail vein injection of the nanofluorescent probe (500 μM, 150 μL) without illumination (Probe-Laser);

[0060] d) Five animals received tail vein injection of the nanofluorescent probe (500 μM, 150 μL) and 980 nm laser irradiation for 5 min (Probe+Laser);

[0061] The day of injection was designated as day 0, and the length (L) and width (W) of the tumor were measured with a vernier caliper. Laser irradiation was performed 24 hours after the injection of the nanofluorescent probe. Tumor size was measured on the day of treatment and recorded every two days thereafter, using the formula V = 0.5 × L × W 2 The tumor volume was calculated and recorded, and the cells were monitored continuously for 14 days.

[0062] This example shows that irradiating a tumor enriched with nanofluorescent probes with a 980 nm laser can generate sufficient heat to ablate the tumor. Figure 10This is a curve showing the volume growth of mouse tumors within 14 days of photothermal therapy. The experimental results demonstrate that the nanofluorescent probe possesses photothermal therapeutic properties and can inhibit tumor growth. Furthermore, 15 days after probe injection, mice in different treatment groups were sacrificed, and their tumors were dissected and weighed. Tumor mass results were consistent with the tumor volume growth trend.

[0063] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, material, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, material, or apparatus.

[0064] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A BODIPY oligomer NIR-II nanofluorescent probe, characterized in that: It is composed of four BODIPY monomers connected by conjugation. Therefore, by increasing the number of repeating units and extending the conjugation, the maximum absorption and emission wavelengths are red-shifted to the NIR-II window, with values ​​of 963nm and 1009nm respectively. The BODIPY tetramer has the following structure:

2. A BODIPY oligomer NIR-II nanofluorescent probe according to claim 1, characterized in that: The BODIPY tetramer is used as the core and polyethylene glycol (PEG) with a molecular weight of 5000 is used as the drug carrier.

3. A method for preparing a BODIPY oligomer NIR-II nanofluorescent probe according to claim 1-2, characterized in that: The specific steps include: Step 1: Dissolve a certain ratio of BODIPY tetramer and PEG in acetonitrile and reflux and stir at room temperature to react; Step 2: The reaction solution is dialyzed and then passed through a 220 nm aqueous membrane to obtain a water-soluble nano fluorescent probe.

4. The method for preparing a BODIPY oligomer NIR-II nanofluorescent probe according to claim 3, characterized in that: The molar ratio of BODIPY tetramer to PEG in step 1 is 1:1-2, and the reaction time is 4-8h.

5. The method for preparing a BODIPY oligomer NIR-II nanofluorescent probe according to claim 3, characterized in that: The molecular weight of PEG in step 1 is 5000.

6. The method for preparing a BODIPY oligomer NIR-II nanofluorescent probe according to claim 3, characterized in that: The dialysis step in step 2 uses a dialysis bag with a molecular weight cutoff range of 4500-7000.

7. The method for preparing a BODIPY oligomer NIR-II nanofluorescent probe according to claim 3, characterized in that: The nano fluorescent probe has the following structure 8. Use of the nano fluorescent probe according to any one of claims 1 to 7 in NIR-II fluorescence imaging and tumor therapy.

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