An aggregation-induced emission photosensitizer with tumor hypoxia alleviating function and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine and photodynamic therapy, and particularly relates to an aggregation-induced emission photosensitizer with tumor hypoxia relief function and a preparation method and application thereof. The photosensitizer is MeTCP-F, which uses methoxy-modified triphenylamine as an electron donor, benzindole as an electron acceptor, and thiophene as a pi bridge to form a D-pi-A structure, and is connected with a perfluorinated compound through an ester bond; and the MeTCP-F NPs are prepared by using CH3-PEG-DSPE through a nano precipitation method. The photosensitizer has near-infrared fluorescence imaging, active oxygen generation and oxygen carrying capacity, can relieve tumor hypoxia, cooperatively realize the generation of type I and type II active oxygen, and improve the photodynamic therapy effect. The MeTCP-F NPs have good water solubility and high biocompatibility, can be enriched in tumor tissues through the EPR effect, can realize efficient tumor killing under the guidance of near-infrared imaging, have low systemic toxicity, and have application value in the preparation of anti-tumor photodynamic therapy drugs.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and photodynamic therapy, specifically relating to an aggregation-induced emission (AIE) photosensitizer with the function of relieving tumor hypoxia, its preparation method, and its application in the preparation of anti-tumor drugs. Background Technology

[0002] Photodynamic therapy (PDT), as a non-invasive tumor treatment strategy, has attracted widespread attention in the field of cancer treatment due to its advantages such as high selectivity, low systemic toxicity, and repeatability. The core element of PDT is a photosensitizer, which, under irradiation with light of a specific wavelength, can transfer energy to surrounding oxygen molecules, generating reactive oxygen species (ROS), including singlet oxygen (¹O2) and superoxide anion radicals (O2⁻). - • and hydroxyl radicals (•OH), which in turn induce tumor cell apoptosis or necrosis.

[0003] However, traditional photosensitizers have several limitations in practical applications. First, their molecules often have planar conjugated structures, making them prone to aggregation in aqueous physiological environments, leading to fluorescence quenching (ACQ) and significantly weakening fluorescence imaging performance and photodynamic therapy efficacy. Second, the tumor microenvironment is generally hypoxic, severely restricting the efficiency of oxygen-dependent type II photodynamic therapy (PDT) and limiting the therapeutic effect of photosensitizers. Studies have shown that the oxygen partial pressure inside tumor tissue is usually below 2.5 mmHg, far lower than the 40 mmHg in normal tissues. This hypoxic environment has become a key bottleneck in improving the efficacy of PDT. In addition, the water solubility, physiological stability, and tumor targeting of traditional photosensitizers also need improvement.

[0004] Aggregation-induced emission (AIE) materials exhibit significantly enhanced fluorescence in the aggregated state, providing an effective approach to overcome the ACQ (acupuncture-induced fluorescence) problem. Since Academician Tang Benzhong's team first reported the AIE phenomenon in 2001, photosensitizers with AIE properties have become a research hotspot in the field of photodynamic therapy due to their unique advantage of simultaneously enhancing fluorescence emission and ROS generation in the aggregated state. Meanwhile, by introducing perfluorinated compounds with oxygen-carrying capacity into the photosensitizer structure, oxygen delivery to hypoxic tumor sites can be achieved, thereby improving the PDT response efficiency under hypoxic conditions. Perfluorinated compounds, due to their high gas solubility and bioinertness, have been widely used to construct oxygen carrier systems.

[0005] Therefore, developing a multifunctional AIE photosensitizer that combines near-infrared fluorescence imaging, efficient ROS generation, and the ability to alleviate tumor hypoxia is of great scientific significance and clinical translational value for achieving precise and efficient tumor photodynamic therapy. Summary of the Invention

[0006] The purpose of this invention is to provide an aggregation-induced emission photosensitizer with the function of relieving tumor hypoxia and its preparation method, and to apply it to anti-tumor photodynamic therapy, so as to solve the problems of aggregation quenching effect, poor photodynamic efficacy caused by tumor hypoxia, and difficulty in integrating near-infrared imaging and treatment functions of traditional photosensitizers.

[0007] To achieve the above objectives, the first aspect of the present invention provides an aggregation-induced emission photosensitizer with the function of alleviating tumor hypoxia, wherein the photosensitizer is a perfluorinated compound-modified methoxytriphenylamine-thiophene-benzoindole derivative, named MeTCP-F, and its structure is shown in the following formula:

[0008] ;

[0009] Among them, R - It is a halide anion, preferably Br - .

[0010] A second aspect of this invention provides a method for preparing the above-mentioned aggregation-induced emission photosensitizer with the function of alleviating tumor hypoxia, comprising the following steps:

[0011] (1) Suzuki coupling reaction: 4-bromo-4',4''-dimethoxytriphenylamine was reacted with 5-aldehyde-2-thiopheneboronic acid under a catalyst and alkaline conditions to obtain the methoxytriphenylamine-thiophene-formaldehyde intermediate, namely compound 1;

[0012] (2) Knoevenagel condensation reaction: Compound 1 obtained in step (1) is condensed with 2,3,3-trimethyl-3H-benzo[g]indole quaternary ammonium salt under catalytic conditions to obtain carboxyl-containing intermediate MeTCP;

[0013] (3) Esterification reaction: The MeTCP obtained in step (2) is esterified with 1H,1H,2H,2H-perfluorooctanol in the presence of a condensing agent to obtain the target product MeTCP-F;

[0014] In step (2), the 2,3,3-trimethyl-3H-benzo[g]indole quaternary ammonium salt is prepared by reacting 2,3,3-trimethyl-3H-benzo[g]indole with p-bromomethylbenzoic acid.

[0015] Further, the Suzuki coupling reaction in step (1) is carried out under alkaline conditions (preferably with the addition of K2CO3) with a palladium catalyst (preferably Pd(PPh3)2Cl2) at a reaction temperature of 70~80℃ and a reaction time of 6~10 h.

[0016] Furthermore, the Knoevenagel condensation reaction in step (2) is carried out under the catalysis of an organic base (preferably piperidine), at a reaction temperature of 70~90℃, and for a reaction time of 10~15 h.

[0017] Further, the esterification reaction in step (3) is carried out at room temperature in the presence of a condensing agent (preferably DMAP and N,N'-dicyclohexylcarbodiimide) for a reaction time of 1 to 3 h.

[0018] The aggregation-induced emission photosensitizer uses methoxylated triphenylamine as an electron donor, benzoindole as an electron acceptor, and thiophene as a π-bridge to form a strong donor-π-acceptor electron push-pull structure. Through the thiophene π-bridge and methoxy modification, the absorption and emission wavelengths are red-shifted, enabling near-infrared fluorescence imaging. By introducing perfluorinated compounds, it is endowed with excellent oxygen-carrying properties, which can deliver oxygen to hypoxic tumor sites, alleviate tumor microenvironment hypoxia, and reduce the dependence of photodynamic therapy on oxygen. Under light irradiation, it can simultaneously generate type I and type II reactive oxygen species, realizing dual-pathway photodynamic therapy and maintaining a highly efficient tumor-killing effect even under hypoxic conditions.

[0019] A third aspect of the present invention provides aggregation-induced emission photosensitizer nanoparticles with the function of relieving tumor hypoxia. The nanoparticles are prepared by the above-mentioned MeTCP-F and amphiphilic polymer by nanoprecipitation method and are named MeTCP-FNPs.

[0020] Furthermore, the amphiphilic polymer is methyl-polyethylene glycol-distearate phosphatidylethanolamine (CH3-PEG-DSPE).

[0021] Furthermore, the mass ratio of the photosensitizer to the amphiphilic polymer is 1:5 to 1:15, resulting in nanoparticles with uniform particle size and good dispersibility. These nanoparticles can be enriched through the tumor EPR effect, while simultaneously improving water solubility, biocompatibility, and in vivo circulation time.

[0022] The fourth aspect of the present invention provides the application of the above-mentioned photosensitizer MeTCP-F or photosensitizer nanoparticles MeTCP-F NPs in the preparation of antitumor drugs.

[0023] Furthermore, the antitumor drug is a near-infrared fluorescence imaging-guided photodynamic therapy drug for the treatment of breast cancer.

[0024] The drug achieves highly efficient tumor killing and elimination by alleviating tumor hypoxia and synergistically generating type I and type II reactive oxygen species. It exhibits low dark toxicity, high phototoxicity, and high in vivo biosafety, and can significantly inhibit tumor growth and achieve tumor clearance in tumor-bearing models.

[0025] In summary, compared with the prior art, the technical solutions conceived by this invention have the following advantages and beneficial effects:

[0026] 1. The photosensitizer MeTCP-F provided by this invention has a typical D-π-A structure. By modifying with methoxy groups and extending the conjugated system with thiophene π bridges, a redshift in absorption and emission wavelengths is achieved. It can be excited by 660 nm near-infrared light and emits near-infrared fluorescence. It has a large tissue penetration depth and is suitable for in vivo tumor imaging and treatment.

[0027] 2. The photosensitizer prepared by this invention has aggregation-induced emission properties, which can overcome the fluorescence quenching problem caused by aggregation of traditional photosensitizers in aqueous phase, and ensure the stability of fluorescence imaging and the effectiveness of photodynamic therapy.

[0028] 3. By introducing perfluorinated compounds into the molecule, the present invention enables the MeTCP-F NP nanoparticles to have excellent oxygen carrying and releasing capabilities, which can effectively improve the hypoxic microenvironment of tumors and significantly enhance the photodynamic therapy effect of hypoxic tumors.

[0029] 4. The MeTCP-F NPs of the present invention can generate reactive oxygen species simultaneously through type I and type II photodynamic pathways. Among them, the type I photodynamic pathway, which is not dependent on oxygen, can continue to function under hypoxic conditions, thereby achieving a highly efficient and stable tumor photodynamic killing effect.

[0030] 5. This invention uses a nanoprecipitation method to prepare water-soluble nanoparticles. The preparation process is simple and the conditions are mild. The resulting nanoparticles have uniform particle size, good dispersibility, high biocompatibility, and good in vivo circulation characteristics, making them suitable for large-scale preparation and application.

[0031] 6. The photosensitizer nanoparticles obtained in this invention integrate near-infrared fluorescence imaging and photodynamic therapy functions, enabling precise treatment under imaging guidance. They exhibit low dark toxicity and strong phototoxicity, and can significantly inhibit tumor growth and even achieve tumor clearance in vivo. They have important application value in the field of preparing anti-tumor photodynamic therapy drugs. Attached Figure Description

[0032] Figure 1 This is the synthesis route of MeTCP-F in a specific embodiment of the present invention.

[0033] Figure 2-1 This is the carbon spectrum of MeTCP-F in a specific embodiment of the present invention.

[0034] Figure 2-2 This is a mass spectrum of MeTCP-F in a specific embodiment of the present invention. Figure 2-3 The image shows the hydrogen NMR spectrum of MeTCP-F in a specific embodiment of the present invention.

[0035] Figure 3 This is a schematic diagram of the structure and function of MeTCP-F nanoparticles in a specific embodiment of the present invention.

[0036] Figure 4 This is a confocal microscope image showing the change in cellular uptake of MeTCP-F NPs over time in a specific embodiment of the present invention.

[0037] Figure 5 The results of cytotoxicity tests on 4T1 cells at different concentrations of MeTCP-F NPs are shown in a specific embodiment of the present invention.

[0038] Figure 6 The diagram illustrates the intracellular reactive oxygen species (ROS) generation in different treatment groups according to specific embodiments of the present invention.

[0039] Figure 7 This illustrates the inhibitory effect of different treatment groups on hypoxia probe signals in specific embodiments of the present invention.

[0040] Figure 8 These are cell live / dead staining images of different treatment groups in specific embodiments of the present invention.

[0041] Figure 9 shows the near-infrared fluorescence imaging results of MeTCP-F NPs in tumor-bearing mice in a specific embodiment of the present invention.

[0042] Figure 10 shows the effect curves of different treatment groups on tumor volume growth in tumor-bearing mice in a specific embodiment of the present invention.

[0043] Figure 11 This is a curve showing the effect of different treatment groups on the body weight of tumor-bearing mice during treatment in a specific embodiment of the present invention.

[0044] Figure 12 The images show H&E stained tissue sections of major organs of mice after treatment in different treatment groups according to a specific embodiment of the present invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0046] Example 1: Synthesis of MeTCP-F

[0047] This embodiment provides a method for synthesizing MeTCP-F, and the synthesis path diagram is shown below. Figure 1 As shown (where the counterion is omitted in the MeTCP and MeTCP-F structural formulas), the specific steps include:

[0048] (1) Synthesis of compound 1:

[0049] Under nitrogen protection, 4-bromo-4',4''-dimethoxytriphenylamine (881 mg, 2.3 mmol), 5-aldehyde-2-thiopheneboronic acid (248 mg), Pd(PPh3)2Cl2 (70 mg, 0.1 mmol), and K2CO3 (691 mg, 5 mmol) were added to 12 mL of a mixed solvent of toluene and methanol (v / v 1:1). The reaction mixture was heated to 75 °C and stirred under reflux for 8 hours. After the reaction was completed, the mixture was cooled to room temperature (25 ± 2 °C), extracted with ethyl acetate (3 × 20 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / petroleum ether = 1:1, v / v) to give compound 1, an orange solid, in 42% (400 mg) yield. 1H NMR (500 MHz, Chloroform-d6) δ9.84 (s, 1H), 7.70 (d, J=4.0 Hz, 1H), 7.47-7.46 (m, 2H), 7.27 (d, J=2.0 Hz, 1H), 7.12-7.08 (m, 4H), 6.91-6.90 (m, 2H), 6.89-6.86 (m, 4H), 3.82 (s, 6H).

[0050] (2) Synthesis of compound 2:

[0051] Under nitrogen protection, 2,3,3-trimethyl-3H-benzo[g]indole (3.5 g, 16.73 mmol) and p-bromomethylbenzoic acid (3.58 g, 16.73 mmol) were added to 24 mL of acetonitrile. The reaction mixture was heated to 80 °C and stirred under reflux for 12 hours. After the reaction was completed by TLC monitoring, it was cooled to room temperature (25 ± 2 °C), the precipitate was filtered off, and the precipitate was washed three times with cold acetone and dried to give compound 2 as a light green solid with a mass of 3.1 g and a yield of 53.82%.

[0052] (3) Synthesis of the target product MeTCP:

[0053] Under nitrogen protection, compound 1 (247 mg, 0.6 mmol) and compound 2 (215 mg, 0.6 mmol) were added to a round-bottom flask containing 10 mL of anhydrous ethanol and 200 μL of piperidine, and sonicated until completely dissolved. The reaction mixture was heated to 80 °C and stirred under reflux for 12 hours, with the reaction progress monitored by thin-layer chromatography. After the reaction was completed, the mixture was cooled to room temperature (25 ± 2 °C), and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50:1, v / v) to give a deep purple solid, MeTCP, with a mass of 300 mg and a yield of 68.02%.

[0054] (4) Synthesis of the target product MeTCP-F:

[0055] Under nitrogen protection, MeTCP (1 g, 1.34 mmol), 1H,1H,2H,2H-perfluorooctanol (490 mg, 1.34 mmol), and 4-dimethylaminopyridine DMAP (82 mg, 0.67 mmol) were added to a round-bottom flask, and 7 mL of DMF was added to dissolve them. N,N'-dicyclohexylcarbodiimide DCC (140 mg, 0.67 mmol) was slowly added dropwise at room temperature, and the reaction was stirred overnight. The reaction was terminated by TLC monitoring until the starting material was completely consumed. The reaction solution was extracted with ethyl acetate and water, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10:1, v / v) to obtain the target product, a green solid, with a mass of 806 mg and a yield of 54.96%. 1 H NMR, 13 The structure was confirmed by C10 NMR and high-resolution mass spectrometry (see Figure 2). The molecular formula is C10. 56 H 44 F 13 N₂O₄SBr, with a molecular weight of 1167.9212 g / mol, has the following structure:

[0056] .

[0057] Example 2: Preparation of MeTCP-F NPs and MeTCP NPs

[0058] This embodiment provides a method for preparing MeTCP-F nanoparticles (MeTCP-F NPs) and MeTCP nanoparticles (MeTCP NPs), specifically including the following steps:

[0059] Using a nanoprecipitation method, MeTCP-F (1 mg) prepared in Example 1 and methyl-polyethylene glycol-distearate phosphatidylethanolamine CH3-PEG-DSPE (MW=2000, 10 mg) were dissolved together in 1 mL of tetrahydrofuran. Under ultrasonic conditions, this solution was slowly added dropwise to 10 mL of deionized water. After the addition was complete, ultrasonication was continued for 5 minutes until homogeneous. Subsequently, the solution was stirred overnight at room temperature (the overnight time in this invention is 12 h) to evaporate and remove the tetrahydrofuran. Finally, the obtained nanoparticle solution was filtered through a 0.22 μm filter membrane to obtain a homogeneous MeTCP-F NPs aqueous solution, which was then lyophilized and stored in the dark for later use. The nanoparticle size was determined using a dynamic light scattering instrument, and the results showed that the average particle size was (85.3±6.2) nm and the polydispersity index was 0.142.

[0060] By replacing the above MeTCP-F with an equal mass of MeTCP, MeTCP nanoparticles (MeTCP NPs) were prepared.

[0061] Example 3: Cell Uptake Experiment

[0062] This embodiment investigated the uptake behavior of MeTCP-F NPs in 4T1 cells.

[0063] 4T1 cells were prepared at 1×10⁴ cells per dish. 5 Cells were inoculated into confocal culture dishes at a seed level of DMEM + 10% fetal bovine serum (FBS) and cultured overnight at 37°C and 5% CO2. The culture medium was discarded, and 2 mL of fresh medium containing MeTCP-FNPs (100 μg / mL) was added. Cells were incubated for 0 min, 30 min, 60 min, 2 h, 3 h, 4 h, 5 h, 6 h, and 7 h, respectively. After incubation, the cells were washed three times with phosphate-buffered saline, and intracellular fluorescence signals were observed using a confocal laser scanning microscope. Results are as follows: Figure 4 As shown, the intracellular red fluorescence signal gradually increased with the extension of incubation time, indicating that MeTCP-FNPs can be effectively taken up by tumor cells, and the take-up is time-dependent.

[0064] Example 4: Cytotoxicity Experiment

[0065] In this embodiment, the CCK-8 assay was used to evaluate the dark toxicity and phototoxicity of MeTCP-F NPs on 4T1 cells.

[0066] 4T1 cells were spaced at 8 × 10⁸ cells per well. 3Cells were seeded at a density of 1000 μg / mL in 96-well plates and incubated overnight at 37°C with 5% CO2 in a cell culture incubator. The culture medium was DMEM + 10% fetal bovine serum (FBS). After the cells had fully adhered to the bottom of the wells, the original culture medium was discarded. MeTCP-F NPs were diluted to a series of concentrations of 0, 10, 20, 40, 60, 80, and 100 μg / mL using culture medium and added to the wells, followed by incubation for 24 h. The cells were washed twice with PBS buffer, and then fresh culture medium containing 10 μL of CCK-8 reagent was added to each well. The cells were incubated in the dark for 30 min. The absorbance of each well at 450 nm was then measured using a microplate reader, and the cell viability was calculated using the appropriate formula.

[0067] Cell viability (%) = (ODsample - ODbackground) / (ODcontrol - ODbackground)

[0068] ODsample: Experimental group (containing cell culture medium, CCK-8, and test substance)

[0069] ODcontrol: Control group (containing cell culture medium, CCK-8, and no analyte)

[0070] ODbackground: Blank group (culture medium and CCK-8 containing no cells or test substances)

[0071] The phototoxicity of MeTCP-F NPs was detected using the CCK-8 method.

[0072] 4T1 cells were fed at 8 × 10 3 Cells were seeded at a density of 1 / 2 well in a 96-well culture plate and incubated overnight at 37°C with 5% CO2. After complete cell adhesion, the original culture medium in the wells was discarded. The culture medium was DMEM + 10% fetal bovine serum (FBS). MeTCP-F NPs solution was serially diluted to 0, 10, 20, 40, 60, 80, and 100 μg / mL using the culture medium as a diluent, and added to the corresponding wells. The wells were then incubated for 3 h. After incubation, the cells were washed twice with PBS buffer, followed by 10 min of laser illumination at 660 nm wavelength and 50 mW / cm² power density. After illumination, the 96-well plates were returned to the cell culture incubator and incubated for another 24 h. Then, fresh culture medium containing 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 30 min. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the formula described above.

[0073] Experimental results are as follows Figure 5 As shown, in the absence of light, MeTCP-F NPs exhibited no significant toxicity to 4T1 cells within the tested concentration range (0–100 μg / mL), with cell viability remaining above 95%, demonstrating good biocompatibility. Under light conditions, MeTCP-F NPs exhibited significant concentration-dependent phototoxicity, with a half-maximal inhibitory concentration (IC50) of approximately 45 μg / mL. At a concentration of 100 μg / mL, cell viability decreased to 10%, indicating its highly efficient photodynamic killing effect.

[0074] Example 5: Detection of intracellular reactive oxygen species

[0075] In this embodiment, the DCFH-DA fluorescent probe was used to detect the ability of MeTCP-F NPs to induce reactive oxygen species generation in 4T1 cells.

[0076] 4T1 cells were stored at 8 × 10⁸ cells per well. 3 Inoculations were performed on confocal culture dishes at a density of 10% DMEM + 10% fetal bovine serum (FBS) and cultured overnight. Then, different treatments were applied: PBS (protected from light), PBS (light), MeTCP NPs (100 μg / mL, no light), MeTCP-F NPs (100 μg / mL, no light), MeTCP NPs (100 μg / mL) + light, and MeTCP-F NPs (100 μg / mL) + light. The light conditions were 660 nm and 50 mW / cm² for all treatments. 2 The samples were prepared using fresh culture medium as the solvent, with an addition volume of 2 mL for 10 min. After treatment, the supernatant was discarded, and fresh culture medium containing the DCFH-DA probe (10 μM) was added. The samples were incubated at 37°C in the dark for 30 min. After washing three times with phosphate-buffered saline (PBS), the green fluorescence signal was observed using a confocal laser scanning microscope. The PBS used in this invention was 10 mM pH 7.4.

[0077] The results are as follows Figure 6 As shown, the MeTCP-F NPs+ illuminated group exhibited the strongest green fluorescence signal, indicating its efficient generation of reactive oxygen species (ROS). The MeTCP NPs+ illuminated group also showed strong fluorescence, but at a lower intensity than the MeTCP-F NPs+ illuminated group. Only weak background fluorescence was observed in the unilluminated group and the PBS group, confirming that ROS generation depends on photoexcitation.

[0078] Example 6: Test of ability to alleviate hypoxia

[0079] This embodiment utilizes a hypoxia probe to detect the oxygen-carrying and hypoxia-alleviating capabilities of MeTCP-F NPs.

[0080] 4T1 cells were loaded at a rate of 1×10⁴ cells per well. 4 Inoculations were performed in confocal culture dishes using DMEM + 10% fetal bovine serum (FBS) for 24 h. After incubation, the samples were placed at 37°C under hypoxic conditions (1% O2, 5% CO2, 94% N2) for 12 h to simulate the hypoxic microenvironment of a tumor. Subsequently, serum-free DMEM containing MeTCP-F NPs and MeTCP NPs (100 μg / mL each) was added, and incubation continued for 6 h. During incubation, 3.75 μmol / L of the oxygen probe [Ru(dpp)3]Cl2 (ruthenium tris(4,7-biphenyl-1,10-o-phenanthroline)dichloride) was dissolved in DMSO and diluted in serum-free medium to prepare a 3.75 μmol / L working solution. After incubation, the drug-containing medium was removed, the sample was washed with PBS, and the [Ru(dpp)3]Cl2 working solution was added for further incubation for 3 h. After washing three times with phosphate buffer, the fluorescence intensity of the hypoxic probe was observed using a fluorescence microscope.

[0081] The results are as follows Figure 7 As shown, the PBS group and the MeTCP NPs group exhibited strong fluorescence signals from hypoxia probes, indicating that the cells were in a state of severe hypoxia. However, the hypoxia probe signal in the MeTCP-F NPs group was significantly weakened, indicating that MeTCP-F NPs could deliver oxygen to the cells and effectively alleviate the hypoxic state.

[0082] Example 7: Cell Viability Staining Experiment

[0083] In this embodiment, the Calcein-AM / PI double staining method was used to evaluate the killing effect of different treatment groups on 4T1 cells.

[0084] 4T1 cells were loaded at a rate of 1×10⁴ cells per well. 4 Inoculations were performed on confocal culture dishes at a density of 10% DMEM + 10% fetal bovine serum (FBS) and cultured overnight. The following treatments were then performed: PBS (no light), PBS (light), MeTCP-F NPs (100 μg / mL, no light), MeTCP NPs (100 μg / mL) + light, and MeTCP-F NPs (100 μg / mL) + light. All light conditions were 660 nm and 50 mW / cm². 2For each group of samples, 2 mL of fresh culture medium was added, and the treatment was carried out for 10 min. After treatment, the supernatant was discarded, and the cells were washed once with PBS. A staining solution containing Calcein-AM (2 μM) and PI (4 μM) prepared with PBS was added, and the cells were incubated at 37°C in the dark for 30 min. The staining solution was discarded, and the cells were washed with phosphate buffer. Live cells (green fluorescence) and dead cells (red fluorescence) were observed using a confocal laser scanning microscope.

[0085] The results are as follows Figure 8 As shown, the MeTCP-F NPs+ light-illuminated group exhibited the strongest red fluorescence and the weakest green fluorescence, indicating significant cell death in this group. A certain degree of cell death was also observed in the MeTCP NPs+ light-illuminated group, but the effect was weaker than that in the MeTCP-F NPs+ light-illuminated group. Other control groups mainly showed green fluorescence, indicating good cell condition. These results further validate the excellent photodynamic therapy effect of MeTCP-F NPs under light irradiation.

[0086] Example 8: In vivo near-infrared fluorescence imaging

[0087] This embodiment examines the biodistribution and accumulation behavior of MeTCP-F NPs in tumor-bearing mice.

[0088] 4T1 tumor-bearing mice were randomly divided into groups and injected via tail vein with MeTCP-F NPs (200 μL, 1 mg / mL, prepared in PBS). Small animal in vivo imaging was performed at 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 120 h post-injection to observe changes in fluorescence signal intensity at the tumor site.

[0089] As shown in Figure 9, after injection, the fluorescence signal of the nanoparticles at the tumor site gradually increased, reaching a maximum at 24 hours and maintaining it until 48 hours. Subsequently, the fluorescence signal gradually weakened, but a detectable signal was still present at 120 hours. This result indicates that MeTCP-F NPs can effectively accumulate in tumor tissue through the EPR effect, providing an optimal irradiation time window for subsequent photodynamic therapy.

[0090] Example 9: Evaluation of in vivo antitumor effect

[0091] This embodiment evaluates the inhibitory effect of MeTCP-F NPs combined with light irradiation on tumor growth in tumor-bearing mice.

[0092] 4T1 tumor-bearing mice were randomly divided into 5 groups (n=5) and given different treatments: (1) PBS group (no light exposure); (2) PBS + light exposure group; (3) MeTCP-F NPs group (no light exposure); (4) MeTCP NPs + light exposure group; (5) MeTCP-F NPs + light exposure group. The dosage for each group was 200 μL (1 mg / mL) administered via tail vein injection. Twenty-four hours after administration, the tumor sites in the light exposure group were irradiated with a 660 nm laser (200 mW / cm²). 2 (10 min). Mouse body weight and tumor volume were measured every 2 days for 14 days. Tumor volume was calculated using the following formula: V = (L × W) 2 ) / 2, where L is the long diameter of the tumor and W is the short diameter of the tumor.

[0093] The experimental results are shown in Figure 10, where V0 represents the initial tumor volume. Tumor volumes in the PBS group, PBS + light irradiation group, and MeTCP-F NPs group (no light irradiation) all increased rapidly, indicating that light irradiation or nanoparticle therapy alone cannot inhibit tumor growth. The MeTCP NPs + light irradiation group showed some tumor inhibition, but the tumor volume continued to increase in the later stages of treatment. In contrast, the MeTCP-F NPs + light irradiation group exhibited the best anti-tumor effect; the tumor volume continued to shrink during treatment, and by day 14, the tumors almost completely disappeared, with some mice showing complete tumor regression.

[0094] Mouse weight changes as follows Figure 11 As shown, the body weight of mice in each treatment group remained stable during the treatment period without significant decrease or fluctuation, indicating that MeTCP-F NPs and related treatments did not produce significant systemic toxicity in mice.

[0095] Example 10: Biosafety Evaluation

[0096] This embodiment assesses the toxicity of MeTCP-F NPs to major organs in mice using histopathological staining.

[0097] After the treatment in Example 9 was completed, mice in each group were sacrificed, and major organs such as heart, liver, spleen, lung, and kidney were taken. After fixation with 4% paraformaldehyde, they were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and their morphological changes were observed under an optical microscope.

[0098] The results are as follows Figure 12 As shown, no significant histopathological abnormalities were observed in the heart, liver, spleen, lungs, and kidneys of mice in each treatment group, and no changes such as inflammatory infiltration, cell necrosis, or fibrosis were observed, with no significant differences compared to the PBS control group. These results indicate that MeTCP-F NPs have good in vivo biocompatibility.

[0099] This invention systematically evaluated the performance of the MeTCP-F NPs prepared in this invention through a series of in vitro and in vivo experiments. In vitro results showed that MeTCP-F NPs possess good cellular uptake capacity, low dark toxicity, high phototoxicity, efficient reactive oxygen species generation, and the ability to alleviate cellular hypoxia. In vivo results showed that MeTCP-F NPs can effectively enrich tumor tissue through the EPR effect and, under near-infrared fluorescence imaging guidance, achieve highly efficient photodynamic therapy through light irradiation, significantly inhibiting tumor growth and even achieving tumor clearance, while showing no significant toxic side effects on major organs in mice. Therefore, the MeTCP-F NPs provided by this invention, as a multifunctional nanoplatform integrating near-infrared imaging and photodynamic therapy, show promising application prospects in the treatment of hypoxic tumors.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An aggregation-induced emission photosensitizer with the function of relieving tumor hypoxia, characterized in that, The photosensitizer is a perfluorinated compound-modified methoxytriphenylamine-thiophene-benzoindole derivative, named MeTCP-F, with the following structure: ； Among them, R - It is a halide anion.

2. The aggregation-induced emission photosensitizer with tumor hypoxia-relieving function according to claim 1, characterized in that, The R - It is a bromide ion.

3. A method for preparing the aggregation-induced emission photosensitizer with tumor hypoxia-relieving function as described in claim 2, comprising the following steps: (1) Suzuki coupling reaction: 4-bromo-4',4''-dimethoxytriphenylamine was reacted with 5-aldehyde-2-thiopheneboronic acid under a catalyst and alkaline conditions to obtain the methoxytriphenylamine-thiophene-formaldehyde intermediate, namely compound 1; (2) Knoevenagel condensation reaction: Compound 1 obtained in step (1) is condensed with 2,3,3-trimethyl-3H-benzo[g]indole quaternary ammonium salt under catalytic conditions to obtain carboxyl-containing intermediate MeTCP; (3) Esterification reaction: The MeTCP obtained in step (2) is esterified with 1H,1H,2H,2H-perfluorooctanol in the presence of a condensing agent to obtain the target product MeTCP-F; The 2,3,3-trimethyl-3H-benzo[g]indole quaternary ammonium salt mentioned in step (2) is prepared by reacting 2,3,3-trimethyl-3H-benzo[g]indole with p-bromomethylbenzoic acid.

4. The preparation method according to claim 3, characterized in that, The 2,3,3-trimethyl-3H-benzo[g]indole quaternary ammonium salt is prepared by reacting 2,3,3-trimethyl-3H-benzo[g]indole with p-bromomethylbenzoic acid.

5. The preparation method according to claim 3, characterized in that, The Suzuki coupling reaction in step (1) is carried out under alkaline conditions with a palladium catalyst, at a reaction temperature of 70-80°C, and for a reaction time of 6-10 h.

6. The preparation method according to claim 3, characterized in that, The Knoevenagel condensation reaction in step (2) is carried out under the catalysis of an organic base, at a reaction temperature of 70~90℃, and for a reaction time of 10~15 h.

7. The preparation method according to claim 3, characterized in that, The esterification reaction described in step (3) is carried out at room temperature for 1 to 3 hours.

8. An aggregation-induced emission photosensitizer nanoparticle with the function of alleviating tumor hypoxia, said nanoparticle being prepared by nanoprecipitation method from the photosensitizer MeTCP-F of claim 1 and an amphiphilic polymer, named MeTCP-F NPs; and / or The amphiphilic polymer is methyl-polyethylene glycol-distearatephosphatidylethanolamine; and / or The mass ratio of the photosensitizer to the amphiphilic polymer is 1:5 to 1:

15.

9. The use of the photosensitizer MeTCP-F according to claim 1 or the photosensitizer nanoparticles MeTCP-F NPs according to claim 8 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The antitumor drug is a near-infrared fluorescence imaging-guided photodynamic therapy drug used to treat breast cancer.

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