A porphyrin-based covalent organic framework nanodispersion, its preparation method, and its application.

Abstract

This invention discloses a porphyrin-based covalent organic framework nanodispersion, its preparation method, and its applications. The nanodispersion contains porphyrin-based covalent organic framework nanoparticles. These nanoparticles comprise an intertwined polymer DSPE-PEG3400-Biotin, and also include porphyrin-based covalent organic frameworks encapsulated within the intertwined polymer DSPE-PEG3400-Biotin. This invention uses an imine condensation method to link porphyrin with tetraaldehyde tetraphenylethylene. The nanosystem modified with DSPE-PEG3400-Biotin is a photosensitizer with high reactive oxygen species generation capacity, high anticancer activity, and low toxicity.

Description

Technical Field

[0001] This invention relates to a nano-dispersion based on a porphyrin covalent organic framework, and also to a method for preparing the above-mentioned nano-dispersion and its application as a photosensitizer in photodynamic therapy. Technical Background

[0002] The microenvironment of solid tumors is generally characterized by severe regional hypoxia. Rapidly proliferating tumor cells and a disordered tumor vascular system together lead to severe local oxygen deficiency, becoming a key obstacle restricting the clinical efficacy of photodynamic therapy (PDT). Traditional type II photosensitizers are highly oxygen-dependent, not only exhibiting significantly reduced activity in hypoxic regions but also further consuming the limited oxygen molecules within the tumor, exacerbating hypoxia and inducing treatment resistance. In contrast, type I photosensitizers achieve a highly efficient reactive oxygen species burst without relying on oxygen, overcoming the hypoxia limitation at its mechanism of action and providing a novel approach for photodynamic therapy of hypoxic tumors.

[0003] Tetra(p-aminophenyl)porphyrin is a classic photosensitizer widely used in photodynamic therapy. It possesses significant advantages such as high molar absorptivity, strong visible light response, easy structural modification, and good biocompatibility, making it an ideal unit for constructing highly efficient photosensitizing systems. However, as a typical type II photosensitizer, its activity is highly dependent on oxygen, and its effectiveness is significantly reduced in hypoxic tumor microenvironments. Furthermore, its planar conjugated structure is prone to aggregation-induced quenching, leading to reduced reactive oxygen species production and poor photostability, severely limiting its therapeutic efficacy. Summary of the Invention

[0004] Objectives of this invention: One objective is to provide a nano-dispersion based on a porphyrin covalent organic framework. This nanosystem effectively solves the problems of easy aggregation and quenching and poor photostability of porphyrin as a photosensitizer. Furthermore, it transforms porphyrin from a type II photosensitizer to a type I photosensitizer, thereby effectively overcoming the influence of tumor microenvironment hypoxia on photodynamic therapy efficacy. Another objective of this invention is to provide a method for preparing the above-mentioned nano-dispersion and its application as a photosensitizer in photodynamic therapy.

[0005] Technical solution: The present invention provides a nano-dispersion based on a porphyrin covalent organic framework, wherein the nano-dispersion contains porphyrin-based covalent organic framework nanoparticles; the porphyrin-based covalent organic framework nanoparticles include an intertwined polymer DSPE-PEG3400-Biotin, and also include a porphyrin-based covalent organic framework encapsulated within the intertwined polymer DSPE-PEG3400-Biotin.

[0006] The nano-dispersion liquid has a solid content of 1 mg / mL, and the porphyrin-based covalent organic framework nanoparticles have a particle size of 134 nm.

[0007] The preparation method of the above-mentioned porphyrin-based covalent organic framework nanodispersion includes the following steps:

[0008] (1) Tetra(p-aminophenyl)porphyrin and tetraaldehyde tetraphenylethylene were added to a reaction solvent containing a catalyst and reacted at high temperature. After the reaction, the mixture was cooled to room temperature to obtain a porphyrin-based covalent organic framework.

[0009] (2) Disperse DSPE-PEG3400-Biotin in ultrapure water by ultrasonication, add the porphyrin-based covalent organic framework from step (1) to it, stir thoroughly at room temperature to obtain a nano-dispersion of porphyrin-based covalent organic framework.

[0010] The structural formula of the tetra(p-aminophenyl)porphyrin (CAS: 22112-84-1) is as follows:

[0011] ;

[0012] The structural formula of the tetraaldehyde tetraphenylethylene (CAS: 2170451-48-4) is as follows:

[0013] ;

[0014] The structural formula of the porphyrin-based covalent organic framework is as follows:

[0015] ;

[0016] The structural formula of the DSPE-PEG3400-Biotin (purchased from Yusi Pharmaceutical, product code YS-D9241) is as follows:

[0017] .

[0018] This invention relates to a photosensitizer based on a porphyrin covalent organic framework that links porphyrin to tetraaldehyde tetraphenylethylene via imine condensation. The resulting nanosystem modified with DSPE-PEG3400-Biotin is a photosensitizer with high reactive oxygen generation capacity, high anticancer activity, and low toxicity.

[0019] In step (1), the molar ratio of tetra(p-aminophenyl)porphyrin to tetraaldehyde tetraphenylethylene is 1:1; the reaction solvent is a mixed solvent of 1,4-dioxane and 1,3,5-trimethylbenzene, in which the volume ratio of 1,4-dioxane to 1,3,5-trimethylbenzene is 1:1; the catalyst is acetic acid, and the concentration of acetic acid is 6M; the amount of 6M acetic acid added is 20% of the volume of the reaction solvent; the reaction temperature is 120℃, and the reaction time is 72h.

[0020] In step (2), the molar ratio of DSPE-PEG3400-Biotin to the porphyrin-based covalent organic framework is 5:1.

[0021] The above-mentioned nanodispersions based on porphyrin covalent organic frameworks are used as photosensitizers in photodynamic therapy.

[0022] This invention uses porphyrin as a functional unit to copolymerize with organic monomers to construct a porphyrin-based covalent organic framework. This not only retains the excellent optical and photosensitizing properties of porphyrin, but also utilizes the skeletal effect of the covalent organic framework to inhibit aggregation quenching, improve photostability, and enhance charge separation. Furthermore, through structural design, the monomeric porphyrin can be transformed from a type II photodynamic pathway to an oxygen-independent type I photodynamic process, significantly improving the therapeutic effect under hypoxic conditions (porphyrin monomers are type II photosensitizers, and their photodynamic effect is limited by the hypoxic microenvironment of tumors; by combining porphyrin with organic molecules to form a covalent organic framework, it can be transformed into an oxygen-independent type I photosensitizer).

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention is based on the covalent organic framework of porphyrin. By introducing aldehyde tetraphenylethylene molecules, the problems of easy aggregation and quenching and poor photostability of porphyrin as a photosensitizer are solved. At the same time, the porphyrin is changed from a type II photosensitizer to a type I photosensitizer, overcoming the influence of hypoxia in the tumor microenvironment on the efficacy of photodynamic therapy. (2) The nanosystem formed by the covalent organic framework of porphyrin modified with DSPE-PEG3400-Biotin has good cellular uptake capacity. It is distributed in the endoplasmic reticulum, mitochondria and lysosomes of tumor cells. It can generate reactive oxygen species under 450nm light irradiation, causing endoplasmic reticulum stress, lysosomal membrane permeability and mitochondrial damage, inducing panapoptosis of tumor cells, specifically manifested as apoptosis, necrotizing apoptosis and pyroptosis, achieving good anti-tumor activity and having a wide range of applications. Therefore, the covalent organic framework of porphyrin in the present invention provides a feasible direction for developing efficient, stable and adapted photodynamic therapy for hypoxic tumor microenvironments. Attached Figure Description

[0024] Figure 1 The image shows the localization of TPCOF@NP obtained in Example 1 in MDA-MB-231 cells;

[0025] Figure 2 The images shown are laser confocal fluorescence imaging and flow cytometry images of reactive oxygen species generated by TPCOF@NP prepared in Example 1 in MDA-MB-231 cells; where a is a confocal fluorescence imaging image and b is a cell flow cytometry image.

[0026] Figure 3The images show confocal fluorescence imaging and immunoblotting protein images of TPCOF@NP prepared in Example 1 in MDA-MB-231 cells after light-induced endoplasmic reticulum stress; where a is a confocal fluorescence imaging image at different concentrations; and b is an immunoblotting protein image.

[0027] Figure 4 This is a laser confocal fluorescence imaging image of TPCOF@NP prepared in Example 1 in MDA-MB-231 cells after light-induced calcium ion overload.

[0028] Figure 5 This is a confocal fluorescence image of lysosomal membrane permeation induced by light exposure in MDA-MB-231 cells prepared in Example 1 using TPCOF@NP.

[0029] Figure 6 The images show confocal fluorescence imaging and flow cytometry of mitochondrial membrane potential changes induced by light exposure in MDA-MB-231 cells prepared in Example 1; where a is a confocal fluorescence imaging image and b is a flow cytometry image at different concentrations.

[0030] Figure 7 This is a flow cytometry diagram showing the apoptosis induced by light exposure in MDA-MB-231 cells using TPCOF@NP prepared in Example 1.

[0031] Figure 8 This is a confocal fluorescence imaging image of the cell membrane morphology of TPCOF@NP prepared in Example 1 after MDA-MB-231 illumination;

[0032] Figure 9 This is an immunoblot protein image of TPCOF@NP prepared in Example 1 induced pan-apoptosis after MDA-MB-231 light irradiation;

[0033] Figure 10 The images show in vivo experiments in mice with TPCOF@NP photodynamic therapy prepared in Example 1; where a is a schematic diagram of the experiment; b is an image after tumor removal; c is a graph showing changes in tumor volume; d is a graph showing changes in tumor mass; e is a graph showing changes in mouse body weight; and f is an H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of the mice after treatment.

[0034] Figure 11 The images show the PXRD (a), FTIR (b), and XPS (c) plots of the TPCOF prepared in Example 1. Detailed Implementation

[0035] Example 1

[0036] The present invention discloses a method for preparing a nano-dispersion based on a porphyrin covalent organic framework, comprising the following steps:

[0037]

[0038] (1) Tetraaldehyde tetraphenylethylene (8.9 mg, 0.02 mmol), tetra(p-aminophenyl)porphyrin (13.5 mg, 0.02 mmol), 1,4-dioxane (0.5 mL), 1,3,5-trimethylbenzene (0.5 mL), and 6M acetic acid (0.2 mL) were added to a 19×65 mm (od×length) Pyrex tube; after sonication for 30 minutes, the reaction tube containing the reactants was degassed three times in liquid nitrogen at 196 °C, and finally evacuated to an internal pressure of approximately 100 m Torr and sealed; after being placed at room temperature, it was heated at 120 °C for 72 hours; after cooling to room temperature, a purple-red precipitate was obtained by filtration, and the precipitate was washed several times with DMF and acetone. Then, the wet sample was transferred to a Soxhlet extractor and washed with THF for 24 h; finally, the product was dried at 120 °C to obtain a porphyrin-based covalent organic framework (yield 70%); through Figure 11 PXRD, FTIR, and XPS confirmed the successful synthesis of TPCOF;

[0039] (2) DSPE-PEG3400-Biotin (5 mg, MW=3400, 0.015 mmol) was ultrasonically dispersed in 5 mL of ultrapure water, and the porphyrin-based covalent organic framework (5 mg, 0.003 mmol) obtained in step (1) was added to it. The mixture was stirred at room temperature for 12 h. The resulting mixture was centrifuged at 3500 rpm for 5 min, and the supernatant was collected. Then, the mixture was dialyzed with water (the permeation molecular weight of the dialysis bag was 5000 kDa) for 24 h to remove unreacted DSPE-PEG3400-Biotin. A nano-dispersion of porphyrin-based covalent organic framework was obtained.

[0040] The solid content of the porphyrin-based covalent organic framework nanoparticles in the nanodispersion obtained in Example 1 was 1 mg / mL, and the particle size of the porphyrin-based covalent organic framework nanoparticles (TPCOF@NP) was 134 nm.

[0041] The crystal structure of TPCOF was characterized using powder X-ray diffraction (PXRD), and theoretical structural simulations were performed using Material Studio 2020 to establish an orthogonal P1 space group model of TPCOF. Further Pawley corrections were applied to this structural model. The refined unit cell parameters of TPCOF are a = 25.3686 Å, b = 19.2827 Å, c = 3.9689 Å, α = β = γ = 90°. Furthermore, the PXRD pattern simulated using the AA packing mode accurately reproduced the experimental profile, with an unweighted profile R-factor (Rp) of 3.25% and a weighted profile R-factor (RWP) of 4.27%, demonstrating the reliability of the theoretical simulation. The significant diffraction peaks at 5.50°, 11.33°, 17.24°, and 23.11° in the PXRD pattern of TPCOF correspond to the (110), (220), (330), and (440) crystal planes, respectively (e.g., ...). Figure 11 (As shown in (a)). Further Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were performed to confirm the successful preparation of TPCOF. The FTIR spectrum at 1620 cm⁻¹... -1 The area exhibits characteristic tensile vibrations of C=N bonds, while simultaneously, C=O tensile bands originating from the original precursor (1700 cm) are present. -1 ) and -NH2 vibration zone (3200~3500 cm) -1 The significant attenuation confirms the successful condensation reaction between these functional groups (e.g.) Figure 11 As shown in (b)). XPS spectroscopy revealed a peak at 398.7 eV indicating the formation of an imine-C=N− bond, collectively demonstrating the formation of the COF material (as shown in [reference]). Figure 11 (as shown in (c)).

[0042] Comparative Example 1

[0043] The preparation method of a nano-dispersion based on a covalent organic framework linked by tetraaminoporphyrin and tetrathiofulvalene tetraaldehyde includes the following steps:

[0044] (1) Tetrathiofulvalenetetraaldehyde (TTF-4CHO, CAS: 1639134-02-3) (12.4 mg, 0.02 mmol), tetra(p-aminophenyl)porphyrin (13.5 mg, 0.02 mmol), 1,4-dioxane (0.5 mL), 1,3,5-trimethylbenzene (0.5 mL), and 6M acetic acid (0.2 mL) were added to a 19×65 mm (od×length) Pyrex tube. After sonication for 30 minutes, the reaction tube containing the reactants was degassed three times in liquid nitrogen at 196 °C. Finally, the tube was evacuated to an internal pressure of approximately 100 mTorr and then sealed. After being placed at room temperature, the tube was heated at 120 °C for 72 hours. After cooling to room temperature, the purplish-red precipitate was obtained by filtration and washed repeatedly with DMF and acetone. Subsequently, the wet sample was transferred to a Soxhlet extractor and washed with THF for 24 hours. h; Finally, the product was dried at 120°C to obtain the final sample (yield 65%).

[0045] (2) DSPE-PEG3400-Biotin (5 mg, MW=3400, 0.015 mmol) was ultrasonically dispersed in 5 mL of ultrapure water, and the porphyrin-based covalent organic framework (5 mg, 0.003 mmol) obtained in step (1) was added to it. The mixture was stirred at room temperature for 12 h. The resulting mixture was centrifuged at 3500 rpm for 5 min, and the supernatant was collected. Then, the mixture was dialyzed with water (supernatant) (the permeation molecular weight of the dialysis bag was 5000 kDa) for 24 h to remove unreacted DSPE-PEG3400-Biotin. A nano-dispersion of porphyrin-based covalent organic framework was obtained.

[0046] The solid content of porphyrin-based covalent organic framework nanoparticles in the nano-dispersion prepared in Comparative Example 1 was 1 mg / mL, and the particle size of the porphyrin-based covalent organic framework nanoparticles (TTCOF@NP) was 134 nm.

[0047] The TPCOF@NP prepared in Example 1 and the TTCOF@NP prepared in Comparative Example 1 were subjected to the following experiments:

[0048] Cytotoxicity against human triple-negative breast cancer cells MDA-MB-231 and normal human breast epithelial cells MCF-10A under hypoxic conditions:

[0049] The MTT assay was used to analyze the antiproliferative effects of porphyrin-based covalent organic framework nanosystems TPCOF@NP, TTCOF@NP, and cisplatin CDDP. MTT (thiazolyl blue) is a tetrazolium salt that can be reduced by succinate dehydrogenase in the mitochondria of living cells to a blue-violet product, formazan (the product is soluble in DMSO), which has an absorption peak at 490 nm. Therefore, it can be used as an indicator. 490nm To analyze cell proliferation.

[0050] The specific experimental steps are as follows:

[0051] (1) First, revive a tube of tumor cells, culture them in fresh culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin and streptomycin), and use them after passage 3 times;

[0052] (2) When the cells reach the logarithmic growth phase, seed them into 96-well plates at a density of 5000 cells / well (100 μL of culture medium per well) and then place them in an incubator (37℃, 21% O2, 5% CO2) for culture.

[0053] (3) After the cells adhered, they were transferred to a hypoxic incubator (37℃, 1% O2, 5% CO2). After 24 hours, 100 μL of fresh culture medium containing different concentration gradients of TPCOF@NP, TTCOF@NP, and cisplatin CDDP was added to each well, and then placed in an incubator for further incubation. The light-illuminated group was incubated for 6 hours and then treated with a 450 nm laser (0.6 W / cm²). 2 Irradiate for 30 minutes and continue incubation;

[0054] (4) After incubation for 48 hours, add 20 μL MTT (5 mg / mL) to each well and continue incubation at 37°C for 4 hours. Then, remove the supernatant and add 150 μL dimethyl sulfoxide (DMSO) to each well. Detect A using an ELISA reader. 490nm Calculate the cell proliferation inhibition rate and determine the IC50. 50 Value (drug concentration corresponding to an inhibition rate of 50%). The MTT test results of TPCOF@NP, TTCOF@NP and cisplatin CDDP are shown in Table 1.

[0055] Table 1 lists the ICs for TPCOF@NP, TTCOF@NP, and cisplatin CDDP. 50 Value (μg / mL; μM)

[0056]

[0057] The results showed that under hypoxic conditions, TPCOF@NP exhibited higher phototoxicity to MDA-MB-231 cells than TTCOF@NP and CDDP, while showing very low dark toxicity and low toxicity to normal cells, indicating that TPCOF@NP had higher antitumor activity.

[0058] Example 2

[0059] Application of TPCOF@NP prepared in Example 1 for the localization of intracellular subcellular organelles (endoplasmic reticulum, lysosomes, and mitochondria):

[0060] Methods: MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density reached 70%, TPCOF@NP at a concentration of 20 μg / mL was added for 6 h. The culture medium was then aspirated, and the cells were washed twice with PBS. Commercial dyes such as ER-Tracker Green, Lyso-Tracker Green, and Mito-Tracker Green were added. The cells were incubated at 37°C for 10–30 minutes, followed by two washes with PBS. Observation was performed using a confocal microscope. TPCOF@NP: λ ex =488 nm, λ em =670 ±20 nm; the excitation wavelengths of ER-Tracker Green, Lyso-Tracker Green, and Mito-Tracker Green are 365~488 nm, respectively. Colocation coefficient analysis was performed using ImageJ software.

[0061] The intracellular localization of the nanoparticles TPCOF@NP prepared in Example 1 after co-incubation with endoplasmic reticulum, lysosomes, and mitochondrial probes is as follows: Figure 1 As shown, the results indicate that the colocalization coefficients of TPCOF@NP with the endoplasmic reticulum, lysosomes, and mitochondria probes were 0.87, 0.62, and 0.69, respectively, indicating that after entering MDA-MB-231 cells, TPCOF@NP can be distributed in the endoplasmic reticulum, lysosomes, and mitochondria.

[0062] Example 3

[0063] Application of TPCOF@NP prepared in Example 1 to generate reactive oxygen species in cells under hypoxic conditions upon photoexcitation:

[0064] Method 1: Confocal microscopy for ROS detection in tumor cells. MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes and incubated overnight under hypoxic (1% O2) atmosphere. Then, different concentrations of TPCOF@NP (0.5–1 μg / mL) and TTCOF@NP (1 μg / mL) were added for 6 h. The light-illuminated group was treated with a 450 nm laser (0.6 W / cm²). 2 Irradiate for 30 minutes, then culture the cells in serum-free culture medium containing 10 μM H2DCFH-DA and stain at 37°C in the dark for 30 min. Wash twice with PBS and then observe immediately with a confocal microscope. The excitation wavelength is 488 nm and the emission wavelength is 530 ± 20 nm.

[0065] Method 2: Flow cytometry detection of ROS in tumor cells. MDA-MB-231 cells were seeded in 6-well plates and grown overnight under hypoxic (1% O2) atmosphere. Different concentrations of TPCOF@NP (0.5–1 μg / mL) and TTCOF@NP (1 μg / mL) were added for 6 h of treatment. The light-illuminated group was treated with a 450 nm laser (0.6 W / cm²). 2 After irradiation for 30 minutes, cells were stained with serum-free culture medium containing 10 μM H2DCFH-DA at 37°C in the dark for 30 minutes. The cells were centrifuged, the supernatant discarded, and the cells were washed three times with serum-free medium to remove any H2DCFH-DA that had not entered the cells. The green fluorescence intensity was measured by flow cytometry within half an hour of cell collection. The excitation wavelength was 488 nm, and the emission wavelength was 530 ± 20 nm. The green fluorescence intensity was analyzed using FlowJo v10.9.0 software.

[0066] The results of reactive oxygen species production by TPCOF@NP and TTCOF@NP under hypoxic conditions in darkness and light are as follows: Figure 2 As shown in the figure. The results indicate that, compared with the control group, the green fluorescence was significantly enhanced by flow cytometry and confocal microscopy after TPCOF@NP was treated with light, indicating that TPCOF@NP can generate a large amount of reactive oxygen species in MDA-MB-231 cells when excited by light, and that TPCOF@NP has a better ability to generate ROS than TTCOF@NP.

[0067] Example 4

[0068] Application of TPCOF@NP prepared in Example 1 to induce intracellular endoplasmic reticulum stress under hypoxic conditions:

[0069] Methods: Confocal microscopy was used to detect endoplasmic reticulum stress in tumor cells. MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes and incubated overnight under hypoxic (1% O2) atmosphere. Then, different concentrations of the nanosystem TPCOF@NP (0.5–1 μg / mL) and TTCOF@NP (1 μg / mL) were added for 6 h. The light irradiation group used a 450 nm laser (0.6 W / cm²). 2 Irradiate for 30 minutes, then incubate in the dark for 1 h. Wash cells with PBS, and incubate in DMEM medium containing ER-Tracker Green (1 µM) without phenol red and serum at 37°C for 30 min. After washing with PBS, observe cells under a confocal microscope. Probe ER-Tracker Green: λex = 488 nm, λem = 500~550 nm.

[0070] The effects of TPCOF@NP on induced intracellular endoplasmic reticulum stress are as follows: Figure 3 As shown in the figure. The results indicate that, compared with the control group, the green fluorescence of the endoplasmic reticulum (ER) was significantly reduced after TPCOF@NP light treatment. Simultaneously, the phosphorylation levels of ER stress-related proteins p-PERK, C / EBP homolog (CHOP), and eukaryotic cell initiation factor 2α (eIF2α) were upregulated, indicating that TPCOF@NP can effectively induce ER stress under photoexcitation. Green fluorescence of the ER was still observed after TPCOF@NP light treatment, indicating that TPCOF@NP cannot effectively induce ER stress under photoexcitation.

[0071] Example 5

[0072] Application of TPCOF@NP prepared in Example 1 in influencing intracellular calcium ion levels in MDA-MB-231 cells under hypoxic conditions:

[0073] Methods: Changes in intracellular calcium ion levels in tumor cells were detected using confocal microscopy. MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes and incubated overnight under hypoxic (1% O2) conditions. Then, different concentrations of TPCOF@NP (0.5–1 μg / mL) and TTCOF@NP (1 μg / mL) were added for 6 h of treatment. The light-illuminated group was treated with a 450 nm laser (0.6 W / cm²). 2 Irradiate for 30 minutes, then stain cells with 2 µM Fluo-4 AM working solution at 37°C in the dark for 30 minutes, wash twice with PBS, and then observe immediately using a confocal microscope. The excitation wavelength is 488 nm, and the emission wavelength is 530 ± 20 nm.

[0074] The results of TPCOF@NP on the effect of hypoxia on intracellular calcium ion levels in MDA-MB-231 cells are as follows: Figure 4 As shown in the figure. The results indicate that, compared with the control group, the green fluorescence of calcium ions was significantly enhanced after TPCOF@NP light treatment, indicating that TPCOF@NP can effectively induce intracellular calcium ion homeostasis imbalance under photoexcitation. Green fluorescence of calcium ions was also observed after TTCOF@NP light treatment, but the fluorescence intensity was lower than that of the TPCOF@NP treatment group.

[0075] Example 6

[0076] Application of TPCOF@NP prepared in Example 1 to induce intracellular lysosomal membrane permeation under hypoxic conditions:

[0077] Methods: Changes in lysosomal integrity within tumor cells were detected using confocal microscopy. MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes and incubated overnight under hypoxic (1% O2) conditions. Then, different concentrations of TPCOF@NP (0.5–1 μg / mL) and TTCOF@NP (1 μg / mL) were added for 6 h of treatment. The light-illuminated group was treated with a 450 nm laser (0.6 W / cm²). 2 Irradiate for 30 minutes, then incubate in the dark for 1 hour. Next, stain cells with 5 µM AO working solution at 37°C in the dark for 30 minutes, wash twice with PBS, and immediately observe using a confocal microscope. The excitation wavelength for the green channel was 488 nm, and the excitation wavelength for the red channel was 561 nm.

[0078] The results of TPCOF@NP under hypoxic conditions on the induction of intracellular lysosomal membrane permeation are as follows: Figure 5 As shown in the figure. The results indicate that, compared with the control group, the red fluorescence of lysosomes was significantly weakened after TPCOF@NP light treatment, indicating that TPCOF@NP can effectively induce lysosomal membrane permeation under photoexcitation. Red fluorescence of lysosomes could also be observed after TPCOF@NP light treatment.

[0079] Example 7

[0080] Application of TPCOF@NP prepared in Example 1 to induce changes in intracellular mitochondrial membrane potential under hypoxic conditions:

[0081] Method 1: Confocal microscopy was used to detect changes in mitochondrial membrane potential in tumor cells. MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes and incubated overnight in a hypoxic (1% O2) atmosphere. Then, different concentrations of TPCOF@NP (0.5–1 μg / mL) and TTCOF@NP (1 μg / mL) were added for 6 h. The light-illuminated group was treated with a 450 nm laser (0.6 W / cm²). 2 Irradiate for 30 minutes, continue incubation in the dark for 18 hours, then stain the cells with pre-prepared JC-1 working solution at 37°C in the dark for 20 minutes, and then observe them immediately with a confocal microscope.

[0082] Method 2: Flow cytometry was used to detect changes in mitochondrial membrane potential in tumor cells. Cell culture media containing different concentrations of TPCOF@NP (0.5–1 μg / mL) and TTCOF@NP at a concentration of 1 μg / mL were added to 6-well plates seeded with well-morphologically shaped and normally growing MDA-MB-231 cells. After 6 h of drug treatment, the light-treated group was treated with a 450 nm laser (0.6 W / cm²). 2Irradiate for 30 minutes, then incubate in the dark for 18 hours. Collect cells, wash with PBS, and then stain with prepared JC-1 working solution for 20 minutes. Wash cells with 1×Incubation buffer and resuspend. Immediately analyze the samples using a BD C6 flow cytometer. Process and analyze the results using FlowJo v10.9.0 software. The detection fluorescence channel is λ. ex = 488 nm, λ em = 530 ± 30 nm; λ ex = 488 nm, λ em = 590 ± 30 nm.

[0083] The results of TPCOF@NP under hypoxic conditions on the induced changes in intracellular mitochondrial membrane potential are as follows: Figure 6 As shown in the figure. The results indicated that, compared with the control group, the red fluorescence in cells was weakened and the green fluorescence was significantly enhanced after TPCOF@NP treatment, indicating that TPCOF@NP can effectively induce a decrease in mitochondrial membrane potential. Flow cytometry results also showed similar conclusions. TTCOF@NP light treatment also induced a decrease in mitochondrial membrane potential (10.0%), but the effect was not as good as that of TPCOF@NP (28.8%).

[0084] Example 8

[0085] Application of TPCOF@NP prepared in Example 1 in inducing apoptosis in MDA-MB-231 cells under hypoxic conditions:

[0086] Methods: Flow cytometry was used to detect intracellular apoptosis in tumor cells. Cell culture medium containing different concentrations of TPCOF@NP (0.5–1 μg / mL) was added to 6-well plates seeded with well-morphologically shaped and normally growing MDA-MB-231 cells. After 6 hours of drug treatment, the light-treated group was treated with a 450 nm laser (0.6 W / cm²). 2 Irradiate for 30 minutes, then incubate in the dark for 18 hours. Collect cells, wash with PBS, resuspend in binding buffer, then stain with 5 µM FITC working solution in the dark for 5 minutes, followed by 5 µM PI staining in the dark for 5-10 minutes. Immediately analyze the samples using a BD C6 flow cytometer, and process and analyze the results using FlowJov 10.9.0 software. The detection fluorescence channel is λ. ex = 488 nm, λ em = 530 ± 30nm; λ ex = 561 nm, λ em = 600 ± 30 nm.

[0087] The experimental results of TPCOF@NP under hypoxic conditions on inducing apoptosis in MDA-MB-231 cells are as follows: Figure 7 As shown in the figure. The results indicate that, compared with the untreated control group, the proportion of apoptotic and necrotic apoptotic cells significantly increased after TPCOF@NP light treatment, indicating that TPCOF@NP can induce apoptosis and necrotic apoptosis in MDA-MB-231 cells.

[0088] Example 9

[0089] Application of TPCOF@NP prepared in Example 1 to induce pyroptosis under hypoxic conditions:

[0090] Methods: Changes in pyroptosis of tumor cells were detected by confocal microscopy. MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes and incubated overnight under hypoxic (1% O2) atmosphere. Then, cells were treated with 1 μg / mL TPCOF@NP and 1 μg / mL TTCOF@NP for 6 h, respectively. The light-illuminated group was treated with a 450 nm laser (0.6 W / cm²). 2 Irradiate the cells for 30 minutes, then incubate in the dark for 12 hours. Wash the cells with PBS and stain with Dio-Green for 30 minutes. After washing with PBS, observe the morphological changes of the cell membrane using a confocal microscope.

[0091] Results of TPCOF@NP inducing pyroptosis under hypoxic conditions as follows Figure 8 As shown in the figure. The results indicate that, compared with the control group, numerous vesicular protrusions appeared around MDA-MB-231 cells after TPCOF@NP light treatment. These protrusions have been confirmed as a typical feature of pyroptosis cells—pyroptotic bodies—indicating that TPCOF@NP can effectively induce pyroptosis under photoexcitation. However, no pyroptotic bodies were generated in the TTCOF@NP light-treated group, indicating that TTCOF@NP cannot effectively induce pyroptosis under photoexcitation.

[0092] Example 10

[0093] Application of TPCOF@NP prepared in Example 1 to induce pan-apoptosis in MDA-MB-231 cells under hypoxic conditions:

[0094] Methods: Western blotting (WB) was used to detect changes in the levels of pan-apoptotic proteins. The primary antibodies used in this application included pan-apoptotic marker proteins Bax, Bcl-2, caspase 3, cleaved caspase 3, GSDMD, N-GSDMD, caspase 1, MLKL, and p-MLKL.

[0095] The experimental results of TPCOF@NP inducing pan-apoptotic protein expression in MDA-MB-231 cells under hypoxic conditions are as follows: Figure 9 As shown in the figure. The results showed that, compared with the control group, after treatment with TPCOF@NP, the expression of apoptosis-related proteins Bax and Cleaved caspase 3 was significantly upregulated, while the expression of Bcl-2 and caspase 3 was significantly downregulated; the expression of pyroptosis-related proteins GSDMD and Pro caspase 1 was significantly downregulated, while the expression of N-GSDMD and Cleaved caspase 1 was significantly upregulated; at the same time, the expression of necrosis-related apoptosis proteins MLKL and p-MLKL was upregulated, indicating that TPCOF@NP can induce pan-apoptosis in MDA-MB-231 cells.

[0096] Example 11

[0097] Antitumor application of TPCOF@NP prepared in Example 1:

[0098] Methods: Cultured human breast cancer MDA-MB-231 cells were collected and a cell suspension was prepared with a cell concentration of 5 × 10⁻⁶ cells / mL. 8 Cells / mL were diluted 1:1 by volume with PBS and matrix gel; 0.1 mL of cell suspension was subcutaneously injected into the left axilla of each mouse to construct a subcutaneous xenograft tumor model. The long and short diameters of the tumor were measured periodically using calipers, and the tumor volume was increased to 80 mm. 3 At approximately 10:00 AM, tumor-bearing mice were randomly divided into four groups of six mice each: Group I: PBS protected from light; Group II: PBS with light exposure; Group III: TPCOF@NP with light exposure; Group IV: TPCOF@NP with light exposure (TPPCOF@NP concentration was 4 mg / kg). After grouping, quantitative administration began, and the long axis (a) and short axis (b) of the tumor were measured periodically according to the formula V = a × b. 2 / 2. Tumor volume was calculated, tumor growth was dynamically monitored, and the in vivo antitumor effect of the material was evaluated. Mice were euthanized after the drug administration cycle for subsequent analysis. After the experiment, mice were euthanized, and tumors and organs such as the heart, liver, lungs, spleen, and kidneys were collected for hematoxylin-eosin staining.

[0099] Experimental results are as follows Figure 10 As shown in the figure. The results indicated that tumor growth was significantly inhibited in the TPCOF@NP+ phototherapy group after irradiation, with a tumor inhibition rate of up to 88%, which was significantly higher than that in the control group. No significant weight loss was observed in the mice throughout the experiment. Furthermore, hematoxylin-eosin (H&E) staining showed no significant organ damage, indicating that the TPCOF@NP+ phototherapy group possessed strong PDT therapeutic efficacy and strong biocompatibility.

Claims

1. A nano-dispersion based on a porphyrin covalent organic framework, characterized in that: The nano-dispersion contains porphyrin-based covalent organic framework nanoparticles; the porphyrin-based covalent organic framework nanoparticles include intertwined polymer DSPE-PEG3400-Biotin, and also include porphyrin-based covalent organic frameworks encapsulated within the intertwined polymer DSPE-PEG3400-Biotin.

2. The nanodispersion based on a porphyrin covalent organic framework according to claim 1, characterized in that: The nano-dispersion liquid has a solid content of 1 mg / mL, and the porphyrin-based covalent organic framework nanoparticles have a particle size of 134 nm.

3. The method for preparing the nano-dispersion based on porphyrin covalent organic framework as described in claim 1, characterized in that, Includes the following steps: (1) Tetra(p-aminophenyl)porphyrin and tetraaldehyde tetraphenylethylene were added to a reaction solvent containing a catalyst and reacted at high temperature. After the reaction, the mixture was cooled to room temperature to obtain a porphyrin-based covalent organic framework. (2) Disperse DSPE-PEG3400-Biotin in ultrapure water by ultrasonication, add the porphyrin-based covalent organic framework from step (1) to it, stir thoroughly at room temperature to obtain a nano-dispersion of porphyrin-based covalent organic framework. The structural formula of the porphyrin-based covalent organic framework is as follows: 。 4. The method for preparing the nanodispersion based on porphyrin covalent organic framework according to claim 3, characterized in that: In step (1), the molar ratio of tetra(p-aminophenyl)porphyrin and tetraaldehyde tetraphenylethylene is 1:

1.

5. The method for preparing the nanodispersion based on porphyrin covalent organic framework according to claim 3, characterized in that: In step (1), the reaction solvent is a mixed solvent of 1,4-dioxane and 1,3,5-trimethylbenzene, wherein the volume ratio of 1,4-dioxane and 1,3,5-trimethylbenzene in the mixed solvent is 1:

1.

6. The method for preparing the nanodispersion based on porphyrin covalent organic framework according to claim 3, characterized in that: In step (1), the catalyst is acetic acid, and the concentration of acetic acid is 6M; the amount of 6M acetic acid added is 20% of the volume of the reaction solvent.

7. The method for preparing the nanodispersion based on porphyrin covalent organic framework according to claim 3, characterized in that: In step (1), the reaction temperature is 120℃ and the reaction time is 72h.

8. The method for preparing the nanodispersion based on porphyrin covalent organic framework according to claim 3, characterized in that: In step (2), the molar ratio of DSPE-PEG3400-Biotin to the porphyrin-based covalent organic framework is 5:

1.

9. The application of the porphyrin-based covalent organic framework nanodispersion as described in claim 1 as a photosensitizer in photodynamic therapy.

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