A porphyrin-based cubic metal-organic cage complex and its application in photodynamic therapy

Abstract

A cubic metal-organic cage complex based on porphyrin and its application in photodynamic therapy, which belongs to the fields of metal-organic supramolecules, photodynamic therapy and bioinorganic chemistry. This application adopts a relatively simple modification method of porphyrin photosensitizers, so that porphyrin avoids the decrease in photodynamic therapy ability caused by aggregation self-quenching, and the construction of porphyrin supramolecular functional materials avoids cumbersome organic synthesis and covalent modification, and adopts a simple coordination self-assembly process for preparation. It is a simple and reliable preparation method of porphyrin photosensitizers that can be applied to the water environment in vivo, and provides a new method for the transformation of porphyrin photosensitizers and the improvement of their photodynamic therapy performance.

Description

Technical Field

[0001] The present invention relates to a cubic metal-organic cage based on porphyrin, which belongs to the technical fields of metal-organic complexes, supramolecular chemistry, photodynamic therapy and bioinorganic chemistry, and in particular to a simple and efficient synthesis method of a porphyrin photosensitizer and the application of the porphyrin photosensitizer nanoparticles in photodynamic therapy of cells. Background Art

[0002] Photodynamic therapy is a type of treatment method that has been widely studied in recent years. It stands out for its excellent anti-cancer potential. It is a newly developed new cancer treatment strategy with excellent targeting and almost no drug resistance. Among them, photosensitizers are carriers of photodynamic therapy. By utilizing the sensitization of photosensitizers to oxygen, cancer cells can be selectively killed, without drug resistance, and have good targeting. It is a type of anti-cancer strategy with excellent therapeutic effects that has developed rapidly in recent years and has been widely studied. Through different methods, the performance of photosensitizers has been improved or given new functions, so that photosensitizers have been widely used in the clinical application of photodynamic therapy. At present, a variety of photosensitizers have been developed in the research field for use in photodynamic therapy. Common photosensitizers include porphyrin compounds, phthalocyanine compounds, indocyanine green compounds, BODIPY compounds, aggregation-induced emission effect compounds (AIE effect compounds), etc. Based on the advantages and disadvantages of different types of photosensitizers, a large number of modified and improved photosensitizer materials have been developed and popularized, and have had a profound impact in the field of photodynamic therapy.

[0003] Porphyrin and its derivatives are classic photosensitizers. Porphyrins have been widely studied due to their widespread presence in nature, easy availability of raw materials, and excellent photophysical and photochemical properties. Porphyrins have a large π-conjugated aromatic structure at their core, a large absorption peak with a large molar extinction coefficient in the visible light region, and a high ROS yield. However, the π-π interaction between porphyrin molecules can lead to aggregation and self-quenching at high concentrations, hindering the generation of ROS. In order to improve the biological activity of porphyrins in photodynamic therapy, it is necessary to increase the solubility of porphyrins and solve the self-quenching problem. Imparting good water solubility to the porphyrin core is usually achieved by appropriate hydrophilic substituents or axial substitution of the core to improve the aggregation self-quenching phenomenon, further improve the ROS generation efficiency and enhance phototoxicity. Developing a strategy that can effectively improve the photosensitivity of porphyrins through a simple preparation process remains a challenging task.

[0004] Photosensitizers in metal-organic cages (MOCs) for biomedical applications have attracted considerable attention in the past few years. Compared with traditional small-molecule compounds and simple metal coordination compounds, MOCs are easily functionalized by ligand modification and possess fixed stereospecific conformations that general metal complexes and ligands never provide. Compared with traditional covalently linked complexes, the presence of various non-covalent interactions, such as hydrogen bonding, π-π stacking, electrostatic interactions, and hydrophobic or hydrophilic effects, endow MOCs with excellent flexibility and more effective applications in molecular identification, drug delivery, imaging, or anticancer activity and photodynamic therapy. In addition, discrete MOCs are prepared at the molecular level and are often distributed in sizes of several nanometers. The larger size provides advantages for MOCs in biological applications, such as promoting the enhanced permeation and retention (EPR) effect in tumors to enhance drug absorption and release, which is in line with the ultimate goal of nanomedicine technology. Through the application of metal-organic cages and the improvement of porphyrin photosensitizers, porphyrins have great potential to improve the efficacy of photodynamic therapy by using them as building blocks in metal-organic cages and as precursors for the construction of metal-organic cages. Summary of the invention

[0005] The purpose of the present invention is to propose a simple and efficient synthesis route of porphyrin-based metal-organic cages, and to synthesize them into water-soluble nanoparticles through simple self-assembly. Nanoparticles derived from porphyrin metal-organic cages can efficiently generate reactive oxygen species in aqueous solution, and can effectively generate reactive oxygen species in tumor cells, leading to apoptosis of tumor cells, thereby achieving the purpose of treating tumors.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A method for preparing a tetraaminometalloporphyrin ligand for coordination self-assembly of a metal-organic cage comprises the following steps:

[0008]

[0009] (1) using p-nitrobenzaldehyde, pyrrole, propionic acid and pyridine as raw materials to prepare intermediate 1; the molar ratio of p-nitrobenzaldehyde to pyrrole is 1:1;

[0010] (2) intermediate 2 is prepared using intermediate 1, stannous chloride and hydrochloric acid as raw materials; the molar ratio of intermediate 1 to stannous chloride is 1:15;

[0011] (3) reacting the intermediate 2 and metal acetate M(OAc)2 in N,N-dimethylformamide to obtain a tetraaminometal porphyrin ligand, wherein the molar ratio of the intermediate 2 to the metal acetate M(OAc)2 is 1:13;

[0012] The metal acetate M(OAc)2 is cobalt acetate, nickel acetate or zinc acetate.

[0013] The tetraamino metal porphyrin ligand is assembled with metal acetate M(OAc)2 and aldehyde compounds to obtain an M8L6 type cubic metal-organic cage-shaped complex.

[0014] The molar ratio of the tetraaminometal porphyrin ligand: metal acetate M(OAc)2: aldehyde compound is 6:8:24.

[0015] The structure of the aldehyde compound is: R-CHO;

[0016] in,

[0017] The tetraaminometal porphyrin ligand, aldehyde compound and metal acetate M(OAc)2 are dissolved in an organic solvent, the solution is fully mixed and stirred, and the metal-organic cage complex is precipitated by solvent evaporation crystallization, solvent diffusion or adding a poor solvent for the product metal-organic cage.

[0018] The nanoparticles include any of the above metal-organic cage-like complexes and amphiphilic molecule polyethylene glycol monomethyl ether-2000-dioctadecylphosphatidylethanolamine (MPEG-2000-DSPE).

[0019] Any of the above metal-organic cage-like complexes and the amphiphilic molecule MPEG-2000-DSPE are self-assembled in a mixed solution of water and acetone to obtain nanoparticles.

[0020] The nanoparticles are used for detecting the active oxygen generation ability in aqueous solution.

[0021] Singlet oxygen probe 1,3-diphenylisobenzofuran or superoxide anion free radical probe dihydrorhodamine 123 is added into the solution of the nanoparticles for ultraviolet and fluorescence detection.

[0022] The nanoparticles are used for preparing reagents for cell photodynamic therapy.

[0023] Porphyrin ligands as coordinated self-assembled organic ligand units of metal-organic cages;

[0024] The preparation method of the metal-organic cage and the preparation method of the nanoparticles include the following steps:

[0025]

[0026] (1) M-TAPP, aldehyde, and zinc trifluoromethanesulfonate were heated and stirred in N,N-dimethylformamide, and ether was added to precipitate the metal-organic complex to obtain a metal-organic cage structure of formula III, wherein the molar ratio of M-TAPP:aldehyde:metal acetate M(OAc)2 was 6:24:8. The structure M8L6 was confirmed by NMR and mass spectrometry data.

[0027] (2) The metal-organic cage (Formula III) and MPEG-2000-DSPE are self-assembled in a mixed solution of water and acetone to obtain the final product, metal-organic cage nanoparticles, wherein the mass ratio of the metal-organic cage (Formula III):MPEG-2000-DSPE is 1:5.

[0028] The metal-organic cage with porphyrin as the core generates active oxygen in the solvent.

[0029] A type of metal-organic cage with porphyrin as the core for efficient generation of reactive oxygen species in solvents. Test method: Add porphyrin ligand (M-TAPP) (6μM) and metal-organic cage (1μM) to the newly configured DMSO solution (0.05mM) of 1,3-diphenylisobenzofuran (singlet oxygen detection probe) / dihydrorhodamine 123 (superoxide anion detection probe) respectively. After 1,3-diphenylisobenzofuran is decomposed in the presence of photosensitizer, the absorption peak at 418nm decreases; after dihydrorhodamine 123 is oxidized, the fluorescence emission at 540nm increases. The ability to generate reactive oxygen species is obtained by the corresponding signal changes. The porphyrin ligand is modified into a metal-organic cage to inhibit the aggregation self-quenching between porphyrins to a certain extent, and the performance of the photosensitizer is improved.

[0030] The metal-organic cage with porphyrin as the core is modified into nanoparticles to detect the generation of reactive oxygen species.

[0031] A type of metal-organic cage with porphyrin as the core and its nanoparticles for efficient generation of reactive oxygen species in solvents. The test method is: metal-organic cage (1μM) and nanoparticles (1μM) are added to the newly configured aqueous solution (0.05mM) of 1,3-diphenylisobenzofuran (singlet oxygen detection probe) / dihydrorhodamine 123 (superoxide anion detection probe). After 1,3-diphenylisobenzofuran is decomposed in the presence of a photosensitizer, the absorption peak at 420nm decreases; after dihydrorhodamine 123 is oxidized, the fluorescence emission at 526nm increases. The ability to generate reactive oxygen species is obtained by the corresponding signal changes. The modification of porphyrin metal-organic cage compounds into nanoparticles makes the metal-organic cage have a higher water solubility than before, and the photosensitizer performance in aqueous solution is improved.

[0032] The metal-organic cage nanoparticles are used for photodynamic therapy in cells.

[0033] A type of metal-organic cage-based nanoparticles for efficiently generating reactive oxygen in a solvent. The test method is as follows: DMEM culture medium (with 10% FBS and 1% double antibody) is prepared for culturing Hela cells. After culturing the cells under normal oxygen concentration conditions for 24 hours, 2 μM metal-organic cage nanoparticles are incubated for 4 hours. After incubation, DCFH-DA and calcein propidium iodide live-dead cell double staining reagent are added respectively. After irradiation with 600-610 nm LED light, laser confocal fluorescence imaging experiments are performed on the cells to determine the phototoxicity of the nanoparticles in the cells.

[0034] Beneficial effects of the present invention:

[0035] The present application adopts a relatively simple modification method of porphyrin photosensitizers, so that porphyrin avoids the decrease in photodynamic therapy ability caused by aggregation self-quenching, and the construction of porphyrin supramolecular functional materials avoids tedious organic synthesis and covalent modification, and adopts a simple coordination self-assembly process for preparation. It is a simple and reliable preparation method of porphyrin photosensitizers that can be applied to the water environment in vivo, and provides a new method for the modification of porphyrin photosensitizers and the improvement of their photodynamic therapy performance.

[0036] Metal-organic cages based on porphyrin core units were synthesized through a coordination self-assembly process to improve the efficacy of photodynamic therapy. Common porphyrin derivative ligands were used as face modifications to the metal-organic cage as the main structure, and the introduction of zinc ions provided attachment sites for the metal-organic cage to self-assemble into the cubic cage. The metal-organic cage has a higher visible light utilization than the original ligand; the design of the metal-organic cage structure provides a fixed spacing for the porphyrin and inhibits the aggregation self-quenching effect. In addition, the modification of nanoparticles gives the metal-organic cage better water solubility, better reactive oxygen generation ability in aqueous solution, and can be effectively internalized by cancer cells and cause cancer cell apoptosis through photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a picture of the metal-organic cage C66 with porphyrin as the core and its nanoparticles used in photodynamic therapy in cells.

[0038] Figure 2 These are the UV-visible absorption and fluorescence emission graphs of the metal-organic cage C66 and the ligand Zn-TAPP.

[0039] Figure 3 This is a comparison chart of the singlet oxygen production effects of the metal-organic cage C66 and the ligand Zn-TAPP.

[0040] Figure 4 This is a comparison chart of the effects of metal-organic cage C66 and ligand Zn-TAPP in producing superoxide anion radicals.

[0041] Figure 5 This is a comparison chart of the particle size, UV-visible absorption, fluorescence emission and active oxygen generation capacity of metal-organic cage C66 after it is prepared into nanoparticles.

[0042] Figure 6 is a graph of the stability of the nanoparticles over seven days.

[0043] Figure 7 It is a dark toxicity diagram of the cell phototoxicity of metal-organic cage nanoparticles and the pre-modified ligands and metal-organic cages.

[0044] Figure 8 This is a confocal laser scanning microscopy image of the DCFH-DA reactive oxygen species generation experiment of metal-organic cage nanoparticles and modified pre-ligands and metal-organic cages.

[0045] Fig. 9 This is a laser confocal microscopy image of the calcein propidium iodide double staining experiment of metal-organic cage nanoparticles and pre-modified ligands and metal-organic cages. DETAILED DESCRIPTION

[0046] Preparation, synthesis and in vivo photodynamic therapy of porphyrin-based cubic metal-organic cages and their nanoparticles, such as Figure 1 shown.

[0047] Among them, the porphyrin ligand, tetraaminometalloporphyrin (M-TAPP), binds to the metal ion M 2+ The M8L6 type metal-organic cage complex was formed, and methyl PEG-DSPE was further used as an amphiphilic molecule to encapsulate the metal-organic cage complex to prepare nanoparticles. The nanoparticles have good solubility in water, can be effectively internalized by tumor cells and released inside the cells, and can generate a large amount of reactive oxygen in the cells through 600-610nm LED light irradiation, causing tumor cell apoptosis.

[0048] The present invention proposes a strategy for easily preparing metal-organic cage nanoparticles based on porphyrin photosensitizers and using them to efficiently generate reactive oxygen species in tumor cells. The nanoparticles can generate a large amount of reactive oxygen species in water, which can be quickly internalized by Hela cells in cell applications and cause apoptosis during intracellular photodynamic therapy, achieving a therapeutic effect on tumors.

[0049] Example 1: Synthesis of metal-organic cages with porphyrin as the core

[0050]

[0051] (1) Synthesis of TNPP using p-nitrobenzaldehyde and pyrrole as raw materials

[0052] In a 250mL round-bottom flask, p-nitrobenzaldehyde (5.5g, 0.0363mol) and acetic anhydride (6mL, 0.0635mol) were dissolved in 150mL propionic acid and the solution was heated to reflux. Pyrrole (2.4mL, 0.036mol) was then slowly added dropwise. After reflux for 30 minutes, the solid was collected by filtration, washed with water and methanol, and then dried under vacuum. The resulting solid was dissolved in 40mL pyridine and refluxed for 1 hour. It was cooled to room temperature and refrigerated overnight. The product was filtered and washed with methanol and acetone to obtain a purple solid. The crude product can be used directly in the next reaction without purification.

[0053] (2) Synthesis of TAPP using TNPP and stannous chloride as raw materials

[0054] TNPP (0.826 g, 1.038 mmol) was dissolved in 56 mL of hydrochloric acid and heated to 80°C. SnCl2·2H2O (3.6 g, 15.94 mmol) was then dissolved in 6 mL of hydrochloric acid and slowly added to the reactor, which was kept at 80°C for 6 hours. After the reaction was completed, it was cooled to room temperature and then the pH was adjusted to 9 in an ice-water bath containing ammonia. The solid product was collected to obtain bright purple crystals. Yield: 0.637 g, 91%. 1 H NMR (400MHz, DMSO) δ8.89 (s, 8H), 7.86 (d, J = 8.1Hz, 8H), 7.01 (d, J = 8.1Hz, 8H), 5.60 (s, 8H), -2.74 (s, 2H). 13 C NMR(101MHz,DMSO)δ149.02,135.95,129.16,121.06,112.98.ESI-MS m / z:[M+H] + Calculated value [C 44 H 35 N8] + 675.2979, experimental value 675.2943.

[0055] (3) Synthesis of Zn-TAPP using TAPP and zinc acetate as raw materials

[0056] TAPP (0.202 g, 0.3 mmol) was dissolved in 37 mL DMF, and Zn(OAc)2·2H2O (0.878 g, 4 mmol) was added to the reaction system, and then the mixture was heated and refluxed for 12 h. After the reaction was completed, the system was cooled to room temperature, then added to 250 mL of water, filtered and washed with water and ethanol to obtain a dark green solid. Yield: 0.208 g, 94%. 1H NMR (400MHz, DMSO) δ8.84(s,8H),7.82(d,J=8.1Hz,8H),6.97(d,J=8.2Hz,8H),5.47(s,8H). 13 C NMR(101MHz,DMSO)δ150.13,148.39,135.64,131.68,130.82,121.26,112.69.MALDI-TOF-MSm / z:[M] + Calculated value [C 44 H 32 N8Zn] + 736.2036, experimental value 736.2030.

[0057] (4) Synthesis of metal-organic cage C66 using Zn-TAPP, 2-pyridinecarboxaldehyde and zinc trifluoromethanesulfonate as raw materials Zn-TAPP (36.8 mg, 0.05 mmol, 6 equiv.), 2-pyridinecarboxaldehyde (20 μL, 0.2 mmol, 24 equiv.), Zn(CF3SO3)2 (25 mg, 0.07 mmol, 8 equiv.) and anhydrous DMF (6 mL) were added to a Schlenk flask, the solution was degassed three times under vacuum, and then the mixture was reacted at 80°C for 24 hours. The product was cooled to room temperature, and the supernatant was centrifuged and filtered to remove the solid. The clear solution was added dropwise to diethyl ether, and the lower solid layer was centrifuged and washed with additional diethyl ether to obtain a brown product. Yield: 40.5 mg, 49.5%. 1 H NMR (400MHz, DMSO) δ9.05(s,4H),8.89(s,12H),8.37(s,4H),8.28(s,8H),8.15(s,4H),7.82(s,8H),7.72(s,4H).

[0058] (5) Synthesis of nanoparticles using metal-organic cage C66 and amphiphilic molecule MPEG-DSPE as raw materials

[0059] Dissolve 2 mg of metal-organic cage C66 in 2 mL of acetone, then dissolve 10 mg of amphiphilic molecule MPEG-2000-DSPE in 10 mL of ultrapure water. Rapidly inject the acetone solution of the metal-organic cage into the ultrapure aqueous solution of the amphiphilic molecule under stirring. After ultrasonic treatment for one minute, evaporate the acetone under stirring in the dark to obtain a nanoparticle solution of the metal-organic cage.

[0060] Example 2: Solvent property test of metal-organic cages with porphyrin as core and their nanoparticles

[0061] The solvent test of metal-organic cages and nanoparticles was carried out according to the literature method, and the porphyrin ligand Zn-TAPP was set as the control group. The specific steps are as follows:

[0062] (1) Photophysical properties of metal-organic cages and ligands Zn-TAPP

[0063] The absorption spectra and fluorescence emission spectra of the ligands Zn-TAPP and C66 in the common solvent DMSO were tested using a UV-Vis spectrophotometer and a fluorescence spectrometer.

[0064] like Figure 2 As shown in the figure, the absorption peaks of the porphyrin ligand Zn-TAPP and the metal-organic cage C66 in DMSO are concentrated at 415nm, 580nm and 610nm, and the emission peak is concentrated at 650nm, with strong red fluorescence and obvious potential to penetrate tissues. When the excitation light wavelength is selected as 365nm, the ligand and the metal cage can still show strong red fluorescence, and there is a group of obvious emission peaks near 650nm, corresponding to the fluorescence of the porphyrin core. In addition, the UV-Vis absorption and fluorescence emission spectra of Zn-TAPP (6 equivalents) and C66 (1 equivalent) in dimethyl sulfoxide are compared. The molar extinction coefficient of C66 (1 equivalent) at its main absorption peak is much higher than that of 6 equivalents of Zn-TAPP. The strong visible light absorption ability provides a solid foundation for the application of metal-organic cage C66. In the comparison of the fluorescence intensities of the two, the luminescence ability of the metal-organic cage is still significantly improved compared with 6 equivalents of Zn-TAPP, indicating that the modification method of the metal-organic cage C66 can improve the aggregation self-quenching phenomenon of porphyrin.

[0065] (2) Test of the reactive oxygen generation ability of metal-organic cage and ligand Zn-TAPP

[0066] Singlet oxygen is a common reactive oxygen species that plays an important role in cell apoptosis in photodynamic therapy. The ability to produce singlet oxygen is, to a certain extent, one of the evaluation criteria for the photodynamic therapy ability of a photosensitizer. 1,3-Diphenylisobenzofuran (DPBF) is used as a measurement of singlet oxygen ( 1 O2) indicator. Determine the release of Zn-TAPP and C66 under red light excitation 1 O2 capacity, in the presence of singlet oxygen, the furan part of DPBF can be oxidized to endoperoxides, which are further decomposed into low-fluorescence 1,2-dibenzoylbenzene. At the same time, the absorption peak at 418nm gradually decreases until it disappears. The performance of the photosensitizer in singlet oxygen generation can be inferred by the degree of change in the absorption peak of DPBF at 418nm. After irradiating the DMSO solution of porphyrin Zn-TAPP and C66 with a 600-610nm LED light for 50 seconds, as shown in Figure 3 As shown in the figure, after irradiating a DMSO solution containing 6 μmol / L Zn-TAPP, the absorption peak of DPBF at 418 nm in the UV-Vis spectrum decreased by about 67%. In contrast, after irradiating a DMSO solution containing 1 μmol / L C66, the DPBF absorption decreased more strongly and was almost completely degraded, indicating that the metal-organic cage C66 has a higher singlet oxygen production effect than the six equivalent ligands, producing more in the same time. 1 In addition, the use of singlet oxygen quencher NaN3 can effectively quench singlet oxygen species. After adding sodium azide to the DMSO solution of C66 metal-organic cage, the C66 1 The O2 production capacity indicates that the metal-organic cage C66 can generate singlet oxygen with high efficiency.

[0067] In order to determine whether the ligand and C66 can release superoxide anion radicals under red light excitation, dihydrorhodamine 123 (DHR123) was used as a detector of superoxide anion radicals (·O2 — ) indicator. In the presence of superoxide anion free radicals, DHR123 can be oxidized to rhodamine 123 with strong green fluorescence, and the photosensitizer produces ·O2 - The ability to detect the fluorescence emission at 540 nm in DMSO can therefore be confirmed by observing the increase in fluorescence emission at 526 nm in water. Figure 4 After irradiating the solution of DHR123 with porphyrin ligand or metal-organic cage C66 dissolved in DMSO with 600-610nm LED light for 50 seconds, the fluorescence emission of DHR123 at 540nm was enhanced by nearly two times for 6μmol / L Zn-TAPP, indicating that the porphyrin ligand can effectively generate ·O2 - active oxygen species, and 1 μmol / L C66 showed better ·O2 than its 6 equivalent porphyrin ligand - The generation efficiency was shown as a three-fold enhancement of fluorescence emission. The generation of superoxide anion radicals was then confirmed, and the addition of ascorbic acid, a superoxide anion radical quencher, significantly inhibited the generation of ·O2 - The ability to produce O2 - Produce ability.

[0068] Example 3: Performance test of metal-organic cages modified into nanoparticles

[0069] The hydrodynamic diameter of the C66-NPs nanoparticles was obtained by DLS analysis, and the diameter distribution of the nanoparticles was found to be about 135 nm. Figure 5 and Figure 6After modification into nanoparticles, the UV-visible absorption and fluorescence emission of metal-organic cage C66 in water were greatly enhanced, which can be attributed to its superior water solubility. 1 O2 and O2 - The production capacity was tested, and the comparison of the degradation rates of DPBF and DHR123 in water showed that the nanoparticles C66-NPs had better singlet oxygen and superoxide anion radical production ability in aqueous solution than the metal-organic cage C66. After the C66 nanoparticle solution was placed for a week, the approximate particle size of the C66 nanoparticles fluctuated stably in the range of 130-150nm without drastic changes. In addition, the ultraviolet absorption spectrum of the C66 nanoparticles was almost unchanged, confirming that the nanoparticles had excellent stability during storage, which laid the foundation for the storage and application of nanoparticles.

[0070] Example 4: Evaluation of the Photodynamic Therapy Effect of Metal-Organic Cage Nanoparticles on Cells

[0071] (1) Cytotoxicity experiments of metal-organic cage nanoparticles

[0072] The dark cytotoxicity and photocytotoxicity of the ligand Zn-TAPP, metal-organic cage C66 and nanoparticle C66-NPs on Hela cells in the concentration range of 0 to 10 μM were evaluated using the MTT method based on 3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyltetrazolium bromide. Figure 7 As shown in Figure 2, nanoparticles C66-NPs exhibited low dark cytotoxicity in the concentration range of 0 to 10 μM, avoiding unnecessary cytotoxicity associated with photodynamic therapy. The nanoparticles successfully produced in situ with strong cytotoxicity under light conditions. 1 O2 / ·O2 - species, causing severe oxidative damage to cancer cells, with a half inhibitory concentration IC 50 In the range of 0.5-1 μmol / L, it indicates that nanoparticles C66-NPs have excellent therapeutic effects.

[0073] (2) Evaluation of intracellular reactive oxygen species production capacity

[0074] The intracellular ROS probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to characterize the ability of intracellular ROS production. Figure 8 As shown, after irradiation with 600-610nm LED light, strong green fluorescence was observed in the Hela cells treated with nanoparticles by CLSM, and the cell morphology changed significantly, indicating that C66 nanoparticles can effectively produce reactive oxygen under intracellular light.

[0075] (3) Evaluation of cell photodynamic therapy ability

[0076] Calcein-AM and propidium iodide (PI) were used to stain the cells to detect the effect of C66-NPs on tumor cells through oxidative damage. Calcein-AM can only stain living cells to produce green fluorescence, and PI, as a nuclear staining dye, only stains dead cells to produce red fluorescence. Fig. 9 As shown in the figure, after irradiation with 600-610nm LED light, Hela cells treated with nanoparticles showed almost no signal in the green channel and significant signal in the red channel, indicating that Hela cells were almost completely dead. In contrast, cells treated with C66-NPs showed strong fluorescence only in the green channel in the absence of light, indicating that cell death was negligible, indicating that the activity of tumor cells in the absence of light was good after the nanoparticles C66-NPs were internalized by cells (low dark toxicity) and that tumor cells were killed in large numbers under light conditions and the cell activity was very low (high phototoxicity).

Claims

1. A metal-organic cage nanoparticle, characterized in that: The nanoparticles are prepared by the following method: (1) Synthesis of TNPP using p-nitrobenzaldehyde and pyrrole as raw materials 0.0363 mol of p-nitrobenzaldehyde and 0.0635 mol of acetic anhydride were dissolved in 150 mL of propionic acid, and the solution was heated to reflux; then 0.036 mol of pyrrole was slowly added dropwise; after reflux for 30 minutes, the solid was collected by filtration, washed with water and methanol, and then dried under vacuum; the obtained solid was dissolved in 40 mL of pyridine and refluxed for 1 hour; it was cooled to room temperature and refrigerated overnight; the product was filtered and washed with methanol and acetone to obtain purple-red solid TNPP; (2) Synthesis of TAPP using TNPP and stannous chloride as raw materials 1.038 mmol of TNPP was dissolved in 56 mL of hydrochloric acid and heated to 80°C. Then 15.94 mmol of SnCl2·2H2O was dissolved in 6 mL of hydrochloric acid, added to the reactor, and kept at 80°C for 6 hours. After the reaction was completed, it was cooled to room temperature, and then the pH was adjusted to 9 in an ice water bath containing ammonia. The solid product was collected to obtain bright purple crystals of TAPP; (3) Synthesis of Zn-TAPP using TAPP and zinc acetate as raw materials 0.3 mmol of TAPP was dissolved in 37 mL of DMF, and 4 mmol of Zn(OAc)2·2H2O was added to the reaction system, and then the mixture was heated and refluxed for 12 h. After the reaction was completed, the system was cooled to room temperature, and then added to 250 mL of water, filtered and washed with water and ethanol to obtain dark green solid Zn-TAPP. (4) Synthesis of metal-organic cage C66 using Zn-TAPP, 2-pyridinecarboxaldehyde and zinc trifluoromethanesulfonate as raw materials Zn-TAPP 0.05 mmol, 2-pyridinecarboxaldehyde 0.2 mmol, Zn(CF3SO3)2 0.07 mmol and anhydrous DMF 6 mL were added to a Schlenk flask, the solution was degassed three times under vacuum, and then the mixture was reacted at 80°C for 24 hours; the product was cooled to room temperature, the supernatant was centrifuged and filtered to remove the solid; the clear solution was added dropwise to ether, the lower solid layer was centrifuged and washed with additional ether to obtain a brown product metal-organic cage C66; (5) Synthesis of nanoparticles using metal-organic cage C66 and amphiphilic molecule MPEG-DSPE as raw materials 2 mg of metal-organic cage C66 was dissolved in 2 mL of acetone, and then 10 mg of the amphiphilic molecule MPEG-2000-DSPE was dissolved in 10 mL of ultrapure water. The acetone solution of the metal-organic cage C66 was injected into the ultrapure aqueous solution of the amphiphilic molecule under stirring. After ultrasonic treatment for one minute, the acetone was evaporated by stirring in the dark to obtain a nanoparticle solution of the metal-organic cage.

2. The use of nanoparticles according to claim 1, characterized in that: The nanoparticles are used for preparing reagents for cell photodynamic therapy.

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