A bismuth chloro-cluster-cucurbit[6]uril-based supramolecular framework material, a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medical materials and radiation medicine, and particularly relates to a bismuth-chloride cluster-cucurbit[6]uril-based supramolecular framework material and a preparation method and application thereof. 10 ] 4‑ Anion clusters) as a building unit and cucurbit[6]uril (CB[6]) are fused and co-constructed through host-guest interaction to prepare a supramolecular framework material. The structure and physicochemical properties of the compound are determined through single crystal structure testing and various spectroscopic characterizations, and the application of the compound as a radiotherapy sensitizer is studied. The results of in-vitro and in-vivo biological experiments prove that the material can be used for inhibiting the growth of various cancer cells by significantly sensitizing radiotherapy.

Description

Technical Field

[0001] This invention belongs to the field of interdisciplinary technology of biomedical materials and radiomedicine, specifically involving a bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material and its preparation method and application. Background Technology

[0002] In the field of anti-tumor research, radiotherapy, as a local treatment method, is an important approach to treating various cancers. Advances in nanomedicine have spurred the development of numerous radiosensitization techniques. Research has found that nanoparticles can promote the uptake of intracellular radiosensitizers and cell cycle arrest. Bismuth, as a low-toxicity and high-atomic-number metallic element, exhibits characteristics such as a significantly high X-ray attenuation coefficient, near-infrared absorbance, efficient photothermal conversion, and prolonged cyclic half-life in bismuth-containing nanomaterials. Therefore, bismuth-containing nanomaterials have immense application value in the field of radiosensitization and anti-tumor therapy.

[0003] Metal clusters are aggregates of atoms formed by two or more metal atoms linked by metal-metal bonds or ligand bridging. Metal halide clusters, in particular, possess unique electronic structures and tunable band structures, along with nanoscale size and structural diversity, making them widely applicable in nanotechnology, catalysis, biomedicine, and energy. Meanwhile, as a special class of organic functional molecules, organic macrocycles have been widely used in supramolecular assembly due to their porous structure and host-guest recognition properties. Cucurbita, with its polar ports and hydrophobic cavities, provides supramolecular framework materials with advantages such as high porosity and biocompatibility. In recent years, the co-construction of framework materials with cucurbita and third-party components has provided new opportunities for the development of this field. Summary of the Invention

[0004] The present invention aims to provide a novel supramolecular framework material based on bismuth chloride clusters and cucurbita[6]urea for radiotherapy sensitization, as well as its preparation method and application.

[0005] In a first aspect, the present invention provides a bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material, the building unit of which is mainly cucurbit[6]urea (CB[6]), and [Bi2Cl] 10 ] 4- Anion clusters are constructed through host-guest interactions, with the anion clusters serving as the guests.

[0006] Furthermore, [Bi2Cl 10 ] 4- The stoichiometric ratio of cucurbita[6]urea is 2:3.

[0007] Furthermore, in the supramolecular framework material, the cation is an alkali metal cation.

[0008] Furthermore, the alkali metal cation is lithium or potassium ion.

[0009] Furthermore, the [Bi2Cl] 10 ] 4− In the anion cluster, bismuth atoms all exhibit a six-coordinate octahedral configuration, with two octahedrons connected by sharing an edge.

[0010] Furthermore, the supramolecular framework material belongs to the monoclinic crystal system, with space group C2 / m and unit cell parameters of: a=24.62 Å, b=29.08 Å, c=15.94 Å, α=90°, β=105.53°, γ=90°.

[0011] Furthermore, the supramolecular framework material belongs to the orthorhombic crystal system with space group Cmcm and cell parameters of a=28.62 Å, b=24.64 Å, c=30.63 Å, α=90°, β=90°, γ=90°.

[0012] In a second aspect, the present invention provides a formulation comprising a bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material, as described herein, dispersed in water after ultrasonic treatment.

[0013] In a third aspect, the present invention provides a method for preparing a bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material as described herein, comprising the following steps:

[0014] (1) Dissolve cucurbit[6]urea, trivalent bismuth salt, and alkali metal chloride in water to form a mixed solution;

[0015] (2) Add an organic structure directing agent to the mixed solution, mix well, and then carry out a hydrothermal reaction;

[0016] (3) After the reaction is completed, the reaction product is collected, washed and dried to obtain the bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material.

[0017] Furthermore, the organic structure directing agent is selected from 4,4'-bipyridine, imidazole-4,5-dicarboxylic acid, cysteine, 4-aminopyridine, tetrabutylammonium bromide, 4-formylphenylboronic acid, or 4,4-diphenyl ether dicarboxylic acid.

[0018] Furthermore, the trivalent bismuth salt is bismuth nitrate or its hydrate.

[0019] Furthermore, the alkali metal chloride is lithium chloride or potassium chloride.

[0020] Furthermore, in step (1), the molar ratio of cucurbit[6]urea to bismuth ions is 3:4-5.

[0021] Furthermore, in step (1), the alkali metal chloride is added in excess of bismuth ions in a stoichiometric amount. For example, the molar amount of alkali metal chloride is at least five times the molar amount of bismuth ions.

[0022] Furthermore, in step (2), the organic structure-directing agent can act as a structure-directing agent, regulating the relationship between CB[6] and [Bi2Cl] in the supramolecular framework. 10 ] 4- The stacking pattern can be used to regulate the size and particle size of the derived nanomaterials. The amount of organic structure directing agent added is not limited; for example, the molar ratio of the organic structure directing agent to CB[6] can be 1-2:1.

[0023] Furthermore, the hydrothermal reaction temperature is 120-180℃, and the reaction time is 2-5 days.

[0024] In a fourth aspect, the present invention provides the use of bismuth chloride cluster-cucurbit[6]urea supramolecular framework materials, or formulations containing the thereof, as described herein, in the preparation of medicaments for radiosensitizing and treating tumors.

[0025] Beneficial effects of the present invention

[0026] This invention selects metal halide clusters (bismuth chloride clusters) as building units and fuses them with cucurbita[6]urea (CB[6]) to prepare supramolecular framework materials. The structure and physicochemical properties of the compounds were determined by single-crystal structure testing and various spectroscopic characterizations, and their application as radiosensitizers was studied. In vivo and in vitro biological experiments confirmed that this type of material can significantly sensitize radiotherapy to inhibit the growth of various cancer cells. Attached Figure Description

[0027] Figure 1 It shows a) Bi2Cl 10 -1 Schematic diagram of the ball-and-stick structure of the unit cell in Figure 1; b) Bi2Cl 10 -2 Schematic diagram of the ball-and-stick structure of the unit cell; c) Bi2Cl 10 -1 Molecular packing diagram from the c-axis perspective; d) Bi2Cl 10 -2 Molecular packing diagram from the c-axis perspective; e) Bi2Cl 10 -1 Molecular packing diagram from the b-axis perspective; f) Bi2Cl 10 -2 Molecular packing diagram from the b-axis perspective.

[0028] Figure 2 Bi2Cl was shown 10 -1 (a) and Bi2Cl 10 XPS plot of -2 (b).

[0029] Figure 3 Bi2Cl was shown 10 -1 and Bi2Cl 10 Infrared spectrum of -2.

[0030] Figure 4 Bi2Cl was shown 10 -1 (a) and Bi2Cl 10 -2 (b) Thermogravimetric analysis diagram.

[0031] Figure 5 Bi2Cl was shown 10 -1 (a) and Bi2Cl 10 -2 (b) PXRD pattern.

[0032] Figure 6 This shows a) different concentrations of Bi₂Cl 10 -1 / 2 material effect on CT26 cell viability; b) Bi2Cl 10 -1 / 2 material combined with radiotherapy flow cytometry apoptosis detection; c) Flow cytometry apoptosis statistics of the four groups in b.

[0033] Figure 7 It shows a) Bi2Cl 10 a) Flow cytometry detection of apoptosis at -1; b) Bi2Cl 10 -1 flow cytometry apoptosis statistics; c) Bi2Cl 10 -2 flow cytometry apoptosis detection; d) Bi2Cl 10 -2 Flow cytometry apoptosis statistics (all concentrations were 10 μg / mL).

[0034] Figure 8 Bi2Cl was shown 10 -1 and Bi2Cl 10 -2 inhibits MOC2 cell migration in a 48-h scratch assay.

[0035] Figure 9 It shows a) Bi2Cl 10 -1 / 2 Transwell assay to inhibit MOC2 cell migration; b) Statistical graph of the number of migrating MOC2 cells per unit field of view; c) Bi2Cl 10 -1 / 2 Colony formation experiment to inhibit MOC2 proliferation; d) Statistical graph of MOC2 clone cell count per unit field of view.

[0036] Figure 10 Bi2Cl was shown 10 -1 and Bi2Cl 10 -2 DNA damage experiment on MOC2 cells.

[0037] Figure 11 Bi2Cl was shown 10 -1 and Bi2Cl 10 -2 DNA damage experiments on MOC2 cells that have established radiotherapy tolerance (cumulative irradiation dose of 70 Gy).

[0038] Figure 12 The Calcein-AM / PI staining pattern is shown (Calcein-AM staining for live cells; PI staining for dead cells).

[0039] Figure 13 This shows a) a schematic diagram of the in vivo experimental process of CDX-MOC2; b) Bi2Cl 10 -2 Comparison of tumor volume in mice after combined radiotherapy; c) Bi2Cl 10 -2 Comparison of mouse weight after combined radiotherapy; d) Comparison of average mouse weight over time after treatment.

[0040] Figure 14 Showing information about Bi2Cl 10 A staining comparison image of a series of materials combined with radiotherapy.

[0041] Figure 15 It shows that after Bi2Cl 10 HE staining control of major internal organs in mice transplanted with MOC2 cancer cells after combined radiotherapy -2.

[0042] Figure 16 This shows a) a schematic diagram of the in vivo experimental process of CDX-CAL33; b) Bi2Cl 10 -2 Comparison of tumor quality in mice after combined radiotherapy; c) Bi2Cl 10 -2 Comparison of tumor volume in mice after combined radiotherapy

[0043] Figure 17 It shows that after Bi2Cl 10 HE staining control of major internal organs in mice transplanted with CAL33 cancer cells after combined radiotherapy -2.

[0044] Figure 18 It shows a) Bi2Cl 10 -1 Particle size distribution of the material; b) Bi2Cl 10 -2 Particle size distribution diagram of the material.

[0045] Figure 19 It shows a) Bi2Cl 10 -1. Overall morphology of the material under SEM; b) Bi2Cl 10-2. Overall morphology of the material under SEM; c) Bi2Cl 10 -1 Individual particle morphology images of the material under SEM; d) Bi2Cl 10 -2 Individual particle morphology images of the material under SEM; eh) Bi2Cl 10 -1 Elemental distribution of the material under EDX; il) Bi2Cl 10 -2 Elemental distribution of the material under EDX. Detailed Implementation

[0046] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0047] Example 1: Li4[(Bi2Cl 10 (CB[6]) 1.5 ]·25H2O (Bi2Cl 10 Preparation of -1)

[0048] CB[6] (0.030 g, 0.030 mmol) and Bi(NO3)3·5H2O (0.020 g, 0.041 mmol) were mixed in LiCl aqueous solution (5 mL, 1 M). After mixing evenly, imidazole-4,5-dicarboxylic acid (0.005 g, 0.032 mmol) was added and stirred vigorously at room temperature for 1 h. The mixture was transferred to a Teflon-lined container and sealed in a stainless steel container, kept under autogenous pressure at 150 °C for 3 days, and then cooled to room temperature at a rate of 10 °C per hour. Colorless blocky crystals were collected, washed with ultrapure water and dried to obtain bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material Li4[(Bi2Cl 10 (CB[6]) 1.5 ·25H2O, named Bi2Cl 10 -1. Yield: 0.030 g (55% yield calculated with CB[6] as the limiting reactant). Elemental analysis theoretical values ​​(%): Li, 1.01; Bi, 15.22; C, 23.62; N, 18.36; Elemental analysis experimental values ​​(%): Li, 0.97; Bi, 15.45; C, 23.50; N, 17.89. Infrared spectrum (KBr pellet / cm) −1): 3483 (s), 1740 (s), 1480 (s), 1377 (m), 1325 (s), 1236 (s), 1192 (s), 986 (w), 965 (s), 820 (m), 802 (s), 761 (w), 676 (w), 632 (w).

[0049] Example 2: Li4[(Bi2Cl 10 (CB[6]) 1.5 ]·41H2O (Bi2Cl 10 Preparation of -2)

[0050] CB[6] (0.030 g, 0.030 mmol) and Bi(NO3)3·5H2O (0.020 g, 0.041 mmol) were mixed in LiCl aqueous solution (5 mL, 1 M). After mixing, 4,4'-bipyridine (0.005 g, 0.032 mmol) was added and stirred vigorously at room temperature for 1 h. The mixture was transferred to a Teflon-lined container and sealed in a stainless steel container. It was kept under autogenous pressure at 150 °C for 3 days and then cooled to room temperature at a rate of 10 °C per hour. Colorless blocky crystals were collected, washed with ultrapure water and dried to obtain bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material Li4[(Bi2Cl 10 (CB[6]) 1.5 ]·41H2O, named Bi2Cl 10 -2. Yield: 0.037 g (61% yield calculated with CB[6] as the limiting reactant). Elemental analysis theoretical values ​​(%): Li, 0.91; Bi, 13.78; C, 21.38; N, 16.62; Elemental analysis experimental values ​​(%): Li, 0.80; Bi, 13.99; C, 21.73; N, 16.84. Infrared spectrum (KBr pellet / cm) −1 ): 3483 (s), 1736 (s), 1478 (s), 1376 (m), 1326 (s), 1236 (s), 1192 (s), 985 (w), 965 (s), 820 (m), 802 (s), 761 (w), 676 (w), 633 (w).

[0051] Examples 3-7

[0052] Bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework materials were prepared by replacing imidazole-4,5-dicarboxylic acid in Example 1 with cysteine, 4-aminopyridine, tetrabutylammonium bromide, 4-formylphenylboronic acid and 4,4-diphenyl ether dicarboxylic acid, respectively, using the same method as in Example 1.

[0053] Comparative Example

[0054] Except for removing imidazole-4,5-dicarboxylic acid from the reaction system, bismuth chloride cluster-cucurbita[6]urea supramolecular framework materials were prepared using the same method as in Example 1. However, it was found that the Bi2Cl obtained in Example 1 could not be obtained. 10 -1.

[0055] Test Example 1: Crystal Structure and Characterization

[0056] 1. Crystal structure

[0057] For compound Bi2Cl 10 -1 and Bi2Cl 10 X-ray single-crystal diffraction tests were performed on the sample, and the crystallographic data are listed in Table 1 below.

[0058] Table 1: Bi2Cl 10 -1 and Bi2Cl 10 Crystallographic data and structural correction parameters of -2

[0059] compound <![CDATA[Bi2Cl 10 -1]]> <![CDATA[Bi2Cl 10 -2]]> Empirical molecular formula <![CDATA[Li4Bi2C 54 N 36 O 43 H 104 Cl 10 ]]> <![CDATA[Li4Bi2C 54 N 36 O 59 H 136 Cl 10 ]]> Molecular weight, g / mol 2745.86 3031.28 Crystal system monoclinic orthogonal Space Group C2 / m Cmcm a, Å 24.6214(10) 28.6233(13) b, Å 29.0753(11) 24.6397(10) c, Å 15.9370(6) 30.6300(12) α, ° 90 90 β, ° 105.531(2) 90 γ, ° 90 90 Unit cell volume, ų 10992.3(7) 21602.4(16) Z 94 186 Calculate density, g / cm3 1.459 1.479 Absorption coefficient, mm⁻¹ 3.507 3.600 F(000) 4580.0 9211.0 Data collection θ angle range, 4.432 to 56.736 4.362 to 56.69 Completeness under Qmax 99.6 % 99.9 % Index range -25 ≤ h ≤ 32-38 ≤ k ≤ 35-21 ≤ l ≤ 21 -33 ≤ h ≤ 38-32 ≤ k ≤ 31-40 ≤ l ≤ 33 Number of diffraction points collected 60919 107525 Independent diffraction point count 13969 13939 R(int) 0.0517 0.0806 Absorption correction Semi-empirical method based on equivalent value Semi-empirical method based on equivalent value Data / Constraints / Parameters 13969 / 0 / 551 13939 / 0 / 568 Goodness of fit (based on F²) 1.076 1.063 R1,[a] wR2[b](I>2σ(I)) R1 = 0.0710w R2 = 0.2069 R1 = 0.0620w R2 = 0.1496 R1,[a] wR2[b](All data) R1 = 0.0782w R2 = 0.2115 R1 = 0.0802w R2 = 0.1580 Maximum residual electron density peaks and valleys, e / Å3 6.32 and -5.09 3.46 and -2.08

[0060] [a] R1 = Σ||F o | – |F c || / Σ|F o | [b] wR2 = [Σw(F o 2 – F c 2 ) 2 / Σw(F o 2 ) 2 ] 1 / 2 .

[0061] From the X-ray single-crystal diffraction data in Table 1, it can be seen that Bi₂Cl 10 -1 belongs to the monoclinic crystal system, space group C2 / m. For example... Figure 1 As shown in a, the supramolecular framework is composed of [Bi2Cl 10 ] 4− It consists of two main components: anionic clusters and CB[6]. [Bi2Cl] 10 ] 4− In the anionic cluster, bismuth atoms all exhibit a six-coordinated octahedral configuration, with two octahedrons connected by sharing edges. In Bi₂Cl… 10 In the -1 framework, [Bi2Cl 10 ] 4−The stoichiometric ratio of CB[6] to CB[6] is 2:3, and six CB[6] molecules are distributed around each anion cluster. [Bi2Cl 10 ] 4− All are located on the outer periphery of the CB[6] equator and exist stably with each other through supramolecular forces such as electrostatic interactions. Figure 1 c and Figure 1 e). It should be noted that the added organic ligand imidazole-4,5-dicarboxylic acid did not appear in the final crystal structure, but removing it from the reaction system failed to produce Bi₂Cl. 10 -1. It is speculated that imidazole-4,5-dicarboxylic acid, in addition to acting as a structure directing agent in the reaction system, can also participate in the regulation of experimental variables such as pH.

[0062] Keep Bi2Cl 10 The hydrothermal synthesis conditions remain basically unchanged; only the external ligand imidazole-4,5-dicarboxylic acid in the reaction raw materials is replaced with 4,4'-bipyridine to obtain orthorhombic Bi₂Cl₃. 10 -2. With Bi2Cl 10 Compared to -1, the supramolecular framework components and their proportions remain unchanged ( Figure 1 b), due to unit cell differences, Bi2Cl 10 -2 is more molecularly packed than Bi2Cl 10 -1 is more compact ( Figure 1 d and Figure 1 f). Based on this, organic molecules such as cysteine, 4-aminopyridine, tetrabutylammonium bromide, 4-formylphenylboronic acid, and 4,4-diphenyl ether dicarboxylic acid were selected as external ligands, and the resulting crystals were all Bi₂Cl. 10 -1. Therefore, it can be inferred that the added organic ligands in CB[6] and [Bi2Cl]... 10 ] 4− The assembly process can affect the stacking pattern of both components.

[0063] 2. X-ray photoelectron spectroscopy

[0064] Bi₂Cl was detected by X-ray photoelectron spectroscopy. 10 -1, Bi2Cl 10 -2 and Bi2Cl 10 -3 Oxidation states of metallic bismuth atoms. For example... Figure 2 As shown, 164.8 eV and 159.5 eV ( Figure 2 a) and 164.8 and 159.5 eV ( Figure 2 b) Corresponding to Bi2Cl 10 -1 and Bi2Cl 10 -2 in Bi III The existence of.

[0065] 3. Infrared Spectroscopy

[0066] For Bi2Cl 10 -1 and Bi2Cl 10 -2. Because the main components are roughly the same, the infrared spectra of the compounds are also quite similar. (Using 1736, 1476, and 1325 cm⁻¹) –1 The peaks centered on the CB[6] are attributed to the vibrations of the C=O, C–H and C–N bonds, respectively. Figure 3 ), of which 987 cm –1 A new peak appeared on the left and right sides compared to CB[6], which may be due to the binding of [Bi2Cl] 10 ] 4– This is caused by the in-plane torsional vibration of the CB[6] octagonal heterocycle.

[0067] 4. Thermogravimetric analysis

[0068] The thermal stability of the two prepared crystal samples was investigated under a nitrogen atmosphere. Figure 4 The weight loss process of this series of framework materials is divided into two stages: the first stage is the removal of water of crystallization molecules, and the second stage is the thermal decomposition of organic components. Experimental data show that this type of supramolecular framework can maintain structural stability at least at 340-360 °C. [Bi2Cl 10 ] 4− The outer surface bond between Bi2Cl and CB[6] is strong and tight, thus giving the relevant skeleton excellent thermal stability. 10 -1, before 200 ℃, the sample lost 16.45% of its weight, compared to a theoretical value of 16.40%. Within the 350~585 ℃ range, the sample lost 61.94% of its weight, corresponding to the thermal decomposition of cucurbita within the supramolecular framework ( Figure 4 a); for Bi2Cl 10 The sample lost 24.32% of its weight before reaching -200 °C, compared to a theoretical value of 24.37%. Figure 4 (b) The above weight loss steps all correspond to the loss of water of crystallization molecules in the structure.

[0069] 5. X-ray powder diffraction pattern

[0070] like Figure 5 As shown, CB-Bi2Cl 10 The measured data and simulated data of the powder XRD patterns of the collected crystals showed good agreement, and the absence of extraneous impurity peaks indicated good phase purity of the crystal material. There were significant differences in the intensity of some measured diffraction peaks compared to the simulated data. This is because the collected solid samples were all single crystals, and the preferred orientation of the crystal samples caused variations in intensity.

[0071] Test Example 2: Study on Radiosensitizing and Anticancer Activity

[0072] 1. Biological experimental conditions and testing methods

[0073] (1) Tumor cell culture

[0074] Mouse colon cancer cells (CT26), mouse oral squamous cell carcinoma cells (MOC2), and human tongue squamous cell carcinoma cells (CAL-33) were selected for radiosensitization studies. These cells were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum (PBS), 1% penicillin, and 1% streptomycin.

[0075] (2) Material preparation and toxicity assessment

[0076] Collect Bi2Cl 10 -1 and Bi2Cl 10 -2 crystal materials were dissolved in water using a non-contact sonicator to prepare solutions with concentrations of 5 / 10 / 20 / 40 / 60 / 80 / 100 μg / mL. CT26 or MOC2 cells were seeded at 5000 cells / well in 96-well plates and incubated for 24 h in a 37 °C incubator containing 5% CO2. Then, different concentrations of Bi2Cl were used as solutions. 10 -1 and Bi2Cl 10 -2 treatment. After 48 h, the cell counting reagent (Cell Counting Kit 8) was added to the culture medium at a ratio of 1:10, and after incubation at a constant temperature for 2 h, the absorbance at 450 nm was measured.

[0077] (3) Flow cytometry apoptosis experiment

[0078] CT26 or MOC2 cells were seeded in 6-well plates and cultured at 37°C for 24 h under a constant temperature condition containing 5% CO2. Bi2Cl was then used to incubate the cells. 10 -1 and Bi2Cl 10 Cells were treated with 5 / 10 / 20 μg / mL samples and washed twice with PBS after 24 h. Flow cytometry analysis of apoptosis was performed after incubation in the dark for 10 min using the Annexin V-PE / 7-AAD Apoptosis Detection Kit (Vazyme, No. A213-02).

[0079] (4) Scratch test

[0080] Seed 500,000 MOC2 cells per well in a 6-well plate, ensuring uniform cell distribution. Incubate the plate at 37°C in a 5% CO2 incubator until a cell monolayer forms. Mark parallel lines on the bottom of the 6-well plate with a marker. Once the cells have reached confluence, use a 200 µL sterile pipette tip to make incisions along the marked lines, ensuring the incisions are perpendicular to the lines and clearly defined. Gently rinse the cells 2-3 times with PBS. Immediately image the incision area under a microscope (0 h time point). Continue imaging at 24 h, 48 h, and other time points, ensuring consistent magnification and exposure conditions for subsequent analysis.

[0081] (5) DNA damage experiment

[0082] DNA damage was detected using a DNA damage detection kit (γ-H2AX immunofluorescence assay). 5000 MOC2 cells / well were seeded in 96-well plates and incubated at 37 °C for 24 h in a 5% CO2 atmosphere. The cells were then inoculated with 10 μg / mL Bi2Cl. 10 -1 and Bi2Cl 10 -2 treatment. After 24 h of treatment, cells were washed with PBS, fixed for 30 min, incubated overnight at 4 °C with γ-H2AX rabbit monoclonal antibody, and then incubated at 25 °C for 1 h with Alexa Fluor 488-conjugated anti-rabbit secondary antibody. Cells were washed twice with PBS, stained with DAPI for 10 min, and imaged using a confocal microscope.

[0083] (6) Transwell experiment

[0084] Based on the scratch assay, the effect of the material on inhibiting MOC2 cell migration was further precisely evaluated using the Transwell assay. A suspension of 20,000 MOC2 cells / mL was added to a Transwell chamber, taking care to avoid air bubbles, and incubated in a 37°C incubator containing 5% CO2. Bi2Cl was used as the incubator. 10 -1 and Bi2Cl 10 After treatment with -2, the cells were incubated for another 24 hours, then fixed and stained, and imaged using a confocal microscope.

[0085] (7) Cloning experiment

[0086] MOC2 cells were seeded at 1000 cells / well in 6-well plates and cultured for 24 h in a 37 °C incubator containing 5% CO2. Then, the cells were treated with 10 μg / mL Bi2Cl... 10 -1 and Bi2Cl 10Cells were treated with -2 and washed twice with PBS after 24 h. They were then incubated with fresh DMEM medium until visible colonies formed. The colonies were fixed, stained with crystal violet, and observed under a microscope.

[0087] (8) Staining experiment

[0088] Staining experiments were performed using the Calcein / PI Cell Viability / Cytotoxicity Assay Kit. For staining with Bi₂Cl at a concentration of 10 μg / mL... 10 -1 and Bi2Cl 10 MOC2 cells treated with -2, with live cells stained with Calcein AM and dead cells stained with Propidium Iodide (PI).

[0089] (9) In vivo experiments in mice

[0090] The mice used in this study were BALB / c nude mice, employing a cell line-derived xenograft tumor (CDX) model. Immunodeficient nude mice aged 4–6 weeks were inoculated with MOC2 or CAL33 cells, and their body weight and condition were closely monitored after inoculation. After an appropriate number of days, they were administered Bi2Cl at a dose of 10 mpk. 10 The mice were treated with a series of materials combined with 6 Gy radiotherapy, and their body weight and condition were closely monitored. Throughout the process, the trends in tumor mass and volume were compared.

[0091] (10) Major organ staining experiments

[0092] After the in vivo CDX experiment, Bi2Cl was administered at a dose of 10 mpk. 10 The series of materials were combined with HE (hematoxylin-eosin) staining of the major organs of mice after 6 Gy radiotherapy to observe changes in organs such as heart, liver, spleen, lung, and kidney.

[0093] 2. Results of radiosensitization test

[0094] First, this invention explored the toxicity of two materials at different concentrations to CT26 cells using the Cell Counting Kit-8 experiment. For example... Figure 6 As shown in Figure a, at concentrations below 20 μg / mL, the selected materials exhibit relatively low cytotoxicity. A sample concentration of 5 μg / mL was selected for Bi₂Cl… 10 Preliminary flow cytometry apoptosis experiments were performed on a series of samples, with NC serving as the control group. The experimental results showed that Bi2Cl... 10 -1 and Bi2Cl 10-2 can increase the apoptosis rate, indicating that the material has the potential to enhance radiosensitization. Figure 6 (bc). Next, the anticancer activity of the material was evaluated by testing the apoptosis rate of mouse oral squamous cell carcinoma cells (MOC2). Flow cytometry apoptosis experiments were conducted on the two materials at concentrations of 10 μg / mL and 20 μg / mL, respectively. It was found that the sensitizing effect of both materials at a concentration of 10 μg / mL was superior to that at 20 μg / mL; therefore, a concentration of 10 μg / mL was selected for subsequent experiments. Regarding Bi2Cl... 10 -1, compared to the control group, under combined radiotherapy (6 Gy), its sensitization effect was 17.02% ( Figure 7 ab); for Bi2Cl 10 -2, its sensitizing effect can reach 28.66% ( Figure 7 cd).

[0095] A more intuitive scratch test shows that, under combined 6 Gy radiotherapy conditions, Bi2Cl... 10 -2 can significantly inhibit the migration of MOC2 cells ( Figure 8 The experimental results for both materials were consistent with the flow cytometry data for apoptosis. (Bi2Cl) 10 -2 has a significantly better anti-cancer effect than Bi2Cl. 10 -1.

[0096] To further evaluate the inhibitory effect of the material on MOC2 cell migration, a Transwell assay was performed. Figure 9 As can be seen from ab, without combined radiotherapy, both materials have limited inhibitory effects on MOC2 cell migration. However, the use of Bi2Cl... 10 MOC2 cells treated with Bi2Cl2 combined with radiotherapy showed the lowest number of cell migrations under a microscope among the six groups, indicating that Bi2Cl2... 10 Bi2Cl significantly inhibited the migration of MOC2 cells. 10 The combined radiotherapy with -1 is less effective than Bi2Cl. 10 -2, the experimental result is consistent with the previous analysis.

[0097] Similarly, in clonogenic experiments, neither material showed significant effect in inhibiting MOC2 cell proliferation without combined radiotherapy. However, the use of Bi2Cl... 10 MOC2 cells treated with combined radiotherapy showed a significantly lower number of bacterial colonies compared to the control group. Figure 9 cd), indicating that Bi2Cl 10 -2 combined with radiotherapy has a strong inhibitory effect on the proliferation of MOC2 cells.

[0098] Based on the above experiments, this invention discovered a method based on Bi2Cl 10 The nanomaterials prepared by -1 / 2 showed excellent effects in combined radiotherapy antitumor experiments. To confirm the site of action of this method on cancer cell apoptosis, related DNA damage experiments and staining experiments were conducted. First, from Bi2Cl... 10 -1 and Bi2Cl 10 The DNA damage experiment on MOC2 cells showed that, under combined radiotherapy at an intensity of 6 Gy, the γ-H2AX content in MOC2 cells treated with both materials was high, and Bi2Cl... 10 -2 is higher than Bi2Cl 10 -1 ( Figure 10 ), indicating Bi2Cl 10 -1 / 2 enhanced the DNA damage effect on MOC2 cells during combined radiotherapy. Furthermore, this invention cultured radioresistant MOC2 cells with a cumulative radiotherapy intensity of 70 Gy and conducted DNA damage experiments; the results were consistent with those of normal MOC2 cells. Figure 11 This indicates that the material, when combined with radiotherapy, remains effective against resistant cells.

[0099] In the Calcein-AM / PI staining experiment, by Figure 12 It can be seen that live cells that show fluorescent staining after Calcein-AM staining indicate good intracellular esterase activity; dead cells with disrupted cell membrane integrity can be stained with PI and show fluorescent staining, indicating that Bi2Cl 10 -2 disrupted the cell membrane of MOC2 cells, and the difference in fluorescence staining can be clearly seen in the overlay image.

[0100] After evaluating Bi2Cl 10 Following the in vitro anticancer activity assay of -1 / 2 on MOC2 cells, this invention was then subjected to in vivo experiments on mice. In the mouse in vivo experiments, all experimental groups received a 10 mpk dose combined with 6 Gy of radiotherapy. For mice transplanted with MOC2 cancer cells, this invention administered Bi2Cl on day seven. 10 -1 and Bi2Cl 10 -2 combined radiotherapy treatment ( Figure 13 a) Subsequently, the present invention analyzed tumors in mice, and it can be seen that Bi2Cl 10 The tumor volume of mice treated with -2 was ( Figure 13 b) and tumor quality ( Figure 13 c) All were at the lowest level among all groups, indicating that Bi2Cl 10 -2 also showed good anti-cancer effects in vivo. The weight of mice in each group remained stable after treatment. Figure 13d).

[0101] This invention performed staining analysis on the major organ tissues of mice undergoing in vivo radiotherapy. For example... Figure 14 As shown, the first row of images (HE) indicates that the tissue morphology is well preserved; in the second row of images (Ki67), Bi2Cl 10 -2 The combined radiotherapy group showed the lowest number of staining cells, indicating the weakest proliferation; in the third row of images (TUNEL), Bi2Cl 10 The group receiving combined radiotherapy with Bi2Cl showed the highest number of fluorescent stainings, indicating the highest number of apoptosis, which also corroborates the presence of Bi2Cl. 10 The combined radiotherapy group (-2) showed the best anti-cancer effect.

[0102] This invention further relates to Bi2Cl 10 HE staining was performed on the major organs (heart, liver, spleen, lung, and kidney) of mice in the combined radiotherapy group -2. It can be seen that the combined radiotherapy did not cause significant damage to the major organs of the mice. Figure 15 ).

[0103] Furthermore, based on in vivo experiments using transplanted MOC2 cancer cells, this invention explores the effects of Bi2Cl... 10 -2 The effect of Bi2Cl on the universality of anti-cancer activity. This invention involved inoculating mice with CAL33 cancer cells and administering Bi2Cl to the mice on day 22. 10 -2 combined radiotherapy ( Figure 16 a) The treatment dose and radiation intensity were consistent with those observed in in vivo studies of MOC2. The study found that Bi2Cl... 10 Tumor weight in mice in the combined radiotherapy group (-2) Figure 16 b) and tumor volume ( Figure 16 c) was also at the lowest level within the group, and showed no significant damage to the major organs of the mice. Figure 17 The above in vivo experiments demonstrate that Bi2Cl 10 -2 The universality of radiosensitization.

[0104] Through the above radiosensitization biological experiments, it was found that Bi2Cl 10 -2 always performs better than Bi2Cl 10 -1, which aroused great research interest in the inventors. Both materials are composed of CB[6] and {Bi2Cl 10} constitutes, from Figure 1 As can be seen, the overall packing of the two framework materials is quite similar. Meanwhile, ICP measurements showed no significant difference in the bismuth content between the two materials. Therefore, this invention focuses on the particle size and morphology of the two materials after ultrasonication.

[0105] This invention selects aqueous solutions of two materials, each with a concentration of 10 μg / mL, and measures the Bi₂Cl concentration using dynamic light scattering. 10 The hydrated particle size of the material is 742.4 nm, Bi₂Cl 10 The hydrated particle size of the material is 582.8 nm, indicating that Bi₂Cl 10 -2 The hydrated particle size of the material is smaller than that of Bi2Cl 10 -1 material ( Figure 18 ab).

[0106] To further confirm the particle size difference between the two materials, aqueous solutions of both materials at a concentration of 10 μg / mL were dropped onto the sample stage. After sample preparation via evaporation, the morphology and size differences were observed under a scanning electron microscope. SEM images showed that the particles of both materials were uniformly distributed at the microscale. Figure 19 ab), Bi2Cl 10 The particle diameter of the material under SEM was approximately 350 nm. (Bi₂Cl) 10 The particle diameter of the material under SEM is approximately 200 nm. Figure 19 The discrepancy between the two materials (cd) further corroborates the difference in particle size. It is speculated that the difference in radiosensitization effects between the two materials may be related to the different particle sizes after ultrasound treatment. Furthermore, the elemental distribution map shows that the overall framework of the nanomaterials prepared by the ultrasound method is completely preserved. Figure 19 el).

[0107] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material, characterized in that, The building blocks of the supramolecular framework material are mainly cucurbit[6]urea (CB[6]), with [Bi2Cl] as the main building block. 10 ] 4- Anion clusters are constructed through host-guest interactions, with the anion clusters serving as guests. Among them, [Bi2Cl 10 ] 4- The stoichiometric ratio of cucurbita[6]urea is 2:3, and the cations in the supramolecular framework material are alkali metal cations; The supramolecular framework material belongs to the monoclinic crystal system and has a space group of [space group number missing]. C 2 / m The unit cell parameters are: a = 24.62 Å, b = 29.08 Å, c = 15.94 Å, α = 90°, β = 105.53°, γ = 90°, or The supramolecular framework material belongs to an orthorhombic crystal system with space group [space group number missing]. Cmcm The unit cell parameters are: a=28.62 Å, b=24.64 Å, c=30.63 Å, α=90°, β=90°, γ=90°.

2. The bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material according to claim 1, characterized in that, The alkali metal cation is lithium or potassium ion; The [Bi2Cl 10 ] 4− The bismuth atoms in the anion cluster all exhibit a six-coordinate octahedral configuration, with two octahedrons connected by sharing an edge.

3. A formulation, characterized in that, The formulation comprises a bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material dispersed in water after being ultrasonically treated.

4. A method for preparing a bismuth chloride cluster-cucurbit[6]urea-based supramolecular framework material according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Dissolve cucurbit[6]urea, trivalent bismuth salt, and alkali metal chloride in water to form a mixed solution; (2) Add an organic structure directing agent to the mixed solution, mix well, and then carry out a hydrothermal reaction; (3) After the reaction is completed, the reaction product is collected, washed and dried to obtain the bismuth chloride cluster-cucurbit[6]urea supramolecular framework material; The organic structure directing agent is selected from 4,4'-bipyridine, imidazole-4,5-dicarboxylic acid, cysteine, 4-aminopyridine, tetrabutylammonium bromide, 4-formylphenylboronic acid, or 4,4-diphenyl ether dicarboxylic acid.

5. The preparation method according to claim 4, characterized in that, The trivalent bismuth salt is bismuth nitrate or its hydrate.

6. The preparation method according to claim 4, characterized in that, The alkali metal chloride is lithium chloride or potassium chloride.

7. The preparation method according to claim 4, characterized in that, The molar ratio of cucurbit[6]urea to bismuth ions is 3:4-5.

8. The preparation method according to claim 4, characterized in that, The hydrothermal reaction is carried out at a temperature of 120-180℃ for 2-5 days.

9. The use of the bismuth chloride cluster-cucurbit[6]urea supramolecular framework material according to any one of claims 1-2 or the formulation according to claim 3 in the preparation of a drug for radiosensitizing and treating tumors.

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