Tetraphenylethylene-based molecular cage as well as preparation method and application thereof

Abstract

The invention discloses a tetraphenylethylene-based molecular cage as well as a preparation method and application thereof, 3, 5-TPE is taken as a carboxylic acid bridging ligand, and is subjected to self-assembly reaction with Co < 2 + > and sulfone bridged calixarene [4] to obtain a coordination molecular cage (CoTPE) with a plurality of cavities. The molecular cage provided by the invention has a specific cavity and skeleton structure, a constructed coordination molecule container is used as a carrier, biomedical applications such as loading of drug molecules and pH responsive release can be realized, and in-vivo and in-vitro experiments prove that the molecular cage has a better tumor inhibition effect compared with free sorafenib.

Description

Technical Field

[0001] This invention relates to the field of molecular cage technology, and in particular to a tetraphenylethylene molecular cage, its preparation method, and its application. Background Technology

[0002] Sorafenib (SOR) is an orally administered small-molecule kinase inhibitor for the treatment of hepatocellular carcinoma (HCC), exhibiting potent anti-tumor and anti-angiogenic effects. However, the presence of numerous hydrophobic groups such as pyridine and halobenzene rings in its molecular structure results in poor water solubility, significantly limiting its clinical application. This is due to systemic skin problems, diarrhea, and hypertension caused by non-specific uptake by systemic tissues; and low tumor absorption efficiency resulting from rapid clearance and metabolism. Therefore, to address these issues and broaden the clinical application of sorafenib, there is an urgent need to develop a novel material loaded with sorafenib that can target and deliver it to tumor tissues and enhance its anti-tumor effect.

[0003] Coordination molecular containers (MOSCs) constructed from metal coordination bonds possess unique cavity structures that can selectively contain guest molecules of specific sizes, configurations, and functional groups to achieve the enrichment of specific compounds. The design and construction of MOSCs fully utilize the diversity of carboxylic acid ligands, the particle size advantages of mesoporous materials, and their unique functional cavities, achieving a combination of drug co-loading, combination therapy, and tumor suppression. Currently, there is little or no research combining molecular cages with sorafenib. Summary of the Invention

[0004] Therefore, based on the above background, this invention provides a tetraphenylethylene molecular cage, its preparation method and application, and develops a novel drug carrier that can load sorafenib, which not only broadens the clinical application of sorafenib, but also provides new ideas for supramolecular materials in the fields of biology and medicine.

[0005] The technical solution provided by this invention is as follows:

[0006] The application of 3,5-tetraphenyldicarboxylic acid ligand in the preparation of drug carriers, wherein the molecular structure of the 3,5-tetraphenyldicarboxylic acid ligand is shown in formula (1):

[0007]

[0008] Based on the same inventive concept, the present invention also provides a tetraphenylethylene molecular cage, the general structural formula of which is shown in the following formula (2):

[0009] {[M4(μ4-H2O)(X)]4Y8}(2);

[0010] in,

[0011] M is selected from metallic elements such as magnesium, zinc, manganese, cobalt, nickel, copper, iron, or ruthenium;

[0012] μ4-H2O represents a 4-coordination mode formed by 4 M atoms with the O atoms in H2O;

[0013] X is a thiacalix[4] aromatic ligand, and its molecular structure is shown in formula (3):

[0014]

[0015] In formula (3), R1, R2, R3, and R4 may be the same or different, and are independently selected from one of hydrogen, alkyl, or aryl groups;

[0016] A1, A2, A3, and A4 may be the same or different, and are independently selected from one of -S-, -SO-, or -SO2-.

[0017] Y is a dicarboxylic acid ligand, and its molecular structure is shown in formula (1):

[0018]

[0019] Furthermore, R1, R2, R3, and R4 in formula (3) are selected from one of tert-butyl, tert-octyl, and phenyl, respectively.

[0020] Furthermore, A1, A2, A3, and A4 in equation (3) are all -SO2-.

[0021] Furthermore, the general structural formula of the tetraphenylethylene molecular cage is {[Co4(μ4-H2O)(TBSC)]4[TPE]8}, where X is a 5,11,17,23-tetratert-butyl-sulfonyl bridged calix[4] ligand and Y is a 3,5-tetraphenyldicarboxylic acid ligand.

[0022] Based on the same inventive concept, the present invention also provides a method for preparing a tetraphenylethylene molecular cage, comprising the following steps: performing a self-assembly reaction of H4X, H2Y and M metal salts in an organic solvent to obtain the cage, wherein X, Y and M are as defined above.

[0023] Further, the molar ratio of X:Y:M is (0.9-1.1):(1.8-4.5):(4.0-7.0). Preferably, the molar ratio of X:Y:M is 1:3:5.

[0024] Furthermore, the M metal salt is a nitrate or a halide salt.

[0025] Further, the organic solvent is preferably an amide solvent or a mixed solvent of amide and alcohol, such as N,N'-dimethylformamide (DMF), a mixed solvent of N,N'-dimethylformamide and methanol; preferably, the organic solvent is a mixed solvent of N,N'-dimethylformamide and methanol, and more preferably, the organic solvent is a mixed solvent of N,N'-dimethylformamide and methanol in a volume ratio of 1:1.

[0026] Furthermore, the self-assembly reaction is carried out at a temperature of 80–140°C. Preferably, the self-assembly reaction is carried out at a temperature of 100–120°C.

[0027] Based on the same inventive concept, the present invention also provides applications of tetraphenylethylene molecular cages, wherein the application is at least one of the following:

[0028] ① Application as a drug carrier for loading drugs;

[0029] ② Application in the preparation of drugs for the treatment of hepatocellular carcinoma.

[0030] Furthermore, the drug loaded is sorafenib.

[0031] Based on the same inventive concept, the present invention also provides a drug for treating hepatocellular carcinoma, comprising a drug carrier and sorafenib loaded on the drug carrier, wherein the drug carrier is a tetraphenylethylene molecular cage of the present invention.

[0032] The beneficial effects of adopting the above technical solution are as follows:

[0033] This invention uses a tetraphenylethylene molecular cage to load sorafenib, which not only exhibits significant pH-responsive drug release but also significantly prolongs the residence time in tissues compared to free sorafenib, thus enhancing the duration of drug action. Animal experiments have verified that loading sorafenib with the tetraphenylethylene molecular cage of this invention can improve its inhibitory effect on tumor cells, resulting in better therapeutic efficacy for tumors.

[0034] This invention is the first to use molecular cages as drug carriers for loading sorafenib, which not only broadens the clinical application of sorafenib, but also provides new ideas for supramolecular materials in the fields of biology and medicine. Attached Figure Description

[0035] Appendix Figure 1 This is a schematic diagram of the crystal structure of the supramolecular cage complex obtained in Example 1.

[0036] Appendix Figure 2 This is a schematic diagram of the crystal structure of the supramolecular cage complex obtained in Example 1. Its inner cavity is filled with virtual yellow spheres, and its outer cavity is filled with virtual purple spheres.

[0037] Appendix Figure 3 The UV spectra of the sorafenib solution before and after immersion in the supramolecular cage complex crystals prepared in Example 2 are shown.

[0038] Appendix Figure 4 The in vitro release curves of sorafenib in PBS buffer solutions of different pH values ​​for the supramolecular cage complex crystals prepared in Example 2 are shown.

[0039] Appendix Figure 5 This is a graph showing the cytotoxicity evaluation of the supramolecular cage complex prepared in Example 2 against HepG2 cells.

[0040] Appendix Figure 6 This is a graph showing the cytotoxicity evaluation of the supramolecular cage complex prepared in Example 2 against LO2 cells.

[0041] Appendix Figure 7 The figure shows the uptake efficiency of NR@CoTPE by HepG2 cells.

[0042] Appendix Figure 8 The figure shows the results of flow cytometry analysis of the level of CoTPE uptake in cells.

[0043] Appendix Figure 9 The images show tumor photographs of mice in different experimental groups after different treatments according to this invention.

[0044] Appendix Figure 10 This is a tumor growth curve of tumor-bearing mice during different treatments in this invention.

[0045] Appendix Figure 11 The H&E staining results of HepG2-bearing mice in each treatment group and control group are shown in the figure.

[0046] Appendix Figure 12 Tunel staining results of HepG2 tumor-bearing mice in each treatment group and control group.

[0047] Appendix Figure 13 This is an H&E staining analysis of sections of the main organs (heart, liver, spleen, lungs, and kidneys) of tumor-bearing mice after different treatments, according to the present invention.

[0048] Appendix Figure 14 This is a graph showing the weight change of tumor-bearing mice during different treatments according to the present invention. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0051] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0052] Example 1: Preparation of tetraphenylethylene molecular cages (CoTPE)

[0053] The structural formula of the tetraphenylethylene molecular cage in this embodiment is shown below:

[0054] {[Co4(μ4-H2O)(TBSC)]4[DC]8}

[0055] TBSC represents 5,11,17,23-tetratert-butyl-sulfonyl-bridged calix[4] ligand, and DC represents 3,5-tetraphenyldicarboxylic acid ligand.

[0056] Figure 1 This is a schematic diagram of the tetraphenylethylene molecular cage crystal structure of this embodiment. Figure 1 It is known that a supramolecular cage complex molecule is obtained by bridging four tetranuclear metal calix[4] aromatic structural units with eight 3,5-tetraphenyldicarboxylic acid ligands, resulting in a supramolecular cage structure with a tetragonal box configuration. This supramolecular cage complex molecule simultaneously contains four outer cavities composed of calixarenes and an inner cavity composed of diacid bridging ligands and tetranuclear metal calixarenes. These inner and outer cavity structures can be used to effectively bind and contain guest molecules ( Figure 2 ).

[0057] The specific preparation steps are as follows:

[0058] Co(NO3)2·6H2O (72.75 mg, 0.25 mmol), 3,5-tetraphenyldicarboxylic acid (74.4 mg, 0.15 mmol), and H4TBSC (42.40 mg, 0.05 mmol) were dissolved in 3 mL of a mixture of N,N'-dimethylformamide (DMF) and 3 mL of methanol. The solution was heated to 100 °C at a rate of 0.5 °C / min and maintained at 100 °C for 24 h. The solution was then cooled at a rate of 0.2 °C / min to obtain red crystals. Yield: 85%.

[0059] The product was characterized by X-ray single-crystal diffraction, and the specific results are as follows:

[0060] Table 1. Crystallographic parameters of {[Co4(μ4-H2O)(TBSC)]4[DC]8}

[0061]

[0062] The above data shows that the target product {[Co4(μ4-H2O)(TBSC)]4[DC]8} was obtained in this embodiment (where TBSC represents 5,11,17,23-tetratert-butyl-sulfonyl-bridged calix[4] aryl ligand, and DC represents 3,5-tetraphenyldicarboxylic acid ligand).

[0063] Example 2: In this example, the tetraphenylethylene molecular cage prepared in Example 1 was used as a drug carrier to load sorafenib to prepare SOR@CoTPE.

[0064] The specific steps are as follows:

[0065] 10 mg of activated (ground and activated) CoTPE crystals were immersed in 10 mL of ethanol solution containing 10 mg of sorafenib and stirred at room temperature for 24 hours. The supernatant was then separated by centrifugation, and the spectra before and after centrifugation were acquired using UV-Vis spectroscopy. The results are as follows: Figure 3 As shown.

[0066] Depend on Figure 3 It can be seen that by comparing the absorbance at the maximum absorption peak of 264 nm, the amount of sorafenib adsorbed by CoTPE is approximately 5.02 mol / mol. Therefore, it can be inferred that CoTPE can encapsulate sorafenib in both the inner and outer cavities.

[0067] Prepare a 0.5 mg / mL SOR@CoTPE PBS solution. Add the solution to a dialysis bag and place it in a beaker containing 50 mL of PBS. Place the beaker in a 37°C water bath shaker at 100 rpm. Take 1 mL of the solution at each of the following time points: 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h, and add the same volume and temperature of PBS solution. Filter the samples through a 0.22 μm microporous membrane and determine the sorafenib concentration at each time point using HPLC under the specified chromatographic conditions. Parallel experiments were conducted using PBS solutions at pH 6 and pH 7.4, following the same procedure. The results are as follows. Figure 4As shown in the figure. The results showed that sorafenib was released immediately from the drug delivery vessel at both pH values, but the release rate slowed significantly after 12 hours and reached equilibrium after 24 hours. The release curves indicate that sorafenib release is gradual, especially in more acidic environments. Therefore, CoTPE exhibits significant pH-responsive drug release, and SOR@CoTPE significantly prolongs its residence time in tissues compared to free sorafenib.

[0068] Example 3: Cell Experiment

[0069] In this embodiment, HepG2 liver cancer cells and normal hepatocytes LO2 were cultured to verify the autotoxicity of CoTPE and its anti-liver cancer effect in vitro.

[0070] Cytotoxicity assay: Cell proliferation toxicity was detected using the CCK8 assay kit.

[0071] ①According to 1×10 per hole 4 HepG2 cells were seeded in 96-well plates at a density of 1,000 cells / well and incubated overnight. CoTPE was then added to prepare different concentrations (1.25, 2.5, 5, 10, 20, 40, 80 μg / mL). A blank control group (Control) was set up, with 5 replicates for each concentration. At 24, 48, and 72 h post-treatment, 10 μL of CCK-8 reagent was added to each well, followed by detection using a microplate reader. The cell viability of each group was calculated by converting the absorbance values ​​measured in the Control group (100% viability). The results are shown below. Figure 5 As shown in the figure, CoTPE has almost no toxicity within 24 hours, and within 48 hours and 72 hours, it does not affect cell viability within the concentration range of 10 μg / mL. However, at concentrations ≥10 μg / mL, the cytotoxicity of CoTPE increases significantly with increasing concentration.

[0072] ② The above steps were repeated for LO2 cells, and the results were as follows: Figure 6 As shown, the results indicate that CoTPE has almost no toxicity to LO2 cells.

[0073] ③ Cell uptake experiment:

[0074] Using Nile Red as a fluorescent probe, NR@CoTPE was prepared, and HepG2 cells were cultured at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 6-well cell culture plates. The prepared NB@CoTPE solution was added to the plates. After incubation for 2, 6, 12, and 24 hours, residual NB@CoTPE was washed away with PBS. Cells were stained with Hoechst 33342 and Lyso-Tracker Green staining solution. The uptake capacity of tumor cells at different time points was examined using laser confocal microscopy. The results are as follows: Figure 7 As shown, the results showed that after 2 h, red fluorescence emitted by NR@CoTPE and blue fluorescence of Hoechst33342 could be clearly seen in most HepG2 cells, demonstrating that HepG2 cells were still viable when taking up CoTPE. Then, the lysosomes of living HepG2 cells were labeled with LysoTracker Green. The results showed that almost no CoTPE was taken up after 2 h of incubation. When the cells were treated for 6 h, 12 h and even longer, green fluorescence could be observed. It was speculated that NR@CoTPE co-localized with lysosomes, indicating that CoTPE entered the cells through the lysosomal pathway.

[0075] Meanwhile, flow cytometry was used to verify the above results. HepG2 cells were seeded in 6-well cell culture plates at a cell density of 5×10 5 per well and cultured in an incubator for 24 h. The final concentration of Nile red was set at 3 μM, and the prepared solution was added to the culture plates. After incubation for 2, 6, 12, and 24 h respectively, the fluorescence intensities of each group were detected by flow cytometry. The results were as Figure 8 shown. The results showed that, consistent with the results obtained by laser confocal microscopy, the fluorescence intensity taken up by the cells increased with time.

[0076] Example 4: Animal experiment

[0077] Experimental animal model: SPF-grade healthy male BALB / c mice (4 - 6 weeks old, weighing 16 - 18 g), purchased from Henan Skbes Biotechnology Co., Ltd. (Henan, China), license number SCXK(Yu)2020 - 0005.

[0078] Tumor model: HepG2 tumor cells were subcutaneously injected into the right anterior axilla of experimental mice; after 48 - 72 h, solid tumor nodules with a diameter of about 5 mm appeared at the injection site, indicating successful model construction.

[0079] Twenty-five SPF-grade male BALB / c mice were selected and grouped by stratified randomization for the experiment: 5 were set as the untreated control group (Blank group), and the remaining 20 tumor model mice were randomly divided into four groups (n = 5 / group) 24 h after inoculation: model control group (Control), CoTPE intervention group (CoTPE), SOR@CoTPE composite group, and sorafenib monotherapy group (SOR).

[0080] Based on the principle of equivalent dose conversion according to body surface area (referring to the specifications of "Laboratory Animal Science"), the dosing dose of sorafenib was set at 20 mg / kg / d, and the doses of CoTPE and its complexes were calculated to obtain the same mass concentration through molar ratio equivalence.

[0081] During the administration period, the general condition of mice in each group was observed and recorded every two days, including eating, drinking, hair changes, mental state and spontaneous activity; the body weight of the mice was monitored using an electronic balance.

[0082] All experimental group animals underwent 14 days of drug intervention, followed by 24-hour fasting (but not water restriction), and their final weight was recorded. Euthanasia was performed using cervical dislocation, and immediate systemic dissection was conducted. Tumor tissue was removed from the mice for photographing, and the results are shown below. Figure 9 The recorded data was plotted as a function graph along the time axis, and the results are shown below. Figure 10 The results showed that the tumor volume of mice in the Control group and CoTPE group increased rapidly. In contrast, the tumor volume of mice treated with free sorafenib and SOR@CoTPE group increased more slowly in the first ten days. After ten days, the tumor volume growth rate of mice in SOR@CoTPE group decreased rapidly. After the sixth administration, the tumor volume of mice began to decrease, proving that SOR@CoTPE can inhibit tumor growth.

[0083] HE and TUNEL staining were performed on various organs and tumor tissues of tumor-bearing mice to detect histological changes in HepG2 subcutaneous ectopic tumors, such as... Figure 11 , 12 As shown, Figure 11 The results showed that, under the microscope, the tumor cell volume in all treatment groups was smaller than that in the Control group. In the Control and CoTPE groups, the tumor cells had larger, irregularly shaped nuclei with a high nucleus-to-cytoplasm ratio, numerous mitotic figures, a small number of focal necrosis, unclear cell structure, condensed, fragmented, and dissolved nuclei, increased eosinophilicity, and a small number of edematous tumor cells surrounding the necrotic cells. The cytoplasm was loose and lightly stained. Both the sorafenib and SOR@CoTPE groups showed cell shrinkage, lighter nuclear staining, and an increase in necrotic foci. However, the SOR@CoTPE group exhibited looser cell arrangement, reduced atypia, and a significantly lower cell number compared to the sorafenib group. There was also a significant reduction in neovascularization within the tumor tissue, and large areas of necrosis were visible. Compared to the Control and CoTPE groups, the sorafenib and SOR@CoTPE groups showed significantly increased tumor cell destruction.

[0084] Figure 12The results showed that tumor cells in the Control and CoTPE groups were randomly distributed, with many mitotic and atypical tumor cells, and a small number of tumor cells necrotized with nuclear pyknosis. In the sorafenib group, mitotic and atypical cells were rare, with many isolated cells and intercellular cavities, and even more tumor cell nuclear pyknosis and necrosis. In the SOR@CoTPE group, a large number of necrotic cells were observed, tumor cells were inhibited and atrophied, accompanied by numerous fragmented and pyknosis nuclei and many intercellular cavities. Apoptosis was rare in the Control and CoTPE groups, while it was significantly increased in the SOR@CoTPE group compared to the sorafenib group.

[0085] H&E staining was performed on the major organs of the harvested mice, including the heart, liver, spleen, lungs, and kidneys, for histological analysis. For example... Figure 13 As shown, no pathological changes occurred in the major organs of any of the experimental groups, indicating that these treatment methods did not cause significant damage to normal organs. Furthermore, throughout the treatment monitoring period, the mice in all experimental groups did not show significant weight loss. Figure 14 This further confirms that the SOR@CoTPE and treatment methods used in this embodiment are not biotoxic.

[0086] The present invention and its embodiments have been described above. This description is not restrictive, and the actual implementation is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and, without departing from the spirit of the invention, design similar implementations and embodiments without creative effort, all such designs should fall within the protection scope of the present invention.

Claims

The application of 1,3,5-tetraphenyldicarboxylic acid ligand in the preparation of drug carriers, characterized in that, The molecular structure of the 3,5-tetraphenyldicarboxylic acid ligand is shown in formula (1): （1）。 2. A tetraphenylethylene molecular cage, characterized in that, The general structural formula of the tetraphenylethylene molecular cage is shown in the following formula (2): {[M4(μ4-H2O)(X)]4Y8} (2); Where M is a metallic element selected from magnesium, zinc, manganese, cobalt, nickel, copper, iron, or ruthenium; μ4-H2O represents a 4-coordination mode formed by 4 M atoms with the O atoms in H2O; X is a thiacalix[4] aromatic ligand, and its molecular structure is shown in formula (3): （3） In formula (3), R1, R2, R3, and R4 may be the same or different, and are independently selected from one of hydrogen, alkyl, or aryl groups; A1, A2, A3, and A4 may be the same or different, and are independently selected from one of -S-, -SO-, or -SO2-. Y is a dicarboxylic acid ligand, and its molecular structure is shown in formula (1): （1）。 3. The tetraphenylethylene molecular cage according to claim 1, characterized in that, In formula (3), R1, R2, R3, and R4 are selected from one of tert-butyl, tert-octyl, and phenyl, respectively.

4. The tetraphenylethylene molecular cage according to claim 2, characterized in that, In equation (3), A1, A2, A3, and A4 are all -SO2-.

5. A tetraphenylethylene molecular cage according to claim 3, characterized in that, The general structural formula of the tetraphenylethylene molecular cage is {[Co4 (μ4-H2O)(TBSC)]4 [TPE]8}, where X is a 5,11,17,23-tetratert-butyl-sulfonyl bridged calix[4] ligand and Y is a 3,5-tetraphenyldicarboxylic acid ligand.

6. A method for preparing a tetraphenylethylene molecular cage according to any one of claims 1 to 4, characterized in that, The method includes the following steps: obtaining the product by self-assembling H4X, H2Y and M metal salts in an organic solvent, wherein X, Y and M are as defined in any one of claims 1-4.

7. The method for preparing a tetraphenylethylene molecular cage according to claim 5, characterized in that, The molar ratio of X:Y:M is (0.9-1.1):(1.8-4.5):(4.0-7.0).

8. The method for preparing a tetraphenylethylene molecular cage according to claim 5, characterized in that, The M metal salt is a nitrate or a halide salt.

9. The application of a tetraphenylethylene molecular cage according to any one of claims 1 to 4, or a tetraphenylethylene molecular cage prepared by the preparation method according to any one of claims 5 to 7, characterized in that, The application is at least one of the following: ① Application as a drug carrier for loading drugs; ② Application in the preparation of drugs for the treatment of hepatocellular carcinoma.

10. The application according to claim 8, characterized in that, The drug loaded is sorafenib.

11. A drug for treating hepatocellular carcinoma, characterized in that, It includes a drug carrier and sorafenib loaded on the drug carrier, wherein the drug carrier is a tetraphenylethylene molecular cage as described in any one of claims 1 to 4, or a tetraphenylethylene molecular cage prepared by the preparation method of any one of claims 5 to 7.

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