A paclitaxel inclusion complex, and a preparation method and application thereof

Abstract

This invention discloses the application of an anionic open-ring cucurbituril derivative in improving the water solubility and antitumor activity of paclitaxel. The inclusion complex is formed by the assembly of anionic open-ring cucurbituril as the host molecule and paclitaxel as the guest molecule through non-covalent host-guest interactions, wherein the molar ratio of paclitaxel to anionic open-ring cucurbituril is 1-10:1. By forming a stable host-guest inclusion complex, the extremely poor water solubility and chiral stability of paclitaxel are significantly improved, effectively enhancing its antitumor efficacy and showing broad application prospects in targeted drug delivery and clinical oncology treatment.

Description

Technical Field

[0001] This invention belongs to the technical field of pharmaceutical formulations and drug delivery systems, specifically relating to an inclusion complex of anionic open-ring cucurbituril and paclitaxel, its preparation method, and its application. Background Technology

[0002] Paclitaxel (PTX), a natural diterpenoid extracted from the yew tree, possesses broad-spectrum and potent antitumor activity. Its primary mechanism of action involves promoting microtubule polymerization and inhibiting its depolymerization, thereby interfering with tumor cell mitosis and ultimately inducing apoptosis. However, paclitaxel's extremely poor room-temperature water solubility (only about 0.13 μg / mL) results in low bioavailability, severely limiting its clinical administration routes and efficacy. To overcome this solubility barrier, first-generation paclitaxel injections (such as Taxol®) were developed, using a mixture of polyoxyethylene castor oil (Cremophor EL) and anhydrous ethanol as a cosolvent. However, this cosolvent system not only easily triggers severe hypersensitivity reactions but also causes serious adverse reactions such as neurotoxicity and myelosuppression. To reduce the toxic side effects of excipients, researchers subsequently developed second-generation paclitaxel formulations, such as albumin-bound paclitaxel (Abraxane®) and micellar paclitaxel (Genexol-PM®). While these formulations have improved drug safety to some extent, they still face technical bottlenecks such as high excipient costs (e.g., limited availability of human serum albumin), complex preparation processes, insufficient physical stability, residual toxicity, and poor in vivo targeted drug release. In recent years, various novel nanodelivery strategies have been explored in this field, such as prodrug micelles (Publication No.: CN113444252A), compound liposomes (Publication No.: CN120093693A), amorphous solid dispersions (Publication No.: CN118948776A), and reactive oxygen species-responsive prodrug nanoparticles (Publication No.: CN119841854A). Although these improved technologies each have their advantages, developing a pure aqueous formulation that can be directly dispersed in physiological saline or water for injection, has a simple preparation process, and exhibits excellent biocompatibility remains a critical technical challenge for the clinical application of paclitaxel. In summary, there is an urgent need in this field to develop a novel paclitaxel delivery system that aims to fundamentally and significantly improve the water solubility and antitumor activity of paclitaxel, completely eliminate traditional solubilizing excipients with toxic side effects, thereby effectively improving the safety of clinical medication, simplifying the dosing process, and enhancing the overall therapeutic index. Summary of the Invention

[0003] This invention provides an inclusion complex of an open-ring cucurbituril derivative and paclitaxel, its preparation method, and its application in the preparation of antitumor drugs. This invention aims to solve the problems of poor water solubility and low bioavailability of paclitaxel, improve its antitumor activity, and fundamentally eliminate the technical risks of severe allergic reactions and other toxic side effects caused by solubilizing excipients (such as polyoxyethylene castor oil) used in traditional preparations (such as paclitaxel injection).

[0004] The open-ring cucurbita derivative and paclitaxel inclusion complex of the present invention is formed by assembling anionic open-ring cucurbita as the host molecule and paclitaxel as the guest molecule through host-guest non-covalent interactions; the molar ratio of paclitaxel to anionic open-ring cucurbita is 1~10:1. The structural formula of the anionic open-ring cucurbituril is shown below:

[0005] In the formula, R is (CH2)3SO3Na, (CH2)3CO3Na, or (CH2)3PO3Na2.

[0006] The chemical structural formula of the paclitaxel is as follows:

[0007] The above-mentioned paclitaxel inclusion complex is prepared by dissolving anionic open-ring cucurbituril and paclitaxel separately in solvents, adding the paclitaxel solution to the anionic open-ring cucurbituril solution at a molar ratio of paclitaxel to anionic open-ring cucurbituril of 1 to 10:1, stirring and reacting at 25 to 45°C in the dark for 24 to 72 hours, filtering through a microporous membrane with a pore size of 0.22 to 0.45 μm, concentrating the filtrate under reduced pressure, and drying the concentrate to obtain the inclusion complex.

[0008] The solvent is selected from water, methanol, ethanol, isopropanol, tert-butanol, acetonitrile, dimethyl sulfoxide, and N,N-dimethylformamide.

[0009] The drying process is selected from vacuum drying, fluidized bed drying, spray drying, and freeze drying.

[0010] Advantages and technical effects of the method of the present invention compared with the prior art: This invention utilizes the flexible hydrophobic cavity of anionic open-ring cucurbituril molecules to form a good cavity match with paclitaxel, increasing the water solubility of paclitaxel. Simultaneously, the anionic open-ring cucurbituril molecules exhibit chiral amplification with paclitaxel, enhancing its chiral stability. The inclusion complex obtained by this invention not only significantly improves the solubility of paclitaxel but also enhances its antioxidant activity and in vitro antitumor activity, improving the drug's inhibitory effect on tumor cells and helping to reduce clinical dosage and related toxic side effects. It solves key technical problems such as poor water solubility, easy aggregation, and insufficient chiral stability of paclitaxel, providing a new technical approach and theoretical basis for developing novel, highly stable, and highly bioavailable paclitaxel injection formulations. It has broad application prospects and industrialization value in the fields of antitumor drug delivery, biopharmaceutical formulation development, and supramolecular drug carriers. Attached Figure Description

[0011] Figure 1 The 1H NMR spectra of paclitaxel, anionic open-ring cucurbituril I-1, anionic open-ring cucurbituril I-1, and the paclitaxel inclusion complex in Example 1 are shown below. 1 (H NMR) Graph; where (a) paclitaxel; (b) anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) and paclitaxel inclusion complex; (c) anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na); Figure 2 The 2D-ROESY spectrum of the anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) and paclitaxel inclusion complex in Example 1; Figure 3 Fourier transform infrared (FT-IR) spectra of the inclusion complex in Example 1; wherein (a) anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na); (b) paclitaxel; (c) a physical mixture of anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) and paclitaxel; and (d) anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) and paclitaxel inclusion complex; Figure 4 The X-ray diffraction (XRD) spectra of the inclusion complexes in Example 1 are shown in the following diagrams: (a) anionic open-ring cucurbituril I-2 (R=(CH2)3CO3Na); (b) paclitaxel; (c) a physical mixture of anionic open-ring cucurbituril I-2 (R=(CH2)3CO3Na) and paclitaxel; and (d) anionic open-ring cucurbituril I-2 (R=(CH2)3CO3Na) and paclitaxel inclusion complex. Figure 5Thermogravimetric (TGA) diagrams of the inclusion complexes in Example 1 are shown; where (a) is anionic open-ring cucurbituril I-2 (R=(CH2)3CO3Na); (b) is paclitaxel; (c) is a physical mixture of anionic open-ring cucurbituril I-2 (R=(CH2)3CO3Na) and paclitaxel; and (d) is an inclusion complex of anionic open-ring cucurbituril I-2 (R=(CH2)3CO3Na) and paclitaxel. Figure 6 Fourier transform infrared (FT-IR) spectra of paclitaxel, anionic open-ring cucurbituril I-3, a physical mixture of anionic open-ring cucurbituril I-3 and paclitaxel, and inclusion complexes of anionic open-ring cucurbituril I-3 and paclitaxel; wherein (a) anionic open-ring cucurbituril I-3 (R=(CH2)3PO3Na2); (b) paclitaxel; (c) a physical mixture of anionic open-ring cucurbituril I-3 (R=(CH2)3PO3Na2) and paclitaxel; and (d) inclusion complexes of anionic open-ring cucurbituril I-3 (R=(CH2)3PO3Na2) and paclitaxel. Figure 7 The 1H NMR spectra of paclitaxel, anionic open-ring cucurbituril II-1, anionic open-ring cucurbituril, and the paclitaxel inclusion complex in Example 2 are shown below. 1 (H NMR) Graph; where (a) paclitaxel; (b) anionic open-ring cucurbituril II-1 (R=(CH2)3SO3Na) and paclitaxel inclusion complex; (c) anionic open-ring cucurbituril II-1 (R=(CH2)3SO3Na); Figure 8 The 2D-ROESY spectrum of the ionized ring-opening cucurbituril II-1 (R=(CH2)3SO3Na) and paclitaxel inclusion complex in Example 2; Figure 9 Scanning electron microscope (SEM) images of anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na) and paclitaxel inclusion complex in Example 2; wherein (a) anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na); (b) paclitaxel; (c) a physical mixture of anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na) and paclitaxel; and (d) anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na) and paclitaxel inclusion complex. Figure 10Thermogravimetric (TGA) diagrams of paclitaxel, anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na), a physical mixture of anionic open-ring cucurbituril II-2 and paclitaxel, and an inclusion complex of anionic open-ring cucurbituril II-2 and paclitaxel in Example 2 are shown below; where (a) anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na); (b) paclitaxel; (c) a physical mixture of anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na) and paclitaxel; and (d) anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na) and paclitaxel inclusion complex. Figure 11 Scanning electron microscope (SEM) images of paclitaxel, anionic ring-opening cucurbituril II-3, a physical mixture of anionic ring-opening cucurbituril II and paclitaxel, and an inclusion complex of anionic ring-opening cucurbituril II and paclitaxel in Example 2; wherein (a) anionic ring-opening cucurbituril II-3 (R=(CH2)3PO3Na2); (b) paclitaxel; (c) a physical mixture of anionic ring-opening cucurbituril II-3 (R=(CH2)3PO3Na2) and paclitaxel; and (d) anionic ring-opening cucurbituril II-3 (R=(CH2)3PO3Na2) and paclitaxel inclusion complex. Figure 12 Comparative X-ray diffraction (XRD) spectra of the inclusion complexes in Example 2; where (a) anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2); (b) paclitaxel; (c) a physical mixture of anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2) and paclitaxel; and (d) anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2) and paclitaxel inclusion complexes. Figure 13 The following is a graph showing the cytotoxicity evaluation results of the inclusion complexes in Example 5 against HeLa cells: (a) shows different concentrations of paclitaxel, (b) shows the inclusion complex of anionic open-ring cucurbituril II-3 with paclitaxel, and (c) shows the inclusion complex of anionic open-ring cucurbituril I-1 with paclitaxel. Figure 14 The following is a graph showing the cytotoxicity evaluation results of the inclusion complex in Example 5 on 4T1 cells: (a) shows different concentrations of paclitaxel, (b) shows the inclusion complex of anionic open-ring cucurbituril II-3 with paclitaxel, and (c) shows the inclusion complex of anionic open-ring cucurbituril I-1 with paclitaxel. Figure 15 The following is a graph showing the cytotoxicity evaluation results of the inclusion complexes in Example 5 against HCT116 cells: (a) shows different concentrations of paclitaxel, (b) shows the inclusion complex of anionic open-ring cucurbituril II-3 with paclitaxel, and (c) shows the inclusion complex of anionic open-ring cucurbituril I-1 with paclitaxel. Figure 16The following is a graph showing the cytotoxicity evaluation results of the inclusion complexes in Example 5 against HepG2 cells: (a) shows different concentrations of paclitaxel, (b) shows the inclusion complex of anionic open-ring cucurbituril II-3 with paclitaxel, and (c) shows the inclusion complex of anionic open-ring cucurbituril I-1 with paclitaxel. Figure 17 The images show Calcein-AM / PI live and dead cell staining patterns of the inclusion complexes in Example 6, where (a) is free paclitaxel, (b) is the inclusion complex of the main anionic open-ring cucurbituril II-3 and paclitaxel, and (c) is the inclusion complex of the main anionic open-ring cucurbituril I-1 and paclitaxel. Detailed Implementation

[0012] The present invention will be further described below with reference to the accompanying drawings and examples. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the methods in this embodiment are operated according to conventional methods, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified. The anionic open-ring cucurbituril in the embodiments is prepared according to the methods in existing literature. Example 1: Preparation of anionic open-ring cucurbituril I-1 and paclitaxel inclusion complex ; 1. Accurately weigh 1.541 g (1 mmol) of anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) solid powder, dissolve it in 20 mL of pure water to prepare an aqueous solution, and stir magnetically until completely dissolved to obtain a clear bulk solution. Weigh 3.419 g (4 mmol) of paclitaxel powder, dissolve it in 25 mL of methanol solution, and slowly add the paclitaxel solution dropwise to the anionic open-ring cucurbituril I-1 solution while continuously stirring. Place the reaction mixture at 30 °C and 150 rpm for 48 hours. Filter the reaction product through a 0.22 μm microporous membrane, and freeze-dry the filtrate to obtain anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) and paclitaxel inclusion complex, with a yield of 60.12%.

[0013] Nuclear magnetic resonance characterization such as Figure 1 As shown, the chemical shift of paclitaxel significantly shifts to a higher field after inclusion, proving that it has been successfully included by the open-ring cucurbituril I-1 (R=(CH2)3SO3Na). Further analysis using two-dimensional rotating frame nuclear Overhauser effect spectroscopy (2D-ROESY) is shown below. Figure 2 The aromatic protons H-10, H-11, H-12, H-14, and H-23 of paclitaxel exhibit strong spatial correlation signals with the internal cavity protons (Ha, Hb, H-c', H-d', and H-e'), confirming the successful inclusion of the host and guest components.

[0014] 2. Accurately weigh 1.521 g (1 mmol) of anionic open-ring cucurbituril I-2 (R=O(CH2)3CO3Na) solid powder, add it to 20 mL of pure water, and magnetically stir at room temperature until completely dissolved to obtain a clear and transparent aqueous solution. Separately weigh 2.559 g (3 mmol) of paclitaxel powder, add it to 15 mL of methanol, and stir at room temperature until completely dissolved to obtain a paclitaxel methanol solution. Under continuous stirring, slowly add the paclitaxel methanol solution dropwise to the aqueous solution. After the addition is complete, continue stirring to mix thoroughly to obtain the reaction system. Place the reaction system at 30℃ and 200 rpm and stir for 72 h to allow the anionic open-ring cucurbituril I-2 and paclitaxel to fully undergo host-guest inclusion. After the reaction is complete, filter the resulting mixture through a 0.22 μm microporous membrane to remove unencapsulated solid impurities. The filtrate was freeze-dried to obtain a white solid anionic open-ring cucurbituril I-2 (R=O(CH2)3CO3Na) and paclitaxel inclusion complex, with a yield of 75.43%. Analysis was performed using X-ray diffraction (XRD). Figure 3 The XRD pattern of free paclitaxel showed numerous sharp and strong characteristic diffraction peaks in the 2θ range of 5-30°. In the XRD pattern of the inclusion complex, the characteristic diffraction peaks of the guest almost disappeared completely, leaving only broad and diffuse diffraction peaks similar to those of anionic open-ring cucurbituril I-2 (R=O(CH2)3CO3Na), proving its successful inclusion.

[0015] 3. The preparation method of the anionic open-ring cucurbituril I-3 (R=O(CH2)3PO3Na2) and paclitaxel inclusion complex is the same as in step 1. The yield of the anionic open-ring cucurbituril I-3 (R=(CH2)3PO3Na2) and paclitaxel inclusion complex is 54.45%. Fourier transform infrared spectroscopy (FT-IR) analysis was performed, as shown... Figure 4 As shown, free paclitaxel exhibits a relatively complex characteristic absorption spectrum. In the 1600-1500 cm⁻¹ range... -1 Strong aromatic skeletal vibrational peaks appeared in the region, and at 1200-1000 cm⁻¹ -1 Strong CO and CN stretching vibration signals were observed within the range, and the spectrum of the inclusion compound showed the disappearance of some peaks of the guest, which further proved the successful inclusion of the host and guest.

[0016] Example 2: Preparation of anionic open-ring cucurbituril II and paclitaxel inclusion complex

[0017] 1. Weigh 1.621 g (1 mmol) of anionic open-ring cucurbituril II-1 (R=(CH2)3SO3Na) and dissolve it in 10 mL of pure water; separately weigh 4.265 g (5 mmol) of paclitaxel and dissolve it in 10 mL of methanol. Slowly add the paclitaxel powder to the aqueous solution of anionic open-ring cucurbituril II-1 (R=(CH2)3SO3Na), mix well, and react at 25 °C and 150 rpm for 36 h to allow for complete inclusion of the host and guest components. After the reaction is complete, filter the reaction solution through a 0.22 μm microporous membrane, freeze-dry the filtrate, and obtain a white solid inclusion complex with a yield of 67.98%.

[0018] Nuclear magnetic resonance analysis, such as Figure 7 The results showed that the chemical shift changed significantly before and after inclusion due to the different chemical environments. The inclusion complex system exhibited the characteristic proton signal of PTX, while free paclitaxel was almost insoluble and difficult to detect in D2O. This was confirmed by two-dimensional rotating frame nuclear Overhauser effect spectroscopy (2D-ROESY). Figure 8 The aromatic protons (H-3, H-4, H-10, H-6, and H-7) of paclitaxel exhibit strong spatial correlation signals with the internal cavity protons (Hk, H-e', H-f', and H-g') of the main body.

[0019] 2. The preparation method of the anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na) and paclitaxel inclusion complex is the same as in step 1. The yield of the anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na) and paclitaxel inclusion complex is 76.23%.

[0020] Scanning electron microscopy (SEM) analysis, such as Figure 9 The original regular crystal structure of paclitaxel has largely disappeared, replaced by a lamellar structure, and the surface morphology has also changed significantly. The morphology of the inclusion compound differs from both free paclitaxel and the anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na), indicating that it is not a simple physical mixture of the two components, but rather a new supramolecular structure formed under host-guest interaction. Thermogravimetric analysis (TGA) is as follows: Figure 10 As shown, the initial decomposition temperature of the inclusion complex was significantly higher than that of free paclitaxel, and the main weight loss phase shifted to the high-temperature region, with a slower decomposition process. These results indicate that paclitaxel is confined and protected within the host cavity after being included by the anionic open-ring cucurbituril II-2 (R=(CH2)3CO3Na), thus restricting its thermal motion and significantly improving its thermal stability.

[0021] 3. Weigh 1.601 g (1 mmol) of anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2) and dissolve it in 30 mL of pure water. Separately weigh 2.132 g (2.5 mmol) of paclitaxel powder and add it to the aqueous solution of anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2). After mixing evenly, react at 27 °C and 200 rpm for 48 h. After the reaction is completed, filter through a filter membrane and freeze dry to obtain the inclusion complex with a yield of 73.21%.

[0022] Perform scanning electron microscopy (SEM) analysis, such as Figure 11 The original crystal structure of free paclitaxel almost completely disappeared, forming a lamellar structure. This demonstrates that the crystal structure of anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2) and paclitaxel was effectively destroyed upon inclusion, suggesting a strong intermolecular interaction between the two and further confirming the successful construction of the inclusion complex. Figure 12 X-ray diffraction (XRD) analysis showed that the disappearance of the characteristic crystal form of paclitaxel and the decrease in crystallinity proved the successful formation of the inclusion complex.

[0023] Example 3: Solubility test of different anionic open-ring cucurbituril with paclitaxel inclusion complexes Paclitaxel has extremely poor water solubility at room temperature (1.30 × 10⁻⁶). -4 The water solubility of the inclusion complex after anionic ring-opening cucurbita contains paclitaxel is shown in the table below;

[0024] As shown in Table 1, the solubility of the inclusion complexes is higher than that of free paclitaxel.

[0025] Example 4: Antitumor experiment on human cervical cancer cells (HeLa) by different anionic open-ring cucurbituril-paclitaxel inclusion complexes. Human cervical cancer cells (HeLa) were used as model cells to evaluate the in vitro antitumor activity of free paclitaxel and paclitaxel inclusion complexes with different anionic open-ring cucurbitacins. Cells were seeded in 96-well plates at a density of 1 × 10⁻⁶ cells / well. 4 Cells were cultured in 37℃, 5% CO2 incubators for 24 h. Different concentrations of paclitaxel and different anionic open-ring cucurbitacin-paclitaxel inclusion complex solutions were added to each well. After 4 h of culture, 20 μL of MTT solution (5 mg / mL) was added to each well. Four h later, 150 μL of DMSO was added to each well to dissolve formazan crystals. OD values ​​were measured at 492 nm using a microplate reader, and cell viability was calculated. The inhibitory effects of different anionic open-ring cucurbitacin-paclitaxel inclusion complexes on tumor cell proliferation were compared, and IC50 was calculated. 50 The value was calculated by taking the average of three parallel experiments.

[0026] As can be seen from Table 2, compared with free paclitaxel, the inclusion complex of the present invention has a higher inhibitory effect on tumors, and the inclusion complex of the present invention can enhance the ability of free paclitaxel to kill tumor cells.

[0027] Example 5: In vitro antitumor activity experiments of the anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) and paclitaxel inclusion complex of Example 1, and the anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2) and paclitaxel inclusion complex of Example 2. Human cervical cancer cells (HeLa), mouse breast cancer cells (4T1), human colon cancer cells (HCT116), and human liver cancer cells (HepG2) were used as model cells to evaluate the in vitro antitumor activities of free paclitaxel, the main anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) with paclitaxel inclusion complex, and the anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2) with paclitaxel inclusion complex. Each cell was seeded in a 96-well plate at a seeding density of 1 × 10⁶ cells / well. 4 Cells were cultured per well at 37°C in a 5% CO2 incubator for 24 hours. Then, different concentrations of paclitaxel, anionic open-ring cucurbituril I-1 and paclitaxel inclusion complex, and anionic open-ring cucurbituril II-3 and paclitaxel inclusion complex were added to each well. The cells were cultured for another 48 hours. After the culture was completed, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for another 4 hours. After discarding the culture medium, 150 μL of DMSO was added to each well to dissolve formazan crystals. The absorbance was measured at 492 nm using a microplate reader, and the cell viability was calculated. The results are as follows Figure 13-16 As shown, with increasing drug concentration, the cell viability of the treatment groups treated with paclitaxel, anionic open-ring cucurbituril I-1 with paclitaxel, anionic open-ring cucurbituril II-3, and paclitaxel inclusion complex all showed a significant dose-dependent decreasing trend. Compared with free paclitaxel, both inclusion complexes showed stronger cell growth inhibition, with the anionic open-ring cucurbituril I-1 with paclitaxel group showing the most significant inhibitory effect on HeLa, 4T1, HCT116, and HepG2 cells.

[0028] The above results indicate that the antitumor activity of paclitaxel is effectively preserved and further enhanced after inclusion by open-ring cucurbituril; in particular, the anionic open-ring cucurbituril I-1 forms a more stable host-guest complex system with paclitaxel, which improves the drug's dispersibility and cell utilization efficiency, thus exhibiting a superior antitumor effect.

[0029] Example 6: Live and dead cell staining experiments of anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na) with paclitaxel inclusion complex and anionic open-ring cucurbituril II-3 (R=(CH2)3PO3Na2) with paclitaxel inclusion complex. Human cervical cancer cells (HeLa) were used as a model cell line to evaluate the ability of free paclitaxel, anionic open-ring cucurbituril I-1 (R=(CH2)3SO3Na), and paclitaxel inclusion complex to induce cell death. HeLa cells were seeded in confocal culture dishes and cultured at 37 ℃ in a 5% CO2 incubator for 24 h. Subsequently, free paclitaxel and the inclusion complex were added to achieve their corresponding IC50 concentrations. 50 The concentration was adjusted, and the cells were cultured for another 24 hours. After culture, the culture medium was discarded, and the cells were gently washed three times with PBS buffer. Then, Calcein-AM / PI live / dead cell staining working solution was added to each culture dish, and the cells were incubated at 37°C in the dark for 20 minutes. After staining, the cells were washed three more times with PBS and observed using a laser confocal microscope. Calcein-AM staining showed green fluorescence, representing live cells; PI staining showed red fluorescence, representing dead cells.

[0030] The results are as follows Figure 17 As shown, the blank control group cells mainly exhibited green fluorescence, indicating that the cells had good growth status and high survival rate. After treatment with free paclitaxel, green fluorescence was significantly reduced and red fluorescence was significantly increased, indicating that paclitaxel can effectively induce HeLa cell death. Compared with the free paclitaxel group, the green fluorescence in the group treated with the anionic open-ring cucurbituril II-3 and paclitaxel inclusion complex was further weakened, and the red fluorescence was further enhanced, indicating that the inclusion complex could induce death in more tumor cells. Furthermore, the group treated with the anionic open-ring cucurbituril I-1 and paclitaxel inclusion complex showed the strongest red fluorescence signal and the least green fluorescence signal, indicating that this inclusion complex has the best tumor cell killing ability. Therefore, the inclusion complex formed by anionic open-ring cucurbituril I-1 and paclitaxel exhibits stronger cytotoxicity and cell death-promoting effects.

[0031] In summary, this invention provides a novel paclitaxel inclusion complex that successfully solves the fundamental problem of poor water solubility of paclitaxel by encapsulating paclitaxel in a biocompatible anionic open-ring cucurbituril, while simultaneously enhancing its antitumor activity. Furthermore, the preparation method provided by this invention is simple and feasible, offering a solid technical foundation for developing safer and more efficient paclitaxel aqueous injections, and possessing significant clinical application prospects.

Claims

1. A paclitaxel inclusion complex, characterized in that: It contains paclitaxel and anionic open-ring cucurbituril, wherein the molar ratio of paclitaxel to anionic open-ring cucurbituril is 1~10:1; The structural formula of the anionic open-ring cucurbituril is as follows: ； In the formula, R is selected from (CH2)3SO3Na, (CH2)3CO3Na, and (CH2)3PO3Na2.

2. The method for preparing the paclitaxel inclusion complex according to claim 1, characterized in that: Anionic open-ring cucurbituril and paclitaxel are dissolved separately in solvents. The paclitaxel solution is added to the anionic open-ring cucurbituril solution at a molar ratio of paclitaxel to anionic open-ring cucurbituril of 1 to 10:

1. The mixture is stirred and reacted at 25 to 45°C in the dark for 24 to 72 hours. The mixture is then filtered through a microporous membrane with a pore size of 0.22 to 0.45 μm. The filtrate is concentrated under reduced pressure and dried to obtain the final product.

3. The preparation method according to claim 2, characterized in that: The solvent is selected from water, methanol, ethanol, isopropanol, tert-butanol, acetonitrile, dimethyl sulfoxide, and N,N-dimethylformamide.

4. The use of the paclitaxel inclusion complex according to claim 1 in the preparation of antitumor drugs.

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