Hybrid nano assembly for dual synergistic chemotherapy as well as preparation method and application of hybrid nano assembly

Abstract

The invention relates to a hybrid nano assembly for dual synergistic chemotherapy as well as a preparation method and application of the hybrid nano assembly, and belongs to the technical field of pharmaceutical preparations. The invention relates to a hybrid nano assembly for dual synergistic chemotherapy, which is formed by co-assembling a cabazitaxel prodrug and gossypol under hydrophobic action and hydrogen bond driving, and is modified by adopting a PEG (Polyethylene Glycol) modifier, the molar ratio of the cabazitaxel prodrug to the gossypol is 1: (1-7), and the ratio of the total mass of the cabazitaxel prodrug and the gossypol to the mass of the PEG modifier is 4: 1. A cabazitaxel prodrug and a sensitizer gossypol are co-assembled to obtain a hybrid nano assembly, the hybrid nano assembly is oxygenated to obtain the oxygen-carrying preparation, the nano assembly and the oxygen-carrying preparation are simple and convenient to prepare and high in drug loading efficiency, and efficient co-loading of drugs can be achieved; meanwhile, the curative effect of chemotherapy can be enhanced through dual mechanisms of relieving tumor hypoxia and promoting tumor apoptosis, and the pharmaceutical composition has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to a hybrid nanoassembly for dual-effect chemotherapy, its preparation method, and its application. Specifically, it relates to a method and application of a nanoassembly based on a dual strategy of alleviating tumor hypoxia through exogenous oxygen supply and simultaneously promoting tumor cell apoptosis by combining it with the sensitizer gossypol, thereby reducing toxic side effects at an equivalent dose of cabazitaxel and thus enhancing the efficacy of chemotherapy. This invention belongs to the field of pharmaceutical formulation technology. Background Technology

[0002] Currently, chemotherapy remains one of the main methods of clinical cancer treatment. In most cases, chemotherapy can slow disease progression, thereby significantly prolonging patient survival. However, chemotherapy usually has serious toxic side effects. Taking the chemotherapy drug cabazitaxel as an example, as a second-generation taxane, its antitumor activity is stronger than that of paclitaxel and docetaxel, but its toxic side effects are also more severe. Therefore, in clinical practice, low-dose cabazitaxel is often used for tumor treatment, and the dose-toxicity contradiction greatly limits the clinical application of cabazitaxel.

[0003] Prodrug strategies, through structural modification of chemotherapeutic drugs, can significantly reduce their systemic toxicity. Therefore, the rational design and synthesis of cabazitaxel prodrug molecules can increase the dosage and further improve therapeutic efficacy without enhancing side effects. However, besides the inherent dose-toxicity contradiction, the clinical application of cabazitaxel in chemotherapy faces multiple challenges. Studies have shown that the hypoxic microenvironment of solid tumors significantly reduces the antitumor effects of various chemotherapeutic drugs, including cabazitaxel. Furthermore, tumor cells often overexpress the anti-apoptotic protein Bcl-2, exhibiting apoptosis resistance, which also severely limits the efficacy of chemotherapy.

[0004] Therefore, it is of great significance to develop a novel pre-race drug strategy for carbazide that can synergistically overcome tumor hypoxia, inhibit Bcl-2 function, and achieve tumor-specific drug release. Summary of the Invention

[0005] To address the dose-toxicity inconsistencies and tumor microenvironment hypoxia and apoptosis resistance issues present in the clinical application of cabazitaxel in existing technologies, this invention provides a hybrid nanoassembly for dual-effect chemotherapy, its preparation method, and its application. Specifically, this invention co-assembles cabazitaxel prodrug molecules and the sensitizer gossypol (GSP) via a one-step nanoassembly method and modifies them with DSPE-PEG. 2kThe mixture is then oxygenated to obtain a hybrid nanoassembly. The hybrid nanoassembly prepared by this invention, utilizing a prodrug strategy, an oxygen supply strategy, and a gossypol sensitization strategy, exhibits good stability, strong oxygen-carrying capacity, excellent tumor-reducing responsive drug release characteristics, and the ability to promote tumor cell apoptosis. It can specifically address three major issues faced by the chemotherapy drug cabazitaxel in clinical tumor treatment: dose-limiting toxicity, hypoxia-limited efficacy, and the upregulation of the anti-apoptotic protein Bcl-2 leading to tumor cell apoptosis resistance.

[0006] A hybrid nanoassembly for dual-effect chemotherapy, the nanoassembly being co-assembled from cabazitaxel and gossypol under hydrophobic interaction and hydrogen bonding driven, and modified with a PEG modifier; The molar ratio of the carbamazepine pre-race drug to gossypol is 1:1~7, and the total mass ratio of the carbamazepine pre-race drug and gossypol to the PEG modifier is 4:1. The structural formula of the carbazita pre-race drug is shown below:

[0007] Where R is or .

[0008] Preferably, the molar ratio of the carbadox predrug and gossypol is 1:5.

[0009] In the above technical solution, the PEG modifier is DSPE-PEG. 2k Cy7-labeled DSPE-PEG 2k Or coumarin-6 labeled DSPE-PEG 2k .

[0010] In the above technical solution, the cabazitaxel prodrug is prepared by the following method: First, acetic anhydride is used as a solvent and initiator to react 2,2'-dithiodiacetic acid to the corresponding anhydride; then, the anhydride is esterified with perfluorohexylethyl alcohol or 1-octanol to generate an intermediate product, and the reaction mixture is separated and purified by column chromatography; finally, cabazitaxel is added to the reaction system to esterify with the intermediate product to generate the final product, which is then separated and purified by preparative liquid chromatography.

[0011] Furthermore, in the reaction of 2,2'-dithiodiacetic acid to anhydride via the dehydration of acetic anhydride, the reaction temperature is 20~40℃ and the time is 2~8 h.

[0012] Furthermore, in the reaction of acid anhydride with perfluorohexylethyl alcohol or 1-octanol, the molar ratio of acid anhydride to perfluorohexylethyl alcohol or 1-octanol is 1~3:1, the catalyst is 4-dimethylaminopyridine, the molar ratio of perfluorohexylethyl alcohol or 1-octanol to the catalyst 4-dimethylaminopyridine is 10:0.1~1, the reaction temperature is 20~40℃, and the time is 10~24 h.

[0013] Furthermore, in the esterification reaction of the intermediate with cabazitaxel, the solvent is dichloromethane, and the catalysts are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine. The molar ratio of cabazitaxel to the intermediate is 1:1~10. The molar numbers of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine are the same, and their molar ratio with cabazitaxel is 1~3:1. The reaction temperature is 20~40℃, and the time is 48~72 h.

[0014] Furthermore, the above reaction is carried out under nitrogen protection.

[0015] Another objective of this invention is to provide a method for preparing the above-mentioned hybrid nano-assemblies, wherein cabazitaxel, gossypol, and PEG modifier are dissolved in organic solvents and mixed evenly, and then the mixed solution is slowly added dropwise to deionized water under stirring, so that it spontaneously assembles into co-assembled nanoparticles with uniform particle size, and the organic solvent is removed by vacuum distillation to obtain the product.

[0016] Furthermore, the organic solvent is one or both of anhydrous ethanol and tetrahydrofuran.

[0017] Preferably, the organic solvent is a combination of tetrahydrofuran and anhydrous ethanol in a volume ratio of 2:1.

[0018] Furthermore, the volume ratio of the organic solvent to deionized water is 1:2~20.

[0019] Preferably, the volume ratio of the organic solvent to deionized water is 1:15.

[0020] Furthermore, the stirring rate is 300~1200 rpm.

[0021] Preferably, the stirring rate is 1000 rpm.

[0022] Another object of the present invention is to provide a method for preparing an oxygen-carrying agent based on the above-mentioned hybrid nanoassemblies, wherein the above-mentioned hybrid nanoassemblies are subjected to oxygenation treatment, wherein the oxygen flow rate is 0.1~10 L / min and the oxygenation time is 5~60 min during the oxygenation process.

[0023] Preferably, the oxygen flow rate is 5 L / min and the oxygenation time is 30 min.

[0024] Another object of the present invention is to provide the application of the above-described hybrid nanoassemblies or oxygen-carrying agents prepared by the above-described methods in the preparation of antitumor drugs.

[0025] Furthermore, the present invention provides the application of the above-described hybrid nanoassemblies or oxygen-carrying agents prepared by the above-described methods in the preparation of drug delivery systems.

[0026] The nanoparticles prepared in this invention exhibit uniform particle size (approximately 90 nm), a polydispersity index (PDI) of less than 0.2, a zeta potential of approximately -25 mV, good stability, and strong oxygen-carrying capacity. Furthermore, in the presence of reducing agents, the bridging disulfide bonds in the cabazitaxel prodrug component break, releasing the parent drug cabazitaxel, demonstrating excellent tumor-reducing responsive drug release characteristics. Simultaneously, the perfluorohexylethyl alcohol side chain in the prodrug CSSF possesses physical oxygen-dissolving capacity, enabling it to carry oxygen to hypoxic tumors, thereby overcoming the limitations imposed by hypoxia on the antitumor effects of chemotherapeutic drugs. More importantly, gossypol can downregulate the expression of anti-apoptotic Bcl-2 in tumor cells, increasing the sensitivity of tumor cells to chemotherapeutic drugs and promoting tumor cell apoptosis.

[0027] The beneficial effects of this invention are: 1. This invention co-assembles a cabazitaxel prodrug and the sensitizer gossypol to obtain a hybrid nanoassembly, and then oxygenates it to obtain an oxygen-carrying formulation to address the challenges faced by the chemotherapy drug cabazitaxel in clinical cancer treatment. Specifically, firstly, cabazitaxel is designed as a prodrug: on the one hand, based on the high redox characteristics of the tumor microenvironment, reduction-sensitive disulfide bonds are selected as the prodrug bridging bonds, enabling the prodrug molecule to specifically release the parent drug cabazitaxel within tumor cells, thereby improving the safe dosage of cabazitaxel; on the other hand, addressing the problem of tumor hypoxia limiting the efficacy of chemotherapy, the prodrug molecule uses perfluorohexylethyl alcohol with oxygen-carrying capacity as a side chain, which can relieve the limitation of tumor hypoxia on the efficacy of cabazitaxel through exogenous oxygen supply. Secondly, based on this, by utilizing the ability of gossypol to downregulate the expression of the anti-apoptotic protein Bcl-2 in tumor cells, the nanoassembly co-assembles the prodrug with gossypol, enabling gossypol to exert a sensitizing effect, increasing the sensitivity of tumor cells to cabazitaxel, thereby promoting tumor cell apoptosis and further enhancing the anti-tumor effect of cabazitaxel.

[0028] 2. The hybrid nanoassemblies prepared in this invention possess advantages such as high drug loading capacity, good colloidal stability, and long blood circulation time, providing a new strategy for designing novel cabazitaxel formulations for clinical cancer treatment. Furthermore, compared to clinically used cabazitaxel solutions, the nanoassemblies exhibit less toxicity and side effects in terms of safety; and in terms of efficacy, they demonstrate significantly superior antitumor effects, meeting the urgent clinical need for novel nano-formulations. Attached Figure Description Figure 1 This is the mass spectrum of the CSSF molecule of the carbamate predrug from Example 1 of the present invention.

[0029] Figure 2 This is the mass spectrum of the carbamate predrug molecule CSSH from Example 1 of the present invention.

[0030] Figure 3 This is a graph showing the cytotoxicity test results of screening the optimal assembly molar ratio of CSSF and gossypol in CSSF / GNAs in Example 2 of the present invention.

[0031] Figure 4 This is a particle size diagram of the nano-assembly of Example 3 of the present invention.

[0032] Figure 5 This is a transmission electron microscope image of the nano-assembly of Example 3 of the present invention.

[0033] Figure 6 The figures show the molecular docking simulation results and molecular force disruption experiment results of CSSF / GNAs in Example 4 of this invention.

[0034] Figure 7 This is a graph showing the results of the oxygen-carrying capacity test of the nano-assembly in Example 5 of the present invention.

[0035] Figure 8 This is a colloidal stability diagram of the nano-assembly in Example 6 of the present invention.

[0036] Figure 9 The figure shows the results of the in vitro activation and drug release behavior of the prodrug in the nano-assembly of Example 7 of the present invention.

[0037] Figure 10 This is a confocal microscope image of the nanoassembly of Example 8 of the present invention, taken under a cellular uptake microscope.

[0038] Figure 11 This is a flow cytometry quantification diagram of cellular uptake of the nanoassembly in Example 8 of the present invention.

[0039] Figure 12 This is a diagram showing the cytotoxicity experiment results of the nano-assembly of Example 9 of the present invention on 4T1 cells.

[0040] Figure 13 This is a graph showing the results of investigating the effect of gossypol on Bcl-2 protein downregulation in Example 10 of the present invention.

[0041] Figure 14 This is a blood drug concentration-time curve for different formulations in Example 11 of the present invention.

[0042] Figure 15 This shows the in vivo tissue distribution of the nano-assembly in Example 12 of the present invention.

[0043] Figure 16 This shows the in vitro tissue distribution of the nano-assembly in Example 12 of the present invention.

[0044] Figure 17 This is a mouse tumor growth curve from the in vivo anti-tumor experiment in Example 13 of the present invention.

[0045] Figure 18 This is a graph showing the tumor bearing rate in mice in Example 13 of the present invention.

[0046] Figure 19 This is a mouse tumor image from Example 13 of the present invention.

[0047] Figure 20 This is an H&E and TUNEL staining image of mouse tumors in Example 13 of the present invention.

[0048] Figure 21 This is a graph showing the change in mouse body weight in Example 13 of the present invention.

[0049] Figure 22 This is a H&E and TUNEL staining image of the main organs of the mouse in Example 13 of the present invention.

[0050] Figure 23 This is a graph showing the results of liver and kidney function analysis in mice in Example 13 of the present invention. Detailed Implementation

[0051] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0052] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0053] Example 1 Synthesis of two carbamazepine pre-race drugs (CSSF and CSSH) (1) Synthesis of the prodrug CSSF 2,2'-Dithiodiacetic acid (2 mmol) was dissolved in 5 mL of acetic anhydride, and the reaction was carried out at 25 °C under N2 protection for 2 h. After the reaction was completed, 20 mL of toluene was added to the reaction system in three portions, and the solvent was removed by vacuum distillation after each addition. Subsequently, 15 mL of dichloromethane was added, followed by 1-octanol (2 mmol) and 4-dimethylaminopyridine (0.2 mmol), and the reaction was carried out at 25 °C under N2 protection for 16 h. The intermediate product was separated and purified by column chromatography (dichloromethane / methanol = 100:1 as eluent). The intermediate (1 mmol), 4-dimethylaminopyridine (1.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5 mmol), 1-hydroxybenzotriazole, and 4-dimethylaminopyridine (1.5 mmol) were dissolved in 15 mL of dichloromethane and reacted under N2 protection in an ice-water bath for 2 h. Then, cabazitaxel (1 mmol) was added, and the reaction was continued at 25 °C under N2 protection for 48 h. The final product CSSH was purified by preparative liquid chromatography (acetonitrile / water = 90:10 as the mobile phase).

[0054] The structure of the above prodrug CSSF was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1 As shown. The results of the 1H NMR spectrum analysis are as follows: 11H-NMR (600 MHz, Methanol-d4): δ: 8.12 (d, J = 7.7 Hz, 2H), 7.67 (t, J = 7.4 Hz, 1H), 7.58 (t, J = 7.7 Hz, 2H), 7.46–7.39 (m, 4H), 7.28 (dd, J = 7.8, 4.9 Hz, 1H), 6.14 (t, J = 9.1 Hz, 1H), 5.61 (d, J = 7.1 Hz, 1H), 5.37 (d, J = 5.1 Hz, 1H), 5.31 (d, J = 5.1 Hz, 1H), 5.02 (d, J = 9.2 Hz, 1H), 4.90 (s, 1H), 4.47 (t, J = 6.3 Hz, 2H), 4.18 (q, J = 8.0 Hz, 2H), 3.92 (dd, J = 10.6, 6.6 Hz, 1H), 3.82 (d, J = 7.1 Hz, 1H), 3.79–3.69 (m, 2H), 3.67–3.56 (m, 2H), 3.40 (s, 3H), 3.29 (s, 3H), 2.74 (ddd, J = 14.2, 9.8, 6.4 Hz, 1H), 2.65 (tt, J = 18.9, 6.4 Hz, 2H), 2.42 (s, 3H), 2.24 (dd, J = 15.3, 9.2 Hz, 1H), 2.04–1.87 (m, 4H), 1.73 - 1.60 (m, 4H), 1.42 (s, 9H), 1.15 (s, 3H), 1.12 (s, 3H). (2) Synthesis of the prodrug CSSH 2,2'-Dithiodiacetic acid (2 mmol) was dissolved in 5 mL of acetic anhydride, and the reaction was carried out at 25 °C under N2 protection for 2 h. After the reaction was completed, 20 mL of toluene was added to the reaction system in three portions, and the solvent was removed by vacuum distillation after each addition. Subsequently, 15 mL of dichloromethane was added, followed by 1-octanol (2 mmol) and 4-dimethylaminopyridine (0.2 mmol), and the reaction was carried out at 25 °C under N2 protection for 16 h. The intermediate product was separated and purified by column chromatography (dichloromethane / methanol = 100:1 as eluent). The intermediate (1 mmol), 4-dimethylaminopyridine (1.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5 mmol), 1-hydroxybenzotriazole, and 4-dimethylaminopyridine (1.5 mmol) were dissolved in 15 mL of dichloromethane and reacted under N2 protection in an ice-water bath for 2 h. Then, cabazitaxel (1 mmol) was added, and the reaction was continued at 25 °C under N2 protection for 48 h. The final product CSSH was purified by preparative liquid chromatography (acetonitrile / water = 90:10 as the mobile phase).

[0055] The structure of the prodrug CSSH was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 2 As shown. The results of the 1H NMR spectrum analysis are as follows: 1H-NMR (600 MHz, Methanol-d4): δ: 8.12 (d, J=7.7 Hz, 2H), 7.67 (t, J=7.4 Hz, 1H), 7.58 (t, J=7.7 Hz, 2H), 7.46–7.39 (m, 4H), 7.28 (dd, J=7.8, 4.9Hz, 1H), 6.14 (t, J=9.1 Hz, 1H), 5.61 (d, J=7.1 Hz, 1H), 5.37 (d, J=5.1 Hz, 1H), 5.31 (d, J=5.1 Hz, 1H), 5.02 (d, J=9.2 Hz, 1H), 4.90 (s, 1H), 4.47 (t, J=6.3 Hz, 2H), 4.18 (q, J=8.0 Hz, 2H), 3.92 (dd, J=10.6, 6.6 Hz, 1H), 3.82 (d,J=7.1 Hz, 1H), 3.79–3.69 (m, 2H), 3.67–3.56 (m, 2H), 3.40 (s, 3H), 3.29 (s, 3H), 2.74 (ddd, J=14.2, 9.8, 6.4 Hz, 1H), 2.65 (tt, J=18.9, 6.4 Hz, 2H), 2.42 (s, 3H), 2.24 (dd, J=15.3, 9.2 Hz, 1H), 2.04–1.87 (m, 4H), 1.73-1.60 (m, 4H), 1.42 (s, 9H), 1.15 (s, 3H), 1.12 (s, 3H). Example 2 Preparation methods of nanoassemblies and screening of the optimal molar ratio of cabazitaxel prodrug CSSF and gossypol (1) The preparation method of the nano-assemblies includes the following steps: setting five assembly molar ratios of CSSF and GSP (1:0, 1:1, 1:3, 1:5 and 1:7), and preparing hybrid nano-assemblies using a one-step nanoprecipitation method. Specifically, the operation involves weighing CSSF, DSPE-PEG... 2k Approximately 10 mg each of CSSF and GSP were dissolved in anhydrous ethanol, and GSP was dissolved in tetrahydrofuran to prepare corresponding solutions with a concentration of 10.0 mg / mL. The total mass of CSSF and GSP was fixed at 1.00 mg, and DSPE-PEG was added. 2kPrepare 5 mixed solutions with a mass of 0.25 mg each, each solution having a volume of 0.1 mL. Add each of the 5 solutions dropwise to 1 mL of deionized water, stir for 3 min, and then remove the organic solvent by vacuum distillation. The resulting solution is then brought to a final volume of 1 mL with deionized water. The particle size and particle size distribution of the hybrid nanoassemblies are determined using a Malvern particle size analyzer.

[0056] (2) MTT assay for screening assembly molar ratio: 4T1 cells in good growth condition were assembled at a ratio of 1.5 × 10⁻⁶. 3 Cells were seeded at a density of [number] cells / well into 96-well cell culture plates and incubated for 16 h. The culture medium was discarded, and a series of drug-containing culture media of varying concentrations were added to each well, with further incubation for 48 h. Subsequently, 0.02 mL of 5 mg / mL MTT solution was added to each well, and the plates were incubated for 4 h. After incubation, the culture medium was discarded, and 0.2 mL of DMSO was added to each well, followed by shaking for 10 min to dissolve the blue-purple crystals. The absorbance of each well was measured at 490 nm using a microplate reader after zeroing. The results are as follows: Figure 3 As shown, the nanoassemblies obtained when the molar ratio of cabazitaxel pre-race drug CSSF and gossypol is 1:5 exhibit the strongest cytotoxicity.

[0057] Example 3 Formulation screening and process optimization of nanoassemblies (1) Screening of organic reagents: Fixed-formulation DSPE-PEG 2k The content was 20%. Approximately 1 mg each of CSSF and GSP were weighed, totaling four portions, and dissolved in four solvents: tetrahydrofuran, tetrahydrofuran / anhydrous ethanol (V / V) = 2:1, tetrahydrofuran / anhydrous ethanol (V / V) = 1:1, and tetrahydrofuran / anhydrous ethanol (V / V) = 1:2, respectively, to obtain corresponding solutions with a concentration of 10 mg / mL. With the total mass of CSSF and GSP fixed at 1 mg and their molar ratio at 1:5, four 100 μL mixed solutions were prepared. Each of the four solutions was added dropwise to 1 mL of deionized water, stirred for 3 min, and the organic reagents were removed by vacuum distillation. The solution was then brought to a final volume of 1 mL with deionized water. The particle size and PDI were determined using a Malvern particle size analyzer. The results are shown in Table 1. It can be seen that the optimal particle size was achieved when using tetrahydrofuran / anhydrous ethanol (V / V) = 2:1 as the solvent to prepare the nanoassemblies.

[0058] Table 1. Particle size and particle size distribution of CSSF / GNAs prepared with different organic solvents (n = 3)

[0059] (2) Screening of the ratio of organic phase to water phase With other conditions kept constant, the organic phase / aqueous phase ratios were set to 1:2, 1:5, 1:10, 1:15, and 1:20. Hybrid nanoassemblies were prepared using the aforementioned method, and their formulation properties were investigated. The results are shown in Table 2. It can be seen that the optimal particle size was achieved when the organic phase to aqueous phase ratio was 1:15.

[0060] Table 2. Particle size and particle size distribution of nanoassemblies with different organic phase and aqueous phase ratios.

[0061] (3) Selection of stirring speed With other conditions kept constant, the stirring speeds during formulation were set to 300, 500, 800, 1000, and 1200 rpm, respectively. Hybrid nanoassemblies were prepared according to the aforementioned method, and their formulation properties were investigated. The results are shown in Table 3. It can be seen that the optimal particle size was achieved when the stirring speed was 1000 rpm.

[0062] Table 3. Particle size and particle size distribution of nanoassemblies at different stirring speeds

[0063] Based on the optimal organic reagents, organic-to-aqueous phase ratio, and stirring speed during formulation, hybrid nanoassemblies CSSH / GNAS or CSSF / GNAS with a total CSSH or CSSF and GSP concentration of 1 mg / mL and a CSSH or CSSF and GSP molar ratio of 1:5 were prepared. Self-assembled nanoassemblies CSSF NAs with a CSSF concentration of 1 mg / mL were also prepared. The results are shown in Table 4. Figure 4 As shown, the nanoassemblies CSSF NAs, CSSH / GNAs, and CSSF / GNAs all have particle sizes of approximately 90 nm and exhibit high drug loading. In Table 4, CTX refers to cabazitaxel, and GSP refers to gossypol. Furthermore, transmission electron microscopy (TEM) reveals that the hybrid nanoassemblies are uniformly spherical. Figure 5 ).

[0064] Table 4. Particle size, particle size distribution, zeta potential, and drug loading of nanoassemblies

[0065] Example 4 Assembly mechanism analysis of CSSF / GNAs nanoassemblies The molecular interaction between CSSF and gossypol was investigated using molecular docking simulation technology on the YInfotek cloud computing platform (http: / / cloud.yinfotek.com / ), exploring the driving mechanism for their assembly into nanoparticles. The chemical structures of CSSF and gossypol were plotted using Chemdraw 17.1 software, then converted to 3D structures. Energy minimization was then performed under an MFF94 force field, and finally, semi-flexible docking was performed using the AutoDock Vina program to output the optimal conformation and corresponding assembly forces.

[0066] Simultaneously, an experiment was conducted to disrupt intermolecular forces. Specifically, 1 mL of CSSF / GNAs with a concentration of 1.0 mg / mL was added to 9 mL of SDS (10 mM) or Urea (10 mM) and incubated in a constant temperature shaker (37°C, 100 rpm) for 2 h. The particle size change of the nanoassemblies before and after incubation was measured.

[0067] like Figure 6 As shown, molecular docking simulation results indicate that the assembly driving forces between CSSF and gossypol are mainly hydrophobic interactions and hydrogen bonds. Furthermore, the particle size of CSSF / GNAs increased in both SDS and Urea solutions, with a more significant increase in SDS solution, suggesting that hydrophobic interactions are the primary intermolecular driving force for their spontaneous assembly into hybrid nanoassemblies.

[0068] Example 5 Oxygen-carrying capacity of nanoassemblies A portable fluorescence dissolved oxygen analyzer was used for the investigation. Specifically, hybrid nanoassemblies (CSSF NAs) self-assembled from the cabazitaxel prodrug molecule CSSF, hybrid nanoassemblies (CSSH / G NAs) co-assembled from the control prodrug CSSH and gossypol were oxygenated for 30 min at an oxygen flow rate of 5 L / min. The corresponding oxygen-carrying agents were named CSSFO NAs, CSSHO / G NAs, and CSSFO / G NAs, respectively. The particle size of the agents before and after oxygenation was measured using a Malvern particle size analyzer. Then, 5 mL each of phosphate buffer (pH 7.4), CSSFO NAs, CSSHO / G NAs, and CSSFO / G NAs (with an equivalent concentration of 2 μmol / mL of perfluorohexylethyl alcohol or 1-octanol) were added to 15 mL of deoxygenated deionized water. Oxygen release was triggered by ultrasound, and the changes in oxygen concentration were quickly recorded using a portable fluorescence dissolved oxygen analyzer. The results are shown in Table 5. Figure 7 As shown in Table 5, the particle size of the formulation did not change significantly before and after oxygenation. Figure 7The results show that CSSFO NAs and CSSFO / G NAs have comparable oxygen-carrying capacity, indicating that co-assembly with gossypol does not affect the oxygen-carrying capacity of the prodrug CSSF. Furthermore, for CSSH / G NAs, which theoretically lack oxygen-carrying capacity, an increase in oxygen concentration was observed after oxygenation treatment, possibly due to slight oxygen adsorption between the colloidal particles and water.

[0069] Table 5. Particle size and particle size distribution of nanoassemblies before and after oxygenation

[0070] Example 6 Colloidal stability study of CSSF / GNAs Particle size was used as the evaluation index to investigate the stability of the formulation in PBS (pH 7.4). The specific procedure was as follows: 1 mL each of 1.0 mg / mL CSSF NAs, CSSH / G NAs, and CSSF / G NAs were added to 9 mL of PBS (pH 7.4) and incubated in a constant temperature shaker (37℃, 100 rpm). The particle size change was measured at 0, 0.5, 1, 2, 4, 6, 8, and 12 h. The results are shown below. Figure 8 As shown, the particle size did not change significantly within 12 hours, indicating that CSSF / GNAs have good colloidal stability.

[0071] Example 7 In vitro activation and drug release behavior of CSSF in CSSF / GNAS Using PBS (pH 7.4) containing 20% ​​tetrahydrofuran (v / v) as the release medium, high performance liquid chromatography (HPLC) was employed to detect the release behavior of the parent drug cabazitaxel from nanoassemblies at different dithiothreitol (DTT) concentrations. Specifically, 1 mL each of CSSF / GNAPs and CSSFO / GNAPs (cabazitaxel equivalent mass 200 μg) were added to 30 mL of release medium containing 0, 1, or 5 mM DTT, respectively, and incubated in a constant temperature shaker (37℃, 100 rpm). At pre-set time points of 0, 0.5, 1, 2, 4, 6, 8, and 12 h, 200 μL of release medium was collected to determine the cabazitaxel release amount, and 0.2 mL of release medium was immediately added after each collection. The chromatographic conditions were as follows: mobile phase: acetonitrile / water (V / V) 70:30; column: Cosmosil-C18 column (150mm×4.6 mm, 5 μm); flow rate: 1 mL / min; column temperature: 30℃; injection volume: 10 μL.

[0072] The results are as follows Figure 9As shown, in the absence of DTT, the cumulative release of cabazitaxel is less than 10%; when the DTT concentration is 1 mM, the cumulative release of cabazitaxel is approximately 75%, and the release rate increases significantly; when the DTT concentration is 5 mM, the cumulative release of cabazitaxel from both formulations exceeds 90%. Therefore, it can be concluded that the release mode of cabazitaxel by CSSF / GNAs and CSSFO / GNAs is reduction-responsive, and oxygenation of the formulation has almost no effect on the release behavior.

[0073] Example 8 Cellular uptake of CSSFO / GNAs 4T1 cells were fed at a rate of 2 × 10 5 Cells were seeded at a density per well in 24-well cell culture plates with built-in crawler slides and cultured at 37°C for 16 h. The cell culture plates were then removed, the culture medium discarded, and fresh drug-containing culture medium containing 200 ng / mL of coumarin-6 (C6) equivalent concentration of C6 Sol and C6-labeled nanoformulations (C6-CSSF NAs, C6-CSSFO NAs, C6-CSSH / GNAs, C6-CSSF / GNAs, and C6-CSSFO / GNAs) was added, respectively. The cells were incubated at 37°C for 0.5 h and 2.0 h, respectively. Discard the culture medium, wash the cells three times with 4°C PBS (pH 7.4), fix the cells with 4% paraformaldehyde for 10 min, then add 0.2 mL of 100 nM Hoechst to stain the cell nuclei for 10 min. Take out the smear, place it upside down on a glass slide with anti-fluorescence attenuation mounting medium, observe the results and take pictures using a laser confocal microscope.

[0074] 4T1 cells were fed at a rate of 2 × 10 5 Cells were seeded at a density of 12-well cell culture plates and cultured at 37°C in a CO2 incubator for 16 h. The culture medium was discarded, and the cells were washed three times with serum-free RPMI-1640 medium. Then, drug-containing culture media containing 200 ng / mL of C6-equivalent C6 Sol, C6-CSSF NAs, C6-CSSFO NAs, C6-CSSH / GNAs, C6-CSSF / GNAs, and C6-CSSF / GNAs were added, and the cells were incubated at 37°C for 0.5 h and 2.0 h, respectively. Discard the culture medium, wash the cells three times with 4°C phosphate buffer (pH 7.4), add trypsin digestion solution to digest the cells and collect the cells, centrifuge (4°C, 1000 rpm) for 3 min, discard the supernatant, add phosphate buffer (pH 7.4) to resuspend the cells, filter through a 70 μm cell filter membrane and add to flow cytometry tubes, and quantitatively detect the uptake of drugs by the cells using flow cytometry.

[0075] The results are as follows Figure 10 and Figure 11 As shown, at 0.5 h and 2.0 h, the fluorescence intensity of cells treated with the nanoassemblies was higher than that of C6 Sol, indicating that tumor cells had a higher uptake efficiency of the nanoassemblies than the solution. Furthermore, the cellular uptake efficiency of the nanoassemblies increased with time, exhibiting a time-dependent effect. Notably, there was no significant difference in cellular uptake efficiency among C6-CSSFNAs, C6-CSSFONAs, C6-CSSF / GNAs, and C6-CSSFO / GNAs. This is because the nanoassemblies have similar particle size and surface properties, and oxygen loading did not affect their uptake. However, the cellular uptake efficiency of C6-CSSH / GNAs was relatively lower. This may be due to the relatively poor stability of CSSH / GNAs; the nanoassemblies were unstable during incubation with cells, resulting in less uptake by tumor cells.

[0076] Example 9 Cytotoxicity of CSSFO / GNAs Using 4T1 cells as a cell model, the MTT assay was used to evaluate the cytotoxicity of CSSFP / GNAs. Cells (1.5 × 10⁻⁶ cells) were... 3 Cells (per well) were seeded in 96-well plates and cultured for 16 h under normoxic or hypoxic conditions. The culture medium was discarded, and a series of drug-containing culture media containing CSSF NAs, CSSFO NAs, CSSF / GNAs, CSSFO / GNAs, and CSSH / GNAs at different concentrations were added. After addition, the normoxic group cells were cultured for another 48 h under normoxic conditions; the hypoxic group cells were cultured for 12 h under hypoxic conditions, followed by another 36 h under normoxic conditions. At the end of culture, 5 mg / mL of the drug was added to each well. -1 0.02 mL of MTT solution was added and incubated for 4 h. After incubation, the culture medium was discarded, and 0.2 mL of DMSO was added to each well. The wells were then shaken for 10 min to dissolve the blue-purple crystals. The absorbance of each well was measured at 490 nm using a microplate reader after zeroing.

[0077] The results are as follows Figure 12 As shown, it can be seen that, for both normoxic and hypoxic tumor cells, the formulation introducing a non-toxic dose of gossypol exhibits stronger cytotoxicity than the corresponding formulation without gossypol. Even though CSSH / G NAs have relatively poor stability, their cytotoxicity is still stronger than CSSF NAs and CSSFO NAs, indicating that the hybrid nanoassemblies of gossypol and cabazitaxel prodrugs (CSSF or CSSH) can effectively exert the sensitizing effect of gossypol on cabazitaxel, thereby significantly improving the antitumor effect of cabazitaxel.

[0078] Example 10 Investigation on the effect of gossypol in downregulating Bcl-2 protein Once the cells in the culture dish have grown to approximately 80% confluence, discard the culture medium. Add 1 mL of trypsin digestion solution to pre-digest the cells, discard the digestion solution, and then add another 1 mL of digestion solution to formally digest the cells. After 1 min, add 4 mL of culture medium to terminate the digestion. Centrifuge at 1000 rpm for 3 min and discard the supernatant. Resuspend the cells in culture medium to obtain a concentration of approximately 2 × 10⁻⁶ cells / mL. 6 Cell suspension at a concentration of cells / mL was used. 2 mL of the cell suspension was added to 10 mL of culture medium and cultured for 24 h. The old culture medium was discarded, and fresh culture medium was added to dilute cabazitaxel solution (CTX Sol), gossypol solution (GSP Sol), CSSF NAs, CSSF / GNAs, and CSSH / GNAs, respectively. The equivalent concentration of cabazitaxel was 10 nM, and the equivalent concentration of gossypol was 50 nM. The cells were cultured for another 48 h. RIPA lysis buffer was added to lyse the cells, and the cells were collected and centrifuged. Protein quantification was performed using the BCA colorimetric method on the supernatant. Proteins were separated using a polyacrylamide gel, then transferred to a 0.45 μm PVDF membrane and blocked with irrelevant proteins for 1 h. The membranes were then incubated overnight at 4°C with Bcl-2 and β-Actin primary antibodies, respectively. After incubation, excess antibody was washed away with 1×TBST, and the membrane was incubated with the corresponding secondary antibody at 37°C for 1 h. After washing with 1×TBST, the membrane was developed with ultrasensitive ECL solution.

[0079] The results are as follows Figure 13 As shown, the expression of Bcl-2 protein was significantly reduced in the GSP Sol, CSSF / GNAs, and CSSH / GNAs groups. Analysis indicates that the GSP introduced into the CSSF / GNAs nanoassemblies did not reach a toxic dose, but it could downregulate the expression of the anti-apoptotic protein Bcl-2 in tumor cells, providing favorable conditions for the anti-tumor effect of cabazitaxel and thus achieving a sensitizing effect against cabazitaxel chemotherapy.

[0080] Example 11 Pharmacokinetic characteristics of CSSFO / GNAs DSPE-PEG 2k Replace with Cy7-DSPE-PEG 2kThe remaining procedures were the same for the preparation of nanoassemblies. Male SD rats (weighing 180-220 g) were fasted for 12 h and then administered Cy7 equivalent at 2 mg / kg via tail vein injection. Cy7 Sol, Cy7-CSSF NAs, Cy7-CSSFO NAs, Cy7-CSSH / G NAs, Cy7-CSSF / G NAs, and Cy7-CSSFO / G NAs were administered at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 12 h post-administration. 0.3 mL of blood was collected from the orbital venous plexus and collected in heparin-coated EP tubes. The tubes were centrifuged at 13000 rpm for 3 min, and the pharmacokinetic behavior of each formulation was then detected using an ELISA reader (excitation 750 nm, emission 773 nm). The experimental results are as follows: Figure 14 As shown in Table 6, it can be seen that the Cy7 solution is eliminated from the blood relatively quickly, while the half-life (t) of various nanoassemblies is relatively short. 1 / 2 The time elapsed between the drug-time curves of Cy7-CSSFO and Cy7-GNAs was longer than that of the solution formulation, indicating that the nano-formulations were eliminated from the body more slowly and had a significantly longer blood circulation time. Specifically, the area under the drug-time curve (AUC0-12 h) of Cy7-CSSFO and Cy7-CSSFO NAs was approximately 3.7 times that of the solution formulation. Furthermore, the time elapsed between the drug-time curves of Cy7-CSSFO and Cy7-CSSFO NAs was significantly longer than that of the solution formulation. 1 / 2 There was no significant difference between AUC0-12 h and both were superior to Cy7-CSSH / GNAs, which had poor stability.

[0081] Table 6. Pharmacokinetic parameters of each formulation (n=5)

[0082] Example 12 In vivo distribution of CSSFO / GNAs 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached 300 mm, 3 At the time of administration, Cy7 Sol, Cy7-CSSF NAs, Cy7-CSSFO NAs, Cy7-CSSH / GNAs, Cy7-CSSF / GNAs, and Cy7-CSSFO / GNAs were administered via tail vein injection. Mice were anesthetized and placed in a small animal in vivo imaging system at 2, 4, 6, 8, and 12 h post-administration, and the fluorescence signal intensity at the tumor site was detected at an excitation wavelength of 750 nm. Furthermore, mice were sacrificed 4 h post-Cy7 Sol administration and 8 h post-nanoform administration, and centrifuges, livers, spleens, lungs, kidneys, and tumors were dissected and their fluorescence signal intensity was detected in a small animal in vivo imaging system.

[0083] The results are as follows Figure 15 and Figure 16 As shown, the strongest fluorescence signal in the tumors of mice in the Cy7 solution (Cy7 Sol) group was observed 4 h after drug administration, while the strongest fluorescence signal in the tumors of mice in the Cy7-labeled nanoassemblies group was observed 8 h after drug administration. Compared to the Cy7 Sol group, the nanoassemblies showed stronger fluorescence intensity and longer detectable time at the tumor site, indicating a higher accumulation of nanoassemblies at the tumor site, which is attributed to the longer blood circulation time of the nanoassemblies. Furthermore, no significant difference was observed in the accumulation of the fluorescently labeled nanoassemblies Cy7-CSSF NAs, Cy7-CSSFO NAs, Cy7-CSSF / GNAs, and Cy7-CSSFO / GNAs at the tumor site, while the less stable Cy7-CSSFO / GNAs showed relatively weaker fluorescence signal at the tumor site, indicating a lower accumulation at the tumor site.

[0084] Example 13 In vivo antitumor efficacy and safety of CSSFO / GNAs Using 4T1 tumor-bearing Balb / c mice (20-22 g) as a model, the in vivo antitumor activity of CSSFO / GNAs was studied. The tumor volume in the mice was increased to 100 mm. 3 Mice were administered PBS (pH 7.4), gossypol solution (GSP Sol), a mixture of CSSF and gossypol (CSSF / G Sol, CSSF to GSP molar ratio of 1:5), cabazitaxel solution (CTX Sol), CSSF NAs, CSSFONAs, CSSH / G NAs, CSSF / G NAs, and CSSFO / G NAs via tail vein injection every two days for a total of four administrations. Mouse body weight and tumor volume changes were measured and recorded daily. From the start of administration, the major axis a (mm) and minor axis b (mm) of the tumor in the tumor-bearing mice were measured daily using calipers. The tumor volume was calculated using the formula V = 0.5 × a × b × b, and a trend graph of tumor volume change over time, i.e., the tumor growth curve (Vt), was plotted. The mouse body weight was measured and recorded one day after the last administration. Mice were euthanized, tumor tissue was dissected, photographed, and the tumor weight was measured. The tumor bearing rate (%) was calculated using the formula: Tumor bearing rate (%) = Tumor weight / Body weight of tumor-bearing mouse × 100%. After dissection, the tumor tissue was simply rinsed with physiological saline and then fixed in 4% paraformaldehyde. Subsequently, TUNEL and H&E staining were used to evaluate tumor cell apoptosis and / or necrosis in mice of different drug administration groups. The results are as follows: Figure 17 , Figure 18 , Figure 19 and Figure 20As shown, the antitumor effects of oxygenated CSSFO NAs and CSSFO / G NAs are stronger than those of their corresponding non-oxygenated counterparts (CSSF NAs and CSSF / G NAs), indicating that the oxygen they carry can alleviate the limitations of hypoxia on the efficacy of cabazitaxel to some extent. Furthermore, CSSFO / G NAs infused with gossypol exhibit better antitumor effects than CSSFO NAs, suggesting that gossypol can increase the sensitivity of tumor cells to cabazitaxel.

[0085] Mouse body weight was used as a preliminary indicator for drug safety evaluation. From the start of administration, the body weight of tumor-bearing mice was measured daily, and a weight-time curve was plotted. Before euthanasia, blood was collected by removing one eyeball with surgical forceps, centrifuged at 13,000 rpm for 10 min, and the supernatant was collected. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine were measured to evaluate the drug's hepatotoxicity and nephrotoxicity. Mice were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) were dissected. After a simple rinse with physiological saline, the organs were fixed in 4% paraformaldehyde, and organ lesions were evaluated by H&E staining. Results are as follows: Figure 21 , Figure 22 and Figure 23 As shown, CSSFO / GNAs have good safety profiles. Mice did not lose weight during treatment, and no significant abnormalities were observed in the sections of major organs (heart, liver, spleen, lungs, and kidneys) or in liver and kidney function indicators.

Claims

1. A hybrid nanoassembly for dual-effect chemotherapy, characterized in that: The nanoassemblies are co-assembled from cabazitaxel and gossypol under hydrophobic interaction and hydrogen bonding, and modified with PEG modifier. The molar ratio of the carbamazepine pre-race drug to gossypol is 1:1~7, and the total mass ratio of the carbamazepine pre-race drug and gossypol to the PEG modifier is 4:

1. The structural formula of the carbazita pre-race drug is shown below: Where R is or .

2. The hybrid nanoassembly according to claim 1, characterized in that: The PEG modifier is DSPE-PEG. 2k Cy7-labeled DSPE-PEG 2k Or coumarin-6 labeled DSPE-PEG 2k .

3. The hybrid nanoassembly according to claim 1, characterized in that: The cabazitaxel prodrug is prepared by the following method: First, acetic anhydride is used as a solvent and initiator to react 2,2'-dithiodiacetic acid to the corresponding anhydride; then, the anhydride is esterified with perfluorohexylethyl alcohol or 1-octanol to generate an intermediate product, and the reaction mixture is separated and purified by column chromatography; finally, cabazitaxel is added to the reaction system to esterify with the intermediate product to generate the final product, which is then separated and purified by preparative liquid chromatography.

4. The hybrid nanoassembly according to claim 3, characterized in that: In the reaction of 2,2'-dithiodiacetic acid to anhydride via the dehydration of acetic anhydride, the reaction temperature is 20~40℃ and the time is 2~8h; In the reaction of acid anhydride with perfluorohexylethyl alcohol or 1-octanol, the molar ratio of acid anhydride to perfluorohexylethyl alcohol or 1-octanol is 1~3:1, the catalyst is 4-dimethylaminopyridine, the molar ratio of perfluorohexylethyl alcohol or 1-octanol to catalyst 4-dimethylaminopyridine is 10:0.1~1, the reaction temperature is 20~40℃, and the time is 10~24 h; In the esterification reaction of the intermediate with cabazitaxel, the solvent was dichloromethane, and the catalysts were 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine. The molar ratio of cabazitaxel to the intermediate was 1:1 to 10. The molar numbers of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine were the same, and their molar ratio with cabazitaxel was 1 to 3:

1. The reaction temperature was 20 to 40 °C, and the reaction time was 48 to 72 h.

5. A method for preparing the hybrid nanoassemblies according to any one of claims 1 to 4, characterized in that: Cabazitaxel, gossypol, and PEG modifier were dissolved in organic solvents and mixed thoroughly. The mixture was then slowly added dropwise to deionized water under stirring, allowing it to spontaneously assemble into co-assembled nanoparticles with uniform particle size. The organic solvent was removed by vacuum distillation to obtain the final product.

6. The preparation method according to claim 5, characterized in that: The organic solvent is one or both of anhydrous ethanol and tetrahydrofuran.

7. The preparation method according to claim 5, characterized in that: The volume ratio of the organic solvent to deionized water is 1:2~20.

8. The preparation method according to claim 5, characterized in that: The stirring speed is 300~1200 rpm.

9. A method for preparing an oxygen-carrying agent, characterized in that: The hybrid nanoassembly according to claim 1 is subjected to oxygenation treatment, wherein the oxygen flow rate is 0.1~10 L / min and the oxygenation time is 5~60 min during the oxygenation process.

10. The use of the hybrid nanoassembly of claim 1 or the oxygen-carrying agent prepared by the method of claim 9 in the preparation of antitumor drugs.

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