Paclitaxel inclusion compound as well as preparation method and application thereof

Abstract

The invention discloses a paclitaxel clathrate compound as well as a preparation method and application thereof. The paclitaxel clathrate compound is formed by clathration of a bovine serum albumin-gamma-cyclodextrin compound and paclitaxel. The preparation method of the paclitaxel inclusion compound comprises the following steps: (1) synthesizing gamma-bromo-amide cyclodextrin; (2) preparing a bovine serum albumin-gamma-cyclodextrin compound; and (3) preparing the BSA-CD-PTX inclusion compound. The invention also discloses application of the paclitaxel clathrate compound in preparation of medicines for treating tumors. According to the paclitaxel inclusion compound, through a dual inclusion synergistic mechanism of a BSA hydrophobic pocket and a gamma-CD cavity, the solubilization limitation of a single carrier is remarkably broken through, the water solubility of paclitaxel is improved to 35 mM, and the drug loading capacity is synchronously optimized to 28%; premature release of drugs can be effectively avoided, explosive release in tumor cells can be potentially achieved, and the tumor cell killing activity of the compound is superior to that of an existing preparation. The preparation method of the paclitaxel clathrate compound is simple to operate and convenient for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a complex that enhances the water solubility of paclitaxel, its preparation method, and its application. Background Technology

[0002] Paclitaxel (PTX), a diterpenoid natural product isolated from the bark of the yew tree, has become a first-line chemotherapy drug for advanced ovarian cancer, breast cancer, and skin cancer due to its unique mechanism of action of accelerating microtubule polymerization and inhibiting mitotic spindle formation. However, paclitaxel's hydrophobic properties are extremely prominent, with a water solubility of only 0.25~0.60 μg / mL, making it almost insoluble in commonly used pharmaceutical solvents. This core defect has become a key bottleneck restricting its clinical application.

[0003] To solve the dissolution problem of paclitaxel, the first clinical formulation, Taxol, was developed. ® Using Cremophor EL ® Prepared with a 1:1 (v / v) mixed solvent of ethanol, but Cremophor EL in this formulation ® It can cause hypersensitivity reactions in up to 40% of patients, requiring pretreatment with corticosteroids and antihistamines, severely impacting medication safety. The subsequently marketed albumin-bound formulation Abraxane™, while achieving targeted accumulation in tumor tissue through albumin nanosuspension technology, suffers from chronic toxicity due to residual chloroform from its preparation process, and single-dose treatment costs tens of thousands of yuan, placing a heavy financial burden on patients. Furthermore, it still carries the risk of myelosuppression due to uneven biodistribution and an excessively long half-life (21.6 h) (binding constant 12000M). -1 Other modified formulations, such as the paclitaxel-glutamate polymer conjugate Xyotax™, have been discontinued due to their inability to effectively dissociate into free drug, resulting in insufficient clinical efficacy and significant hematologic toxicity. This demonstrates that a single carrier system cannot simultaneously resolve the multiple contradictions between solubility, toxicity, and bioavailability.

[0004] Cyclodextrin (CD), a classic molecular complexing agent, uses its conical cavity (hydrophobic core / hydrophilic surface) to encapsulate hydrophobic molecules through van der Waals forces and hydrogen bonds, and has been widely used for the solubilization and modification of poorly soluble drugs. γ-Cyclodextrin (γ-CD), containing eight glucose units, has a cavity size (inner diameter 0.75 nm) that closely matches the molecular structure of paclitaxel, enabling complete drug molecule embedding. However, the binding constant of the inclusion complex formed by unmodified γ-CD and paclitaxel is only 785 M. -1 Modifiers such as hydroxypropyl-β-cyclodextrin (HP-β-CD) can increase the solubility of paclitaxel by 2 × 10⁻⁶.3 CN103920163A discloses a paclitaxel inclusion complex, its preparation method, and its application. It utilizes the acylation between cyclodextrin and acryloyl chloride to first synthesize acrylated cyclodextrin. Then, cyclodextrin-modified polyacrylic acid is prepared directly in aqueous solution via free radical copolymerization. The modified polyacrylic acid, when combined with paclitaxel, rapidly increases the solubility of paclitaxel in water, raising it from 0.25 mg / mL to 3.6 mg / mL, a solubility increase of 14,400 times. However, this type of single cyclodextrin carrier still suffers from unsatisfactory biodistribution and insufficient targeting.

[0005] Serum albumin, as the main protein in vertebrate plasma, has advantages such as strong conformational adaptability, biodegradability, and mature preparation processes. Among them, bovine serum albumin (BSA) has become the preferred material for drug carrier research due to its low cost, availability, 17 stable disulfide bonds, high lysine content, and stable physicochemical properties. BSA nanoparticles prepared by Kono et al. via intermolecular disulfide bonds exhibited superior colloidal stability and sustained drug release characteristics compared to Abraxane™. However, the solubilization capacity of albumin carriers alone is limited, and they lack a specific targeting mechanism for tumor tissues. Although linear oligosaccharide-albumin complexes (such as dextran-BSA) can synergistically solubilize, the linear glycan chains lack specific cavitary structures and only bind through hydrogen bonds and hydrophobic interactions. The solubilization factor is only 1.8-2.3 times higher than that of albumin alone, which is insufficient to meet clinical needs.

[0006] Currently, there are no in-depth reports on the solubilizing effect of "cyclic sugar-protein" complex carriers on paclitaxel. As a cyclic oligosaccharide, cyclodextrin's unique cavity structure synergistically interacts with the hydrophobic binding sites of albumin, and it is expected to construct a "double inclusion" system, breaking through the solubilization bottleneck of single carriers. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a paclitaxel inclusion complex with a simple preparation method and significant solubilizing effect, as well as its preparation method and application.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] A paclitaxel inclusion complex is formed by the inclusion of paclitaxel in a bovine serum albumin-γ-cyclodextrin complex (abbreviated as BSA-CD complex).

[0010] The bovine serum albumin-γ-cyclodextrin complex and paclitaxel are mixed in equal volumes and then encapsulated in an ice bath with a concentration ratio ≥1:80. Preferably, the concentration ratio of bovine serum albumin-γ-cyclodextrin complex to paclitaxel is 1:40 to 1:25. The applicant verified through experiments that when bovine serum albumin-γ-cyclodextrin complex and paclitaxel were mixed in equal volumes for inclusion, the drug loading of the paclitaxel inclusion complex was greater than 20% when the concentration ratio of bovine serum albumin-γ-cyclodextrin complex to paclitaxel was 1:40-1:25, and its maximum water solubility of PTX was greater than 30 mM, which is much greater than the maximum water solubility of PTX of inclusion complexes obtained by encapsulating paclitaxel with bovine serum albumin or γ-cyclodextrin alone (20 mM). Secondly, when the concentration ratio of serum albumin-γ-cyclodextrin complex to paclitaxel was 3:50 and the mixture was mixed in equal volumes for inclusion, the maximum water solubility of the product BSA-CD-PTX inclusion complex was as high as 46 mM, indicating that the bovine serum albumin-γ-cyclodextrin complex at 0.1 M... Under the weak alkalinity of NaHCO3 buffer, paclitaxel can fully enter the hydrophobic pocket of BSA and the hydrophobic cavity of γ-CD, exhibiting the combined inclusion effect of BSA and γ-CD, resulting in a synergistic "double inclusion" effect, which significantly improves the inclusion effect of paclitaxel and the maximum water solubility of PTX.

[0011] The bovine serum albumin-γ-cyclodextrin complex has a molecular weight of 68 kDa, and the molar ratio of bovine serum albumin to γ-cyclodextrin is 1:9.

[0012] Another technical solution adopted by the present invention to solve its technical problem is:

[0013] A method for preparing a paclitaxel inclusion complex includes the following steps:

[0014] 1) Synthesis of γ-bromoamide cyclodextrin (γ-CD-Br)

[0015] The 6-hydroxyl group of γ-CD was sulfonated with p-toluenesulfonyl chloride, then the p-toluenesulfonyl group was replaced with the azide group of sodium azide, the azide group was hydrolyzed to an amino group, and finally a short alkyl chain γ-CD-Br was synthesized by bromination reaction.

[0016] 2) Preparation of bovine serum albumin-γ-cyclodextrin complex (BSA-CD complex)

[0017] Add BSA (25 mg, 0.375 μmol) to the reaction flask and dissolve it in 2.5 mL of 10 mM~0.1 M NaHCO3 buffer under ice bath. Add TCEP·HCl (0.3 mg, 1.125 μmol) and react under N2 on ice bath for 10 min. Add solid γ-CD-Br (5 mg, 3.375 μmol) and react gently with stirring under N2 protection in ice bath and darkness for 12 h. Monitor the reaction for completeness with Ellman's reagent. After lyophilization, obtain the product BSA-CD complex. Ultrafilter the BSA-CD complex once using a 3 kDa ultrafiltration tube (6000 x g × 25 min), leaving approximately 20 mM NaHCO3 buffer for later use.

[0018] 3) Preparation of BSA-CD-PTX inclusion complex

[0019] A 1 mM paclitaxel (PTX) solution dissolved in ethanol was mixed with a 0.1 mM BSA-CD complex dissolved in water at an equal volume ratio (e.g., a volume ratio of 1:1). The mixture was shaken in the dark at 30°C and 200 rpm for more than 60 hours to achieve equilibrium. After the mixture was purged with nitrogen to remove the ethanol, the free paclitaxel was removed by centrifugation, and the supernatant was freeze-dried to obtain a white powder, which is the prepared BSA-CD-PTX inclusion complex.

[0020] In step 1), the specific operation for synthesizing the γ-cyclodextrin derivative is as follows: First, γ-CD is dissolved in pyridine and added to a reaction flask. A solution of p-toluenesulfonyl chloride dissolved in pyridine is added dropwise under N2. After the addition is complete, the reaction is carried out at room temperature. After the reaction, 10 times the volume of acetone is added to precipitate the product. The product is then washed more than twice with acetone, ethanol, and diethyl ether, respectively. Finally, it is dried in a vacuum drying oven at 50°C to obtain γ-p-toluenesulfonated cyclodextrin (γ-CD-OTs).

[0021] Then, γ-CD-OTs were dissolved in 4 mL of N,N'-dimethylformamide (DMF) and added to a reaction flask. Sodium azide was added, and the mixture was refluxed under N2. Precipitation, washing, and drying were performed as in the previous step to obtain γ-azidocyclodextrin (γ-CD-N3).

[0022] γ-CD-N3 was dissolved in DMF and added to the reaction flask. Triphenylphosphine dissolved in DMF was added dropwise under N2. After the addition was complete, the reaction was carried out at room temperature for more than 3 hours. Deionized water was added dropwise while the temperature was raised to the specified temperature. After the addition was complete, the reaction was refluxed for more than 6 hours. Acetone was added to the reaction solution to precipitate the product. The product was then washed twice with acetone and ether, respectively, and dried in a vacuum drying oven at 50°C to obtain γ-aminocyclodextrin (γ-CD-NH2).

[0023] Finally, γ-CD-NH2 was dissolved in DMF solution, and the acid-binding agent triethylamine was added and stirred in an ice bath. Then, bromoacetyl bromide was added and stirred in an ice bath. The reaction was carried out at room temperature in the dark for 5 hours. The precipitation, washing and drying were the same as in the previous step to obtain the product γ-CD-Br.

[0024] The technical solution further adopted by the present invention to solve its technical problem is as follows:

[0025] Application of paclitaxel inclusion complexes in the preparation of drugs for treating tumors.

[0026] The beneficial effects of this invention, including a paclitaxel inclusion complex, its preparation method, and its application, are as follows:

[0027] The present invention uses the BSA-CD complex as a drug carrier for paclitaxel. A four-step site-specific synthesis strategy is used to prepare γ-bromoamide cyclodextrin (γ-CD-Br), and the reactive thiol at the Cys34 site on the BSA surface is used to achieve efficient grafting (grafting rate up to 82%), forming a "cyclic sugar-protein" hybrid system that combines protein biocompatibility and cyclodextrin molecule inclusion properties.

[0028] This BSA-CD complex, used as a drug carrier, utilizes a synergistic "dual inclusion" mechanism between the hydrophobic pocket of BSA and the cavity of γ-CD. Under optimized processing (0.1 M NaHCO3 buffer as solvent, single ultrafiltration with a 3 kDa ultrafiltration tube, and 60 h of inclusion), it significantly overcomes the solubility limitations of single carriers, increasing the water solubility of paclitaxel to 35 mM, far superior to the clinical formulation Abraxane™, while simultaneously optimizing the drug loading to 28%. Structural characterization confirmed the successful construction of the BSA-CD complex and the stable inclusion of paclitaxel. In vitro release experiments revealed its unique pH-responsive characteristics, which can effectively avoid premature drug release and potentially achieve "burst release" within tumor cells. MTT assays verified the biocompatibility of the carrier and the superior tumor cell killing activity of the inclusion complex compared to free paclitaxel and Abraxane™.

[0029] The preparation method of this paclitaxel inclusion complex is simple and easy to carry out on a large scale for industrial production.

[0030] The paclitaxel inclusion complex prepared by this invention provides a new and feasible approach for the clinical application of paclitaxel as an anticancer / antitumor drug, and also provides a new preparation method for the preparation of antitumor drugs containing paclitaxel. Attached Figure Description

[0031] Figure 1 —This is a synthetic route diagram of γ-bromoamide cyclodextrin in the preparation method of a paclitaxel inclusion complex in Example 1;

[0032] Figure 2—A structural characterization diagram of the γ-bromoamide cyclodextrin in Example 1;

[0033] Among them: (A) MALDI-TOF-MS images of (a) γ-CD-OTs, (b) γ-CD-N3, (c) γ-CD-NH2, and (d) γ-CD-Br;

[0034] (B) FTIR images of (a) γ-CD-OTs, (b) γ-CD-N3, (c) γ-CD-NH2, and (d) γ-CD-Br;

[0035] (C) Nuclear magnetic resonance 1 H spectra of (a) γ-CD-OTs, (b) γ-CD-N3, (c) γ-CD-NH2, and (d) γ-CD-Br;

[0036] (D) Nuclear magnetic resonance 13 C spectra of (a) γ-CD-OTs, (b) γ-CD-N3, (c) γ-CD-NH2, and (d) γ-CD-Br.

[0037] Figure 3 —This is a flowchart of the preparation method of the paclitaxel inclusion complex (BSA-CD-PTX) of the present invention;

[0038] Figure 4 —These are the MALDI-TOF-MS and FTIR characterization diagrams of the BSA-CD complex in this invention;

[0039] Among them: (A) MALDI-TOF-MS image of the BSA-CD complex;

[0040] (B) FTIR spectrum of the BSA-CD complex, (a) γ-CD; (b) BSA; (c) BSA-CD.

[0041] Figure 5 —This is a graph showing the effect of different raw materials' aqueous solutions and NaHCO3 solutions on the encapsulation efficiency, drug loading, and water solubility of paclitaxel after being mixed with them.

[0042] Figure 6 —This is a graph showing the effect of the BSA-CD complex obtained by the preparation method of a paclitaxel inclusion complex in Example 1 on the encapsulation efficiency, drug loading and water solubility of paclitaxel.

[0043] Among them: (A) Encapsulation efficiency and drug loading of BSA-CD containing different concentrations of paclitaxel;

[0044] (B) Maximum water solubility of PTX containing different concentrations of paclitaxel in BSA-CD;

[0045] Figure 7 —Example 1: A method for preparing paclitaxel inclusion complexes. Structural characterization diagram of the paclitaxel inclusion complexes obtained (I).

[0046] Among them: (A) UV absorption spectra of BSA-CD-PTX inclusion complexes at different reaction times;

[0047] (B) Fluorescence emission spectra of BSA-CD-PTX inclusion complexes at different reaction times;

[0048] (C) Circular dichroism chromatogram of the BSA-CD-PTX inclusion complex;

[0049] (D) DSC diagram of the BSA-CD-PTX inclusion complex.

[0050] Figure 8 —Example 1: A method for preparing paclitaxel inclusion complexes. Structural characterization diagram of the paclitaxel inclusion complexes obtained (II).

[0051] Among them: (A) FTIR spectrum of BSA-CD-PTX inclusion complex;

[0052] (a) γ-CD; (b) BSA; (c) PTX; (d) BSA-CD; (e) a physical mixture of BSA-CD and PTX; (f) a BSA-CD-PTX inclusion complex;

[0053] (B) FTIR spectra of γ-CD-PTX and BSA-PTX inclusion complexes;

[0054] (a) γ-CD; (b) BSA; (c) PTX; (d) a physical mixture of γ-CD and PTX; (e) a physical mixture of BSA and PTX; (f) γ-CD-PTX (water); (g) BSA-PTX (water); (h) γ-CD-PTX (0.1 M NaHCO3); (i) BSA-PTX (0.1 M NaHCO3);

[0055] (C) NMR of BSA-CD-PTX inclusion complex 1 H spectrum; (a) PTX; (b) γ-CD-Br; (c) BSA-CD-PTX inclusion complex;

[0056] (D) 2D ROSEY plot of BSA-CD-PTX inclusion complex.

[0057] Figure 9 - Scanning electron micrograph of the paclitaxel inclusion complex (BSA-CD-PTX) of the present invention; wherein:

[0058] (A) PTX; (B) BSA-CD; (C) Physical mixture of BSA-CD complex and PTX; (D) BSA-CD-PTX inclusion complex.

[0059] Figure 10 - For the BSA-CD-PTX inclusion complex and Abraxane of the present invention TM In vitro release curves of PTX at 37°C for 72 h in PBS buffer (pH 7.4) and acetate buffer (pH 5.5);

[0060] Figure 11 - For the BSA-CD-PTX inclusion complex, free PTX and Abraxane of the present invention TM In vitro cytotoxicity after 48 hours of incubation in B16F10 cells;

[0061] Figure 12 - Microscopic images (×10) of 10 μM drug after 48 hours of incubation in B16F10 cells;

[0062] Among them: (A) negative control; (B) BSA-CD-PTX inclusion complex; (C) free PTX; (D) Abraxane TM .

[0063] Figure 13 - The maximum water solubility of the BSA-CD complex containing 1mM paclitaxel at different reaction times obtained by the preparation methods of paclitaxel inclusion complexes in Examples 1-4;

[0064] Among them: (A) Maximum water solubility of the BSA-CD complex prepared in Examples 2 and 3 containing 1mM paclitaxel at different reaction times;

[0065] (B) Maximum water solubility of the BSA-CD complexes prepared in Examples 1 and 4 containing 1 mM paclitaxel at different reaction times.

[0066] Figure 14 - This is an analytical graph showing the encapsulation efficiency, drug loading, and maximum water solubility of paclitaxel encapsulated in the BSA-CD complex of this invention at different concentrations; wherein:

[0067] (A) Encapsulation efficiency and drug loading of paclitaxel encapsulated in BSA-CD complex at different concentrations;

[0068] (B) Maximum water solubility of PTX containing paclitaxel in different concentrations of BSA-CD complex. Detailed Implementation

[0069] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0070] I. Materials of this invention: Bovine serum albumin was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); γ-cyclodextrin was obtained from Myriel Biochemical Technology Co., Ltd. (Shanghai, China); tris(2-carbonylethyl)phosphohydrochloric acid was purchased from Great Pharmaceutical Technology Co., Ltd. (Suzhou, China); paclitaxel was purchased from Titan Technology Co., Ltd. (Shanghai, China); and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0071] II. Structural characterization methods for γ-CD derivatives, BSA-CD complexes, and BSA-CD-PTX inclusion complexes:

[0072] 1) MALDI-TOF-MS analysis

[0073] γ-CD derivative: 1 μL of water-soluble γ-CD derivative was spotted onto the target plate at the fixed position. After drying, 1 μL of matrix (2,5-dihydroxybenzoic acid, DHB containing Na) was added dropwise. + After drying, the sample is placed on the machine in reflective cation mode; the molecular weight range is set to 1000~2000 Da.

[0074] BSA-CD complex: 1 μL of water-soluble BSA-CD complex sample and 1 μL of matrix (2,5-dihydroxyacetanilide, DHAP) were spotted at the same position on the target plate, mixed thoroughly, dried, and then processed by mass spectrometry. Mass spectrometry conditions: reflectance cation mode; molecular weight range: 25000~75000 Da.

[0075] 2) Ultraviolet spectrophotometer analysis

[0076] All absorption spectra were measured using a UV spectrophotometer (Shimadzu UV-2600) with deionized water as a blank. The UV-vis spectra of BSA-CD-PTX inclusion complexes at different reaction times in the range of 200–330 nm were determined in quartz cuvettes (frosted walls, 1 mm slit, 0.4 mL).

[0077] 3) Fluorescence spectrophotometer analysis

[0078] The fluorescence emission spectra of BSA-CD-PTX inclusion complexes with different reaction times were measured using an F-7000 fluorescence spectrometer (Hitachi, Japan). The excitation wavelength was 270 nm, and a 5 nm excitation and emission slit was used to obtain emission spectra in the range of 290-500 nm.

[0079] 4) Circular dichroism (CD) analysis

[0080] Circular dichroism spectroscopy analysis of 0.2 mg / mL free paclitaxel and its inclusion complex with BSA-CD-PTX in a methanol / water solution (30:70, v / v) was performed using a Jasco J-600D recording spectrophotometer. The circular dichroism spectrum was scanned at wavelengths of 250–350 nm, with a quartz tube path length of 1 mm.

[0081] 5) Differential Scanning Calorimetry (DSC) Analysis

[0082] The thermodynamic stability of the BSA-CD-PTX inclusion complex was analyzed using Mettler Toledo (Switzerland) differential scanning calorimetry (DSC). Approximately 2 mg of sample was placed in an aluminum dish, with a blank aluminum crucible used as a reference. For DSC analysis, measurements were performed in flowing N2 gas at a rate of 50 mL / min, with the sample heated from 10 °C to 300 °C at a rate of 10 °C / min.

[0083] 6) Fourier Transform Infrared (FTIR) Spectroscopy

[0084] A small amount of dry sample powder was placed directly onto the ATR crystal and secured with a pressure bar to ensure tight contact. Subsequently, FTIR (Thermo Nicolet, USA) was used to analyze the sample at 400–4000 cm⁻¹. -1 Infrared spectral analysis was performed within the specified spectral range using a diamond ATR accessory. Acquisition parameters included 32 scans at a resolution of 4 cm⁻¹. -1 Each measurement was performed with a blank spectrum collected against an air background, and the raw data were processed with baseline correction and atmospheric compensation.

[0085] 7) Nuclear magnetic resonance hydrogen spectrum and carbon spectrum ( 1 H NMR, 13 C NMR and 2D ROSEY NMR analysis

[0086] 30 mg of γ-CD derivative and PTX were dissolved in 0.5 mL of deuterated dimethyl sulfoxide (DMSO-d6), and 20 mg of BSA-CD-PTX inclusion complex was dissolved in 0.5 mL of deuterated water (D2O). The solution was obtained at 25 °C. 1 H NMR, 13C10 NMR spectra (Avance III 60, Bruker Instruments Inc., Billerica, MA, USA). To determine the spatial conformation and proximity relationships of intramolecular groups, two-dimensional ROESY spectra were further acquired for the BSA-CD-PTX inclusion complex using a roesyph pulse sequence with a mixing time set to 300 ms. Data processing was then performed using MestReNova version 14.

[0087] 8) Scanning electron microscopy (SEM) analysis

[0088] The morphology of PTX, BSA-CD complex, physical mixture of BSA-CD complex and PTX and BSA-CD-PTX inclusion complex were observed using a scanning electron microscope (FEI, USA). The samples were fixed on a metal plate and sputtered with a 100° thick gold-palladium mixture at an accelerating voltage of 10 kV.

[0089] III. In vitro release studies

[0090] The release behavior of the BSA-CD-PTX inclusion complex was studied using dialysis. In short, due to the different microenvironments of blood circulation and tumors, the release medium consisted of PBS buffer (pH 7.4) containing 0.1% (v / v) Tween 80 and 0.1M acetate buffer (pH 5.5). At 37°±0.5°C, BSA-CD-PTX inclusion complex containing 2 mg of PTX (0.5 mg / mL) and Abraxane™ were placed in dialysis bags (MWCO 10000 Da) and immersed in 200 mL of medium. The mixture was stirred at 150 rpm, and 0.5 mL samples were extracted at 0.25, 0.5, 1, 2, 4, 8, 12, 16, 24, 48, and 72 h. Fresh release medium was added after each sampling. The in vitro release behavior was monitored by HPLC, and the amount of PTX released was calculated.

[0091] IV. In vitro cytotoxicity studies

[0092] In vitro cytotoxicity assays were performed using the MTT assay. Melanoma B16F10 cells were seeded at a density of 5 x 10³ cells / well in 100 μl of 1640++ medium containing 10% FBS and 1% penicillin-streptomycin in 96-well cell culture plates and cultured at 37°C and 5% CO2 for 24 h to allow complete cell adhesion.

[0093] After removing the old culture medium, 100 μl of BSA-CD-PTX inclusion complex, free PTX and Abraxane at concentrations of 0.001, 0.01, 0.1, 1 and 10 μM, prepared with 1640-- medium, and 10 μM drug carrier BSA-CD were added respectively.

[0094] After 48 hours of incubation, 100 μl (5 mg / mL PBS) of MTT solution was added, and the cells were incubated for another 2 hours. At the predetermined time point, the culture medium was aspirated. The generated formazan crystals were dissolved in 100 μl of DMSO. Because the amount of formazan in each well is directly related to the number of viable cells, the absorbance of the sample was measured at 490 nm using a microplate reader. Untreated cells served as a negative control with 100% viability. Cell viability was calculated using the following formula.

[0095]

[0096] Example 1

[0097] Reference Figures 1-7 The method for preparing the paclitaxel inclusion complex in this embodiment includes the following steps:

[0098] 1) Synthesis of γ-bromoamide cyclodextrin (γ-CD-Br)

[0099] according to Figure 1 The synthetic route shown describes the synthesis of a γ-bromoamide cyclodextrin (γ-CD-Br). The 6-hydroxyl group of γ-CD was sulfonated with p-toluenesulfonyl chloride, the p-toluenesulfonyl group was replaced with the azide group of sodium azide, the azide group was hydrolyzed to an amino group, and finally a short alkyl chain γ-CD-Br was synthesized by bromination.

[0100] Specifically, γ-CD (200 mg, 154 μmol) was first dissolved in 2 mL of pyridine and added to a reaction flask. Then, a solution of p-toluenesulfonyl chloride (234 mg, 1232 μmol) dissolved in 2 mL of pyridine was added dropwise under N2. After the addition was complete, the reaction was carried out at room temperature for 5 h. After the reaction, 10 times the volume of acetone was added to precipitate the product. The product was then washed twice with acetone, ethanol, and diethyl ether, respectively, and then dried in a vacuum drying oven at 50 °C to obtain γ-p-toluenesulfonated cyclodextrin (γ-CD-OTs).

[0101] Then, γ-CD-OTs (200 mg, 140 μmol) were dissolved in 4 mL of N,N'-dimethylformamide (DMF) and added to a reaction flask. Sodium azide (90.8 mg, 1400 μmol) was added, and the mixture was refluxed at 100 °C under N2 for 7 h. Precipitation, washing, and drying were performed as in the previous step to obtain γ-azidocyclodextrin (γ-CD-N3).

[0102] γ-CD-N3 (200 mg, 150 μmol) was dissolved in 5 mL of DMF and added to the reaction flask. Triphenylphosphine (80 mg, 300 μmol) dissolved in 5 mL of DMF was added dropwise under N2. After the addition was complete, the reaction was carried out at room temperature for 3 h. The temperature was raised to 90 °C and 2 mL of deionized water was added dropwise. After the addition was complete, the reaction was refluxed for 6 h. Ten times the volume of acetone was added to the reaction solution to precipitate the product. The product was then washed twice with acetone and ether, respectively, and dried in a vacuum drying oven at 50 °C to obtain γ-aminocyclodextrin (γ-CD-NH2).

[0103] Finally, γ-CD-NH2 (100 mg, 77 μmol) was dissolved in 10 mL of DMF solution. Triethylamine (60 μl, 460 μmol), an acid-binding agent, was added and stirred in an ice bath for 15 min to deprotonate the amino group. Then, bromoacetyl bromide (40 μl, 460 μmol) was added and stirred in an ice bath for 10 min. The reaction was carried out at room temperature in the dark for 5 h. The precipitation, washing, and drying were performed as in the previous step to obtain the product γ-CD-Br.

[0104] The product γ-CD-Br prepared above was analyzed using MALDI-TOF-MS, FTIR, and other methods. 1 H NMR and 13 C NMR analysis, results are as follows Figure 2 As shown.

[0105] The primary hydroxyl group at the 6-position of cyclodextrin outside the cavity is highly reactive and can introduce a bromoacetamide group (-NHCO-CH2-Br). The synthesis of γ-CD-Br begins with sulfonation of the 6-position hydroxyl group of γ-cyclodextrin in a pyridine solvent with p-toluenesulfonyl chloride (TsCl) to obtain γ-CD-OTs. Then, reflux at 100°C allows the azide group of sodium azide (NaN3) to replace the p-toluenesulfonyl group, yielding γ-CD-N3. The azide group is then reacted with triphenylphosphine (PPh3) to generate a phosphine imine intermediate, which is hydrolyzed upon heating to obtain an amino group, yielding γ-CD-NH2. Finally, a short-chain γ-CD-Br is synthesized via bromination, with a yield of 35.2%.

[0106] like Figure 2 As shown in -A, the molecular weight of the γ-CD derivatives is consistent with the theoretical value. The γ-CD derivatives are mainly monosubstituted, with small amounts of disubstituted and trisubstituted derivatives. The FTIR spectra are shown below. Figure 2 As shown in Figure -B, γ-CD-OTs exhibit a characteristic peak at 1340 cm⁻¹. -1 and 1025cm -1 Corresponding to the asymmetric stretching vibration of the sulfonyl group S=O and the stretching vibration of SO. γ-CD-N3, 2039 cm⁻¹ -1The characteristic peak is due to the asymmetric stretching vibration of the N=N azide group. The characteristic peak of azide disappears in γ-CD-NH2, and the characteristic peak is at 1646 cm⁻¹. -1 The characteristic of the NH bending vibration indicates that the azide group has been hydrolyzed to an amino group. In γ-CD-Br at 1652 cm⁻¹ -1 and 1543 cm -1 The characteristic peaks correspond to the C=O stretching vibration and NH bending vibration of the amide bond, respectively.

[0107] pass 1 H NMR and 13 C10 NMR analysis further confirmed the structure of the γ-CD derivative.

[0108] like Figure 2 As shown in Figure -C, the signal peaks at δ 7.12–7.49 in γ-CD-OTs correspond to the signal peaks of hydrogen atoms in the benzene ring. γ-CD-N3 contains a strongly electronegative azide group, and the strong electron-withdrawing effect shifts the chemical shift of the hydrogen atom (H-6) attached to the azide group to a higher field, reaching δ 1.05. In γ-CD-NH2, the electron-donating effect of the amino group causes the hydrogen atom (H-6) attached to the amino group to shift to a lower field, reaching δ 1.75. The signal peaks at δ 7.12 and 3.87 in γ-CD-Br correspond to the signal peaks of the secondary amine group and the ortho-alkyl hydrogen atom of bromine, respectively.

[0109] like Figure 2 As shown in Figure -D, γ-CD-OTs exhibit signal peaks at δ 125-145 for the carbon atom of the benzene ring. The strong electron-withdrawing effect of the azido group in γ-CD-N3 shifts the chemical shift of the methylene carbon (C-6) attached to the azido group to a higher field, reaching δ 51. In γ-CD-NH2, the electron-donating effect of the amino group further shifts the chemical shift of the methylene carbon (C-6) attached to the amino group to a higher field, reaching δ 41.7. In γ-CD-Br, the signal peaks at δ 45.8 and 29.5 represent the methylene carbon (C-6) attached to the secondary amine group and the methylene carbon of the bromomethyl group, respectively. Therefore, MALDI TOF-MS mass spectrometry, 1 HNMR, 13 CNMR and FT-IR spectral results confirmed the successful synthesis of γ-CD-Br.

[0110] 2) Preparation of bovine serum albumin-γ-cyclodextrin complex (BSA-CD complex)

[0111] Like cyclodextrin (CD), bovine serum albumin (BSA) is a hydrophilic polymer with excellent properties, making it a preferred delivery carrier for water-insoluble molecules. BSA was chosen as the protein scaffold for binding to γ-CD-Br because its surface contains an active thiol (Cys34), and the grafting reaction is a nucleophilic substitution reaction (SN2) of the thiol group under alkaline conditions, exhibiting site specificity.

[0112] The nucleophilic substitution reaction (SN2) of BSA's active thiol with γ-CD-Br produces the byproduct HBr, requiring a weakly alkaline buffer solution. This buffer solution also enhances the nucleophilicity of the thiol group and accelerates the cross-linking reaction rate. Initially, 1M MEPES (pH 7.5), PBS (pH 7.4) phosphate buffer, and 0.1M sodium acetate (pH 7.7) buffer were tried as reaction solvents. While these solutions synthesized the BSA-CD complex, they failed to effectively encapsulate paclitaxel. After screening, the applicant ultimately selected NaHCO3 buffer as the reaction solvent.

[0113] The synthetic route of the BSA-CD complex is as follows: Figure 3 As shown, BSA (25 mg, 0.375 μmol) was added to the reaction flask and dissolved in 2.5 mL of 0.1 M NaHCO3 buffer under ice bath conditions. TCEP·HCl (0.3 mg, 1.125 μmol) was added, and the reaction was carried out under N2 protection in an ice bath for 10 min. Solid γ-CD-Br (5 mg, 3.375 μmol) was added, and the reaction was carried out under N2 protection in an ice bath and in the dark with gentle stirring for 12 h. The Ellman reagent was used to monitor the completion of the reaction. After lyophilization, the product BSA-CD complex was obtained. The BSA-CD complex was ultrafiltered once using a 3 kDa ultrafiltration tube (6000 x g × 25 min), leaving approximately 20 mM NaHCO3 buffer for later use.

[0114] In this embodiment, the synthesized γ-CD-Br was grafted onto the Cys34 reactive thiol on the surface of BSA. The reaction was carried out in an ice bath and in the dark in 0.1 M NaHCO3 (pH=8.86) buffer solution, with only a small amount of TCEP·HCl reducing agent added. The mixture was gently stirred under N2 for 12 h, and the grafting rate of the BSA-CD complex reached 82%. The TCEP·HCl reducing agent did not break a large number of disulfide bonds, but mainly maintained the reduced state of Cys34-SH to avoid oxidation to disulfide bond deactivation, and only broke a small number of disulfide bonds.

[0115] The BSA solution without added γ-bromoamide cyclodextrin after reduction was used as the control group, and deionized water was used as the blank group.

[0116]

[0117] (OD) a ODb OD c (The absorbance values ​​at 412 nm are for the reaction group, blank group, and control group, respectively).

[0118] BSA-CD complex ultrafiltration completely removes salt.

[0119] The BSA-CD complex needs to be completely desalted by ultrafiltration using Tris-HCl buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.5) before characterization such as MALDI-TOF-MS analysis can be performed.

[0120] First, pre-equilibrate the 30 kDa ultrafiltration tube with 0.45 mL of Tris-HCl buffer and centrifuge twice (1800 x g × 15 min, with the same speed and time for subsequent centrifugations). Then, add 0.45 mL of BSA-CD complex, centrifuge once, add more Tris-HCl buffer, and repeat the centrifugation three times. Wash three times with pure water, and finally add 0.45 mL of pure water, invert and centrifuge for 5 min. Collect the filtrate as completely desalted BSA-CD complex.

[0121] like Figure 4 As shown in -A, MALDI-TOF-MS quality analysis revealed that the molecular weight of the product was basically consistent with the theoretical molecular weight. The main product was a single graft of γ-CD onto Cys34 reactive thiols of BSA, and a small amount of product was an additional thiol group of BSA grafted with 2-5 γ-CDs.

[0122] See Figure 4 -B, FTIR plots observed at 1645, 1541 cm⁻¹ -1 The characteristic peaks correspond to the C=O stretching vibration and NH bending vibration of the amide bond, respectively, and are also present at 837.5 cm⁻¹. -1 The presence of characteristic peaks of the SC group after the thiol reaction confirmed the successful grafting of γ-bromoamide cyclodextrin with bovine serum albumin, synthesizing a novel "cyclic oligosaccharide-protein" complex (BSA-CD complex) as a delivery carrier for hydrophobic drugs.

[0123] 3) Preparation of BSA-CD-PTX inclusion complex

[0124] A 1 mM paclitaxel (PTX) solution dissolved in ethanol was mixed with a 0.1 mM BSA-CD complex dissolved in water at a volume ratio of 1:1. The mixture was shaken in the dark at 30°C and 200 rpm for 60 h to achieve equilibrium. After nitrogen blowing, the ethanol was removed from the mixture, and the free paclitaxel was removed by centrifugation. The supernatant was freeze-dried to obtain a white powder, which is the prepared BSA-CD-PTX inclusion complex.

[0125] Comparative Example 1

[0126] A 1 mM γ-CD (1.3 mg, 1 μmol) solution dissolved in 1 mL of water was mixed with an equal volume of 1 mL paclitaxel solutions dissolved in 1, 2, and 4 mM paclitaxel solutions. The mixture was incubated for 60 h, and the maximum water solubility, encapsulation efficiency, and drug loading of PTX were measured. The γ-CD aqueous solution showed almost no encapsulation of paclitaxel.

[0127] Comparative Example 2

[0128] A 0.1 mM BSA (6.6 mg, 0.1 μmol) solution dissolved in 1 mL of water was mixed with an equal volume of 1 mL paclitaxel solutions dissolved in 1, 2, and 4 mM ethanol. After encapsulation for 60 h, the maximum water solubility, encapsulation efficiency, and drug loading of PTX were measured. The BSA aqueous solution showed almost no encapsulation of paclitaxel.

[0129] Comparative Example 3

[0130] 1 mM γ-CD was prepared by dissolving in 0.1 M NaHCO3 buffer (specifically, γ-CD (1.3 mg, 1 μmol) was added to the reaction flask and dissolved in 1 mL of 0.1 M NaHCO3 buffer under ice bath). After stirring gently under ice bath for 6 h, it was then mixed with an equal volume of 1 mL of paclitaxel solution dissolved in 1, 2, and 4 mM ethanol (γ-CD molecular weight was too small to be desalted by ultrafiltration). After inclusion for 60 h, the maximum water solubility, encapsulation efficiency, and drug loading of PTX were measured.

[0131] Comparative Example 4

[0132] 0.1 mM BSA was prepared by dissolving in 0.1 M NaHCO3 buffer (specifically, BSA (6.6 mg, 0.1 μmol) was added to the reaction flask and dissolved in 1 mL of 0.1 M NaHCO3 buffer under ice bath). After stirring gently under ice bath for 6 h, the BSA was ultrafiltered once (3 kDa ultrafiltration tube, 6000 x g, 25 min). Then, it was mixed with an equal volume of 1 mL of paclitaxel solution dissolved in 1, 2, and 4 mM ethanol and encapsulated for 60 h. The maximum water solubility, encapsulation efficiency, and drug loading of PTX were measured.

[0133] Comparative Example 5

[0134] BSA-CD complex and paclitaxel physical mixture

[0135] 1) Synthesis of γ-cyclodextrin (γ-CD) derivatives

[0136] The preparation of γ-bromoamide cyclodextrin (γ-CD-Br) was the same as in Example 1, except that the 6-hydroxyl group of γ-CD was sulfonated with p-toluenesulfonyl chloride, the p-toluenesulfonyl group was replaced with the azide group of sodium azide, the azide group was hydrolyzed to an amino group, and finally a short alkyl chain γ-CD-Br was synthesized by bromination reaction.

[0137] 2) Preparation of bovine serum albumin-γ-cyclodextrin grafted complex (BSA-CD complex)

[0138] The synthesis of the BSA-CD complex was the same as in Example 1. BSA (25 mg, 0.375 μmol) was added to a reaction flask and dissolved in 2.5 mL of 10 mM NaHCO3 buffer under ice bath conditions. TCEP·HCl (0.3 mg, 1.125 μmol) was added, and the reaction was carried out under N2 protection in an ice bath for 10 min. Solid γ-CD-Br (5 mg, 3.375 μmol) was added, and the reaction was carried out under N2 protection in an ice bath and in the dark with gentle stirring for 12 h. The Ellman reagent was used to monitor the completeness of the reaction. The BSA-CD complex was ultrafiltered once using a 3 kDa ultrafiltration tube (6000 x g × 25 min), leaving approximately 20 mM NaHCO3 buffer. After lyophilization, the product BSA-CD complex was obtained.

[0139] 3) A physical mixture of BSA-CD complex and paclitaxel

[0140] A 1 mM paclitaxel solution dissolved in ethanol was mixed with a 0.1 mM BSA-CD solution dissolved in water at a volume ratio of 1:1. The ethanol was then removed by nitrogen blowing, and the mixture was freeze-dried to obtain a white powder, which is the prepared physical mixture of BSA-CD complex and paclitaxel.

[0141] The effects of different raw material aqueous solutions and NaHCO3 solutions on the encapsulation efficiency, drug loading, and water solubility of paclitaxel after being miscible with each other are shown in the following figures. Figure 5 As shown.

[0142] Comparative Examples 1 and 2 were dissolved in water to dissolve BSA and γ-CD respectively, and then mixed with an equal volume of paclitaxel solution dissolved in ethanol. After 60 hours of inclusion, almost no paclitaxel was included.

[0143] Comparative Examples 3 and 4 were prepared by dissolving BSA and γ-CD in 0.1M NaHCO3 buffer, respectively, and gently stirring under ice for 6 h. Then, they were mixed with an equal volume of paclitaxel solution in ethanol. After 60 h of encapsulation, 1 mM paclitaxel was completely encapsulated. The maximum water solubility of PTX for 4 mM paclitaxel encapsulated by BSA and γ-CD were 18 mM and 20 mM, respectively. BSA and γ-CD could only encapsulate approximately 90% of 2 mM paclitaxel, with drug loadings of approximately 18.6% and 54.4%, respectively.

[0144] The effects of the BSA-CD complex and paclitaxel inclusion complex prepared in Example 1 of this invention on the encapsulation efficiency, drug loading, and water solubility of paclitaxel are as follows: Figure 6 As shown.

[0145] See Figure 6 -B, In Example 1 of this invention, the maximum water solubility of PTX after encapsulation with 0.1 mM BSA-CD complex and 4 mM paclitaxel reached 33.67 mM, and the maximum water solubility of PTX in the paclitaxel inclusion complex first increased and then decreased with the extension of encapsulation time (e.g. Figure 13 As shown in -B), the water solubility of PTX is extremely low in the initial stage of inclusion (equivalent to a physical mixture of BSA-CD complex and PTX), but reaches maximum water solubility of PTX after 60 hours of inclusion.

[0146] The mechanism of action of the BSA-CD complex in this invention for encapsulating paclitaxel is as follows: BSA is usually bound to endogenous fatty acids and other small molecules. These ligands occupy its hydrophobic binding pockets, competitively inhibiting the binding of paclitaxel. The 0.1 M NaHCO3 buffer is weakly alkaline. Under these conditions, fatty acids deprotonate to form water-soluble fatty acid salts, which dissociate from the binding sites of BSA. The conformation of BSA undergoes a moderate adjustment, exposing more hydrophobic regions and enhancing its affinity for paclitaxel. Ice bath conditions help maintain the native folded state of the protein, preventing irreversible thermal denaturation. Secondly, cyclodextrin molecules easily form aggregates through hydrogen bonds, causing their hydrophobic cavities to be masked, reducing their inclusion capacity. The ions in the NaHCO3 buffer (Na+, Na+, and Na+) further contribute to this effect. + and HCO3 - It can disrupt the hydrogen bond network between cyclodextrin molecules, inhibit self-aggregation, and allow individual cyclodextrin molecules to be fully dispersed, thus improving cavity accessibility; the presence of appropriate ionic strength can enhance the hydrophobic effect and promote the entry of paclitaxel into the cyclodextrin cavity.

[0147] Under the weakly alkaline conditions of 0.1 M NaHCO3 buffer, the BSA-CD complex allows paclitaxel to fully enter the hydrophobic pocket of BSA and the hydrophobic cavity of γ-CD, exhibiting a combined inclusion effect of BSA and γ-CD, resulting in a synergistic "double inclusion" effect. This significantly improves the inclusion effect of paclitaxel and the maximum water solubility of PTX.

[0148] The characterization results of the paclitaxel inclusion complex are as follows: Figure 7 As shown.

[0149] See Figure 7-A shows the UV absorption spectra of BSA-CD-PTX inclusion complexes at different reaction times. Paclitaxel has a maximum UV absorption peak at 230 nm and another at 273 nm. BSA shows a strong peptide bond absorption peak at 223 nm, and double bond absorption peaks from tryptophan and tyrosine at 280 nm. The peptide bond absorption peak of the BSA-CD complex exhibits a 2 nm blue shift. Under the interaction between the BSA-CD complex and paclitaxel, the UV absorption intensity of the BSA-CD-PTX inclusion complex around 230 nm and 280 nm gradually increases with increasing inclusion reaction time, and a gradual transverse shift occurs, indicating that the two compounds form a BSA-CD-PTX inclusion complex.

[0150] Fluorescence emission spectra of BSA-CD-PTX inclusion complexes at different reaction times are shown below. Figure 7 As shown in Figure -B, due to the relatively low quantum yield of paclitaxel, Raman scattering occurs at 315 nm in the emission spectrum and makes a significant contribution. Therefore, the contribution of Raman scattering is considered in the interpretation of the data. Tryptophan in BSA has an absorption peak at 340 nm, while the tryptophan absorption peak of the BSA-CD complex shifts to 335 nm. The absorption peaks of the BSA-CD-PTX inclusion complex represent the average signal from the BSA and paclitaxel fluorophores in two environments with different polarities. Compared to paclitaxel, the BSA-CD-PTX inclusion complex significantly increases fluorescence intensity, broadens the emission spectrum, and shifts the absorption peak to 330 nm due to the interaction between the BSA-CD complex and paclitaxel. With increasing inclusion reaction time, the fluorescence intensity of the BSA-CD-PTX inclusion complex gradually increases, confirming that PTX enters the hydrophobic microenvironment formed by BSA and γ-CD, forming the BSA-CD-PTX inclusion complex.

[0151] Circular dichroism spectroscopy is highly sensitive to minute changes in drug structure and is a valuable technique for detecting conformational changes in drugs. PTX exhibits circular dichroism activity due to its chiral centers linked to multiple chromophores. Therefore, this technique was used to determine the interaction and complexation between PTX and the BSA-CD complex. The interaction between paclitaxel and the BSA-CD complex in aqueous solution was investigated by evaluating the effect of complexation on the spectral properties of the drug.

[0152] The circular dichroism chromatogram of the BSA-CD-PTX inclusion complex is shown below. Figure 7-C is shown. Since it does not absorb ultraviolet light of a specific wavelength, the BSA-CD complex does not exhibit any bands. Paclitaxel exhibits two CD bands at 264 nm and 310 nm. The shoulder band at 264 nm is the aromatic ring on the benzoyl group at position 2 of the main ultraviolet ring, while the negative band at 310 nm is due to the π-π* transition of the aromatic ring in PTX. Compared to the spectrum of paclitaxel, the negative band of BSA-CD-PTX shifts 15 nm to lower wavelengths. The relatively large increase in signal intensity is due to the change in the aromatic group environment caused by the complexation of paclitaxel with the BSA-CD complex, confirming the interaction between PTX and the BSA-CD complex.

[0153] The DSC diagram of the BSA-CD-PTX inclusion complex is shown below. Figure 7 As shown in Figure -D, the DSC thermal analysis of PTX, the BSA-CD complex, the physical mixture of the BSA-CD complex and PTX in Comparative Example 5, and the BSA-CD-PTX inclusion complex in Example 1 is presented. PTX shows a sharp endothermic peak at around 221°C, indicating its characteristic melting point. The BSA-CD complex has a melting endothermic peak at around 225°C. The physical mixture of the BSA-CD complex and PTX only shows an endothermic peak of PTX at around 221°C. The BSA-CD-PTX inclusion complex does not show an endothermic peak of PTX at around 221°C, indicating that the inclusion complex does not contain free crystalline paclitaxel and may exist in an amorphous form.

[0154] The FTIR spectrum of the BSA-CD-PTX inclusion complex is shown below. Figure 8 As shown in -A, the physical mixture of BSA-CD and PTX in Comparative Example 5 and the BSA-CD-PTX inclusion complex prepared in Example 1 both exhibit a characteristic peak at 1243 cm⁻¹. -1 and 709 cm -1 These correspond to the C–O–C stretching vibration and CH out-of-plane bending vibration of the aromatic ring of paclitaxel, respectively, while the physical mixture of BSA-CD and PTX did not appear at 832.6 cm⁻¹. -1 The characteristic peak of the SC group after the thiol reaction is 838 cm⁻¹ in the BSA-CD-PTX inclusion complex. -1 The characteristic peaks of the SC group indicate the formation of the BSA-CD-PTX inclusion complex.

[0155] FTIR spectra of γ-CD-PTX and BSA-PTX inclusion complexes are shown below. Figure 8 As shown in Figure B, the physical mixtures of γ-CD and PTX in Comparative Example 5, and the physical mixtures of BSA and PTX, all exhibit characteristic peaks in the 600-1300 cm⁻¹ range. -1 The range is similar to that of paclitaxel, both having a characteristic peak at 1243 cm⁻¹. -1 and 709 cm-1 These correspond to the C–O–C stretching vibration of the aromatic ring of paclitaxel and the out-of-plane bending vibration of the CH group of the aromatic ring, respectively.

[0156] The infrared spectra of γ-CD-PTX (water) in Comparative Example 1 and BSA-PTX (water) in Comparative Example 2 are basically consistent with those of the raw materials γ-CD and BSA, indicating that no inclusion complex was formed with paclitaxel after dissolving the raw materials γ-CD and BSA in water.

[0157] The γ-CD-PTX (0.1 M NaHCO3) in Comparative Example 3 and the BSA-PTX (0.1 M NaHCO3) in Comparative Example 4 both exhibit a characteristic peak at 1434 cm⁻¹. -1 Corresponding to the C=C stretching vibration of the aromatic ring of paclitaxel, 830 cm -1 and 709 cm -1 This corresponds to the out-of-plane bending vibration of the aromatic ring CH of paclitaxel. This indicates that after dissolving the raw materials γ-CD and BSA in 0.1 M NaHCO3 buffer, both can form BSA-PTX and γ-CD-PTX inclusion complexes with paclitaxel.

[0158] NMR of BSA-CD-PTX inclusion complex 1 H spectrum as shown Figure 8 As shown in Figure -C, when BSA-CD and paclitaxel are complexed, signal peaks of PTX are observed at δ 8.9 for NH and δ 7.0–8.0 for A–C aromatic ring protons. The signal peaks of the BSA-CD complex at δ 5.7 for the secondary amine group and δ 3.5–5.0 for the sugar chain protons show significant changes, while the non-aromatic protons of PTX are not observed. This is because this part of the surface does not interact with the hydrophilic outer part of γ-cyclodextrin.

[0159] like Figure 8 The BSA-CD complex shown in -D and the 2D ROESY complexed with paclitaxel were observed to have strong correlations between the protons of the secondary amine group at δ 4.36 and δ 5.38 (δ=5.38) in the cyclodextrin cavity and the meta proton of the C aromatic ring of paclitaxel at δ 7.40. This indicates that the cyclodextrin cavity encapsulates the C aromatic ring of paclitaxel.

[0160] Scanning electron microscope image of BSA-CD-PTX inclusion complex as follows Figure 9 As shown, PTX in 9-A is a long needle-like crystal with a wide particle size distribution, while the BSA-CD complex in 9-B is amorphous. The physical mixture of the BSA-CD complex and PTX in 9-C shows long needle-like crystals of PTX, and the BSA-CD-PTX inclusion complex in 9-D is amorphous; the typical needle-like crystal structure of PTX is not present in the complex. Therefore, this visually confirms the complexation effect between the BSA-CD complex and PTX.

[0161] like Figure 10 As shown, BSA-CD-PTX inclusion complexes and Abraxane were obtained at 37°C in PBS (pH 7.4) buffer and acetate buffer (pH 5.5). TM The in vitro PTX release process at different reaction times was observed. We used PBS buffer (pH 7.4) and acetate buffer (pH 5.5) to simulate the blood circulation and tumor tissue / lysosomal environment. The release process showed that drug release was relatively rapid in the first 12 hours, with the release rate gradually decreasing thereafter. In PBS (pH 7.4) buffer, Abraxane... TM The release of [the compound] is slow, with a cumulative release rate of approximately 27% over 72 hours. The release rate accelerates in acetate buffer (pH 5.5), reaching a cumulative release rate of 55% over 72 hours, exhibiting pH-responsive release characteristics. The BSA-CD-PTX inclusion complex releases slowly in acetate buffer (pH 5.5), with a cumulative release rate of approximately 23% over 72 hours, while it releases more rapidly in PBS (pH 7.4) buffer, with a cumulative release rate of approximately 40% over 72 hours.

[0162] At the physiological pH of PBS (pH 7.4), the weakly alkaline state of the NaHCO3 buffer used in the preparation of the inclusion complex was maintained. During release, the hydrophobic pockets of BSA and the hydrophobic cavities of cyclodextrin remained open, facilitating drug release. In acetate buffer (pH 5.5), the inclusion complex solution changed from weakly alkaline to weakly acidic. This drastic conformational change may lead to some aggregation of protein molecules, and cyclodextrin molecules are also prone to forming aggregates through hydrogen bonds. This partially masks the hydrophobic pockets of BSA and the hydrophobic cavities of cyclodextrin, thus slowing drug release. The release characteristics of the BSA-CD-PTX inclusion complex can prevent premature and excessive drug release in the extracellular space (pH 6.5-7.0) after reaching tumor tissue. After active uptake by tumor cells via albumin receptors (such as gp60 / SPARC-mediated) or endocytosis, the drug may be rapidly degraded by abundant proteases in lysosomes, leading to the instantaneous release of the encapsulated paclitaxel. This could enable "intracellular burst release," producing extremely high local concentrations of the drug where it needs to work (cytoplasm), resulting in a stronger killing effect.

[0163] like Figure 11 As shown, after incubation for 48 hours in the presence of formulations ranging from 0.001 to 10 μM, the cytotoxicity of blank nanoparticles to B16 F10 cells was assessed by MTT assay. No cytotoxicity was observed in the blank carrier containing cells after 48 hours of culture, indicating that the carrier is fully biocompatible and suitable for further in vivo evaluation.

[0164] Free PTX and Abraxane TM Both exhibited cytotoxicity within the concentration range corresponding to predicted plasma levels in humans (0.001-10 μM), while the BSA-CD-PTX inclusion complex showed measurable cytotoxicity at a dose up to 1 μM, and a concentration of 10 μM was highly effective in killing tumor cells. The killing effect of the BSA-CD-PTX inclusion complex on tumor cells was dose- and time-dependent.

[0165] Comparative analysis Figure 12 Cell images taken under a microscope at 10x magnification (-A and 12-B) clearly show that the BSA-CD-PTX inclusion complex has strong toxicity to tumor cells. The cells become smaller, rounder, and deformed, with a large number of black spots caused by melanin aggregation after apoptosis. Filamentous structures of the cytoskeleton, which appear to be disintegrating, are also observed around the cells. This is consistent with the mechanism of action of paclitaxel as a microtubule stabilizer.

[0166] Figure 12 In the 12-C and 12-D regions, only partial regions of free PTX and Abraxane showed apoptosis, indicating that at a concentration of 10 μM, the BSA-CD-PTX inclusion complex was more cytotoxic than free PTX and Abraxane. TM .

[0167] Based on the high PTX water solubility and high drug loading of the paclitaxel inclusion complex of the present invention, the applicant proposes the application of the paclitaxel inclusion complex in the preparation of anti-tumor drugs.

[0168] Example 2

[0169] Compared with Example 1, the preparation method of the paclitaxel inclusion complex in this example differs in the following ways:

[0170] 2) Preparation of bovine serum albumin-γ-cyclodextrin grafted complex (BSA-CD complex)

[0171] BSA (25 mg, 0.375 μmol) was added to a reaction flask and dissolved in 2.5 mL of 10 mM NaHCO3 buffer under ice bath conditions. TCEP·HCl (0.3 mg, 1.125 μmol) was added, and the reaction was carried out under N2 protection in an ice bath for 10 min. Solid γ-CD-Br (5 mg, 3.375 μmol) was added, and the reaction was carried out under N2 protection in an ice bath and in the dark with gentle stirring for 12 h. The reaction was monitored for completeness using Ellman's reagent. The product, the BSA-CD complex, was obtained after lyophilization.

[0172] Example 3

[0173] Compared with Example 1, the preparation method of the paclitaxel inclusion complex in this example differs in the following ways:

[0174] 2) Preparation of bovine serum albumin-γ-cyclodextrin grafted complex (BSA-CD complex)

[0175] BSA (25 mg, 0.375 μmol) was added to a reaction flask and dissolved in 2.5 mL of 10 mM NaHCO3 buffer containing 2% (v / v) glycerol under ice bath conditions. TCEP·HCl (0.3 mg, 1.125 μmol) was added, and the reaction was carried out under N2 protection in an ice bath for 10 min. Solid γ-CD-Br (5 mg, 3.375 μmol) was then added, and the reaction was carried out under N2 protection in an ice bath and in the dark with gentle stirring for 12 h. The reaction was monitored for completeness using Ellman's reagent. The product, the BSA-CD complex, was obtained after lyophilization.

[0176] Considering that the weakly alkaline solution and TCEP·HCl reducing agent may cause partial irreversible damage to the three-dimensional structure of BSA, leading to denaturation and inactivation, 2% v glycerol was added to the 10 mM NaHCO3 buffer system to protect the protein's three-dimensional structure. The differences in solubility of paclitaxel between the BSA-CD complex obtained by different preparation methods in Examples 2 and 3 and the BSA-CD-PTX inclusion complex containing PTX are shown below. Figure 13 As shown in -A, although the addition of 2% glycerol shortened the inclusion time, it failed to significantly improve the solubility, indicating that the reaction conditions of weak alkaline solution and TCEP·HCl reducing agent did not irreversibly destroy the BSA structure.

[0177] Example 4

[0178] Compared with Example 1, the preparation method of the paclitaxel inclusion complex in this example differs in the following ways:

[0179] 2) Preparation of bovine serum albumin-γ-cyclodextrin grafted complex (BSA-CD complex)

[0180] BSA (25 mg, 0.375 μmol) was added to a reaction flask and dissolved in 2.5 mL of 0.1 mM NaHCO3 buffer under ice bath conditions. TCEP·HCl (0.3 mg, 1.125 μmol) was added, and the mixture was reacted under N2 conditions in an ice bath for 10 min. Solid γ-CD-Br (5 mg, 3.375 μmol) was added, and the mixture was reacted with gentle stirring under N2 protection in an ice bath and in the dark for 12 h. The reaction was monitored for completeness using Ellman's reagent. The product, the BSA-CD complex, was obtained after lyophilization.

[0181] The differences in the solubility of paclitaxel between the BSA-CD complex obtained by different preparation methods in this embodiment and Example 1 and the BSA-CD-PTX inclusion complex containing PTX are as follows: Figure 13 As shown in -B, in this embodiment, the maximum solubility of PTX in the BSA-CD-PTX inclusion complex sample prepared using the unfiltered BSA-CD complex can reach 8.1 mM. However, the high salt content and weak alkalinity have a continuous destructive effect on the protein structure and cannot meet the low salt content requirements for experiments such as antitumor activity.

[0182] The applicant also found that after completely desalting the BSA-CD complex using a 30 kDa ultrafiltration tube, it was found that almost no paclitaxel could be included. However, in Example 1, using a 3 kDa ultrafiltration tube to ultrafilter the BSA-CD complex once (8000 rpm x 50 min), leaving approximately 20 mM NaHCO3 buffer, significantly improved the inclusion efficiency. After 60 h of inclusion, the maximum water solubility reached 13 mM. However, the inclusion efficiency decreased significantly after two ultrafiltrations. Specifically, when the NaHCO3 buffer was 0.1 M, the appropriate salt concentration provided sufficiently opened the hydrophobic pocket of BSA and the hydrophobic cavity of γ-CD. During paclitaxel inclusion, the excessively high salt concentration in the un-ultrafiltration-treated BSA-CD complex may have caused the BSA-CD complex to remain in a slightly aggregated state, inhibiting inclusion stability. The 20 mM NaHCO3 buffer remaining after one ultrafiltration significantly improved the inclusion efficiency. The ionic strength of the NaHCO3 buffer solution provides a stable conformation for open hydrophobic pockets and cavities, preventing conformational instability caused by charge repulsion and avoiding hydrophobic aggregation due to excessive salt concentration. After dilution, it also meets the low-salt requirements for subsequent intravenous injection experiments. In contrast, the BSA-CD complex obtained through two ultrafiltrations, or a completely desalted BSA-CD complex, cannot provide a sufficiently weakly alkaline environment with adequate salt concentration when encapsulating paclitaxel. This also hinders the full opening of the hydrophobic pockets of BSA and the hydrophobic cavities of γ-CD, thus affecting the encapsulation effect.

[0183] Combination Figure 13The red lines in -A and 13-B show the water solubility curves of paclitaxel for the BSA-CD complex and the BSA-CD-PTX inclusion complex obtained under different preparation methods in this example and Example 2. It can be seen that the maximum water solubility of paclitaxel in the BSA-CD-PTX inclusion complex prepared with 10 mM NaHCO3 buffer (1.52 mM) is significantly lower than that in the BSA-CD-PTX inclusion complex prepared with 0.1 M NaHCO3 buffer (8.1 mM). The salt concentration provided by the NaHCO3 buffer directly affects the opening degree of the BSA hydrophobic pocket and γ-CD hydrophobic cavity in the inclusion complex, and thus affects the effect of the paclitaxel inclusion complex on the water solubility of paclitaxel. Combined with the difference in the maximum PTX solubility between the BSA-CD complex prepared with 0.1M NaHCO3 buffer and the paclitaxel inclusion after one ultrafiltration and the paclitaxel inclusion without ultrafiltration, it can be concluded that the salt concentration provided by 10mM NaHCO3 buffer is insufficient to fully open the BSA hydrophobic pocket and γ-CD hydrophobic cavity.

[0184] Example 5

[0185] In this embodiment, 1 mL of 0.1 mM BSA-CD complex prepared in Example 1 was encapsulated with 1 mL of paclitaxel solutions of different concentrations (0.5, 1, 2, 4, 6, 8 mM) dissolved in ethanol. The maximum content of paclitaxel encapsulated by the 0.1 mM BSA-CD complex was then analyzed. The results are as follows: Figure 14 As shown.

[0186] See Figure 6 According to -A, 0.5, 1, and 2 mM paclitaxel can be completely encapsulated, 4 mM paclitaxel has an encapsulation efficiency of about 80% and a drug loading of about 28%, 6 and 8 mM paclitaxel only slightly improve the encapsulation efficiency of the inclusion complex, and the maximum encapsulation capacity of the 0.1 mM BSA-CD complex is about 3.5 mM paclitaxel.

[0187] like Figure 6 As shown in -B, the maximum water solubility of PTX in the BSA-CD-PTX inclusion complex under the inclusion condition of 4 mM paclitaxel can reach 33.67 mM. The inclusion effect of the BSA-CD complex is only slightly lower than that of the sum of the inclusion effects of 0.1 mM BSA and 1 mM γ-CD on 4 mM paclitaxel (e.g., ...). Figure 5 -B (18mM + 20mM). The albumin-bound formulation Abraxane™ has a maximum water solubility of 7 ± 0.5 mM for PTX and a drug loading of approximately 10%. The BSA-CD complex in this formulation encapsulates paclitaxel with approximately 5 times higher water solubility and approximately 2.8 times higher drug loading.

[0188] As the concentration of paclitaxel increases, the maximum water solubility of PTX in the BSA-CD-PTX inclusion complex obtained by encapsulating paclitaxel in 1 mL of 0.1 mM BSA-CD complex increases accordingly. When the concentration of paclitaxel is greater than or equal to 4 mM, the increase in paclitaxel concentration has little effect on the maximum water solubility of PTX in the BSA-CD-PTX inclusion complex. The maximum water solubility of PTX in the BSA-CD-PTX inclusion complex can reach 35 mM under the inclusion condition of 8 mM paclitaxel, and the drug loading can reach 29%.

[0189] Example 6

[0190] In this embodiment, 1 mL of paclitaxel solution of 5 mM dissolved in ethanol was encapsulated with 1 mL of BSA-CD complex of different concentrations (0.01, 0.025, 0.05, 0.1, 0.15, 0.2 and 0.3 mM) obtained by the preparation method in Example 1. The highest concentration of paclitaxel completely encapsulated by the BSA-CD complex of different concentrations was then analyzed, which provides guidance for scale-up experiments.

[0191] The reason for choosing 5 mM paclitaxel as the highest concentration is that when the concentration of paclitaxel is higher than 5 mM, the decrease in ethanol concentration after adding the paclitaxel solution dissolved in ethanol to the aqueous solution of the BSA-CD complex will significantly reduce the solubility of paclitaxel. Shaking will cause a large amount of paclitaxel suspension to precipitate out, which will waste expensive paclitaxel.

[0192] See Figure 14 According to -A, 0.3 mM of the BSA-CD complex is required to completely encapsulate 5 mM paclitaxel, with a drug loading of approximately 17%. Figure 14 -B indicates that the maximum water solubility of PTX can reach approximately 46 mM.

Claims

1. A paclitaxel inclusion complex, characterized in that, The paclitaxel inclusion complex is composed of bovine serum albumin-γ-cyclodextrin complex and paclitaxel.

2. The paclitaxel inclusion complex according to claim 1, characterized in that, The bovine serum albumin-γ-cyclodextrin complex and paclitaxel were mixed in equal volumes and then encapsulated under ice bath conditions, with a concentration ratio ≥1:

80.

3. The paclitaxel inclusion complex as described in claim 1 or 2, characterized in that, The concentration ratio of bovine serum albumin-γ-cyclodextrin complex to paclitaxel is 1:40 to 1:

25.

4. The paclitaxel inclusion complex according to claim 1 or 2, characterized in that, The bovine serum albumin-γ-cyclodextrin complex has a molecular weight of 68 kDa, and the molar ratio of bovine serum albumin to γ-cyclodextrin is 1:

9.

5. The method for preparing the paclitaxel inclusion complex according to any one of claims 1 to 4, characterized in that, Includes the following steps: 1) Synthesis of γ-bromoamide cyclodextrin The 6-hydroxyl group of γ-CD was sulfonated with p-toluenesulfonyl chloride, then the p-toluenesulfonyl group was replaced with the azide group of sodium azide, the azide group was hydrolyzed to an amino group, and finally a short alkyl chain γ-CD-Br was synthesized by bromination reaction. 2) Preparation of bovine serum albumin-γ-cyclodextrin complex Add BSA to the reaction flask, dissolve it in 10mM~0.1M NaHCO3 buffer under ice bath, add TCEP·HCl, and react under N2 in an ice bath; add the solid γ-CD-Br prepared in step 1), and react gently in an ice bath and in the dark under N2 protection. Monitor the reaction with Ellman reagent to see if it is complete. After lyophilization, the product BSA-CD complex is obtained and set aside for later use. 3) Preparation of BSA-CD-PTX inclusion complex A solution of paclitaxel (PTX) dissolved in ethanol was mixed with a BSA-CD complex dissolved in water at an equal volume ratio. The mixture was shaken in the dark for more than 60 hours to reach equilibrium. After the mixture was purged with nitrogen to remove the ethanol, the free paclitaxel was removed by centrifugation, and the supernatant was freeze-dried to obtain a white powder, which was the BSA-CD-PTX inclusion complex.

6. The method for preparing the paclitaxel inclusion complex as described in claim 5, characterized in that, In step 2), the BSA is dissolved in 0.1M NaHCO3 buffer.

7. The method for preparing the paclitaxel inclusion complex as described in claim 6, characterized in that, Step 2) further includes: using a 3 kDa ultrafiltration tube to ultrafilter the BSA-CD complex once, leaving a small amount of NaHCO3 buffer solution.

8. The method for preparing the paclitaxel inclusion complex as described in claim 7, characterized in that, The residual NaHCO3 buffer after ultrafiltration was 20 mM NaHCO3.

9. The method for preparing the paclitaxel inclusion complex as described in claim 5, characterized in that, In step 1), the specific operation for synthesizing the γ-cyclodextrin derivative is as follows: First, γ-CD is dissolved in pyridine and added to a reaction flask. A solution of p-toluenesulfonyl chloride dissolved in pyridine is added dropwise under N2. After the addition is complete, the reaction is carried out at room temperature. After the reaction, 10 times the volume of acetone is added to precipitate the product. The product is then washed more than twice with acetone, ethanol, and diethyl ether, respectively. Finally, it is dried in a vacuum drying oven at 50°C to obtain γ-p-toluenesulfonated cyclodextrin (γ-CD-OTs). Then, γ-CD-OTs were dissolved in 4 mL of N,N'-dimethylformamide (DMF) and added to a reaction flask. Sodium azide was added, and the mixture was refluxed under N2. Precipitation, washing, and drying were performed as in the previous step to obtain γ-azidocyclodextrin (γ-CD-N3). γ-CD-N3 was dissolved in DMF and added to the reaction flask. Triphenylphosphine dissolved in DMF was added dropwise under N2. After the addition was complete, the reaction was carried out at room temperature for more than 3 hours. Deionized water was added dropwise while the temperature was raised to the specified temperature. After the addition was complete, the reaction was refluxed for more than 6 hours. Acetone was added to the reaction solution to precipitate the product. The product was then washed twice with acetone and ether, respectively, and dried in a vacuum drying oven at 50°C to obtain γ-aminocyclodextrin (γ-CD-NH2). Finally, γ-CD-NH2 was dissolved in DMF solution, and the acid-binding agent triethylamine was added and stirred in an ice bath. Then, bromoacetyl bromide was added and stirred in an ice bath. The reaction was carried out at room temperature in the dark for 5 hours. The precipitation, washing and drying were the same as in the previous step to obtain the product γ-CD-Br.

10. The use of the paclitaxel inclusion complex according to any one of claims 1 to 4 in the preparation of a tumor-treating drug.

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