Preparation and application of spaced disulfide bridge-linked dimer prodrugs and their self-assembled nanoparticles

Abstract

The preparation and application of spaced double disulfide bond bridged dimer prodrug and self-assembled nanoparticles thereof belong to the technical field of medicine, and relate to the synthesis of the connection of the spaced double disulfide bond bridged dimer prodrug shown in structural general formula (I) and the construction of the spaced double disulfide bond bridged dimer prodrug shown in structural general formula (II) and self-assembled nanoparticles thereof, and the application thereof in drug delivery. The preparation method is simple and easy to implement, the spaced double disulfide bond bridged dimer prodrug can be self-assembled to form nanoparticles, and has the ability of redox dual response drug release, so that the intelligent response activation of the prodrug in tumor cells can be realized, and the anti-tumor effect and safety of the prodrug are ensured. The anti-tumor effect, pharmacokinetic parameters and safety thereof are superior to those of the prodrug nanoparticles bridged by a single disulfide bond. The present application provides a new strategy for developing a high-efficiency low-toxicity drug delivery system, and meets the urgent needs of high-end chemotherapy preparations in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a spaced disulfide bond-bridged dimer prodrug linker, the preparation and application of spaced disulfide bond-bridged dimer prodrugs and their self-assembled nanoparticles. Specifically, it relates to the synthesis of dicarboxylic acid linkers for prodrug synthesis, the construction of a redox-sensitive paclitaxel dimer prodrug bridged by spaced disulfide bonds and the self-assembled nanoparticles of the dimer prodrug containing the prodrug, and its application in the preparation of antitumor drugs. Background Technology

[0002] Redox-responsive homodimeric prodrugs, composed of two identical drug molecules bridged by redox-sensitive linkages, can self-assemble into nanoparticles in water without the need for additional carriers. Compared to carrier-dependent drug delivery systems such as liposomes and micelles, redox-responsive homodimeric prodrug nanoassemblies (RHPNs) combine the advantages of prodrugs and nanotechnology, exhibiting ultra-high drug loading efficiency (over 60%) while avoiding carrier-related toxicity. Furthermore, redox substances in the aberrant tumor microenvironment can trigger redox-responsive breaking of linkages, thereby achieving selective drug release. Recently, RHPNs have attracted considerable attention due to their significant potential in improving the efficacy and safety of chemotherapeutic drugs. Despite these advantages, RHPNs are currently only in the preclinical research stage, and their antitumor efficacy is challenged by simultaneously enhancing assembly capacity and drug release rate. Therefore, constructing RHPNs with suitable linkages and simultaneously improving their assembly capacity and drug release rate is crucial to ensuring their stability in systemic circulation and rapid response in tumors.

[0003] Disulfide bonds are widely used in the design of smart drug delivery systems due to their high redox sensitivity. Furthermore, the near 90° dihedral angle of a disulfide bond can introduce "structural defects," preventing excessive aggregation of prodrug molecules and promoting the formation of RHPNs. Previous studies have shown that the position of the disulfide bond relative to the adjacent ester bond in the linker significantly affects the assembly capability and drug release efficiency of RHPNs. Regarding assembly capability, a larger distance between the disulfide and ester bonds results in a more flexible structure, effectively maintaining assembly stability. Regarding drug release efficiency, a shorter distance between the disulfide and ester bonds typically accelerates the hydrolysis of the ester bond. This contradiction between assembly capability and drug release rate makes constructing RHPNs that can both stably assemble and rapidly release drugs a challenge. Summary of the Invention

[0004] To resolve the contradiction between drug assembly and release, we first designed carbon-spaced double-disulfide bonds (CSDDs) and selected paclitaxel (PTX) as a typical model drug to construct three CSDD-bridged RHPNs (CSDD-RHPNs). In CSDDs, the two disulfide bonds are located at the α-position of the ester bond, maintaining the same or extended chemical bond length as the homodimeric prodrug bridged by the γ-position monodisulfide bond, aiming to improve drug release rate and assembly stability. The purpose of this invention is to design and synthesize dimeric prodrugs with spaced double-disulfide bonds, prepare self-assembled nanodrug delivery systems of these dimeric prodrugs, and explore their application in the preparation of antitumor drugs. Using RHPNs bridged by α- and γ-position monodisulfide bonds as a control, this study investigated the effects of different chemical linkages on the assembly stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of self-assembled dimer prodrug nanoparticles. By comprehensively screening out the linkages with the best effects, this study provides new strategies and more options for developing intelligent responsive drug delivery systems for the tumor microenvironment, thus meeting the urgent clinical demand for highly efficient chemotherapy agents.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides a linking bond for a disulfide-bridged dimer prodrug represented by general structural formula (I):

[0007]

[0008] This invention provides a spaced disulfide-bridged dimer prodrug of general formula (II) or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Where n = 1 to 5;

[0011] X is a straight-chain carbon chain, a carbon chain containing substituents, a carbon chain containing heteroatoms, or a carbon chain containing substituents and heteroatoms.

[0012] The drug is a drug containing a hydroxyl, amino, or carboxyl group. The drug containing a hydroxyl, amino, or carboxyl group is selected from antitumor drugs, antimetabolites, and anti-inflammatory drugs. The antitumor drugs are selected from taxanes, anthraquinones, nucleosides, camptothecins, platinum compounds, vincristine alkaloids, piosides, and artemisinin compounds; the antimetabolites are selected from pyrimidines, purines, thiabendazoles, and folic acid derivatives; and the anti-inflammatory drugs are selected from halofanthracene, griseofulvin, cyclosporine A, and their derivatives.

[0013] Preferably, n = 1 to 3;

[0014] X is a straight-chain carbon chain.

[0015] Drugs are taxanes or anthraquinones.

[0016] More preferably, n = 1 to 3;

[0017] X is a C2, C4, or C6 straight-chain carbon chain.

[0018] Drugs are taxanes or anthraquinones.

[0019] Specifically, the spaced disulfide-bridged dimer prodrug of this invention uses paclitaxel as a model drug, and synthesizes linking bonds by reacting 1,2-ethanedithiol, 1,4-butanedithiol, and 1,6-hexanedithiol with thioglycolic acid, respectively. Simultaneously, paclitaxel dimer prodrugs with 2, 4, and 6 carbon atoms spaced by the synthesized linking bonds are prepared by linking the drug with these three types of links. These are compared with α-monosulfide-bridged and γ-monosulfide-bridged paclitaxel dimer prodrugs, which are named PTX-SS(α)-PTX and PTX-SS(γ)-PTX, respectively, and their structural formulas are as follows:

[0020]

[0021] A two-carbon-spaced disulfide-bridged dimer prodrug linker was prepared by reacting 1,2-ethanedithiol with mercaptoacetic acid, named SSCCSS, with the following structural formula:

[0022]

[0023] A four-carbon-spaced disulfide-bridged dimer prodrug linker was prepared by reacting 1,4-ethanedithiol with mercaptoacetic acid, named SS4CSS, with the following structural formula:

[0024]

[0025] A 6-carbon-spaced disulfide-bridged dimer prodrug linker was prepared by reacting 1,6-ethanedithiol with mercaptoacetic acid, named SS6CSS, with the following structural formula:

[0026]

[0027] The paclitaxel dimer prodrug prepared using SSCCSS as the linking bond is named PTX-SSCCSS-PTX, and its structural formula is as follows:

[0028]

[0029] The paclitaxel dimer prodrug prepared using SS4CSS as the linking bond is named PTX-SS4CSS-PTX, and its structural formula is as follows:

[0030]

[0031] The paclitaxel dimer prodrug prepared using SS6CSS as the linking bond is named PTX-SS6CSS-PTX, and its structural formula is as follows:

[0032]

[0033] This invention provides a method for synthesizing paclitaxel dimer prodrugs containing spacer disulfide bonds bridging the dimer prodrugs, comprising the following steps:

[0034] Accurately weigh 1,2-ethanedithiol, diethyl azodicarbonate, and mercaptoacetic acid, and dissolve them separately in methanol. Transfer the dissolved 1,2-ethanedithiol to a reaction flask, add the dissolved mercaptoacetic acid dropwise, and then add the dissolved diethyl azodicarbonate dropwise. The reaction is carried out at room temperature under nitrogen protection. After the reaction is complete, evaporate the solvent, and then dissolve the product in a methanol solution containing 30-50% water and 0.2% formic acid. Centrifuge to discard the precipitate, separate the supernatant, and obtain the product.

[0035] Accurately weigh 1,4-butanedithiol, diethyl azodicarbonate, and thioglycolic acid, and dissolve them separately in methanol. Transfer the dissolved 1,4-butanedithiol to a reaction flask, add the dissolved thioglycolic acid dropwise, and then add the dissolved diethyl azodicarbonate dropwise. The reaction is carried out at room temperature under nitrogen protection. After the reaction is complete, evaporate the solvent, and then dissolve the product in a methanol solution composed of 30-50% water and 0.2% formic acid. Centrifuge to discard the precipitate, separate the supernatant, and obtain the product.

[0036] Accurately weigh 1,6-hexanedithiol, diethyl azodicarbonate, and thioglycolic acid, and dissolve them separately in methanol. Transfer the dissolved 1,6-hexanedithiol to a reaction flask, add the dissolved thioglycolic acid dropwise, and then add the dissolved diethyl azodicarbonate dropwise. The reaction is carried out under nitrogen protection at room temperature. After the reaction is complete, evaporate the solvent, and then dissolve the product in a methanol solution composed of 30-50% water and 0.2% formic acid. Centrifuge to discard the precipitate, separate the supernatant, and obtain the product.

[0037] In the above preparation method, the 1,2-ethanedithiol, 1,4-butanedithiol, and 1,6-hexanedithiol can also be other dithiol compounds. The diethyl azodicarbonate can also be selected from other catalysts capable of linking monosulfide bonds into disulfide bonds. Mercaptoacetic acid can also be selected from mercaptopropionic acid, mercaptobutyric acid, mercaptovalerate, and mercaptohexanoic acid.

[0038] The molar ratio of dithiol (1,2-ethanedithiol, 1,4-butanedithiol, 1,6-hexanedithiol) to diethyl azodicarbonate and thioglycolic acid is 1:2:2.

[0039] This invention also provides two methods for synthesizing paclitaxel dimer prodrugs containing spacer disulfide-bridged dimer prodrugs, comprising the following steps:

[0040] Method 1: Accurately weigh the following synthesized components: the linker (1 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (3 equivalents), 4-dimethylaminopyridine (DMAP) (0.2 equivalents), and PTX (2 equivalents). Add the linker to a reaction flask and dissolve it in dichloromethane. Then, add EDCI (2 equivalents) and DMAP (0.1 equivalents) sequentially. Activate the mixture in an ice bath at 0°C. Then, add PTX (2 equivalents), EDCI (1 equivalent), and DMAP (0.1 equivalents) dropwise. React at room temperature and purify the product.

[0041] Method 2: Accurately weigh the following synthesized components: linker (1 equivalent), EDCI (4.8 equivalents), 1-hydroxybenzotriazole (HOBT) (4.8 equivalents), triethylamine (8 equivalents), and PTX (2.0 equivalents). Add the linker to a reaction flask and dissolve it in dichloromethane. Then, add EDCI and HOBT dropwise. After activation in an ice bath at 0°C, add PTX and triethylamine dropwise. React at room temperature and separate the products.

[0042] In the above preparation method, the paclitaxel can be replaced by other anticancer drugs containing active hydroxyl, amino, or carboxyl groups. The anticancer drugs containing active hydroxyl, amino, or carboxyl groups are selected from other taxanes, anthraquinones, nucleosides, platinum compounds, vincristine alkaloids, piosides, artemisinin derivatives, and camptothecin derivatives.

[0043] The present invention also provides a pharmaceutical composition comprising the spaced disulfide-bridged dimer prodrug or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and excipient.

[0044] The present invention also provides the use of the spaced disulfide bond-bridged dimer prodrug or a pharmaceutical composition containing the prodrug in the preparation of antitumor drugs.

[0045] The present invention also provides the use of the spaced disulfide-bridged dimer prodrug or a pharmaceutical composition containing the prodrug in the preparation of a drug delivery system.

[0046] The present invention also provides the use of the spaced disulfide-bridged dimer prodrug or a pharmaceutical composition containing the prodrug in the preparation of injectable, oral or topical delivery systems.

[0047] This invention also provides self-assembled nanoparticles of the aforementioned spaced bis(disulfide)-bridged dimer prodrug, comprising non-PEGylated spaced bis(disulfide)-bridged dimer prodrug self-assembled nanoparticles, PEG-modified spaced bis(disulfide)-bridged dimer prodrug self-assembled nanoparticles, spaced bis(disulfide)-bridged dimer prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances, and spaced bis(disulfide)-bridged dimer prodrug self-assembled nanoparticles loaded with other drugs. The preparation method is a nanoprecipitation method, including high-speed stirring and ultrasonication.

[0048] The preparation method of the spaced disulfide bond-bridged dimer prodrug self-assembled nanoparticles is as follows:

[0049] When a non-PEGylated spaced disulfide-bridged dimer prodrug self-assembles into nanoparticles, the spaced disulfide-bridged dimer prodrug is dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water, and the prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent.

[0050] When PEGylated spacer bis-disulfide bond-bridged dimer prodrugs self-assemble into nanoparticles, the spacer bis-disulfide bond-bridged dimer prodrug and PEG are dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water. The prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent.

[0051] When self-assembling nanoparticles of spaced disulfide bond-bridged dimer prodrugs loaded with hydrophobic fluorescent substances or other drugs, the spaced disulfide bond-bridged dimer prodrug, fluorescent substances or drugs, and PEG are dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water, and the prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent.

[0052] The PEG modifier is selected from TPGS, DSPE-PEG, PLGA-PEG, and PE-PEG, with DSPE-PEG being the preferred PEG modifier. The molecular weight of the PEG is 1000-5000, preferably 1000, 2000, or 5000, and more preferably 2000.

[0053] The solvent is selected from ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran, and acetone.

[0054] The mass ratio of the disulfide-bridged dimer prodrug to the PEG modifier is 90:10 to 60:40. Under these conditions, the prodrug nanoparticles can exert a good anti-tumor effect.

[0055] The spaced disulfide bond-bridged dimer self-assembled nanoparticles have a particle size of 80-120 nm, a particle size distribution of less than 0.2, and a drug loading of up to 70%.

[0056] The present invention also provides the application of the spaced disulfide bond-bridged dimer prodrug self-assembled nanoparticles in the preparation of drug delivery systems.

[0057] The present invention also provides the application of the spaced disulfide bond-bridged dimer prodrug self-assembled nanoparticles in the preparation of antitumor drugs.

[0058] The present invention also provides the application of the spaced disulfide bond-bridged dimer prodrug self-assembled nanoparticles in the preparation of injection, oral or topical drug delivery systems.

[0059] The technical problem solved by this invention is to introduce spacer disulfide bonds into dimer prodrugs and self-assembled nanoparticles, designing redox-sensitive dimer prodrugs bridged by spacer disulfide bonds, and using these dimer prodrugs in the construction of self-assembled nanoparticles. This achieves good chemical stability, high drug loading, good assembly stability, low toxicity, and rapid drug release specific to tumor sites, thereby improving therapeutic efficacy. Simultaneously, using α- and γ-monosulfide bond-bridged dimer prodrugs as controls, the differences in self-assembly, redox-sensitive response, and antitumor activity caused by different chemical bridging methods were investigated, as well as their effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of the prodrug self-assembled nanoparticles.

[0060] The advantages of this invention are:

[0061] (1) A linker bond of a dimer prodrug bridged by a spaced disulfide bond and a dimer prodrug bridged by a spaced disulfide bond were designed and synthesized. The synthesis method is simple and easy to implement.

[0062] (2) Uniform dimer prodrug self-assembled nanoparticles were prepared. The preparation method is simple and easy to implement, achieving efficient drug loading with a drug loading capacity of up to 70%. The particle size remained basically unchanged after being placed at 4℃ for 40 days.

[0063] (3) The differences in self-assembly, redox-sensitive response, and antitumor activity of different chemical bridging agents were investigated, as well as their effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of the prodrug self-assembled nanoparticles. Based on the combined experimental results, the prodrug bridged by a spacer disulfide bond exhibited better chemical stability and higher redox-sensitive properties, enabling specific activation in the tumor redox microenvironment. Furthermore, the prodrug nanoparticles bridged by the spacer disulfide bond also demonstrated the best assembly ability. This invention provides a new strategy and more options for developing intelligent responsive drug delivery systems for the tumor microenvironment, meeting the urgent clinical need for highly effective chemotherapeutic agents. Attached Figure Description

[0064] Figure 1 This confirms the structure of the bis-disulfide bond (SSCCSS) bridging the paclitaxel dimer prodrug linker in Example 1 of this invention, which is spaced two carbon atoms apart.

[0065] A: Mass spectrum of SSCCSS.

[0066] B: SSCCSS 1 H-NMR spectrum.

[0067] Figure 2 This confirms the structure of the paclitaxel dimer prodrug (PTX-SSCCSS-PTX) with a 2-carbon-atom-spaced disulfide bond bridge in Example 2 of the present invention.

[0068] A: NMR MRI of PTX-SSCCSS-PTX.

[0069] B: Mass spectrum of PTX-SSCCSS-PTX.

[0070] C: High performance liquid chromatography purity chromatogram of PTX-SSCCSS-PTX.

[0071] Figure 3 The particle size diagram and transmission electron microscope image of the self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spaced disulfide bonds prepared in Example 3 of the present invention are shown (figure bar: 100 nm).

[0072] A: Particle size, PDI, Zeta potential, drug loading, and transmission electron microscopy images of PTX-SSCCSS-PTX.

[0073] B: Particle size, PDI, Zeta potential, drug loading, and transmission electron microscopy images of PTX-SS4CSS-PTX.

[0074] C: Particle size, PDI, Zeta potential, drug loading, and transmission electron microscopy images of PTX-SS6CSS-PTX.

[0075] Figure 4 This is a diagram illustrating the competition of assembly forces in the self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spaced disulfide bonds in Example 4 of the present invention.

[0076] A: The particle size to initial particle size ratio of paclitaxel dimer prodrug self-assembled nanoparticles after incubation with 0.1 mol / L hydrophobic interaction competing reagent SDS.

[0077] B: The ratio of particle size to initial particle size of paclitaxel dimer prodrug self-assembled nanoparticles after incubation with 0.1 mol / L hydrogen bonding competing reagent Urea.

[0078] C: The ratio of the particle size to the initial particle size of the paclitaxel dimer prodrug self-assembled nanoparticles after incubation with 0.1 mol / L NaCl, a reagent competing for electrostatic interaction.

[0079] Figure 5 This is an in vitro release test diagram of the paclitaxel dimer prodrug self-assembled nanoparticles bridged by spaced disulfide bonds in Example 5 of the present invention.

[0080] A: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 0.1 mM H2O2 conditions.

[0081] B: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 1mM H2O2 conditions.

[0082] C: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 10mM H2O2 conditions.

[0083] D: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 0.05 mM DTT conditions.

[0084] E: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 0.1 mM DTT conditions.

[0085] F: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 1mM DTT conditions.

[0086] Figure 6 This is a cytotoxicity diagram of the self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spacer disulfide bonds in Example 6 of the present invention.

[0087] A: The toxicity of nanoparticles to mouse breast cancer (4T1) cells.

[0088] B: The toxicity of nanoparticles to mouse melanoma (B16-F10) cells.

[0089] C: The toxicity of nanoparticles to mouse fibroblasts (3T3) cells.

[0090] Figure 7 This is a plasma drug concentration-time curve of the self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spaced disulfide bonds in Example 7 of the present invention.

[0091] A: Plasma drug concentration-time curve of paclitaxel dimer prodrug self-assembled nanoparticles.

[0092] B: Plasma drug concentration-time curve of paclitaxel in self-assembled nanoparticles of paclitaxel dimer prodrug.

[0093] Figure 8This is an experimental diagram showing the maximum safe dose assessment of the self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spacer disulfide bonds in Example 8 of the present invention.

[0094] A: Daily body weight / initial body weight ratio of Balb / C mice after administration of 30 mg / kg paclitaxel dimer prodrug to self-assembled nanoparticles.

[0095] B: Daily body weight / initial body weight ratio of Balb / C mice after administration of 60 mg / kg paclitaxel dimer prodrug self-assembled nanoparticles.

[0096] C: Daily body weight / initial body weight ratio of Balb / C mice after administration of 90 mg / kg paclitaxel dimer prodrug self-assembled nanoparticles.

[0097] Figure 9 This is an in vivo antitumor experiment diagram (maximum safe dose) of paclitaxel dimer prodrug self-assembled nanoparticles bridged by disulfide bonds in Example 9 of the present invention.

[0098] A: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on the growth of subcutaneous breast cancer tumors in Balb / C mice.

[0099] B: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on body weight in Balb / C mice.

[0100] Figure 10 This is an in vivo antitumor experiment diagram of the self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spacer disulfide bonds in Example 10 of the present invention (90 mg / kg paclitaxel equivalent dose).

[0101] A: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on the growth of melanoma in C57BL / 6 mice.

[0102] B: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on body weight of C57BL / 6 mice. Detailed Implementation

[0103] The present invention will be further illustrated by way of embodiments below, but the invention is not limited to the scope of the embodiments described herein.

[0104] Example 1: Synthesis of paclitaxel dimer prodrug linker bridged by a 2-carbon-spaced disulfide bond

[0105] Accurately weigh 1,2-ethanedithiol (305.51 μL, 3.6503 mmol), diethyl azodicarbonate (1148.93 μL, 7.3006 mmol), and thioglycolic acid (507.13 μL, 7.3006 mmol). Dissolve each compound in 5 mL of methanol. Transfer the dissolved 1,2-ethanedithiol to a 50 mL round-bottom flask. Add the dissolved thioglycolic acid dropwise, followed by the dissolved diethyl azodicarbonate. Incubate the reaction under nitrogen at room temperature for 12 h. After rotary evaporation, dissolve the product in a methanol solution containing 50% water and 0.2% formic acid (v / v). Centrifuge to discard the precipitate, and then separate the supernatant using high-performance liquid chromatography (HPLC) to obtain the product.

[0106] The structure of the SSCCSS prepared in Example 1 was determined using mass spectrometry and proton nuclear magnetic resonance (HMR) spectroscopy. The mass spectrum, HMR spectrum, and analysis results of the prepared liquid-phase purified SSCCSS are shown below. Figure 1 As shown, the results indicate that the linker bond was successfully synthesized and can be used for subsequent research.

[0107] Synthesis of paclitaxel dimer prodrug linker bridged by 4-carbon-spaced disulfide bonds

[0108] Accurately weigh 1,4-butanedithiol (368.205 μL, 3 mmol), diethyl azodicarbonate (944.756 μL, 6 mmol), and thioglycolic acid (417.016 μL, 6 mmol). Dissolve each compound in 5 mL of methanol. Transfer the dissolved 1,4-butanedithiol to a 50 mL round-bottom flask. Add the dissolved thioglycolic acid dropwise, followed by the dissolved diethyl azodicarbonate. Incubate the reaction under nitrogen at room temperature for 12 h. After rotary evaporation, dissolve the product in a methanol solution composed of 50% water and 0.2% formic acid. Centrifuge to discard the precipitate, and separate the supernatant by high-performance liquid chromatography to obtain the product SS4CSS.

[0109] Synthesis of paclitaxel dimer prodrug linker bridged by 6-carbon-spaced disulfide bonds

[0110] Accurately weigh 1,6-hexanedithiol (463.33 μL, 3.03 mmol), diethyl azodicarbonate (953.70 μL, 6.06 mmol), and thioglycolic acid (420.96 μL, 6.06 mmol). Dissolve each compound in 5 mL of methanol. Transfer the dissolved 1,6-hexanedithiol to a 50 mL round-bottom flask. Add the dissolved thioglycolic acid dropwise, followed by the dissolved diethyl azodicarbonate. Incubate the reaction under nitrogen at room temperature for 12 h. After rotary evaporation, dissolve the product in a methanol solution composed of 50% water and 0.2% formic acid. Centrifuge to discard the precipitate, and separate the supernatant by high-performance liquid chromatography to obtain the product SS6CSS.

[0111] Example 2: Synthesis of a paclitaxel dimer prodrug bridged by a 2-carbon-atom-spaced disulfide bond. The following components were precisely weighed: the linker bond synthesized in Example 1 (1 equivalent), EDCI (4.8 equivalents), 1-hydroxybenzotriazole (HOBT) (4.8 equivalents), triethylamine (8 equivalents), and PTX (2.0 equivalents). The linker bond was added to a round-bottom flask and dissolved in dichloromethane. Then, a dichloromethane solution of EDCI and HOBT was added dropwise. After activation in an ice bath at 0°C for 2 h, PTX and triethylamine were added dropwise. The reaction was allowed to proceed for 24 h at room temperature. The product PTX-SSCCSS-PTX was separated by high-performance liquid chromatography.

[0112] The structure of the PTX-SSCCSS-PTX prepared in Example 2 was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 2 As shown. This meets the requirements for subsequent experiments.

[0113] The above-described synthetic methods were used to prepare paclitaxel dimer prodrugs PTX-SS4CSS-PTX (bridged by disulfide bonds with a 4-carbon interval), PTX-SS6CSS-PTX (bridged by disulfide bonds with a 6-carbon interval), PTX-SS(α)-PTX, and PTX-SS(γ)-PTX.

[0114] Example 3: Preparation and characterization of self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spaced disulfide bonds

[0115] 2 mg of the paclitaxel dimer prodrug prepared in Example 2 and DSPE-mPEG were added. 2000 (0.5 mg) was dissolved in 0.5 mL of anhydrous ethanol. This anhydrous ethanol solution was slowly added dropwise to 2 mL of deionized water with stirring. The ethanol was removed by rotary evaporation under reduced pressure to obtain a nanocolloidal solution free of organic reagents. The results are as follows... Figure 3 As shown, the particle size of each group of nanoparticles is approximately 80-120 nm, with PTX-SSCCSS-PTX NPs having the smallest particle size. The particle size distribution of each group is less than 0.2 mm, the surface charge is approximately -30 mV, and the drug loading is higher than 68%. Transmission electron microscopy images show that the drug-loaded nanoparticles are uniformly spherical.

[0116] Example 4: Assembly force competition test of self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spaced disulfide bonds

[0117] The spaced disulfide-bridged paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 3 were incubated with 0.1 mol / L sodium dodecyl sulfate (SDS), a hydrophobic interaction competing agent; urea, a hydrogen bonding competing agent; and sodium chloride (NaCl), an ionic interaction competing agent. The particle size was measured at specific time points, and the ratio to the initial particle size was calculated. The results are as follows: Figure 4 As shown, the five nanoparticles are mainly assembled through hydrophobic interactions. The hydrophobic interactions driven by the three nanoparticles that are bridged by disulfide bonds for paclitaxel dimer prodrug self-assembly are stronger than those of the nanoparticles that are bridged by monosulfide bonds for paclitaxel dimer prodrug self-assembly, showing enhanced assembly stability compared to the latter.

[0118] Example 5: In vitro release assay of self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spaced disulfide bonds

[0119] The in vitro release of the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 3 was investigated using phosphate-buffered saline (PBS) containing 30% ethanol at pH 7.4 as the release medium. 0.2 mL of the prodrug self-assembled nanoparticles (paclitaxel content 1 mg / mL) prepared in Example 3 was added to 30 mL of the release medium. Certain concentrations of hydrogen peroxide (H₂O₂, 0.1 mM, 1 mM, 10 mM) or dithiothreitol (DTT, 0.05 mM, 0.1 mM, 1 mM) were added to the release medium. Samples were taken at set time points at 37°C, and the percentage of paclitaxel released was determined by high-performance liquid chromatography (HPLC) to investigate the release of the nanoparticles under oxidizing and reducing conditions. The results are as follows: Figure 5 As shown, PTX-SSCCSS-PTX NPs exhibit extremely high redox dual responsiveness, while PTX-SS4CSS-PTX NPs and PTX-SS6CSS-PTX NPs have weaker redox-responsive drug release capabilities than PTX-SSCCSS-PTX NPs.

[0120] Example 6: Cytotoxicity of self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spacer disulfide bonds

[0121] The MTT assay was used to investigate the toxicity of the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 3 to two types of tumor cells and one type of normal cell: mouse breast cancer (4T1) cells, mouse melanoma (B16-F10) cells, and mouse fibroblasts (3T3) cells. First, morphologically sound cells were digested, diluted with culture medium to 2000 cells / mL, and then 200 μL of cell suspension was added to each well of a 96-well plate. The plates were incubated for 24 h to allow cell adhesion. After cell adhesion, either paclitaxel or the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 3 were added. In this experiment, the drug solution and nanoparticle formulation were prepared and diluted using the corresponding cell culture medium and aseptically filtered through a 0.22 μm filter membrane. 200 μL of the test solution was added to each well, with three parallel wells for each concentration. The control group, i.e., without the test drug solution, was supplemented with 200 μL of culture medium and incubated with the cells in an incubator. Forty-eight hours after drug addition, the 96-well plate was removed, and 35 μL of 5 mg / mL MTT solution was added to each well. The plate was incubated for 4 hours, then the culture medium was discarded. The 96-well plate was inverted onto filter paper to thoroughly absorb any remaining liquid. Then, 150 μL of DMSO was added to each well, and the plate was shaken for 10 minutes to dissolve the blue-purple crystals. Well A1 (containing only 150 μL of DMSO) was designated as the zeroing well. The absorbance of each well after zeroing was measured at 570 nm using a microplate reader.

[0122] The results are as follows Figure 6 As shown, since the prodrugs require activation to exert their effects in cells, the cytotoxicity of the three dimeric prodrug nanoassemblies was weaker than that of paclitaxel. The cytotoxicity of the dimeric prodrug nanoassemblies was closely related to their redox activation capacity. The order of antitumor activity of the three dimeric prodrug nanoassemblies was: PTX-SSCCSS-PTXNPs > PTX-SS6CSS-PTX NPs > PTX-SS4CSS-PTX NPs > PTX-SS(γ)-PTX NPs. PTX-SSCCSS-PTXNPs, due to their dual redox hypersensitivity, could effectively cope with the redox microenvironment of tumor cells, thus exhibiting the strongest in vitro antitumor activity.

[0123] Example 7: Pharmacokinetic Study of Self-Assembled Nanoparticles of Paclitaxel Dimer Prodrug Bridged by Spacer Disulfide Bonds

[0124] SD rats weighing 180-220g were randomly divided into groups and fasted for 12 hours before administration, with free access to water. Taxol and the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 3 were administered intravenously, respectively. The dosage was 5 mg / kg (paclitaxel equivalent). Blood was collected from the orbital sinus at specified time points, and plasma was obtained. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0125] Experimental results are as follows Figure 7 As shown, due to its short half-life, paclitaxel is rapidly cleared from the bloodstream. In contrast, the cycling time of paclitaxel dimer prodrug self-assembled nanoparticles is significantly prolonged. Furthermore, different spacing of disulfide bonds significantly affects the pharmacokinetic behavior of the dimer prodrug nanoparticles. Compared to PTX-SSCCSS-PTX NPs and PTX-SS4CSS-PTX NPs, PTX-SS6CSS-PTX NPs exhibit stronger colloidal stability, prolonging their in vivo retention. The area under the curve (AUC) of the three disulfide-bridged prodrug nanoparticles is larger than that of the monosulfide-bridged prodrug nanoparticles (PTX-SS(α)-PTX NPs and PTX-SS(γ)-PTX NPs). This enhanced pharmacokinetic characteristic reflects the excellent chemical and assembly stability of the disulfide-bridged prodrug nanoparticles.

[0126] Example 8: Maximum safe dose experiment of self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spaced disulfide bonds

[0127] Female BALB / c mice were randomly divided into five groups: a paclitaxel dimer prodrug self-assembled nanoparticle group and a paclitaxel group. The self-assembled nanoparticle group received doses of 30, 60, and 90 mg / kg (PTX equivalent dose, n=3), respectively; the paclitaxel group received a dose of 30 mg / kg (n=3). Administered once daily for 5 consecutive days. Mouse weight and survival status were recorded during the treatment period. A 10% decrease in body weight indicated that the dose exceeded the tolerated dose; a 15% decrease in body weight led to discontinuation of treatment; and a 20% decrease in body weight was considered death. Experimental results are as follows: Figure 8 As shown, the order of maximum safe doses is (i) >90 mg / kg: PTX-SS4CSS-PTX NPs and PTX-SS6CSS-PTX NPs; (ii) 60 mg / kg-90 mg / kg: PTX-SS(γ)-PTX NPs; (iii) 30 mg / kg-60 mg / kg: PTX-SSCCSS-PTX NPs > PTX-SS(α)-PTX NPs; (iv) <30 mg / kg: Taxol.

[0128] At the same dosage, PTX-SSCCSS-PTX NPs showed a superior maximum safe dose compared to PTX-SS(α)-PTX NPs, while PTX-SS4CSS-PTX and PTX-SS6CSS-PTX NPs exhibited higher safe doses and better efficacy than PTX-SS(γ)-PTX NPs. These results indicate that dimeric prodrug nanoparticles bridged by spaced bisdisulfide bonds offer a greater safety advantage compared to monodisulfide-bridged dimeric prodrug nanoparticles.

[0129] Example 9: In vivo antitumor experiment of self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spaced disulfide bonds at the maximum safe dose (BALB / C)

[0130] A 4T1 nude mouse xenograft model was established. 4T1 tumor-bearing BALB / c mice were randomly divided into 5 groups (n=5 per group). The paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 3 were administered via tail vein. The nanoparticles were administered paclitaxel 10 mg / kg, saline, PTX-SSCCSS-PTX NPs 60 mg / kg (PTX equivalent dose), PTX-SS4CSS-PTX NPs 120 mg / kg (PTX equivalent dose), and PTX-SS6CSS-PTX NPs 120 mg / kg (PTX equivalent dose), respectively. Results are as follows: Figure 9 As shown, compared with the saline group, all preparation groups significantly slowed tumor growth to some extent.

[0131] Example 10: In vivo antitumor experiment of self-assembled nanoparticles of paclitaxel dimer prodrug bridged by spacer disulfide bonds (C57BL / 6)

[0132] Using B16-F10 tumor-bearing C57BL / 6 mice as a model, paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 3 were administered via tail vein. Taxol and saline intravenous injection groups were set as control groups. The following dosing regimens were used: (i) paclitaxel 30 mg / kg; (ii) nanoparticles 90 mg / kg (paclitaxel equivalent dose). Results are as follows... Figure 10 As shown, compared with the saline group, all formulations slowed tumor growth to some extent. The dimeric prodrug nanoparticles bridged by disulfide bonds exhibited better efficacy than those bridged by monosulfide bonds. In contrast, PTX-SSCCSS-PTX NPs showed higher antitumor efficacy. This is because PTX-SSCCSS-PTX NPs possess good colloidal stability, which improves their pharmacokinetic behavior and results in a higher area under the curve. Simultaneously, PTX-SSCCSS-PTX NPs exhibit a faster drug release rate in tumor cells, enhancing their cytotoxicity. Furthermore, the disulfide-bridged paclitaxel dimeric prodrug nanoparticle group did not show significant weight loss, demonstrating better safety while exhibiting considerable tumor-suppressive activity. In summary, the stability, cytotoxicity, pharmacokinetic distribution, and tumor-site response to drug release all affect the final antitumor effect. The above results further demonstrate the advantage of disulfide-bridged paclitaxel dimeric prodrug self-assembled nanoparticles in simultaneously improving assembly capacity and drug release rate.

Claims

1. A linker bond for bridging a disulfide-spaced dimer prodrug, characterized in that, The connection key structure is shown in equation (I): Where n = 1~5, and X is a C2, C4 or C6 straight-chain carbon chain.

2. The linking bond of the disulfide-bridged dimer prodrug as described in claim 1, characterized in that, n=1。 3. A disulfide-bridged dimer prodrug or a pharmaceutically acceptable salt thereof, characterized in that, The drug is prepared by reacting it with a drug using the linking bonds of the spaced disulfide-bridged dimer prodrug as described in claim 1 or 2, and its structure is shown in formula (II): The drug is paclitaxel.

4. The spaced disulfide-bridged dimer prodrug or a pharmaceutically acceptable salt thereof as described in claim 3, characterized in that, The spaced disulfide-bridged dimer prodrug is selected from the following structural formulas: 。 5. A method for synthesizing a spaced disulfide-bridged dimer prodrug of claim 3 or 4, or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: Synthesis of the linker bond: Dithiol, diethyl azodicarbonate, and thioglycolic acid were accurately weighed and dissolved separately in methanol. The dissolved dithiol was transferred to a reaction flask, and the dissolved thioglycolic acid was added dropwise, followed by the addition of the dissolved diethyl azodicarbonate. The reaction was carried out under nitrogen protection at room temperature. After the reaction was completed, the solvent was evaporated, and the product was dissolved in a methanol solution containing 30-50% water and 0.2% formic acid. The precipitate was discarded by centrifugation, and the supernatant was separated to obtain the product. The dithiol was selected from 1,2-ethanedithiol, 1,4-butanedithiol, and 1,6-hexanedithiol. Dimeric prodrugs or their pharmaceutically acceptable salts can be prepared using the obtained linker bonds: Method 1: Accurately weigh the linker, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and the anticancer drug. Place the linker in a reaction flask, and add a portion of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine sequentially. Activate in an ice bath at 0°C. Add the anticancer drug dropwise, and another portion of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine dropwise. React at room temperature, and separate and purify the product. Method 2: Accurately weigh the linker, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, triethylamine, and the anticancer drug. Add the linker to a reaction flask, dissolve it in solvent, and then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole dropwise. After activation in an ice bath at 0°C, add the anticancer drug and triethylamine dropwise. React at room temperature and separate the products.

6. The spaced disulfide bond-bridged dimer prodrug self-assembled nanoparticles according to claim 3 or 4, characterized in that, The nanoparticles include unPEGylated disulfide-bridged dimer prodrug self-assembled nanoparticles, PEG-modified disulfide-bridged dimer prodrug self-assembled nanoparticles, disulfide-bridged dimer prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances, and disulfide-bridged dimer prodrug self-assembled nanoparticles loaded with other drugs; the preparation method includes the following steps: When a non-PEGylated disulfide-bridged dimer prodrug self-assembles into nanoparticles, the disulfide-bridged dimer prodrug is dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water. The prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent. When PEGylated disulfide-bridged dimer prodrugs self-assemble into nanoparticles, the disulfide-bridged dimer prodrug and PEG modifier are dissolved in an organic solvent. The solution is then slowly added dropwise to water while stirring. The prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent. When self-assembling nanoparticles of disulfide-bridged dimer prodrugs loaded with hydrophobic fluorescent substances or other drugs, the disulfide-bridged dimer prodrug, fluorescent substance or drug, and PEG modifier are dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water. The prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent. The PEG modifier is selected from TPGS, DSPE-PEG, PLGA-PEG and PE-PEG, and the molecular weight of the PEG is 1000-5000; the solvent is selected from ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran and acetone; the weight ratio of the disulfide-bridged dimer prodrug to the PEG modifier is 90:10 to 60:

40.

7. A pharmaceutical composition comprising the spaced disulfide-bridged dimer prodrug of claim 3 or 4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. The use of the spaced disulfide-bridged dimer prodrug of claim 3 or 4, or a pharmaceutically acceptable salt thereof, or the self-assembled nanoparticles of the dimer prodrug of claim 6, or the pharmaceutical composition of claim 7, in the preparation of a drug delivery system.

9. The use of the spaced disulfide-bridged dimer prodrug of claim 3 or 4, or a pharmaceutically acceptable salt thereof, or the self-assembled nanoparticles of the dimer prodrug of claim 6, or the pharmaceutical composition of claim 7, in the preparation of an antitumor drug.

10. The use of the spaced disulfide-bridged dimer prodrug of claim 3 or 4, or a pharmaceutically acceptable salt thereof, or the self-assembled nanoparticles of the dimer prodrug of claim 6, or the pharmaceutical composition of claim 7, in the preparation of an injectable, oral, or topical delivery system.

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