Tripterine-sophocarpidine self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a tripterine-sophocarpidine self-assembled nanoparticle and application thereof in preparation of an anti-tumor drug, and belongs to the field of biological medicine. The nanoparticles are formed by self-assembly of the tripterine and the sophocarpidine through supramolecular interaction, the molar ratio of the tripterine to the sophocarpidine is 1: 1-1: 3, and the self-assembly system effectively solves the problems of low Cst solubility, poor bioavailability, rapid in-vivo removal and the like; the inhibition effect on triple negative breast cancer (TNBC) is remarkably enhanced through the synergistic effect of Cst and Mar, and a new strategy is provided for expanding the application of a traditional Chinese medicine carrier-free self-assembly nano drug delivery system in the anti-tumor field.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a tripterygium wilfordii-matrine self-assembled nanoparticle, a preparation method thereof, and an application thereof. Background Art

[0002] Celastrol (Cst) is a pentacyclic triterpenoid monomer compound extracted from Tripterygium wilfordii. It has multiple pharmacological effects such as anti-inflammatory, antioxidant, immunosuppressive and anti-tumor, and shows a wide range of anti-cancer activities against various cancers such as cervical cancer, lung cancer, breast cancer, liver cancer, etc. Studies have found that Cst causes the loss of mitochondrial membrane potential (MMP) and the release of cytochrome C into the cytoplasm of human triple-negative breast cancer cells MDA-MB-231, inducing cell apoptosis through mitochondrial dysfunction and PI3K / Akt signaling pathway, thereby exerting anti-TNBC efficacy (SHRIVASTAVA S, JEENGAR MK, REDDY VS, et al. Anticancer effect of celastrol on human triple-negative breast cancer: possible involvement of oxidative stress, mitochondrial dysfunction, apoptosis and PI3K / Akt pathways [J]. Experimental and molecular pathology, 2015, 98(3): 313-27.).

[0003] Although Cst has therapeutic potential for TNBC, it has many limitations, such as poor water solubility (only 13µg / mL at 37°C), low bioavailability, narrow therapeutic window, and multiple adverse reactions (HOU W, LIU B, XU H. Celastrol: Progresses instructure-modifications, structure-activity relationships, pharmacology and toxicology [J]. European journal of medicinal chemistry, 2020, 189: 112081.).

[0004] To overcome the above-mentioned shortcomings, modern pharmaceutical formulation technology can be used to solve the water solubility problem and construct a targeted drug delivery system to reduce systemic toxicity. For example, the applicant previously encapsulated Cst in galactose-modified PEGylated liposomes. This system improved the water solubility of Cst and exhibited good sustained release and active liver targeting properties. The applicant also synthesized a cholesterol derivative modified with triphenylphosphonium bromide (TPP) and constructed it together with natural soybean lecithin into a cationic liposome with mitochondrial targeting function. The negatively charged hyaluronic acid was further coated on the outer layer of the liposome through electrostatic interaction, thereby achieving dual targeting of mitochondria and tumor cells. This dual-targeting system not only significantly improved the anti-liver cancer therapeutic effect of Cst, but also reduced the toxic side effects such as weight loss caused by Cst (CN 116284197 A).

[0005] While targeted delivery systems can improve Cst's water solubility, prolong its circulation in the body, reduce systemic toxicity, and enhance anticancer efficacy, the ingestion of targeted materials may cause certain in vivo toxicity, and the metabolic mechanism is still unclear. Furthermore, single-drug use is not only highly toxic but also prone to drug resistance.

[0006] Supramolecular self-assembly refers to the process by which molecules spontaneously form ordered structures through non-covalent interactions (such as hydrogen bonding, electrostatic interactions, hydrophobic interactions, π-π stacking, and van der Waals forces). This self-assembly property can be used to construct nano-drug delivery systems, not only to improve drug solubility and enhance drug stability, but also to protect drugs from degradation through the self-assembled nanostructures, extending their circulation time in the body. Furthermore, through the design of specific targeting ligands, targeted delivery to tumor cells or other specific cells can be achieved. Zhao et al. used an antisolvent precipitation method to construct dual-functional carrier-free nanoparticles through the hydrophobic and electrostatic interactions between 10-hydroxycamptothecin and the photosensitizer chlorine. These nanoparticles not only showed high cellular uptake but also significantly enhanced cell activity and efficiently generated reactive oxygen species under laser irradiation (ZHAO Y, ZHAO Y, MAQ, et al. Carrier-free, dual-functional nanorods via self-assembly of puredrug molecules for synergistic chemo-photodynamic therapy [J]. Internationaljournal of nanomedicine, 2019, 14: 8665-83.). Clofarabine and raltitrexed are two purine core analogs with different properties. These two analogs can spontaneously self-assemble in water to form supramolecular nanoparticles. This unique nanostructure not only prolongs the half-life of the drug in the body, but also improves the accumulation efficiency of the drug at the tumor site, thereby enhancing the anti-tumor effect of the drug in the body (WANG D, YU C, XU L, et al. Nucleoside analogue-based supramolecular nanodrugs driven by molecular recognition for synergistic cancer therapy [J]. Journal of the American Chemical Society, 2018, 140(28): 8797-806.).

[0007] CN 107929290 A discloses a self-assembling nanomedicine. By spontaneously assembling the hydrophobic drug tripterine and the hydrophilic drug doxorubicin hydrochloride in an aqueous solution to form nanoparticles, the drug significantly improves its bioavailability and effectively prolongs its in vivo circulation time. The nanoparticles also exhibit excellent permeability and retention (EPR) effect in solid tumors, allowing them to specifically target cancer sites, achieving the therapeutic goals of reducing drug toxicity and providing highly effective anti-tumor effects.

[0008] Inspired by this, we envisioned combining Cst with another active Traditional Chinese Medicine molecule through supramolecular self-assembly to form nanoparticles. This could be an effective approach to address Cst's poor water solubility, low bioavailability, and significant toxic side effects. Alkaloids are a class of nitrogen-containing heterocyclic compounds found widely in nature, exhibiting a wide range of pharmacological activities, including anti-tumor, anti-inflammatory, and antiviral properties. Most alkaloids contain multiple nitrogen atoms in their molecular structure and are typically alkaline. These structural features give alkaloids unique behavior in intermolecular interactions, giving them great potential for self-assembly with Cst. Summary of the Invention

[0009] In order to achieve supramolecular self-assembly of tripterygium wilfordii and traditional Chinese medicine alkaloid components, the present invention constructed a tripterygium wilfordii-matrine self-assembly nanoparticle. The construction of the nanoparticle not only solves the problem of poor drugability of Cst, but also achieves the synergistic effect of alkaloids and Cst, thereby enhancing the anti-tumor therapeutic effect.

[0010] To achieve this objective, the applicants previously screened alkaloids from traditional Chinese medicines (TCMs), including matrine, oxymatrine, berberine, and sinomenine, through molecular thermodynamic and kinetic simulations and in vitro tumor inhibition assays. They identified matrine (Mar), an alkaloid that undergoes supramolecular self-assembly with Cst and produces a synergistic anti-tumor effect. In the present invention, the applicants prepared supramolecular self-assembled nanoparticles of tripterygium wilfordii-matrine (Cst-Mar-NPs) using a solvent precipitation method. The particle size and Cst binding efficiency (BE) of the Cst-Mar-NPs were then investigated. The morphology and structural elucidation of the Cst-Mar-NPs were performed using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Furthermore, the stability and in vitro drug release behavior of the Cst-Mar-NPs were investigated, and the effect of the self-assembled nanoparticles on enhancing the water solubility of Cst was evaluated.

[0011] Furthermore, the applicants conducted in vitro cytological studies using human breast cancer cells, MDA-MB-231. First, the MTT assay was used to evaluate the synergistic inhibitory effect of Cst-Mar-NPs on TNBC. Next, coumarin 6 (C6) was used as a fluorescent probe to prepare C6@Cst-Mar-NPs loaded with C6, and the intracellular uptake of Cst-Mar-NPs was observed.

[0012] Finally, by establishing a triple-negative breast cancer heterotopic transplant tumor model, the in vivo pharmacodynamics of Cst, Mar, Cst+Mar (a physical mixture of Cst and Mar), and Cst-Mar-NPs after tail vein administration were investigated. The changes in tumor volume of mice during administration were monitored and the tumor inhibition rate was calculated, thereby evaluating the anti-tumor efficacy of Cst-Mar-NPs.

[0013] The specific technical solutions are as follows: A type of tripterine-matrine self-assembled nanoparticles, formed by the self-assembly of tripterine and matrine through supramolecular interaction, has a particle size of 100-200 nm and a zeta potential of -20 mV to -30 mV. The molar ratio of tripterine to matrine is 1:1 to 1:3. In particular, when the molar ratio of Cst to Mar is 1:3, the self-assembled nanoparticles (Cst-Mar-NPs) exhibit superior assembly properties and produce a stronger synergistic anti-tumor effect.

[0014] The present invention further provides a method for preparing the tripterine-matrine self-assembled nanoparticles, comprising the following steps: (a) dissolving triptolide and matrine in an organic solvent to form an organic phase; (b) adding the organic phase dropwise to the aqueous phase and stirring to form a pre-assembled mixture; (c) placing the mixed solution into a dialysis bag and dialyzing it against water to remove the organic solvent; (d) After dialysis, the supernatant was collected by centrifugation to obtain the tripterygium wilfordii-matrine self-assembled nanoparticle solution.

[0015] The present invention uses a solvent precipitation method to prepare nanoparticles. After comparison, the nanoparticles prepared by this method have a more moderate particle size, a more uniform distribution and a higher Cst binding rate than the thin film dispersion method and the ultrasonic crushing method.

[0016] Wherein, the organic solvent in step (a) is selected from methanol, tetrahydrofuran or DMSO, preferably methanol.

[0017] Wherein, the stirring speed in step (b) is 300-700 r / min, and the stirring time is 30-60 min. More preferably, the stirring speed is 500 r / min, and the stirring time is 60 min.

[0018] The molecular weight cut-off of the dialysis bag in step (c) is 3500 Da, which is set according to the molecular size of Cst-Mar-NPs.

[0019] Leveraging the self-assembly properties of active small molecules from traditional Chinese medicine, this invention, through various technical approaches, discovered and confirmed that Mar and Cst can undergo supramolecular self-assembly to form a stable nanodrug delivery system (Cst-Mar-NPs). This self-assembly system not only effectively addresses the issues of Cst's low solubility, poor bioavailability, and rapid clearance from the body, but also significantly enhances the inhibitory effect against TNBC through the synergistic effect of Cst and Mar. While the invention does not incorporate a targeting ligand, it still achieves passive targeted accumulation of the drug at the tumor site, significantly improving anti-tumor efficacy. More importantly, Cst-Mar-NPs achieve both enhanced efficacy and reduced toxicity, significantly mitigating Cst-induced weight loss, hepatotoxicity, renal toxicity, and other side effects. This invention provides new insights and strategies for optimizing carrier-free self-assembly nanodrug delivery systems from traditional Chinese medicine and expanding their application in the anti-tumor field. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Appearance, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of Cst-Mar-NPs.

[0021] Figure 2 : Fourier transform infrared spectrum of Cst-Mar-NPs.

[0022] Figure 3 : Changes in particle size and binding rate of Cst-Mar-NPs after storage at 4°C for 60 days.

[0023] Figure 4 : Changes in particle size and binding rate of Cst-Mar-NPs after incubation with 10% fetal bovine serum (FBS).

[0024] Figure 5 : Changes in particle size (A) and binding rate (B) of Cst-Mar-NPs before and after freeze-drying and reconstitution.

[0025] Figure 6 : In vitro drug release profiles of Cst-Mar-NPs.

[0026] Figure 7 : Comparison of Cst solubility in water.

[0027] Figure 8 : Inhibitory effects of different preparations on the growth of MDA-MB-231 cells.

[0028] Figure 9 : Combined index of the growth inhibitory effect of Cst+Mar (1:3) and Cst-Mar-NPs on MDA-MB-231 cells.

[0029] Figure 10: Intracellular distribution of free C6 and C6@Cst-Mar-NPs after incubation with MDA-MB-231 cells for 4 h.

[0030] Figure 11 : Uptake of Cst and Mar by free Cst, Mar, and Cst-Mar-NPs after incubation with MDA-MB-231 cells for 4 h.

[0031] Figure 12 : Changes in tumor volume in tumor-bearing nude mice after intravenous injection of different preparations.

[0032] Figure 13 : Tumor body mass index and tumor inhibition rate in tumor-bearing nude mice after drug administration. DETAILED DESCRIPTION

[0033] The present invention will be described in further detail below with reference to specific examples. It should be noted that the specific examples are intended only to explain the present invention and are not intended to limit the present invention. In addition, the various experimental procedures involved in the examples are conventional in the art unless otherwise stated. For any part not specifically described herein, those skilled in the art can refer to various commonly used reference books, scientific literature, or relevant specifications, manuals, etc. before the filing date of the present invention for implementation.

[0034] Celastrol (Cst) has potential therapeutic effects against triple-negative breast cancer (TNBC), but its poor water solubility, low bioavailability, and rapid clearance from the body, coupled with the potential for systemic toxicity and side effects from oral formulations, severely restrict its clinical application. Alkaloids from traditional Chinese medicines, such as matrine, oxymatrine, berberine, and sinomenine, exhibit not only significant antitumor activity but also protective effects on organs such as the liver and kidneys. These alkaloids contain multiple nitrogen atoms in their molecular structure, potentially allowing for supramolecular self-assembly with Cst. Therefore, using a supramolecular self-assembly strategy, we screened these alkaloids for their self-assembly properties and synergistic antitumor effects, namely matrine (Mar). In this study, we constructed self-assembled nanoparticles combining matrine and Cst. This strategy addresses the poor drugability of Cst and, through the synergistic action of matrine and Cst, enhances the therapeutic efficacy against TNBC while mitigating Cst-induced hepatotoxicity and renal toxicity.

[0035] Example 1 6.6 mg of Mar and 4 mg of Cst (molar ratio 3:1) were accurately weighed and dissolved in 400 μL of methanol. The solution was then added dropwise to 10 mL of aqueous phase at room temperature. After stirring at 500 rpm for 60 minutes, the solution was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed against water for 24 hours, changing the water three times. Finally, the solution in the dialysis bag was centrifuged at 3000 rpm for 10 minutes. The supernatant was the Cst-Mar-NPs solution.

[0036] Example 2 4.4 mg of Mar and 4 mg of Cst (molar ratio 2:1) were accurately weighed and dissolved in 400 μL of tetrahydrofuran. The mixture was then added dropwise to 10 mL of aqueous phase at room temperature and stirred at 500 rpm for 30 minutes. The mixed solution was then placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in water for 24 hours, changing the water three times. Finally, the solution in the dialysis bag was centrifuged at 3000 rpm for 10 minutes. The supernatant was the Cst-Mar-NPs solution.

[0037] Example 3 2.2 mg of Mar and 4 mg of Cst (1:1 molar ratio) were accurately weighed and dissolved in 400 μL of tetrahydrofuran. The mixture was then added dropwise to 10 mL of aqueous phase at room temperature and stirred at 500 rpm for 30 minutes. The mixed solution was then placed in a dialysis bag (molecular weight cutoff, 3500 Da) and dialyzed in water for 24 hours, changing the water three times. Finally, the solution in the dialysis bag was centrifuged at 3000 rpm for 10 minutes. The supernatant was the Cst-Mar-NPs solution.

[0038] Example 4 11 mg of Mar and 4 mg of Cst (molar ratio 5:1) were accurately weighed and dissolved in 400 μL of tetrahydrofuran. The mixture was then added dropwise to 10 mL of aqueous phase at room temperature and stirred at 500 rpm for 30 minutes. The mixed solution was then placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in water for 24 hours, changing the water three times. Finally, the solution in the dialysis bag was centrifuged at 3000 rpm for 10 minutes. The supernatant was the Cst-Mar-NPs solution.

[0039] Example 5 6.6 mg of Mar and 4 mg of Cst (molar ratio 3:1) were accurately weighed and dissolved in 400 μL of methanol. The mixture was then added dropwise to 10 mL of aqueous phase at room temperature and stirred at 500 rpm for 15 minutes. The mixed solution was then placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in water for 24 hours, changing the water three times. Finally, the solution in the dialysis bag was centrifuged at 3000 rpm for 10 minutes. The supernatant was the Cst-Mar-NPs solution.

[0040] Example 6 6.6 mg of Mar and 4 mg of Cst (molar ratio 3:1) were accurately weighed and dissolved in 400 μL of DMSO. The mixture was then added dropwise to 10 mL of aqueous phase at room temperature and stirred at 500 rpm for 30 minutes. The mixed solution was then placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in water for 24 hours, changing the water three times. Finally, the solution in the dialysis bag was centrifuged at 3000 rpm for 10 minutes. The supernatant was the Cst-Mar-NPs solution.

[0041] Example 7 6.6 mg of Mar and 4 mg of Cst (molar ratio 3:1) were accurately weighed and dissolved in 400 μL of methanol. The mixture was then added dropwise to 10 mL of aqueous phase at room temperature and stirred at 700 rpm for 30 minutes. The mixed solution was then placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in water for 24 hours, changing the water three times. Finally, the solution in the dialysis bag was centrifuged at 3000 rpm for 10 minutes. The supernatant was the Cst-Mar-NPs solution.

[0042] Example 8 Accurately weigh 5 mg of Mar and 3 mg of Cst (molar ratio 3:1), dissolve in 400 μL of methanol, and add dropwise to 10 mL of aqueous phase at room temperature. After stirring at 500 rpm for 30 minutes, transfer the solution to a dialysis bag (molecular weight cutoff 3500 Da) and dialyze against water for 24 hours, changing the water three times. Centrifuge the solution at 3000 rpm for 10 minutes. The supernatant is the Cst-Mar-NPs solution.

[0043] Example 9 (Thin Film Dispersion Method) Accurately weigh 6.6 mg of Mar and 4 mg of Cst (molar ratio 3:1), add 400 μL of tetrahydrofuran, sonicate and dissolve, and remove the organic solvent by rotary evaporation under reduced pressure at 37°C. After a thin film is formed at the bottom of the bottle, add 10 mL of water and hydrate at 37°C for 3 h. Centrifuge at 3000 r / min for 10 min to remove unbound free Cst. The supernatant is the Cst-Mar-NPs solution.

[0044] Example 10 (Ultrasonic Disintegration Method) Accurately weigh 6.6 mg of Mar and 4 mg of Cst (molar ratio 3:1), add 10 mL of distilled water, and then use a cell ultrasonic disruptor to sonicate in an ice bath at 225 W intensity for 10 min, centrifuge at 3000 rpm for 10 min, and the supernatant is the Cst-Mar-NPs solution.

[0045] Test example Physical and Chemical Property Measurements: After diluting the Cst-Mar-NPs solution to a certain multiple, the particle size distribution, polydispersity index (PDI), and zeta potential were measured using a Malvern laser particle size analyzer. Each sample was measured in triplicate. The drug binding efficiency (BE) was determined using low-speed centrifugation. The nanoparticle solution before and after centrifugation was precisely aspirated, and the supernatant was collected for HPLC determination of the free Cst content. The Cst binding efficiency was calculated according to the following formula.

[0046] , where W is the amount of Cst in the supernatant after centrifugation, and W0 is the amount of Cst in the nanoparticles before centrifugation.

[0047] Table 1: Particle size, PDI, Zeta potential and Cst binding rate of Cst-Mar-NPs

[0048] Results showed that the majority of the prepared Cst-Mar-NPs had a uniform particle size distribution within the 100-200 nm range, making them easily targeted and enriched at tumor sites via the EPR effect. Furthermore, the negative surface charge and high drug binding efficiency of the nanoparticles effectively reduced reticuloendothelial uptake, significantly prolonging their residence time in the bloodstream and improving drug delivery efficiency at the target site.

[0049] 2. Micromorphology of Cst-Mar-NPs Transmission electron microscopy: Cst-Mar-NPs were dropped onto a 100-mesh copper grid. After adsorption for 10 minutes, excess solution was removed with filter paper. The grid was then negatively stained with 2% phosphotungstic acid for 1 minute, excess dye was removed, and the grid was allowed to air dry. The morphology of the nanoparticles was observed using a transmission electron microscope at an accelerating voltage of 80 kV. Scanning electron microscopy observation: Cst-Mar-NPs were diluted to a certain concentration, freeze-dried, and coated with gold for 6 minutes before observing the morphology of the nanoparticles under a scanning electron microscope at an accelerating voltage of 3 kV.

[0050] like Figure 1 As shown, the Cst-Mar-NPs solution is orange-red and exhibits a distinct Tyndall effect under opalescent pen illumination. TEM images show that the Cst-Mar-NPs are spherical and uniform in size, with a particle size of approximately 100-200 nm, consistent with the results obtained using a Malvern particle size analyzer. Scanning electron microscopy (SEM) also reveals that the Cst-Mar-NPs are spherical, with a smooth surface and uniform size distribution.

[0051] 3. Fourier transform infrared spectroscopy (FT-IR) Appropriate amounts of Cst, Mar, and Cst-Mar-NPs freeze-dried powder were weighed and mixed with dry KBr powder, respectively. After grinding and mixing evenly, the mixture was pressed into uniform thin slices using a tablet press. The Fourier transform infrared spectrometer was used to analyze the morphology of the nanostructured ... -1 Its infrared absorption spectrum was measured in a wavenumber range.

[0052] like Figure 2 As shown, Mar at 3420cm -1 The stretching vibration absorption peak of the NH group appears at 1632 cm -1 The typical C=O stretching vibration absorption peak is shown at 3390cm -1 The stretching vibration absorption peak of OH appears at 1701cm -1 The vibration absorption peak of COOH appeared. In the spectrum of Cst-Mar-NPs, the NH characteristic absorption peak of Mar and the OH absorption peak of Cst merged to 3358 cm -1 This change indicates that the NH and OH groups may participate in the formation of intermolecular hydrogen bonds, resulting in the broadening and red shift of the absorption peak. At the same time, the COOH absorption peak of Cst shifted from 1701 cm -1 Red shift to 1695 cm -1 , the C=O absorption peak of Mar is from 1632 cm -1 Red shift to 1595 cm -1 , suggesting that the COOH of Cst and the C=O of Mar may participate in hydrogen bonding. These results indicate that there is an interaction between Mar and Cst.

[0053] 4. Stability of Cst-Mar-NPs (1) Low temperature storage stability The Cst-Mar-NPs solution was stored in a refrigerator at 4°C away from light. Samples were taken at predetermined time points (0d, 1d, 3d, 7d, 15d, 30d, 45d, and 60d) to determine the particle size and Cst binding rate.

[0054] During storage at 4°C for 60 days, the particle size of Cst-Mar-NPs increased slightly, but did not show significant changes (P>0.05) ( Figure 3 Dynamic light scattering analysis showed that the system maintained a narrow, single-peak distribution (PDI < 0.200). The Cst binding rate decreased slightly but remained above 90% after 60 days of storage, demonstrating that Cst-Mar-NPs possessed excellent low-temperature storage stability. (2) Serum stability The Cst-Mar-NPs solution was dispersed in PBS containing 10% FBS to a final concentration of Cst of 200 μg / mL, and then incubated in a 37°C shaking incubator for 24 h. Samples were taken at predetermined time points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h) to determine the particle size and Cst binding rate. like Figure 4 As shown in the data, since the potential of Cst-Mar-NPs is negative, its particle size does not change significantly due to the influence of substances such as proteins in the serum, and only slightly increases from 125.80±0.80nm to 131.30±3.90nm. The binding rate of Cst is not affected by serum and is still above 95% after incubation for 48h, indicating that Cst-Mar-NPs have good serum stability. It also indicates that Cst-Mar-NPs may have good stability in the blood circulation in the body and will not leak drugs.

[0055] (3) Freeze-dried and reconstituted stability The Cst-Mar-NPs solution was placed in a vacuum freeze dryer for freeze drying. After freeze drying, an appropriate amount was taken and reconstituted with distilled water. The particle size and Cst binding rate before and after freeze drying and reconstitution were measured respectively.

[0056] like Figure 5 As shown in Figure 2, the particle size of Cst-Mar-NPs was almost unaffected by freeze-drying. The particle size after freeze-drying was 105.80±2.80nm, which was not significantly different from that before freeze-drying (111.60±1.60nm). Figure 5 A), at the same time, the binding rate of Cst decreased slightly after reconstitution, but still remained above 90% ( Figure 5 B), indicating that Cst-Mar-NPs exhibited good stability during freeze-drying and reconstitution.

[0057] 5. In vitro drug release behavior of Cst-Mar-NPs 1 mL of free Cst (200 μg / mL), free Mar (330.7 μg / mL), and Cst-Mar-NPs (Cst at 200 μg / mL and Mar at 330.7 μg / mL) were placed in a dialysis bag (molecular weight cutoff 3500 Da) and tied at both ends. The samples were then placed in 20 mL of PBS (pH 7.4) containing 1% Tween 80. Release was investigated in a 37°C water bath under stirring at 100 rpm. At 1, 2, 4, 6, 8, 12, and 24 h, 1 mL of release medium was aspirated and replaced with the same volume and temperature of release medium to maintain a constant dialysis environment. Each group underwent three replicates. The drug content in the release medium was determined, the cumulative percentage of drug released (Q) was calculated, and release curves for Cst and Mar were plotted.

[0058] like Figure 6 As shown in the figure, free Cst was completely released within 24 hours, and free Mar was almost completely released within 2 hours. However, Cst-Mar-NPs cumulatively released less than 57% of Cst and 60% of Mar in the same time, indicating that Cst-Mar-NPs have a sustained release property.

[0059] 6. Solubility of Cst in water Excess Cst and Cst-Mar-NPs powders were weighed separately and placed in centrifuge tubes. 2 mL of water was added. The tubes were placed in an incubator shaker at 25°C and shaken at a constant frequency for 48 hours to allow the solution to reach equilibrium. Subsequently, the tubes were centrifuged at 12,000 rpm for 15 minutes. After centrifugation, the supernatant was carefully removed and the Cst content was determined by HPLC.

[0060] like Figure 7 As shown in the results, the solubility of free Cst in water was only 12.96±1.23 μg / mL at 25°C, but after forming self-assembled nanoparticles with Mar, the solubility of Cst increased to 236.18±1.89 μg / mL, an increase of about 20 times, indicating that self-assembled nanoparticles can significantly improve the water solubility of Cst.

[0061] 7. Cell Proliferation Inhibition Test The MTT assay was used to evaluate the inhibitory effect of Cst and Mar supramolecular self-assembled nanoparticles on the proliferation of MDA-MB-231 cells. MDA-MB-231 cells in the logarithmic growth phase were collected and plated at 2×10 4Cells were plated evenly in a 96-well plate at a density of 10 cells / well. Blank, control, and drug-treated groups (free Cst, free Mar, Cst+Mar physical mixture, and Cst-Mar-NPs) were set up, with six replicates per group. After the cells attached and grew, culture medium containing different drug concentrations (0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, and 10 μg / mL, calculated based on the Cst concentration) was added and cultured for a further 24 hours. The culture medium was then aspirated, and the cells were washed twice with PBS. 100 μL of 1 μg / mL MTT reagent was added to each well and cultured for a further 4 hours. The MTT solution was aspirated, and 150 μL of DMSO was added and shaken to completely dissolve the purple crystals. The absorbance (A) of each well was measured at 570 nm using a microplate reader to calculate cell viability.

[0062] Data analysis was performed using CompuSyn software. Developed based on the Chou-Talalay median-effect mathematical model, CompuSyn correlates the dose-effect relationships of monotherapy and combination therapy, and then calculates the Combination Index (CI) to assess drug-drug interactions. Specifically, a CI = 1 indicates an additive effect, a CI < 1 indicates a synergistic effect, and a CI > 1 indicates an antagonistic effect. This method was used to analyze the effects of the combined use of Cst and Mar on MDA-MB-231 cell proliferation.

[0063] GraphPad Prism 8.0 software was used to analyze the experimental data and plot them. Independent sample t-test and one-way analysis of variance (ANOVA) were used to evaluate the differences among the groups. All experimental data were expressed as mean ± standard deviation (SD).

[0064] like Figure 8 As shown in Figure 2, free Cst, free Mar, Cst+Mar physical mixture (Cst to Mar molar ratio of 1:3) and Cst-Mar-NPs all showed a certain degree of concentration-dependent inhibitory effect. The combination index CI was obtained by CompuSyn software analysis. The results are shown in Figure 2. Figure 9 As shown, at IC50 concentration, the CI of Cst+Mar (1:3) is 0.89 ( Figure 9 a), the CI of Cst-Mar-NPs is 0.44 ( Figure 9 b), are all less than 1, indicating that Cst+Mar and Cst-Mar-NPs have a synergistic inhibitory effect on breast cancer cells MDA-MDB-231.

[0065] 8. Cellular Uptake Assay Since Cst itself does not have fluorescent properties, this study selected coumarin 6 (C6) as a fluorescent probe, prepared C6-loaded C6@Cst-Mar-NPs, and observed its cellular uptake.

[0066] MDA-MB-231 cells in the logarithmic growth phase were taken and 2×10 5 Cells were evenly plated in a 6-well plate at a density of 10 cells / well. After the cells adhered and grew, the culture medium was removed and the cells were washed twice with 4°C PBS. Then, 1 mL of free C6 and C6@Cst-Mar-NPs, diluted with complete culture medium to a C6 concentration of 200 ng / mL, were added, respectively, and incubated in a cell culture incubator for another 4 hours. The culture medium was removed, and the cells were washed twice with 4°C PBS. The cells were fixed with 4% paraformaldehyde for 10 minutes, washed with PBS, and then stained with Hoechst 33342 (10 μg / mL) for 10 minutes at room temperature in the dark. After washing with PBS, the cells were resuspended in PBS and observed for cellular uptake using an inverted fluorescence microscope.

[0067] Next, in order to clarify the uptake of Cst and Mar in Cst-Mar-NPs in MDA-MB-231 cells, the HPLC method was used to detect the content of Cst and Mar in the cells after Cst-Mar-NPs were incubated with MDA-MB-231 cells for 4 hours, and the protein concentration in the cells was used as the standard to calculate the cellular uptake of Cst and Mar relative to the protein concentration.

[0068] like Figure 10 As shown in the figure, the green fluorescence representing C6 in the C6@Cst-Mar-NPs group is distributed around the cell nucleus stained blue, while the green fluorescence around the cell nucleus in the free C6 group is extremely weak, indicating that C6@Cst-Mar-NPs can more easily enter the cells. Figure 11 As shown in the results, after 4 h of incubation with cells, the uptake of Cst in the Cst-Mar-NPs group was 1.7 times that of the free Cst group (P<0.001), and the uptake of Mar was 2 times that of the free Mar group (P<0.001), indicating that Cst-Mar-NPs significantly increased the uptake of Cst and Mar in tumor cells.

[0069] 9. Evaluation of drug efficacy in triple-negative breast cancer Experimental animals: Forty female BALB / c-Nu mice (18-22 g) were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., animal production license number: SCXK (Su) 2020-0009. All animal procedures were performed in accordance with the relevant animal experimental procedures regulations of Hubei University of Chinese Medicine. This study was approved by the Animal Experimentation Ethics Committee of Hubei University of Chinese Medicine. Animal ethics approval number: HUCMS 53562747.

[0070] Establishment of ectopic tumor mouse model: 0.2 mL of MDA-MB-231 cell suspension (5×10 7 Cells / mL) were injected into the right axilla of 5-week-old female BALB / c nude mice to establish a xenograft tumor model. The size of the tumor at the subcutaneous injection site of the nude mice was measured with a vernier caliper. The tumor volume was calculated as V = L / 2 × W 2 , V is the tumor volume, L is the long diameter of the tumor, and W is the short diameter of the tumor. When the tumor size reaches about 100 mm 3 When, conduct the test.

[0071] Preparation of reagents: Appropriate amounts of Cst, Mar, and Cst+Mar (molar ratio 1:3) were weighed and dissolved in anhydrous ethanol. An equal volume of polyoxyethylene castor oil was added, and the volume was adjusted to 0.2 mg / mL with normal saline injection. The Cst-Mar-NPs solution was prepared by dissolving the lyophilized powder in normal saline to a 0.2 mg / mL solution.

[0072] Dosage regimen: Six BALB / c mice were randomly selected as the control group (not inoculated with tumor cells), and the remaining 34 mice were inoculated with MDA-MB-231 cells. Seven days after inoculation, four mice that failed to successfully form tumors were culled, and the tumor volume reached approximately 100 mm. 3 The remaining mice were randomly divided into five groups of six. On day 8 of inoculation, mice in each group were injected via the tail vein with different formulations (normal saline, free Cst, free Mar, Cst+Mar, and Cst-Mar-NPs). The Cst dose was 3 mg / kg, and the Mar dose was 5 mg / kg, administered once daily for a total of seven doses. During the dosing period, the nude mice had free access to food and water. Survival was monitored and sacrificed by cervical dislocation on day 21 (calculated from the day of inoculation).

[0073] Results Calculation: Nude mice were weighed and tumor length and width were measured every day. Tumor volume was plotted against time to create a tumor growth curve. After sacrificing the nude mice, tumor tissue was excised, washed with PBS, and then dehydrated with filter paper. The tissue was photographed and weighed. The tumor index was calculated as the ratio of tumor weight to the body weight of the tumor-bearing nude mice. The tumor inhibition rate was also calculated as (average tumor weight of the model group - average tumor weight of the treatment group) / average tumor weight of the treatment group × 100%.

[0074] like Figure 12 As shown in Figure 2, different preparation groups can effectively inhibit the growth of tumor volume. Among them, the tumor growth rate of Cst-Mar-NPs is the slowest, indicating that it has significant anti-tumor activity. Figure 13A It can be found that the tumor index of the Cst-Mar-NPs group is the smallest, which is significantly different from not only the free Cst and Mar groups (P<0.001), but also from the physical mixture group of the two drugs Cst+Mar (P<0.001), indicating that the self-assembly of Cst and Mar helps to improve the anti-tumor effect and can better synergistically inhibit the growth of triple-negative breast tumors. Figure 13 The results of tumor inhibition rate B also confirmed this. The tumor inhibition rate of the Cst-Mar-NPs group was 68.30±0.32%, which was significantly higher than that of the free Cst group (53.65±0.47% (P<0.001) and the Cst+Mar group (55.03±0.35% (P<0.001).

Claims

1. A tripterygium wilfordii-matrine self-assembled nanoparticle, characterized in that: The nanoparticles are formed by self-assembly of tripterine and matrine through supramolecular action. The particle size of the nanoparticles is 100-200 nm, the Zeta potential is -20 mV--30 mV, and the molar ratio of tripterine to matrine is 1:1-1:

3.

2. The tripterygium wilfordii-matrine self-assembled nanoparticles according to claim 1, characterized in that: The molar ratio of triptolide to matrine is 1:

3.

3. A method for preparing the tripterygium wilfordii-matrine self-assembled nanoparticles according to claim 1 or 2, characterized in that The following steps are involved: (a) dissolving triptolide and matrine in an organic solvent to form an organic phase; (b) adding the organic phase dropwise to the aqueous phase and stirring to form a pre-assembled mixture; (c) placing the mixed solution into a dialysis bag and dialyzing it against water to remove the organic solvent; (d) After dialysis, the supernatant was collected by centrifugation to obtain the tripterygium wilfordii-matrine self-assembled nanoparticle solution.

4. The method according to claim 3, wherein: The organic solvent in step (a) is selected from methanol, tetrahydrofuran or DMSO.

5. The method according to claim 3, wherein: The stirring speed in step (b) is 300-700 r / min, and the stirring time is 30-60 min.

6. The method according to claim 3, wherein: The molecular weight cut-off of the dialysis bag in step (c) is 3500 Da.

7. Use of the tripterine-matrine self-assembled nanoparticles according to claim 1 or 2 in the preparation of an anti-tumor drug, wherein the tumor is triple-negative breast cancer.

8. A pharmaceutical preparation comprising the tripterine-matrine self-assembled nanoparticles according to claim 1 or 2 and a pharmaceutically acceptable excipient, wherein the preparation is used to treat triple-negative breast cancer.

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