A hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells, and its preparation method and application

Abstract

The present invention discloses a hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells, and its preparation method and application, which belongs to the technical field of new excipients and new dosage forms for combined treatment of pharmaceutical preparations. The hybrid nanoassembly of the present invention is formed by co-assembly of a dimeric prodrug and a tumor stem cell inhibitor through intermolecular forces, and is surface-modified with a polyethylene glycol modifier. The preparation method of the hybrid nanoassembly of the present invention is simple and reliable, can achieve specific prodrug activation and on-demand drug release at the tumor site, has effective synergistic tumor killing and tumor stemness inhibition effects, and has high safety. The co-assembled nanoformulation of the present invention provides new strategies and more options for the development of drug delivery and multi-drug combination therapy, meeting the urgent clinical demand for new and efficient tumor cell / tumor stem cell dual-effect killing treatment strategies in formulations.

Description

Technical Field

[0001] The present invention belongs to the technical field of new excipients and new dosage forms for combined treatment of pharmaceutical preparations, and specifically relates to a hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells, and a preparation method and application thereof. Background Art

[0002] Cancer is a major threat to human health and life. Currently, chemotherapy remains the mainstay of clinical cancer treatment. However, while conventional chemotherapy can kill most tumor cells, many cancer patients still develop chemoresistance, metastasis, and recurrence. Increasing evidence indicates that cancer stem cells (CSCs), which possess the capacity to self-renew and generate heterogeneous tumor cells, play a crucial role in these processes. Although CSCs, also known as tumor-initiating cells (TICs) or tumor-regenerating cells, comprise only a tiny fraction of tumor tissues, they possess a high degree of drug resistance and are considered the driving force behind tumor development and progression. Furthermore, due to the robust drug efflux and DNA damage repair capabilities of CSCs, conventional chemotherapeutics not only fail to effectively target CSCs but can also trigger phenotypic plasticity in tumor cells, allowing non-CSCs to acquire CSC characteristics. Therefore, developing strategies that simultaneously target both tumor cells and CSCs is crucial for tumor eradication.

[0003] In recent years, drugs that selectively inhibit CSCs, such as salinomycin (SAL), metformin (MET), thioridazine (THZ), disulfiram (DSF), and all-trans-retinoic acid (ATRA), have garnered widespread attention in cancer therapy. However, most CSC inhibitors are hydrophobic and rapidly cleared from the bloodstream. Due to their lack of tumor-targeting ability, they can have significant toxic side effects on normal tissues following systemic administration. Furthermore, while most CSC inhibitors effectively eradicate CSCs, they exhibit limited inhibitory effects on tumor cells, remaining a significant challenge in clinical cancer treatment. Therefore, the development of novel therapeutic strategies that achieve dual-action cytotoxicity against both tumor cells and CSCs is urgently needed to address the dual challenges of inadequate antitumor efficacy of monotherapy and chemotherapy-induced upregulation of tumor stemness. Combining chemotherapeutic agents with CSC inhibitors has been recognized as a promising therapeutic approach. However, traditional small-molecule chemotherapeutics face delivery barriers such as low solubility, a narrow therapeutic window, poor targeting, and poor pharmacokinetic properties. Furthermore, different therapeutic agents have different physicochemical properties and targets, making it difficult to achieve synergistic treatment through staggered dosing. These factors significantly limit the widespread application of multidrug combination therapy strategies.

[0004] With the rapid development of modern bio-nanotechnology and in-depth research on the tumor microenvironment, smart responsive nano-drug delivery systems based on small molecule drugs / prodrugs have established a favorable platform for the combined delivery of anti-tumor multi-drugs. The rational design of nano-drug delivery systems (nano-DDS) is expected to improve the solubility of hydrophobic drugs, increase the in vivo stability of drugs and prolong the blood half-life of drugs. It can also increase the accumulation of drugs in the tumor site through the EPR effect (Enhanced permeability and retention effect), that is, the high permeability and retention effect of solid tumors, thereby reducing toxic side effects. However, traditional carrier-based nano-drug delivery systems still have many "bottleneck" problems, such as complex preparation processes, potential toxicity risks caused by carrier materials, and too low drug loading (generally less than 10%). Summary of the Invention

[0005] To address the above issues, the present invention provides a hybrid nanoassembly for dual-action killing of tumor cells and tumor stem cells, as well as its preparation method and application. The hybrid nanoassembly has low toxicity risk, high drug loading capacity, and is simple to prepare.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] In a first aspect, the present invention provides a hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells. The hybrid nanoassembly is formed by co-assembling a dimeric prodrug and a tumor stem cell inhibitor through intermolecular forces and is surface-modified with a polyethylene glycol modifier.

[0008] Each molecule of the dimer prodrug is formed by two molecules of the anti-tumor parent drug connected by a redox-sensitive chemical bond, and the redox-sensitive chemical bond is selected from at least one of an oxalate bond, a thiol ketal bond, a monosulfide bond, a disulfide bond, a trisulfide bond, a monoselenide bond or a diselenide bond.

[0009] The molar ratio of the dimer prodrug to the tumor stem cell inhibitor is (1-5):(1-5); preferably the molar ratio is 1:1.

[0010] The mass ratio of the sum of the masses of the dimer prodrug and the tumor stem cell inhibitor to the mass of the polyethylene glycol modifier is (10-90):(10-90).

[0011] Furthermore, the intermolecular forces include electrostatic interaction forces and hydrophobic forces.

[0012] Furthermore, the antitumor mother drug is a compound containing active hydroxyl or amino groups.

[0013] Furthermore, the anti-tumor mother drug is selected from one of taxanes, camptothecins, anthracyclines or nucleoside compounds.

[0014] Preferably, the anti-tumor parent drug is docetaxel (DTX); and the redox-sensitive chemical bond is a disulfide bond. Specifically, two DTX drug molecules are coupled together via a disulfide bond to obtain a docetaxel homodimer prodrug. Its structural formula is:

[0015]

[0016] Furthermore, the tumor stem cell inhibitor is selected from one of representative compounds having the effect of inhibiting CSCs, such as salinomycin, metformin, thioridazine, disulfiram, and all-trans retinoic acid, preferably salinomycin.

[0017] Furthermore, the polyethylene glycol modifier is selected from one or more of DSPE-PEG, PLGA-PEG, PCL-PEG, and PE-PEG, and the molecular weight of the polyethylene glycol modifier is 200-20000.

[0018] Preferably, the polyethylene glycol modifier is DSPE-PEG 2K .

[0019] In a second aspect, the present invention provides a method for preparing a hybrid nanoassembly for dual-action killing of tumor cells and tumor stem cells, comprising the following steps:

[0020] The dimer prodrug, tumor stem cell inhibitor and polyethylene glycol modifier are dissolved in an organic solvent, and the mixed solution is added dropwise to water under stirring to spontaneously form uniform co-assembled nanoparticles. Finally, the organic solvent is removed to obtain a hybrid nanoassembly.

[0021] Furthermore, the preparation method of the tumor site reduction-responsive docetaxel dimer prodrug DSSD comprises the following steps:

[0022] Docetaxel and 4,4'-dithiodibutyric acid were dissolved in dichloromethane and stirred evenly. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) were dissolved in an appropriate amount of dichloromethane and stirred evenly. The mixture was slowly added dropwise to the above system and stirred at room temperature for 1 hour under nitrogen protection. EDCI and DMAP were dissolved in an appropriate amount of dichloromethane and slowly added to the reaction system. Stirring was continued at room temperature under nitrogen protection for 24 hours. The resulting product was separated and purified using preparative liquid chromatography.

[0023] In a third aspect, the present invention provides an application of a hybrid nanoassembly in preparing a drug delivery system.

[0024] In a fourth aspect, the present invention provides a use of a hybrid nanoassembly in the preparation of anti-tumor drugs.

[0025] In a fifth aspect, the present invention provides a use of a hybrid nanoassembly in the preparation of an injection, oral administration or topical administration system.

[0026] The present invention has the following beneficial effects compared to the prior art:

[0027] (1) The present invention utilizes prodrug synthesis technology and molecular co-assembly nanotechnology to deliver tumor intelligent responsive chemotherapy prodrugs (preferably docetaxel dimer prodrug DSSD) and anti-CSCs drugs (preferably SAL) together, thereby constructing a hybrid nanoassembly that can achieve dual-effect killing of tumor cells / CSCs. In the absence of any carrier material, the two drugs can be co-assembled to form nanoparticles through intermolecular forces (electrostatic interaction and hydrophobic interaction). In addition, DSPE-PEG is used. 2KThe nanoparticle surface was modified to further improve its colloidal stability and pharmacokinetic properties. This hybrid nanoassembly enables on-demand prodrug activation and site-specific drug release under conditions of high glutathione (GSH) at the tumor site. The SAL in this system not only effectively eliminates CSCs but also increases the sensitivity of tumor cells / CSCs to chemotherapeutic drugs, achieving a synergistic therapeutic effect.

[0028] (2) The hybrid nanoassembly formed by the co-assembly of DSSD and SAL of the present invention can effectively avoid the related toxicity caused by excipients, overcome the affinity differences between different drugs and carrier materials, and can achieve simultaneous delivery of the two drugs in vivo by flexibly adjusting the drug dosage ratio, thereby obtaining the best synergistic therapeutic effect. This hybrid nanoassembly achieves technical effects such as high drug loading, good stability, and low toxic side effects, meeting the urgent clinical demand for high-efficiency and low-toxicity anti-tumor preparations, providing a new idea for combined treatment based on prodrug strategies and CSCs inhibitors, and providing a promising nano-platform for the development of a new and efficient dual-effect tumor cell / CSCs killing treatment model. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The mass spectrum of the reduction-responsive docetaxel dimer prodrug (DSSD) of Example 1 of the present invention and 1 HNMR spectrum.

[0030] Figure 2 This is the Malvern particle size distribution diagram of DS nanoparticles and PEGylated PDS nanoparticles of Example 2 of the present invention.

[0031] Figure 3 Transmission electron microscopy images of DS nanoparticles and PDS nanoparticles according to Example 2 of the present invention.

[0032] Figure 4 This is a stability diagram of DS nanoparticles and PDS nanoparticles in Example 3 of the present invention under PBS (pH 7.4) conditions.

[0033] Figure 5 This is a graph showing the long-term storage stability of the PDS nanoparticles of Example 3 of the present invention at 4°C.

[0034] Figure 6 This is the molecular docking diagram of DSSD and SAL in Example 4 of the present invention.

[0035] Figure 7 This is a diagram showing the molecular force destruction of DS nanoparticles in Example 4 of the present invention under the conditions of sodium chloride (10 mM), sodium dodecyl sulfate (10 mM) and urea (10 mM).

[0036] Figure 8This is a diagram showing the cumulative release of DTX from the PDS nanoparticles of Example 5 of the present invention in release media containing different concentrations of DTT.

[0037] Figure 9 These are confocal microscopy images of the cellular uptake of the C-6 solution and C-6 labeled PDS nanoparticles of Example 6 of the present invention at 0.5 h and 2 h.

[0038] Figure 10 Flow cytometric quantitative graphs of the cellular uptake of the C-6 solution and C-6 labeled PDS nanoparticles of Example 6 of the present invention at 0.5 h and 2 h.

[0039] Figure 11 This is a graph showing the cytotoxicity results of the SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles of Example 7 of the present invention on 4T1 cells.

[0040] Figure 12 This is a graph showing the cytotoxicity of SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles in Example 7 of the present invention on MCF-7 cells.

[0041] Figure 13 This is a graph showing the cytotoxicity results of the SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles of Example 7 of the present invention on L02 cells.

[0042] Figure 14 CD133 treated with SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles according to Example 8 of the present invention + Quantification of CSCs ratio.

[0043] Figure 15 CD44 treated with SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles according to Example 8 of the present invention + CD24 - Quantification of CSCs ratio.

[0044] Figure 16 1 is a blood concentration-time curve of the DiR solution and DiR-labeled PDS nanoparticles of Example 9 of the present invention.

[0045] Figure 17 This is an in vivo imaging diagram of mice administered with DiR solution and DiR-labeled PDS nanoparticles according to Example 10 of the present invention.

[0046] Figure 18 These are in vitro fluorescence imaging images of major organs and tumors of mice administered with DiR solution and DiR-labeled PDS nanoparticles in Example 10 of the present invention.

[0047] Figure 19 This is an in vitro fluorescence quantitative image of the main organs and tumors of mice administered with DiR solution and DiR-labeled PDS nanoparticles in Example 10 of the present invention.

[0048] Figure 20 This is a graph showing the tumor growth curve of mice in the in vivo anti-tumor experiment of Example 11 of the present invention.

[0049] Figure 21 This is a photo of mouse in vitro tumors from the in vivo anti-tumor experiment of Example 11 of the present invention.

[0050] Figure 22 This is a graph showing the in vitro tumor weight of mice in the in vivo anti-tumor experiment of Example 11 of the present invention.

[0051] Figure 23 This is a graph showing the tumor inhibition rate in mice from the in vivo anti-tumor experiment of Example 11 of the present invention.

[0052] Figure 24 Statistical graph of mouse tumor-bearing rate in the in vivo anti-tumor experiment of Example 11 of the present invention.

[0053] Figure 25 This is a graph showing changes in mouse body weight in the in vivo anti-tumor experiment of Example 11 of the present invention.

[0054] Figure 26 This is a graph showing liver and kidney function analysis of the in vivo anti-tumor experiment of Example 11 of the present invention.

[0055] Figure 27 This is a histopathological section diagram of the in vivo anti-tumor experiment of Example 11 of the present invention.

[0056] Figure 28 These are H&E, Ki67, and TUNEL staining images of mouse in vitro tumors from the in vivo anti-tumor experiment of Example 11 of the present invention.

[0057] Figure 29 This is an immunofluorescence image of pluripotent stemness factors in the in vivo anti-tumor experiment of Example 11 of the present invention. DETAILED DESCRIPTION

[0058] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0059] In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0060] Example 1: Synthesis of disulfide-bridged DTX dimer prodrug (DSSD)

[0061] DTX (323.2 mg, 0.4 mmol) and 4,4'-dithiodibutyric acid (47.7 mg, 0.2 mmol) were dissolved in dichloromethane, followed by the addition of EDCI (153.4 mg, 0.8 mmol) and DMAP (4.9 mg, 0.04 mmol). After stirring at room temperature under nitrogen for 1 hour, EDCI (76.7 mg, 0.4 mmol) and DMAP (4.9 mg, 0.04 mmol) were added to the system, and the reaction was continued at room temperature under nitrogen for 24 hours. The reaction product was purified by preparative liquid chromatography to obtain the target product, DSSD.

[0062] The prodrug structures were confirmed by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy ( 1 H NMR) confirmed. The results are as follows Figure 1 As shown. The solvent used for NMR is deuterated DMSO, and the NMR spectrum analysis results are as follows:

[0063] 1H NMR (600MHz, DMSO-d6) δ7.99 (d, J=7.1Hz, 2H, H-23, H-27), 7.88 (d, J=9.3Hz, 1H, 3'-NH), 7.73 (t, J=7.4Hz, 1H ,H-25),7.66(t,J=7.6Hz,2H,H-24,H-26),7.42(t,J=7.6Hz,2H,H-11',H-13'),7.36(d,J=7.6Hz,2H,H-10', H-14'),7.17(t,J=7.4Hz,1H,H-12'),5.78(t,J=9.6Hz,1H,H-13),5.40(d,J=7.2Hz,1H,H-2),5.11(d,J=7.9 Hz,1H,H-2'),5.10-5.04(m,2H,H-3',10-OH),5.02(d,J=7.2Hz,1H,H-3'),4.94(d,J=2.4Hz,1H,H-5),4.90( dd,J=9.5,2.3Hz,1H,H-7),4.44(s,1H,H-10),4.08-3.98(m,3H,H-20α,H-20β,7-OH),3.63(d,J=7.1Hz,1H,H -3),2.71(t,J=7.1Hz,2H,H-1”),2.54(t,J=7.2Hz,2H,H-3”),2.28(dd,J=9.0,6.0Hz,1H,H-6α),2.24(s,3H, H-29),1.94(p,J=6.9Hz,2H,H-2”),1.81(dd,J=15.3,9.3Hz,1H,H-6β),1.70(s,3H,H-19),1.65(t,J=12.4Hz ,1H,H-14α),1.52(m,1H,H-14β),1.51(s,3H,H-19),1.38(s,9H,H-6',H-7',H-8'),0.98(s,6H,H-16,H-17).

[0064] Example 2: Preparation of DSSD / SAL co-assembled nanoparticles

[0065] In this example, a hybrid nanoassembly composed of DSSD and SAL was prepared using a one-step nanoprecipitation method. Briefly, DSSD and SAL were dissolved separately in a mixed solution of tetrahydrofuran and anhydrous ethanol to prepare a 5 mg / mL drug-containing solution. To further optimize the formulation, DSSD and SAL were mixed in different molar ratios (5:1-1:5) to obtain a series of DSSD / SAL mixed solutions (the total volume of the solution was fixed at 200 μL). Under magnetic stirring (1500 rpm), 200 μL of the mixed solution was slowly added dropwise to 2 mL of deionized water and continued to stir evenly for 2 minutes. DSSD and SAL were able to spontaneously form uniform nanoparticles. The organic solvent in the nanoformulation was then removed by rotary evaporation at 34°C for 3 minutes. The nanoformulation was then fixed to 2 mL with deionized water to obtain a nanocolloidal solution free of any organic solvent.

[0066] In addition, the MTT assay was used to further evaluate the synergistic effect of DSSD and SAL. Briefly, 4T1 cells were plated at 2 × 10 3 Cells were seeded at a density of 100 μg / well in a 96-well plate. After culturing for 24 h, 4T1 cells were treated with a series of concentrations of DSSD solution, SAL solution, or hybrid nanoparticles at different molar ratios (5:1-1:5). 48 h after administration, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated in a 37°C incubator for 4 h. Then, the liquid in the well plate was aspirated and replaced with 200 μL of DMSO to dissolve the formazan crystals produced by living cells. Finally, the UV absorbance value at 490 nm was measured by a microplate reader. The synergistic effect between DSSD and SAL was evaluated by calculating cell viability and synergy index (CI).

[0067] The particle size, polydispersity index (PDI) and synergy index of DSSD and SAL of the hybrid nanoparticles with different molar ratios were compared and analyzed, and the results are shown in Table 1.

[0068] Table 1. Particle size, PDI, and synergistic index of DSSD and SAL of the prepared nanoparticles

[0069]

[0070] The synergistic index can be divided into synergistic effect (CI < 1), additive effect (CI = 1), and antagonistic effect (CI > 1). As shown in Table 1, DSSD and SAL exhibited excellent synergistic effects in the molar ratio range of 5:1 to 1:5, with CI values ​​all less than 1. Among them, when DSSD / SAL = 1:1, DSSD and SAL exhibited the most significant synergistic effect, with a CI value of 0.294. In addition, the nanoparticles formed by the two drugs at this molar ratio had a smaller particle size and a more uniform distribution, so the preferred ratio of DSSD to SAL was 1:1. Subsequent experiments were all carried out under the condition of DSSD / SAL = 1:1.

[0071] Preparation method of non-PEGylated nanoparticles (DS nanoparticles): Accurately weigh 0.71 mg of DSSD and 0.29 mg of SAL, dissolve them in 200 μL of a mixed solution of tetrahydrofuran and anhydrous ethanol, and slowly add 200 μL of the mixed solution dropwise to 2 mL of deionized water under magnetic stirring (1500 rpm). Continue to stir evenly for 2 minutes. DSSD and SAL can spontaneously form uniform DS nanoparticles. Then, the organic solvent in the nanoformulation is removed by rotary evaporation at 34°C for 3 minutes, and the nanoformulation is diluted to 2 mL with deionized water to obtain a nanocolloidal solution without any organic solvent.

[0072] DSPE-PEG 2K Preparation method of modified PEGylated nanoparticles (PDS nanoparticles): accurately weigh 0.71 mg DSSD and 0.29 mg SAL, dissolve them in 375 μL of a mixture of tetrahydrofuran and anhydrous ethanol; accurately weigh 0.25 mg DSPE-PEG 2K , dissolve it in 25 μL of tetrahydrofuran and anhydrous ethanol mixed solution to a 10 mg / mL stock solution. 2K The solution was mixed evenly, and 400 μL of the mixed solution was slowly added dropwise to 2 mL of deionized water under magnetic stirring (1500 rpm). Stirring was continued for 2 minutes. DSSD and SAL were able to spontaneously form uniform PDS nanoparticles. The organic solvent in the nanoformulation was then removed by rotary evaporation at 34°C for 5 minutes. The nanoformulation was then fixed to 2 mL with deionized water to obtain a nanocolloidal solution free of any organic solvent. The particle size, particle size distribution, zeta potential, and morphology of the prepared DS nanoparticles and PDS nanoparticles were investigated by dynamic light scattering and transmission electron microscopy (Table 2, Figure 2-3 ), and the drug loading was calculated (Table 3).

[0073] Table 2. Particle size, PDI and Zeta potential of DS nanoparticles and PDS nanoparticles

[0074] Nanoparticles Particle size (nm) PDI Zeta potential (mV) DS nanoparticles 120.9±2.052 0.099±0.052 -24.6±0.64 PDS nanoparticles 81.66±1.153 0.095±0.051 -30.1±1.20

[0075] Table 3. Drug loading of DS nanoparticles and PDS nanoparticles

[0076] Nanoparticles Drug loading (DTX) Drug loading (SAL) Total drugs DS nanoparticles 63％ 29％ 92％ PDS nanoparticles 50％ 23％ 73％

[0077] As shown in Table 2 and Figure 2 As shown, the particle size of DS nanoparticles is about 121 nm, and the Zeta potential is about -25 mV; the particle size of PDS nanoparticles is about 82 nm, and the Zeta potential is about -30 mV.

[0078] The morphology of the DS nanoparticles and PDS nanoparticles prepared in Example 2 was determined by transmission electron microscopy. Figure 3 As shown, the transmission electron microscopy image shows that the nanoparticles are uniformly spherical in shape.

[0079] Example 3: Colloidal stability experiment of nanoparticles

[0080] The DS nanoparticles and PDS nanoparticles (0.5 mg / mL) prepared in Example 2 were incubated in phosphate buffered saline (PBS, pH 7.4), and the changes in their particle size were measured at predetermined time points (0, 0.5, 1, 2, 4, 6, 8, 10, and 12 h). Figure 4 As shown in the figure, the colloidal stability of non-PEG-modified DS nanoparticles was poor, and the particle size increased significantly during the incubation process. Under the same conditions, the particle size of PDS nanoparticles did not change significantly within 12 hours. In addition, the long-term storage stability of PDS nanoparticles at 4°C was further investigated. The results are shown in the figure. Figure 5 As shown in the figure, the particle size of PDS nanoparticles did not change significantly after being stored at 4°C for 30 days, indicating that they have good long-term storage stability at low temperatures. PEG-modified PDS nanoparticles are preferred.

[0081] Example 4: Analysis of DSSD and SAL assembly mechanism

[0082] Computer simulation technology was used to explore the assembly mechanism between DSSD and SAL, and the molecular docking calculation was completed using the Vina program of Yinfu cloud computing platform. Figure 6 As shown in Figure 2, electrostatic interaction and hydrophobic interaction jointly drive the nanoassembly process of DSSD and SAL. In addition, sodium chloride (10mM), sodium dodecyl sulfate (10mM) and urea (10mM) were used to destroy the interaction force to further verify the intermolecular force between DSSD and SAL. The results are shown in Figure 2. Figure 7As shown in the figure, the particle size of the nanoparticles incubated with urea (10mM) remained stable, indicating that the effect of hydrogen bonding on the formation of nanoparticles was negligible. It is worth noting that under the conditions of sodium chloride (10mM) and sodium dodecyl sulfate (10mM), the particle size of the nanoparticles increased sharply in a short period of time, further confirming that electrostatic interactions and hydrophobic forces play a dominant role in the nanoassembly process.

[0083] Example 5: In vitro drug release

[0084] The in vitro release behavior of DTX was investigated using PBS (pH 7.4) containing 30% anhydrous ethanol as the release medium and dithiothreitol (DTT) as a reducing stimulant. PDS nanoparticles (1 mL) were added to a release medium (30 mL) containing different concentrations of DTT (0, 1, 2, and 5 mM) and incubated at 37 ° C in a shaker. At predetermined time intervals (1, 2, 4, 6, 8, and 12 h), the same volume of release medium (200 μL) was taken out and an equal volume of fresh release medium was added. The cumulative release rate of DTX was determined by high performance liquid chromatography (HPLC). The results are shown in Figure 2. Figure 8 As shown in Figure 3, the nanoparticles exhibited a DTT concentration-dependent DTX release behavior. Notably, in the release medium containing 5 mM DTT, approximately 90% of DTX was released within 2 h. In contrast, in the blank release medium (0 mM DTT), almost no DTX was released.

[0085] Example 6: Cellular Uptake of Nanoparticles

[0086] Confocal laser scanning microscopy (CLSM) and flow cytometry were used to evaluate the uptake of PDS nanoparticles prepared in Example 2 in mouse breast cancer (4T1) cells. For CLSM qualitative analysis, 4T1 cells were plated at 5×10 4 Cells were seeded at a density of 100 cells / well in a 12-well plate and cultured for 24 hours to allow them to adhere. The old culture medium was then discarded and replaced with fresh culture medium containing coumarin 6 (C-6) solution or C-6-labeled PDS nanoparticles (C-6 / PDS nanoparticles), with an equivalent C-6 concentration of 200 ng / mL. After incubation for 0.5 hours or 2 hours, the cells were washed and fixed, and the intracellular fluorescence signal was observed by CLSM. The experimental results are shown in Figure 2. Figure 9 For flow cytometry quantitative analysis, 4T1 cells were plated at 1 × 10 5 Cells were seeded at a density of 100 μg / well in a 12-well plate and cultured for 24 h until adhered. The old culture medium was then discarded and replaced with fresh culture medium containing C-6 solution or C-6 / PDS nanoparticles (C-6 equivalent concentration was 200 ng / mL). After incubation for 0.5 h or 2 h, respectively, the cells were washed, collected, and resuspended in PBS for flow cytometry analysis. Figure 10 The above experimental results all indicate that cellular uptake is time-dependent, and at equivalent C-6 concentrations, C-6 / PDS nanoparticles exhibit higher intracellular fluorescence signals than cells treated with C-6 solution. Therefore, the prepared C-6 / PDS nanoparticles have higher cellular uptake efficiency than the free drug solution.

[0087] Example 7: Cytotoxicity of Nanoparticles

[0088] The cytotoxicity of SAL solution, DSSD solution, DSSD / SAL (DS) solution, DTX solution and PDS nanoparticles on mouse breast cancer (4T1) cells, human breast cancer (MCF-7) cells and human normal liver (L02) cells was investigated by MTT assay. 3 Cells were seeded into 96-well plates at a density of 100 μg / well and placed in an incubator for 24 h to adhere to the plate. The original culture medium was then replaced with fresh culture medium containing different concentrations of SAL solution, DSSD solution, DS solution, DTX solution, and PDS nanoparticles. Cells cultured in fresh blank culture medium served as a negative control. After incubation for 48 h, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated in an incubator for 4 h. The liquid in the 96-well plate was discarded, and 200 μL of DMSO was added to each well and shaken on a shaker for 10 min to dissolve the generated formazan crystals. The UV absorbance value at 490 nm was measured using a microplate reader.

[0089] Cytotoxicity results such as Figure 11-13 As shown in Figure 2, in 4T1 cells and MCF-7 cells, DS solution and PDS nanoparticles have a synergistic effect compared with SAL solution and DSSD solution, and the inhibitory effect on tumor cell growth is more significant. It is worth noting that PDS nanoparticles not only show stronger cytotoxicity than DS solution, but even slightly better than DTX solution at high drug concentrations. In addition, as shown in the cytotoxicity results of L02 cells ( Figure 13 ), the DTX solution still exhibited significant cytotoxicity against L02 cells. Under the same conditions, all DSSD-containing groups (DSSD solution, DS solution, and PDS nanoparticles) exhibited negligible cytotoxicity against L02 cells. These results demonstrate the superiority of dimeric prodrug design in enhancing tumor-specific antitumor effects and reducing off-target toxicity.

[0090] Example 8: In vitro anti-tumor stem cell activity

[0091] A 3D microsphere model rich in CSCs was established using a serum-free suspension culture method. 4T1 cells were cultured at a density of 1×10 4Cells were seeded at a density of 100 cells / mL into 6-well ultra-low attachment plates (Corning) and suspended in serum-free DMEM / F12 (1:1) medium supplemented with 1× B27 solution, 20 ng / mL epidermal growth factor, 20 ng / mL basic fibroblast growth factor, 5 μg / mL insulin, 0.4% (w / v) bovine serum albumin, and 1% penicillin-streptomycin solution. As the culture time increased, the formation of cell mammospheres was observed. During the culture period, an appropriate amount of fresh serum-free suspension medium containing growth factors was added to the wells every 3-4 days. After 7-10 days of culture, a 3D mammosphere model rich in CSCs was successfully established.

[0092] Next, flow cytometry was used to evaluate the in vitro anti-CSCs activities of SAL solution, DSSD solution, DS solution, DTX solution, and PDS nanoparticles. Dissociated 4T1 CSCs were cultured at 2×10 5 Cells were seeded at a density of 1000 cells / well in 6-well ultra-low attachment plates and incubated overnight. They were then treated with SAL solution, DSSD solution, DS solution, DTX solution, and PDS nanoparticles, respectively. After 48 hours of incubation, cells were stained with APC-labeled anti-mouse CD133 antibody, FITC-labeled anti-mouse / human CD44 antibody, and PE-labeled anti-mouse CD24 antibody, and then analyzed by flow cytometry.

[0093] The results are as follows Figure 14-15 As shown in the figure, the proportion of CSCs in all SAL-containing groups (SAL solution, DS solution and PDS nanoparticles) was significantly reduced. It is worth noting that the proportion of CSCs in the DS solution and PDS nanoparticle groups was lower than that in the SAL solution group, which indicates that SAL can not only synergistically enhance the toxic effect on tumor cells, but also has obvious advantages in synergistically killing CSCs. In contrast, DTX solution did not reduce the proportion of CSCs, but instead triggered a higher proportion of CD133 + CSCs and CD44 + CD24 - CSCs, this result shows that DTX treatment alone not only fails to eliminate CSCs, but also further leads to the enrichment of CSCs. The above results show that the hybrid nanoassembly constructed by the present invention has significant advantages in dual-effect killing of tumor cells and CSCs.

[0094] Example 9: Pharmacokinetic Study of Nanoparticles

[0095] The near-infrared fluorescent dye DiR was used as a fluorescent probe to prepare DiR-labeled PDS nanoparticles (DiR / PDS nanoparticles). Male Sprague-Dawley (SD) rats weighing 180-220 g were randomly divided into groups and fasted for 12 hours before administration, with free access to water. DiR solution and DiR / PDS nanoparticles (DiR equivalent dose of 1 mg / kg) were injected intravenously. Blood was collected from the orbitals at predetermined time points (0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours), and plasma was isolated. DiR was extracted by protein precipitation, and the plasma concentration was measured using a multifunctional microplate reader (excitation wavelength: 748 nm, emission wavelength: 780 nm).

[0096] The experimental results are as follows Figure 16 As shown in the figure, DiR solution is cleared from the blood quickly due to its short half-life. In contrast, DiR / PDS nanoparticles have obvious advantages in prolonging blood circulation time, and the area under the blood drug concentration-time curve (AUC 0-24 h ) was significantly improved, which laid a good foundation for the effective accumulation of drugs in the tumor site.

[0097] Example 10: Tissue distribution experiment of nanoparticles

[0098] A 4T1 tumor-bearing BALB / c mouse model was constructed to study the in vivo biodistribution of PDS nanoparticles. DiR was also used as a fluorescent probe to prepare DiR / PDS nanoparticles. In short, DiR solution or DiR / PDS nanoparticles (DiR equivalent dose of 0.8 mg / kg) were injected into mice via the tail vein. At predetermined time points (1, 2, 4, 6, 8, 12, and 24 hours), the mice were anesthetized and subjected to in vivo imaging analysis. The results are shown in Figure 2. Figure 17 24 hours after administration, mice were sacrificed and major organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected for in vitro fluorescence imaging, and the ROI tool was used to quantify the fluorescence signal. Figure 18-19 shown.

[0099] These results demonstrate that the fluorescence intensity of DiR / PDS nanoparticles at the tumor site is significantly increased compared to the DiR solution. The in vitro imaging results are consistent with the in vivo imaging results, indicating that DiR / PDS nanoparticles can effectively accumulate at the tumor site, which can be attributed to the significant advantages of DiR / PDS nanoparticles in colloidal stability and pharmacokinetic behavior.

[0100] Example 11: In vivo anti-tumor experiment of nanoparticles

[0101] The 4T1 tumor-bearing BALB / c mouse model was used to further evaluate the anti-tumor effect in vivo. 3 At the same time, the mice were randomly divided into 6 groups, with 5 mice in each group, and were given normal saline, SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles respectively. The drug was administered once every 1 day for a total of 5 times, with a dose of 5 mg / kg equivalent to DTX. The tumor size and body weight changes of the mice were recorded every day during the entire treatment process. After the treatment, blood samples were collected from the mice for liver and kidney function analysis. The mice were killed, and the main organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected for H&E staining. At the same time, Ki67 and TUNEL staining were used to further evaluate the proliferation and apoptosis of tumor cells. In addition, immunofluorescence staining was used to examine the expression of Sox2, Oct4 and Nanog pluripotent stemness factors in tumors to evaluate the anti-CSCs effect in vivo.

[0102] Mouse tumor growth curve, in vitro tumor photos, tumor weight, tumor inhibition rate and tumor bearing rate Figure 20-24 As shown in the results, both SAL solution and DSSD solution showed weak antitumor efficacy when treated alone, which may be attributed to their rapid clearance from the blood and difficulty in achieving effective accumulation in the tumor site. It is worth noting that DS solution showed stronger antitumor activity than SAL solution and DSSD solution, indicating that DSSD / SAL has synergistic antitumor potential. In addition, PDS nanoparticles with tumor-specific drug release properties showed the most significant tumor inhibition effect among all groups, with the highest tumor inhibition rate and the lowest mouse tumor-bearing rate. Although DTX solution also showed effective antitumor activity at the same dose, it also caused a decrease in mouse body weight ( Figure 25 In addition, mice treated with SAL solution and DS solution also showed a certain degree of weight loss, liver toxicity and lung toxicity ( Figures 25-27 ). In contrast, PDS nanoparticles not only showed strong anti-tumor efficacy, but also showed good therapeutic safety. H&E, TUNEL and Ki67 staining results also confirmed the excellent anti-tumor activity of PDS nanoparticles ( Figure 28 ).

[0103] The anti-CSCs effect of PDS nanoparticles in vivo was further evaluated by examining the expression of Sox2, Oct4, and Nanog pluripotent stemness factors in tumors. Figure 29As shown in the figure, SAL solution and DS solution showed only limited anti-CSCs effect due to their faster elimination rate in the body and less accumulation in the tumor site. It is worth noting that a significant downregulation of stemness factors can be observed in the PDS nanoparticle group, indicating that the introduction of SAL in the hybrid nanoassembly can effectively enhance the effect of killing CSCs. In contrast, DTX treatment resulted in a significant increase in the fluorescence intensity of the three pluripotent stemness factors, which indicates that chemotherapy alone not only cannot eliminate CSCs, but also triggers the phenotypic plasticity of tumor cells, leading to an increase in the proportion of CSCs. The above results show that the hybrid nanoassembly (PDS nanoparticles) designed by the present invention can not only dual-effect kill tumor cells and CSCs, but also show good therapeutic safety.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells, characterized in that: The hybrid nanoassembly is formed by co-assembly of a dimeric prodrug and a tumor stem cell inhibitor through intermolecular forces and is surface-modified with a polyethylene glycol modifier; Each molecule of the dimer prodrug is formed by two molecules of the anti-tumor parent drug connected by a redox-sensitive chemical bond, and the redox-sensitive chemical bond is a disulfide bond; The anti-tumor parent drug is docetaxel; The molar ratio of the dimer prodrug to the tumor stem cell inhibitor is (1-5): (1-5); The mass ratio of the sum of the mass of the dimer prodrug and the tumor stem cell inhibitor to the mass of the polyethylene glycol modifier is (10-90): (10-90); The tumor stem cell inhibitor has the function of inhibiting CSCs and is selected from salinomycin.

2. The hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells according to claim 1, characterized in that: The intermolecular forces include electrostatic interaction and hydrophobic interaction.

3. The hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells according to claim 1, characterized in that: The polyethylene glycol modifier is selected from one or more of DSPE-PEG, PLGA-PEG, PCL-PEG, and PE-PEG, and the molecular weight of the polyethylene glycol modifier is 200-20000.

4. The method for preparing a hybrid nanoassembly for dual-action killing of tumor cells and tumor stem cells according to any one of claims 1 to 3, characterized in that: The steps include: The dimer prodrug, tumor stem cell inhibitor and polyethylene glycol modifier are dissolved in an organic solvent, and the mixed solution is added dropwise to water under stirring to spontaneously form uniform co-assembled nanoparticles. Finally, the organic solvent is removed to obtain a hybrid nanoassembly.

5. Use of the hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells according to any one of claims 1 to 3 or the hybrid nanoassembly prepared by the preparation method according to claim 4 in preparing a drug delivery system.

6. Use of the hybrid nanoassembly for dual-effect killing of tumor cells and tumor stem cells according to any one of claims 1 to 3 or the hybrid nanoassembly prepared by the preparation method according to claim 4 in the preparation of anti-tumor drugs.

7. Use of a hybrid nanoassembly for dual-action killing of tumor cells and tumor stem cells as claimed in any one of claims 1 to 3 or a hybrid nanoassembly prepared by the preparation method according to claim 4 in the preparation of an injection, oral administration or local administration system.

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