CN117919435A

CN117919435A - Hybrid nano-assembly for killing tumor cells and tumor stem cells by double effect, and preparation method and application thereof

Info

Publication number: CN117919435A
Application number: CN202410117079.XA
Authority: CN
Inventors: 罗聪; 赵桐瑶; 陈瑶; 张洪源; 何仲贵
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2024-01-26
Filing date: 2024-01-26
Publication date: 2024-04-26

Abstract

The invention discloses a hybrid nano-assembly for killing tumor cells and tumor stem cells by double effects, and a preparation method and application thereof, belonging to the technical field of novel auxiliary materials and novel dosage forms for combined treatment of pharmaceutical preparations. The hybrid nano-assembly is formed by co-assembling a dimer prodrug and a tumor stem cell inhibitor through intermolecular forces, and is subjected to surface modification by a polyethylene glycol modifier. The preparation method of the hybrid nano-assembly is simple and reliable, can realize specific prodrug activation and drug release according to needs at tumor sites, has the effects of effectively synergistically killing tumors and inhibiting tumor dryness, and has high safety. The co-assembled nano preparation provides a new strategy and more choices for developing drug delivery and multi-drug combined treatment, and meets the urgent need of a novel efficient tumor cell/tumor stem cell double-effect killing treatment strategy in clinic on the preparation.

Description

Hybrid nano-assembly for killing tumor cells and tumor stem cells by double effect, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical preparation combined treatment new auxiliary materials and new dosage forms, and particularly relates to a hybrid nano assembly for killing tumor cells and tumor stem cells by double effects, and a preparation method and application thereof.

Background

Cancer is one of the major diseases that threatens human life and health. Currently, chemotherapy is still the primary means of clinical treatment of tumors. However, traditional chemotherapy, while killing most tumor cells, still has many cancer patients with chemotherapy resistance, focal metastasis and recurrence. There is growing evidence that tumor stem cells (CANCER STEM CELLS, CSCs) that have self-renewing capacity and are capable of producing heterogeneous tumor cells play a critical role in these processes. Tumor stem cells, also known as Tumor initiating cells (Tumor INITIATING CELLS, TICs) or Tumor regenerating cells, are extremely resistant to a very small proportion of Tumor tissue, but are considered "power springs" for tumorigenesis and progression. In addition, because CSCs have strong drug efflux activity and DNA damage repair capability, traditional chemotherapeutic drugs cannot play a good killing role on CSCs, and can also become a cause for triggering the phenotypic plasticity of tumor cells, so that non-CSCs obtain the characteristics of CSCs. Therefore, developing a strategy capable of killing both tumor cells and CSCs is critical to eradicating tumors.

In recent years, drugs capable of selectively inhibiting CSCs, such as salinomycin (Salinomycin, SAL), metformin (Metformin, MET), thioridazine (Thioridazine, THZ), disulfiram (Disulfiram, DSF), all-trans retinoic acid (All-trans-retinoicacid, ATRA), and the like, have received attention in the field of tumor treatment. However, most CSCs inhibitors are hydrophobic drugs, which are easily and rapidly cleared during blood circulation, and have strong toxic and side effects on normal tissues of the body after systemic administration due to lack of targeting ability to tumor tissues. In addition, most CSCs inhibitors are effective in eradicating CSCs, but have poor tumor cell inhibition and remain difficult to address the great challenges of clinical tumor therapy. In view of this, there is a need to develop new therapeutic strategies to achieve dual-effect killing of tumor cells and CSCs, thus addressing the dual challenges of insufficient anti-tumor effects of monotherapy and up-regulation of tumor stem caused by chemotherapy. The use of chemotherapeutic agents in combination with CSCs inhibitors has been considered a promising therapeutic approach. However, traditional small molecule chemotherapeutic drugs have drug delivery barriers with low solubility, narrow therapeutic window, poor targeting property, poor pharmacokinetic properties and the like, and different therapeutic agents have different physicochemical properties and action targets, so that the aim of synergistic treatment is difficult to achieve through intermittent administration. These factors greatly limit the wide application of multi-drug combination therapy strategies.

With the rapid development of modern biological nanotechnology and the deep research of tumor microenvironment, an intelligent response type nano-drug delivery system based on small molecular drugs/prodrugs builds a favorable platform for combined delivery of anti-tumor multi-drugs. Reasonable design of the nano drug delivery system (Nanoparticulate drug DELIVERY SYSTEM, nano-DDS) is expected to improve the solubility of the hydrophobic drug, increase the in vivo stability of the drug and prolong the blood half-life of the drug, and can increase the accumulation of the drug at the tumor part through the EPR effect (Enhanced permeability and retention effect), namely the high permeability and retention effect of the solid tumor, thereby reducing the toxic and side effects. However, the conventional nano-drug delivery system based on carrier still has various "bottleneck" problems, such as complex preparation process, potential toxicity risk caused by carrier material, and too low drug loading (generally less than 10%).

Disclosure of Invention

Aiming at the problems, the invention provides a hybrid nano-assembly for killing tumor cells and tumor stem cells by double effect, and a preparation method and application thereof. The hybrid nano assembly has low toxicity risk and high drug loading capacity, and the manufacturing method of the hybrid nano assembly is simple.

The invention realizes the aim through the following technical scheme:

In a first aspect, the present invention provides a hybrid nano-assembly for dual effect killing of tumor cells and tumor stem cells, the hybrid nano-assembly being formed by co-assembly of a dimer prodrug and a tumor stem cell inhibitor by intermolecular forces and surface modification with a polyethylene glycol modifier.

Each molecule of the dimer prodrug is formed by connecting two molecules of antitumor parent drugs through a redox sensitive chemical bond, wherein the redox sensitive chemical bond is at least one of oxalate bonds, ketal bonds, monosulfur bonds, disulfide bonds, trisulfur bonds, monoselenium bonds or diselenium bonds.

The molar ratio of the dimeric prodrug to the tumor stem cell inhibitor is (1-5): (1-5); the preferred molar ratio is 1:1.

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

Further, the intermolecular forces include electrostatic interactions and hydrophobic forces.

Further, the antitumor parent drug is a compound containing an active hydroxyl group or an amino group.

Further, the antitumor parent drug is selected from one of taxane, camptothecine, anthracycline or nucleoside compounds.

Preferably, the anti-tumor drug parent is Docetaxel (DTX); the redox-sensitive chemical bond is a disulfide bond. Specifically, two DTX drug molecules are coupled together by disulfide bonds to give a docetaxel homodimer prodrug. The structural formula is as follows:

further, the tumor stem cell inhibitor is selected from one of representative compounds with the function of inhibiting CSCs, such as salinomycin, metformin, thioridazine, disulfiram, all-trans retinoic acid, and the like. Salinomycin is preferred.

Further, the polyethylene glycol modifier is selected from one or more than two of DSPE-PEG, PLGA-PEG, PCL-PEG and PE-PEG, and the molecular weight of the polyethylene glycol modifier is 200-20000.

Preferably, the polyethylene glycol modifier is DSPE-PEG _2K.

In a second aspect, the present invention provides a method for preparing a hybrid nano-assembly for dual effect killing of tumor cells and tumor stem cells, comprising the steps of:

Dissolving the dimer prodrug, the tumor stem cell inhibitor and the polyethylene glycol modifier into an organic solvent, dropwise adding the uniformly mixed solution into water under stirring to spontaneously form uniform co-assembled nanoparticles, and finally removing the organic solvent to obtain the hybrid nano-assembly.

Further, the preparation method of the tumor site reduction response type docetaxel dimer prodrug DSSD comprises the following steps:

Docetaxel and 4,4' -dithio-dibutyl acid are respectively dissolved in dichloromethane and stirred uniformly, 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride (EDCI) and 4-Dimethylaminopyridine (DMAP) are dissolved in a proper amount of dichloromethane and stirred uniformly, the mixture is slowly added dropwise into the system, and the mixture is stirred for 1h at room temperature under the protection of nitrogen. And (3) dissolving EDCI and DMAP in a proper amount of dichloromethane, slowly adding the mixture into a reaction system, continuously stirring the mixture at room temperature for 24 hours under the protection of nitrogen, and separating and purifying the obtained product through a preparation liquid phase.

In a third aspect, the present invention provides the use of a hybrid nano-assembly for the preparation of a drug delivery system.

In a fourth aspect, the invention provides an application of the hybrid nano-assembly in preparing an anti-tumor drug.

In a fifth aspect, the present invention provides the use of a hybrid nano-assembly for the preparation of an injectable, oral or topical delivery system.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention utilizes prodrug synthesis technology and molecular co-assembly nano technology to jointly deliver tumor intelligent response type chemotherapy prodrug (preferably docetaxel dimer prodrug DSSD) and anti-CSCs drug (preferably SAL), and constructs a hybrid nano assembly capable of realizing double-effect killing of tumor cells/CSCs. In the absence of any carrier material, the two drugs can co-assemble to form nanoparticles by intermolecular forces (electrostatic interactions and hydrophobic forces). In addition, the surface of the nanoparticle is modified with DSPE-PEG _2K to further improve its colloidal stability and pharmacokinetic profile. The hybrid nano-assembly can realize on-demand prodrug activation and site-specific drug release under the condition of Gao Guguang Galanin (GSH) at a tumor site. SAL in the system not only can effectively remove the CSCs, but also can improve the sensitivity of tumor cells/CSCs to chemotherapeutic drugs, thereby achieving the synergistic treatment effect.

(2) The hybrid nano-assembly formed by co-assembly of the DSSD and the SAL can effectively avoid the related toxicity caused by the excipient, overcome the affinity difference between different drugs and carrier materials, and realize the synchronous delivery of the two drugs in the body by flexibly adjusting the dosage ratio of the drugs so as to obtain the optimal synergistic treatment effect. The hybrid nano assembly achieves the technical effects of high drug loading capacity, good stability, low toxic and side effects and the like, meets the urgent requirements of high-efficiency low-toxicity anti-tumor preparations in clinic, provides a new thought for combined treatment based on a prodrug strategy and a CSCs inhibitor, and provides a promising nano platform for developing a novel high-efficiency tumor cell/CSCs double-effect killing treatment mode.

Drawings

FIG. 1 is a mass spectrum and ¹ HNMR spectrum of the reduced responsive docetaxel dimer prodrug (DSSD) of example 1 of the present invention.

FIG. 2 is a Markov particle size distribution plot of DS nanoparticles and PEGylated PDS nanoparticles of example 2 of the invention.

FIG. 3 is a transmission electron microscope image of DS nanoparticles and PDS nanoparticles of example 2 of the present invention.

FIG. 4 is a graph showing the stability of DS and PDS nanoparticles of example 3 of the present invention under PBS (pH 7.4) conditions.

FIG. 5 is a graph showing the long-term storage stability of PDS nanoparticles of example 3 of the present invention at 4 ℃.

FIG. 6 is a molecular docking diagram of DSSD and SAL of example 4 of the present invention.

FIG. 7 is a graph showing the molecular force breakdown of DS nanoparticles of example 4 of the present invention under sodium chloride (10 mM), sodium dodecyl sulfate (10 mM) and urea (10 mM).

Fig. 8 is a graph showing cumulative release of DTX from PDS nanoparticles of example 5 of the present invention in release media containing different concentrations of DTT.

FIG. 9 is a confocal microscopy image of cell uptake at 0.5h and 2h for the C-6 solution and C-6 labeled PDS nanoparticles of example 6 of the invention.

FIG. 10 is a quantitative plot of cell uptake flow cytometry at 0.5h and 2h for the C-6 solution and C-6 labeled PDS nanoparticles of example 6 of the invention.

FIG. 11 is a graph of the cytotoxicity results of SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles of example 7 of the invention on 4T1 cells.

FIG. 12 is a graph of the cytotoxicity results of SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles of example 7 of the invention on MCF-7 cells.

FIG. 13 is a graph showing the cytotoxicity results of SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles of example 7 of the present invention on L02 cells.

FIG. 14 is a graph showing quantitative ratios of CD133 ⁺ CSCs after SAL solutions, DSSD solutions, DS solutions, DTX solutions, and PDS nanoparticles treatment in example 8 of the present invention.

FIG. 15 is a graph showing quantitative ratios of CD44 ⁺CD24^- CSCs after SAL solutions, DSSD solutions, DS solutions, DTX solutions, and PDS nanoparticles treatment in example 8 of the present invention.

FIG. 16 is a graph of plasma concentration versus time for DiR solutions and DiR-labeled PDS nanoparticles of example 9 of the invention.

FIG. 17 is a photograph of an in vivo image of mice administered with DiR solution and DiR-labeled PDS nanoparticles in example 10 of the invention.

FIG. 18 is an in vitro fluorescence imaging of major organs and tumors of mice administered with DiR solutions and DiR-labeled PDS nanoparticles in example 10 of the invention.

FIG. 19 is an in vitro fluorescence quantification of major organs and tumors of mice administered with DiR solutions and DiR labeled PDS nanoparticles in example 10 of the invention.

FIG. 20 is a graph showing the tumor growth of mice in an in vivo antitumor assay of example 11 of the present invention.

FIG. 21 is an in vitro tumor photograph of a mouse of the in vivo anti-tumor experiment of example 11 of the present invention.

FIG. 22 is an in vitro tumor weight plot of mice from an in vivo anti-tumor assay of example 11 of the present invention.

FIG. 23 is a graph showing the tumor suppression rate of mice in an in vivo antitumor test of example 11 of the present invention.

FIG. 24 is a graph showing the statistics of tumor-bearing rate of mice in an in vivo antitumor experiment of example 11 of the present invention.

FIG. 25 is a graph showing the weight change of mice in an in vivo antitumor test of example 11 of the present invention.

FIG. 26 is a graph showing liver and kidney function analysis of in vivo antitumor test in example 11 of the present invention.

FIG. 27 is a histopathological section of the in vivo anti-tumor test of example 11 of the present invention.

FIG. 28 is an in vitro tumor H & E, ki67 and TUNEL staining chart of mice from an in vivo anti-tumor assay of example 11 of the present invention.

FIG. 29 is a diagram showing the immunofluorescence of pluripotent stem factor in an in vivo anti-tumor assay according to example 11 of the present invention.

Detailed Description

The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.

In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Example 1: synthesis of disulfide bridged DTX dimer prodrugs (DSSD)

DTX (323.2 mg,0.4 mmol) and 4,4' -dithiodibutyric acid (47.7 mg,0.2 mmol) were dissolved in methylene chloride, then EDCI (153.4 mg,0.8 mmol) and DMAP (4.9 mg,0.04 mmol) were added sequentially. After stirring for 1h under nitrogen at room temperature, EDCI (76.7 mg,0.4 mmol) and DMAP (4.9 mg,0.04 mmol) were additionally added to the above-mentioned system, and the reaction was continued under nitrogen at room temperature for 24h. The reaction product is purified by adopting a preparation liquid chromatography method to obtain a target product DSSD.

The prodrug structure was confirmed by Mass Spectrometry (MS) and nuclear magnetic resonance hydrogen spectroscopy (¹ H NMR), respectively. The results are shown in FIG. 1. The solvent selected for nuclear magnetic resonance is deuterated DMSO, and the nuclear magnetic spectrum analysis result is as follows:

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＝7.9Hz,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).

Example 2: preparation of DSSD/SAL co-assembled nanoparticles

In this example, a hybrid nanoassembly co-assembled from DSSD and SAL was prepared using a one-step nano-precipitation process. Briefly, DSSD and SAL were dissolved in a mixed solution of tetrahydrofuran and absolute ethanol, respectively, to prepare a 5mg/mL drug-containing solution. To further optimize the formulation, DSSD and SAL were mixed in varying molar ratios (5:1-1:5) to give a series of DSSD/SAL mixed solutions (total volume of solution fixed at 200 μl). Under the condition of magnetic stirring (1500 rpm), 200 mu L of the mixed solution is slowly dropped into 2mL of deionized water, the uniform stirring is continued for 2min, DSSD and SAL can spontaneously form uniform nano particles, then the organic solvent in the nano preparation is removed by spin evaporation for 3min at 34 ℃, and the nano preparation is fixed to 2mL of deionized water, so that the nano colloid solution without any organic solvent is obtained.

Furthermore, the synergy of DSSD and SAL was further evaluated using the MTT method. Briefly, 4T1 cells were seeded at a density of 2x 10 ³ cells/well in 96-well plates and after 24h incubation, 4T1 cells were treated with DSSD solutions, SAL solutions, or hybrid nanoparticles at different molar ratios (5:1-1:5), respectively. After 48h of dosing, 20. Mu.L MTT solution (5 mg/mL) was added to each well and incubated for 4h in a thermostated incubator at 37 ℃. The liquid in the well plate is then blotted and replaced with 200 μl DMSO to lyse the formazan crystals produced by the living cells. Finally, the ultraviolet absorbance at 490nm was measured by a microplate reader. The synergy between DSSD and SAL was assessed by calculating cell viability and synergy index (CI).

Comparative analyses of particle sizes, polydispersity index (PDI), and synergy index of DSSD and SAL were performed for the above-described hybrid nanoparticles in different molar ratios, respectively, and the results are shown in table 1.

TABLE 1 particle size, PDI, synergy index of DSSD and SAL of prepared nanoparticles

Synergy index can be divided into synergy (CI < 1), additive (ci=1) and antagonistic (CI > 1). As shown in table 1, DSSD and SAL each showed excellent synergy in the molar ratio range of 5:1 to 1:5, with CI values of less than 1. Wherein DSSD and SAL exhibit the most pronounced synergistic effect when DSSD/SAL = 1:1, with a CI value of 0.294. In addition, the nanoparticles formed by the two drugs at this molar ratio have smaller particle sizes and are distributed more uniformly, so that the ratio of DSSD to SAL is preferably 1:1. Subsequent experiments were all performed under DSSD/sal=1:1 conditions.

A method for preparing non-PEGylated nanoparticles (DS nanoparticles): precisely weighing 0.71mg of DSSD and 0.29mg of SAL, dissolving the DSSD and the SAL in 200 mu L of a mixed solution of tetrahydrofuran and absolute ethyl alcohol, slowly dripping 200 mu L of the mixed solution into 2mL of deionized water under the condition of magnetic stirring (1500 rpm), continuously uniformly stirring for 2min, enabling the DSSD and the SAL to spontaneously form uniform DS nanoparticles, then removing an organic solvent in the nano preparation by spin-steaming for 3min at 34 ℃, and adopting the deionized water to fix the volume of the nano preparation to 2mL to obtain a nano colloid solution without any organic solvent.

The preparation method of the DSPE-PEG _2K modified PEGylated nanoparticle (PDS nanoparticle) comprises the following steps: accurately weighing 0.71mg of DSSD and 0.29mg of SAL, and dissolving the DSSD and the SAL by 375 mu L of tetrahydrofuran and absolute ethyl alcohol mixed solution; 0.25mg of DSPE-PEG _2K was precisely weighed out and dissolved in a 10mg/mL mother liquor with 25. Mu.L of a mixed solution of tetrahydrofuran and absolute ethanol. Mixing 375 mu L of the drug mixed solution with 25 mu LDSPE-PEG _2K solution uniformly, slowly dripping 400 mu L of the mixed solution into 2mL of deionized water under the condition of magnetic stirring (1500 rpm), continuously uniformly stirring for 2min, enabling DSSD and SAL to spontaneously form uniform PDS nanoparticles, then removing organic solvents in the nano preparation by spin-steaming for 5min at 34 ℃, and adopting deionized water to fix the volume of the nano preparation to 2mL to obtain a nano colloid solution without any organic solvents. The particle size, particle size distribution, zeta potential and morphology of the prepared DS and PDS nanoparticles were examined using a dynamic light scattering method and a transmission electron microscope (Table 2, FIGS. 2-3), and the drug loading was calculated (Table 3).

TABLE 2 particle size, PDI and Zeta potential of DS and PDS nanoparticles

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

TABLE 3 drug loading of DS nanoparticles and PDS nanoparticles

Nanoparticles	Medicine carrying capacity (DTX)	Medicine loading (SAL)	Total medicine
				DS nanoparticles	63％	29％	92％
PDS nanoparticles	50％	23％	73％

As shown in Table 2 and FIG. 2, the DS nanoparticles had a particle diameter of about 121nm and a Zeta potential of about-25 mV; the particle size of the PDS nanoparticles is about 82nm, and the Zeta potential is about-30 mV.

The morphology of the DS nanoparticles and PDS nanoparticles prepared in example 2 was measured by a transmission electron microscope, and the result is shown in FIG. 3, which shows that the nanoparticles were uniformly spherical in shape.

Example 3: colloidal stability experiment of nanoparticles

The DS nanoparticles and PDS nanoparticles (0.5 mg/mL) prepared in example 2 were incubated in phosphate buffer (PBS, pH 7.4), and their particle size change was measured at predetermined time points (0, 0.5, 1,2,4, 6, 8, 10, and 12 h). As shown in fig. 4, the colloidal stability of the non-PEG modified DS nanoparticles was poor and the particle size increased significantly during incubation. Under the same conditions, the particle size of the PDS nanoparticles does not change significantly within 12 hours. In addition, the long-term storage stability of PDS nanoparticles at 4 ℃ was further examined. As shown in FIG. 5, the PDS nanoparticles are placed for 30 days at the temperature of 4 ℃, have no obvious change in particle size, and have good low-temperature long-term storage stability. PEG modified PDS nanoparticles are preferred.

Example 4: DSSD and SAL assembly mechanism analysis

The assembly mechanism between DSSD and SAL was explored using computer simulation techniques, and molecular docking calculations were completed using the Vina protocol of Yan Fuyun computing platform. As a result, the electrostatic interaction force and the hydrophobic force together drive the DSSD and SAL nano-assembly process, as shown in fig. 6. In addition, force disruption was performed using sodium chloride (10 mM), sodium dodecyl sulfate (10 mM) and urea (10 mM), respectively, to further verify intermolecular forces between DSSD and SAL. The results are shown in FIG. 7, where the particle size of nanoparticles incubated with urea (10 mM) remained stable, indicating that hydrogen bonding interactions had negligible effect on nanoparticle formation. Notably, the particle size of the nanoparticles increased sharply in a short time under the conditions of sodium chloride (10 mM) and sodium dodecyl sulfate (10 mM), further confirming that electrostatic interaction force and hydrophobic force play a dominant role in the nano-assembly process.

Example 5: in vitro drug delivery

In vitro release behavior of DTX was examined using PBS (pH 7.4) containing 30% absolute ethanol as release medium and Dithiothreitol (DTT) as a reduction stimulator. PDS nanoparticles (1 mL) were added to release medium (30 mL) containing different concentrations of DTT (0, 1,2 and 5 mM) and incubated at 37℃in a shaker. The same volume of release medium (200 μl) was removed and an equal volume of fresh release medium was added at predetermined time intervals (1, 2, 4, 6, 8 and 12 h). The cumulative release rate of DTX was determined by High Performance Liquid Chromatography (HPLC). The results are shown in fig. 8, where the nanoparticles exhibit DTT concentration-dependent DTX release behavior. Notably, in the release medium containing 5mM DTT, about 90% of DTX was released within 2 hours. In contrast, in the blank release medium (0 mM DTT), there was almost no DTX release.

Example 6: cellular uptake of nanoparticles

Uptake of PDS nanoparticles prepared in example 2 in mouse breast cancer (4T 1) cells was assessed using Confocal Laser Scanning Microscopy (CLSM) and flow cytometry. For CLSM qualitative analysis, 4T1 cells were seeded at a density of 5 x 10 ⁴ cells/well in 12-well plates and cultured for 24h to adhere. The old medium was then discarded and replaced with fresh medium containing coumarin 6 (C-6) solution or C-6 labeled PDS nanoparticles (C-6/PDS nanoparticles) at an equivalent concentration of 200ng/mL, and after incubation for 0.5h or 2h, the cells were washed and fixed for cell fixation, and intracellular fluorescent signals were observed by CLSM. The experimental results are shown in FIG. 9. For flow cytometry quantification, 4T1 cells were seeded at a density of 1 x 10 ⁵ cells/well in 12-well plates for 24h until adherent. The old medium was then discarded and replaced with fresh medium of C-6 solution or C-6/PDS nanoparticles (C-6 equivalent concentration 200 ng/mL), after incubation for 0.5h or 2h, respectively, cells were washed, collected and resuspended in PBS for flow cytometry analysis. The experimental results are shown in FIG. 10. The experimental results show that the cell uptake shows time dependence, and the C-6/PDS nanoparticles have higher intracellular fluorescence signals than the cells treated by the C-6 solution at the equivalent C-6 concentration. Thus, the prepared C-6/PDS nanoparticles have higher cellular uptake efficiency than the free drug solution.

Example 7: cytotoxicity of nanoparticles

The cytotoxicity of SAL solution, DSSD/SAL (DS) solution, DTX solution and PDS nanoparticles on mouse breast cancer (4T 1) cells, human breast cancer (MCF-7) cells and human normal liver (L02) cells, respectively, was examined using the MTT method. Briefly, 4T1 cells, MCF-7 cells and L02 cells were seeded at a density of 2X 10 ³ cells/well in 96-well plates and placed in an incubator for incubation for 24h to allow adherence. The original medium was then replaced with fresh medium containing SAL solutions, DSSD solutions, DS solutions, DTX solutions and PDS nanoparticles at different concentrations. Cells cultured in fresh blank medium served as negative control. After 48h incubation, 20 μl of MTT solution (5 mg/mL) was added to each well, incubated in an incubator for 4h, 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 10min to dissolve the formazan crystals generated. Ultraviolet absorbance at 490nm was measured using a microplate reader.

The cytotoxicity results are shown in FIGS. 11-13, and in 4T1 cells and MCF-7 cells, DS solution and PDS nanoparticles have a synergistic effect and more remarkable inhibition effect on tumor cell growth compared with SAL solution and DSSD solution. Notably, PDS nanoparticles not only exhibit greater cytotoxicity than DS solutions, but are slightly better than DTX solutions even at high drug concentrations. In addition, as shown by the cytotoxicity results of L02 cells (fig. 13), DTX solutions still exhibited significant cytotoxicity to L02 cells. While all DSSD-containing groups (DSSD solutions, DS solutions and PDS nanoparticles) exhibited negligible cytotoxicity to L02 cells under the same conditions. These results well demonstrate the superiority of dimeric prodrug design in enhancing tumor-specific anti-tumor effects and reducing off-target toxicity.

Example 8: in vitro anti-tumor stem cell Activity

And a serum-free suspension culture method is adopted to establish a 3D microsphere model rich in CSCs. 4T1 cells were seeded at a density of 1X 10 ⁴ cells/mL into 6 well ultra low attachment plates (Corning) and cultured in suspension using serum free DMEM/F12 (1:1) medium supplemented with 1 XB 27 solution, 20ng/mL epidermal growth factor, 20ng/mL basic fibroblast growth factor, 5. Mu.g/mL insulin, 0.4% (w/v) bovine serum albumin and 1% penicillin-streptomycin solution. With prolonged incubation time, the formation of cellular microspheres was observed. During the culture period, every 3-4 days, a proper amount of fresh serum-free suspension medium containing growth factors is added into the well plate. After 7-10 days of culture, the 3D microsphere model rich in CSCs was successfully established.

Next, the in vitro anti-CSCs activity of SAL solutions, DSSD solutions, DS solutions, DTX solutions and PDS nanoparticles were evaluated separately using flow cytometry. Dissociated 4t1 CSCs were seeded at a density of 2x 10 ⁵ cells/well in 6 well ultra low adhesion plates for overnight incubation and treated with SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles, respectively. After 48h incubation, cells were stained with APC-labeled anti-mouse CD133 antibody or FITC-labeled anti-mouse/human CD44 antibody and PE-labeled anti-mouse CD24 antibody, followed by detection using a flow cytometer.

The results are shown in figures 14-15, where the CSCs ratio was significantly reduced in all SAL-containing groups (SAL solutions, DS solutions, and PDS nanoparticles). Notably, the lower proportion of CSCs in the DS solution and PDS nanoparticle groups than in the SAL solution group suggests that SAL is not only capable of synergistically enhancing the toxic effects on tumor cells, but also has significant advantages in synergistically killing CSCs. In contrast, DTX solutions did not decrease CSCs ratio, but rather triggered higher ratios of CD133 ⁺ CSCs and CD44 ⁺CD24^- CSCs, indicating that DTX alone treatment failed to eliminate CSCs, but also resulted in further CSCs enrichment. The above results show that the hybridized nano-assembly constructed by the invention has remarkable advantages in the aspects of killing tumor cells and CSCs by double effect.

Example 9: pharmacokinetic study of nanoparticles

Near infrared fluorescent dye DiR was used as fluorescent probe to prepare DiR-labeled PDS nanoparticles (DiR/PDS nanoparticles). Male Sprague-Dawley (SD) rats weighing between 180-220g were randomized and fasted for 12h prior to dosing, with free water. DiR solutions and DiR/PDS nanoparticles (DiR equivalent dose 1 mg/kg) were respectively intravenously injected, and blood was collected from the orbit at predetermined time points (0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours), and plasma was obtained by separation. DiR was extracted by the protein precipitation method, and finally the concentration of DiR in plasma was measured by a multifunctional microplate reader (excitation wavelength: 748nm, emission wavelength: 780 nm).

The experimental results are shown in fig. 16, where DiR solutions cleared faster from the blood due to the short half-life. In contrast, diR/PDS nanoparticles have obvious advantages in the aspect of prolonging the blood circulation time, and the area under the blood concentration-time curve (AUC _0-24 _h) is obviously improved, which lays a good foundation for the effective accumulation of the drug in tumor parts.

Example 10: tissue distribution experiment of nanoparticles

A4T 1 tumor-bearing BALB/c mouse model is constructed to study the in vivo biodistribution of PDS nanoparticles. DiR/PDS nanoparticles were also prepared using DiR as a fluorescent probe. Briefly, diR solutions or DiR/PDS nanoparticles (DiR equivalent dose of 0.8 mg/kg) were injected into mice via tail vein, respectively. Mice were anesthetized at predetermined time points (1, 2, 4, 6, 8, 12 and 24 h) for in vivo imaging analysis. The results are shown in FIG. 17. 24h after dosing, mice were sacrificed and major organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected for ex vivo fluorescence imaging, ROI tools were used to quantify fluorescence signals. The results are shown in FIGS. 18-19.

The above results indicate that the fluorescence intensity of DiR/PDS nanoparticles at tumor sites is significantly increased compared to the DiR solution. The in vitro imaging results are consistent with the in vivo imaging results, which show that the DiR/PDS nanoparticles can be effectively accumulated at the tumor site, and the DiR/PDS nanoparticles have remarkable advantages in terms of colloid stability and pharmacokinetic behavior.

Example 11: in vivo anti-tumor experiments of nanoparticles

The in vivo antitumor effect was further evaluated using a 4T1 tumor-bearing BALB/c mouse model. When the tumor volume of the mice reached about 100mm ³, the mice were randomly divided into 6 groups of 5 mice each and respectively given physiological saline, SAL solution, DSSD solution, DS solution, DTX solution and PDS nanoparticles. 1 time every 1 day, and 5 times in total, the administration dose is 5mg/kg of DTX equivalent dose. Tumor size and body weight changes were recorded daily throughout the course of treatment. After the treatment was completed, a blood sample of the mice was collected for liver and kidney function analysis. Mice were sacrificed and major organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected for H & E staining. Meanwhile, ki67 and TUNEL staining was used to further evaluate tumor cell proliferation and apoptosis. In addition, immunofluorescent staining was used to examine the expression of Sox2, oct4 and Nanog pluripotent stem factors in tumors to evaluate anti-CSCs effects in vivo.

Mouse tumor growth curves, in vitro tumor photographs, tumor weights, tumor inhibition rates, and tumor loading rates as shown in fig. 20-24, SAL solutions and DSSD solutions alone exhibited poor anti-tumor efficacy, probably due to their easy rapid clearance from the blood and difficulty in achieving effective accumulation at tumor sites. Notably, DS solutions exhibited stronger antitumor activity than SAL solutions and DSSD solutions, suggesting that DSSD/SAL has synergistic antitumor potential. In addition, PDS nanoparticles with tumor-specific drug release profile showed the most significant tumor inhibition effect in all groups, with the highest tumor inhibition rate and lowest tumor-bearing rate in mice. Although DTX solutions also showed potent antitumor activity at the same dose, they also resulted in weight loss in mice (fig. 25). In addition, mice treated with SAL solutions and DS solutions also exhibited some degree of weight loss, liver toxicity, and lung toxicity (fig. 25-27). In contrast, PDS nanoparticles not only exhibit strong antitumor efficacy, but also exhibit good therapeutic safety. The H & E, TUNEL and Ki67 staining results also confirm the excellent antitumor activity of PDS nanoparticles (fig. 28).

And further evaluating the anti-CSCs effect in the PDS nanoparticles by examining the expression conditions of Sox2, oct4 and Nanog multipotential dryness factors in tumors. As shown in fig. 29, SAL solutions and DS solutions showed only limited anti-CSCs effects due to the faster rate of in vivo elimination and less accumulation at tumor sites. Notably, significant dry factor downregulation can be observed in the PDS nanoparticle group, indicating that the introduction of SAL in the hybrid nano-assemblies can effectively enhance CSCs killing effects. In contrast, DTX treatment resulted in a significant increase in fluorescence intensity of the three multipotent stem factors, indicating that chemotherapy alone not only failed to eliminate CSCs, but also triggered the phenotypic plasticity of the tumor cells, resulting in an increase in CSCs proportion. These results demonstrate that the hybrid nano-assemblies (PDS nanoparticles) designed by the invention are not only capable of dual-effect killing of tumor cells and CSCs, but also exhibit good therapeutic safety.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. The hybridized nano assembly for killing tumor cells and tumor stem cells is characterized by being formed by co-assembling a dimer prodrug and a tumor stem cell inhibitor through intermolecular forces, and carrying out surface modification by using a polyethylene glycol modifier;

Each molecule of the dimer prodrug is formed by connecting two molecules of antitumor parent drugs through a redox sensitive chemical bond, wherein the redox sensitive chemical bond is at least one of oxalate bonds, ketal bonds, monosulfur bonds, disulfide bonds, trisulfur bonds, monoselenium bonds or diselenium bonds;

The molar ratio of the dimeric prodrug to the tumor stem cell inhibitor is (1-5): (1-5);

2. The hybrid nano-assembly for dual purpose killing tumor cells and tumor stem cells of claim 1, wherein the intermolecular forces comprise electrostatic interactions and hydrophobic forces.

3. The hybrid nano-assembly for dual purpose killing tumor cells and tumor stem cells according to claim 1, wherein the anti-tumor parent drug is a compound containing an active hydroxyl group or an amino group.

4. The hybrid nano-assembly for dual purpose killing tumor cells and tumor stem cells according to claim 3, wherein the anti-tumor parent drug is selected from one of taxanes, camptothecins, anthracyclines or nucleoside compounds.

5. The hybrid nano-assembly for dual effect killing of tumor cells and tumor stem cells according to claim 1, wherein the tumor stem cell inhibitor has an effect of inhibiting CSCs, and is selected from one of salinomycin, metformin, thioridazine, disulfiram and all-trans retinoic acid.

6. The hybrid nano-assembly for dual-effect killing of tumor cells and tumor stem cells according to claim 1, wherein the polyethylene glycol modifier is selected from one or more of DSPE-PEG, PLGA-PEG, PCL-PEG, PE-PEG, and the molecular weight of the polyethylene glycol modifier is 200-20000.

7. The method of preparing a hybrid nano-assembly for dual purpose killing tumor cells and tumor stem cells according to any one of claims 1-6, comprising the steps of:

8. Use of a hybrid nano-assembly for dual effect killing of tumor cells and tumor stem cells according to any one of claims 1-6 or prepared by the preparation method according to claim 7 in the preparation of a drug delivery system.

9. Use of the hybrid nano-assembly for dual effect killing of tumor cells and tumor stem cells according to any one of claims 1-6 or the hybrid nano-assembly prepared by the preparation method according to claim 7 in the preparation of antitumor drugs.

10. Use of a hybrid nano-assembly for dual effect killing of tumor cells and tumor stem cells according to any one of claims 1-6 or prepared by the preparation method according to claim 7 for preparing an injectable, oral or topical delivery system.