Cancer stem cell and cancer cell fusion membrane-coated drug-loaded nanoparticles, and preparation method and application thereof

Abstract

The application relates to a cancer stem cell and cancer cell fusion membrane-coated drug-loaded nanoparticle and a preparation method and application thereof. The cancer stem cell and cancer cell fusion membrane-coated drug-loaded nanoparticle comprises a cancer stem cell and cancer cell fusion membrane and hydroxyapatite nanoparticles loaded with a chemotherapeutic drug and coated in the cancer stem cell and cancer cell fusion membrane; wherein the mass ratio of the chemotherapeutic drug to the hydroxyapatite nanoparticles is (0.1-0.6):1; and the mass ratio of the cancer stem cell and cancer cell fusion membrane to the hydroxyapatite nanoparticles loaded with the chemotherapeutic drug is 1:(0.5-1.5).

Description

Technical Field

[0001] This invention belongs to the field of tumor chemotherapy drugs, specifically relating to a drug-loaded nanoparticle encapsulated in a fusion membrane of cancer stem cells and cancer cells, its preparation method, and its application. Background Technology

[0002] The continuous metastasis and spread of malignant tumors after treatment is a significant obstacle to successful clinical cancer treatment. Cancer stem cells (CSCs) are a subset of tumor cells with the ability to self-renew and differentiate, and are highly correlated with tumor metastasis and recurrence. Although strategies based on nanodelivery technology to target solid tumors have made some progress, the efficacy of chemotherapy is still limited by the low targeting efficiency and insufficient uptake of CSCs.

[0003] The specific antigens expressed on the cell membranes of tumor cells play a crucial role in the adhesion of homologous cells within the tumor microenvironment. Biomimetic NPs, obtained by encapsulating extracted cancer cell membranes on nanoparticles (NPs), can retain the antigenic structure and function of parental cells, achieving homologous targeting. However, current cell membrane encapsulation techniques cannot target cancer cell stem cells (CSCs) or effectively reduce tumor stemness, remaining a challenge for the treatment of highly metastatic malignant tumors. Summary of the Invention

[0004] In view of the above-mentioned technical challenges, the present invention aims to provide a drug-loaded nanoparticle fused with a fusion membrane of cancer stem cells and cancer cells, its preparation method, and its application. This invention utilizes the homologous targeting effect of cell membranes, using nanoparticles coated with a fusion membrane of cancer stem cells (CSCs) and cancer cells (CCs) to target occult cancer stem cells in the body, in addition to targeting solid tumors, thereby weakening or even reversing tumor stemness and blocking tumor occurrence and development.

[0005] In a first aspect, the present invention provides a drug-loaded nanoparticle encapsulated in a cancer stem cell and cancer cell fusion membrane, the drug-loaded nanoparticle encapsulating in the cancer stem cell and cancer cell fusion membrane comprising: a cancer stem cell and cancer cell fusion membrane, and hydroxyapatite nanoparticles loaded with chemotherapeutic drugs encapsulated inside the cancer stem cell and cancer cell fusion membrane. The mass ratio of the chemotherapy drug to the hydroxyapatite nanoparticles is (0.1-0.6):1; the mass ratio of the cancer stem cell and cancer cell fusion membrane to the hydroxyapatite nanoparticles loaded with the chemotherapy drug is 1:(0.5-1.5).

[0006] Preferably, the hydroxyapatite nanoparticles have a short rod-shaped structure with a length of 30–100 nm and a width of 10–50 nm; more preferably, the length is 50 nm and the width is 30 nm.

[0007] Preferably, the chemotherapy drug is doxorubicin or paclitaxel, with doxorubicin being the most preferred.

[0008] Secondly, the present invention provides a method for preparing drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells, the preparation method comprising the following steps: (1) The cancer stem cell membrane and the cancer cell membrane were mixed in a solution and then sonicated on an ice bath to obtain a fusion membrane of cancer stem cells and cancer cells. (2) The chemotherapy drug and hydroxyapatite nanoparticles were mixed in solution and stirred at room temperature and in the dark. After the reaction was completed, the precipitate was collected, washed and dried to obtain hydroxyapatite nanoparticles loaded with chemotherapy drug. (3) The cancer stem cell and cancer cell fusion membrane and hydroxyapatite nanoparticles loaded with chemotherapeutic drugs were ultrasonically mixed in an ice bath, and then the mixture was extruded using an extrusion device to obtain the cancer stem cell and cancer cell fusion membrane coated with drug-loaded nanoparticles.

[0009] Preferably, the mass ratio of the cancer stem cell membrane to the cancer cell membrane is (0.5-1.5):1, more preferably 1:1.

[0010] Preferably, the ultrasonic power of the mixture of cancer stem cell membrane and cancer cell membrane on an ice bath is 50-300W, and the ultrasonic time is 5-15min; more preferably, the ultrasonic power is 100W and the ultrasonic time is 10min.

[0011] Preferably, the mass ratio of the chemotherapy drug to the hydroxyapatite nanoparticles is controlled to be (0.3-1):1, more preferably (0.5-1):1, and even more preferably (0.6-0.8):1.

[0012] Preferably, the ultrasonic mixing power of the cancer stem cell and cancer cell fusion membrane with hydroxyapatite nanoparticles loaded with chemotherapeutic drugs on an ice bath is 50-300W, and the ultrasonic time is 10-50s; more preferably, the ultrasonic power is 100W, the ultrasonic time is 30s, each ultrasonic session is 1s, and the interval between two ultrasonic sessions is 2s.

[0013] Thirdly, the present invention provides an application of the above-mentioned cancer stem cell and cancer cell fusion membrane-coated drug-loaded nanoparticles in the preparation of targeted tumor therapeutic drugs.

[0014] Beneficial effects 1) The preparation method of drug-loaded nanoparticles coated with fusion membrane provided by the present invention is simple, easy to operate, does not require complex and expensive equipment, and is easy to realize industrial production; 2) The drug-loaded nanoparticles coated with the fusion membrane provided by this invention can not only inhibit the growth of solid tumors of breast cancer, but also inhibit the deterioration and metastasis of breast cancer, and have good application prospects. 3) The drug-loaded nanoparticles coated with the fusion membrane provided by this invention have high targeting efficiency for cancer cells and cancer stem cells, and are suitable for the treatment of malignant tumors. Attached Figure Description

[0015] Figure 1 Characterization diagram of CSC / CC@DHAp NPs prepared in Example 1; where (A) SDS-PAGE analysis, (BD) transmission electron microscopy images of HAp NPs, DHAp NPs, and CSC / CC@DHAp NPs (scale bar: 100 nm), (EF) Zeta potential and hydration particle size distribution, and (G) cumulative DOX release of DHAp and CSC / CC@DHAp within 48 h at different pH levels; Figure 2 The image shows the targeting characterization of CSC / CC@DHAp nanoparticles prepared in Example 1 on cancer stem cells. (A) Schematic diagram of cell uptake using laser confocal microscopy after 4 hours of pretreatment with FITC-labeled DHAp, CSC / CC@DHAp on L-02, MCF-7, and cancer stem cells (scale bar: 20 μm); (B) Schematic diagram of cell uptake of FITC-labeled CSC / CC@DHAp analyzed by flow cytometry; (C) Schematic diagram of Ca2+ uptake detected by ICP-OES. 2+ Intake, error bars represent standard deviation (n=3), *P<0.05, **P<0.01, ***P<0.001, bioelectron microscopy images of L-02(D), MCF-7(E), and cancer stem cells (F) after 4 hours of treatment with CSC / CC@DHAp, where arrows indicate CSC / CC@DHAp NPs (scale bar: 4μm), (G) biodistribution of DOX in MCF-7 tumor-bearing nude mice at different time points after intravenous injection of DOX, DHAP, and CSC / CC@DHAp NPs, circles indicate tumor sites (dose: 5mg / kg), mice were sacrificed after 48h to investigate the in vivo biodistribution of DOX, DHAP, and CSC / CC@DHAp NPs in tumors and major organs (heart, liver, spleen, lung, and kidney), (H) and (G) quantification of DOX fluorescence intensity in fluorescence images, (I) measurement of Ca in various organs and tumors by ICP-OES. 2+ The concentration was investigated to determine the biodistribution of CSC / CC@DHAp NPs in mice 48 hours after injection. Figure 3Characterization of the effect of CSC / CC@DHAp nanoeffectors on the bioactivity of breast cancer CSCs; (A) Cell viability of CSCs after 24 hours of incubation with different concentrations of free DOX, DHAp and CSC / CC@DHAp NPs (n=4); (B) Effect of CSC / CC@DHAp NPs on the apoptosis level of CSCs assessed by Annexin V-FITC / PI double staining method; (C) Apoptosis ratio statistically analyzed from Figure (B); (D) Intracellular ATP levels of CSCs after 24 hours of incubation with free DOX, DHAp and CSC / CC@DHAp NPs. Relative ATP levels and fluorescence intensity are expressed as a percentage of untreated control cells. Error bars represent standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001. (E) Relative activities of caspase-9 (left) and caspase-3 (right) (n=3), with untreated cells as a control, relative caspase activities were calculated. *p<0.05, **p<0.01, ***p<0.001. (F) Western blot analysis of the expression levels of caspase 3, cleaved caspase 3, and Bcl-2 proteins. (G) Cell viability of MCF-7 breast cancer cells after incubation with different concentrations of free DOX, DHAp, and CSC / CC@DHAp NPs for 24 hours (n=4); Figure 4 This diagram illustrates the effect of the CSC / CC@DHAp nanoeffector on inhibiting stem-mediated tumor growth and deterioration. (A) Drug treatment regimen in a subcutaneous breast cancer mouse model (n=4); (B) Tumor growth curves of tumor-bearing nude mice during drug administration; tumor volume in each group was normalized to the initial volume on day 1; statistical analysis was performed using the non-parametric Kruskal-Wallis test (*P<0.05, **P<0.01, ***P<0.001); tumor weight after drug administration (C) and tumor tissue photographs (D); (E) Tumor sections from tumor-bearing nude mice after different treatments. (F) H&E, TUNEL, and Ki67 staining images (scale bar: 50 μm), (G) Changes in body weight of tumor-bearing nude mice during drug administration (n=6), (H) Representative images of metastatic nodules in lung tissue on day 62, (H) H&E staining of lung tissue, with arrows indicating metastatic nodules, (I) CD44, CD133, and ALDH1 staining images of tumor tissue collected after treatment (scale bar: 50 μm), (J) and (K) ELISA measurements of the expression levels of metastasis-related proteins CXCR4, VEGF, TGF-β, and MMP9 in tumor tissue (D) and lung tissue (E), n=4. *p<0.05, **p<0.01, ***p<0.001; Figure 5(A) H&E staining images (scale bar: 50 μm) of heart, liver, spleen, lung, and kidney tissue sections from MCF-7 tumor-bearing nude mice treated with saline, free DOX, DHAp, and CSC / CC@DHAp NPs, respectively. (BF) Blood biochemical parameters, including alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE). Data are expressed as mean ± SD (n = 3). Figure 6 This is a TEM image of the control particles prepared in Comparative Example 1. Detailed Implementation

[0016] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0017] This invention utilizes the homology targeting effect of cell membranes, employing nanoparticles coated with a fusion membrane of cancer stem cells (CSCs) and cancer cells (CCs). This targets not only solid tumors but also occult cancer stem cells within the body, weakening or even reversing tumor stemness and blocking tumor occurrence and development. More specifically, this invention obtains biomimetic nanoeffectors CSC / CC@DHAp NPs by encapsulating a fusion membrane of CSCs and CCs on the surface of doxorubicin-loaded hydroxyapatite nanoparticles (DHAp NPs) loaded with chemotherapeutic drugs such as doxorubicin. These nanoeffectors are used to inhibit tumor growth and tumor stemness-mediated deterioration and metastasis.

[0018] First, this invention provides a drug-loaded nanoparticle encapsulated in a cancer stem cell and cancer cell fusion membrane. The drug-loaded nanoparticle encapsulated in the cancer stem cell and cancer cell fusion membrane comprises: a cancer stem cell and cancer cell fusion membrane, and hydroxyapatite nanoparticles loaded with chemotherapeutic drugs encapsulated within the cancer stem cell and cancer cell fusion membrane.

[0019] In some embodiments, the cancer stem cell-cancer cell fusion membrane can be obtained by fusing MCF-7 human breast cancer stem cells and MCF-7 human breast cancer cells.

[0020] The fusion membrane of the cancer stem cells and cancer cells has the structural characteristics of the parent cell membrane and is loaded with a variety of antigens on its surface.

[0021] In some embodiments, the hydroxyapatite nanoparticles can be short rod-shaped structures with a length of 30–100 nm and a width of 10–50 nm; preferably, the length is 50 nm and the width is 30 nm.

[0022] In some embodiments, the chemotherapeutic drug may be doxorubicin or paclitaxel. Doxorubicin is preferred because it is readily soluble in water, easy to detect, and has a high drug loading capacity.

[0023] In some embodiments, the mass ratio of the chemotherapeutic drug to the hydroxyapatite nanoparticles can be (0.1-0.6):1, preferably (0.3-0.4):1, at which point the drug loading is relatively high.

[0024] In some embodiments, the mass ratio (membrane-to-core mass ratio) of the cancer stem cell fusion membrane and the hydroxyapatite nanoparticles loaded with chemotherapeutic drugs can be 1:(0.5-1.5), preferably 1:1. A membrane-to-core mass ratio that is too high or too low will reduce the membrane efficiency.

[0025] This invention is the first to employ a strategy of fusing cancer stem cells and cancer cells into a membrane to coat nanoparticles. This allows the synthesized biomimetic nanomaterials to inherit the antigenic structure and function of the parent cells, enabling simultaneous targeting of occult cancer stem cells and solid tumor cells in vivo, reversing stemness, and inhibiting the growth and deterioration of highly metastatic malignant tumors.

[0026] The following is an exemplary description of a method for preparing drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells. The preparation method may include the following steps.

[0027] (1) Preparation of cancer stem cell and cancer cell fusion membrane. The cancer stem cell membrane and cancer cell membrane were mixed in a solution and then sonicated on an ice bath to obtain the cancer stem cell and cancer cell fusion membrane.

[0028] As an example, the MCF-7 breast cancer stem cells can be obtained by culturing MCF-7 breast cancer cells to enrich cancer stem cell microspheres. Specifically, the following process can be used: MCF-7 breast cancer cells are suspended and cultured at a concentration of 1000 cells / mL in serum-free DMEM / F12 containing 2% B27, 20 ng / mL basic fibroblast growth factor (bFGF), 20 ng / mL epidermal growth factor (EGF), 0.4% low-endotoxin bovine serum albumin (BSA), and 4 mg / mL insulin to induce the production of MCF-7 breast cancer stem cells.

[0029] As an example, the extraction of the cancer stem cell membrane and cancer cell membrane can be performed using the following process: [The process involves] extracting 1×10 [cell membranes]. 7 Add 1 mL of hypotonic lysis buffer containing 0.1 mM protease inhibitor (excluding ethylenediaminetetraacetic acid (EDTA)), 10 mM Tris, and 10 mM MgCl2 to each MCF-7 cancer stem cell or cancer cell, and place on ice for 3 hours to obtain cell homogenate. Then, repeatedly freeze and thaw the cell homogenate (freeze in liquid nitrogen and thaw in a water bath at 37°C, at least 5 times). Next, centrifuge at 850 g for 10 minutes at 4°C to remove precipitated intracellular components, collect the supernatant, centrifuge at 18000 g for 60 minutes, and the precipitate is the purified CSCs and CCs membrane. Furthermore, the protein content can be analyzed using a 2,2-biquinoline-4,4-dicarboxylate disodium (BCA) protein assay kit.

[0030] In some embodiments, the mass ratio of the cancer stem cell membrane to the cancer cell membrane can be (0.5 to 1.5):1, preferably 1:1.

[0031] In some embodiments, the ultrasonic power of the mixture of cancer stem cell membrane and cancer cell membrane on an ice bath can be 50-300W, and the ultrasonic time can be 5-15min; preferably, the ultrasonic power is 100W and the ultrasonic time is 10min.

[0032] (2) Preparation of hydroxyapatite nanoparticles loaded with chemotherapeutic drugs. The chemotherapeutic drugs and hydroxyapatite nanoparticles are mixed in solution and stirred at room temperature and in the dark for 24-72 h, preferably 72 h. After the reaction is completed, the precipitate is collected, washed and dried to obtain the hydroxyapatite nanoparticles loaded with chemotherapeutic drugs.

[0033] In some embodiments, the mass ratio of chemotherapy drugs to hydroxyapatite nanoparticles can be controlled to be (0.3-1):1, preferably (0.5-1):1, and more preferably (0.6-0.8):1.

[0034] (3) Preparation of drug-loaded nanoparticles coated with fusion membrane of cancer stem cells and cancer cells. The fusion membrane of cancer stem cells and cancer cells and hydroxyapatite nanoparticles loaded with chemotherapeutic drugs were ultrasonically mixed in an ice bath, and then the mixture was extruded using an extrusion device to obtain the drug-loaded nanoparticles coated with fusion membrane of cancer stem cells and cancer cells.

[0035] In some embodiments, the mass ratio of the cancer stem cell and cancer cell fusion membrane to the hydroxyapatite nanoparticles loaded with chemotherapeutic drugs can be controlled to be 1:(0.5-1.5), preferably 1:1.

[0036] In some embodiments, the ultrasonic mixing power of the cancer stem cell and cancer cell fusion membrane with hydroxyapatite nanoparticles loaded with chemotherapeutic drugs on an ice bath can be 50-300W, and the ultrasonic time can be 10-50s; preferably, the ultrasonic power is 100W, the ultrasonic time is 30s, each ultrasonic session is 1s, and the interval between two ultrasonic sessions is 2s.

[0037] Extruding the mixture can promote vesicle formation. In some embodiments, the mixture can be repeatedly extruded 5-15 times using a micro extruder.

[0038] The method for preparing drug-loaded nanoparticles coated with a fusion membrane provided by this invention is simple, convenient, and does not require complex or expensive equipment, making it easy to scale up for industrial production. The drug-loaded nanoparticles coated with a fusion membrane obtained by this method can not only inhibit the growth of solid breast cancer tumors but also inhibit the deterioration and metastasis of breast cancer, showing promising application prospects. The drug-loaded nanoparticles coated with a fusion membrane provided by this invention exhibit highly efficient targeting of cancer cells and cancer stem cells, making them suitable for the treatment of malignant tumors.

[0039] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0040] Example 1

[0041] The preparation method of drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells provided in Example 1 includes the following steps: (1) Preparation of fusion membranes for cancer stem cells and cancer cells. MCF-7 cells were cultured in serum-free DMEM / F12 containing 2% B27, 20 ng / mL bFGF, 20 ng / mL EGF, 0.4% low-endotoxin bovine serum albumin (BSA), and 4 mg / mL insulin at a concentration of 1000 cells / mL to induce the formation of MCF-7 breast cancer stem cells; subsequently, 1×10 71 mL of hypotonic lysis buffer containing 0.1 mM EDTA-free protease inhibitor, 10 mM Tris, and 10 mM MgCl2 was added to MCF-7 cancer stem cells (CSCs) and cancer cells (CCs). The mixture was placed on ice for 3 hours to obtain a cell homogenate. The cell homogenate was then subjected to repeated freeze-thaw cycles (freezing in liquid nitrogen and thawing in a 37°C water bath, at least 5 times). Next, the mixture was centrifuged at 850 g for 10 minutes at 4°C to remove intracellular components, and the supernatant was collected. The mixture was then centrifuged at 18000 g for 60 minutes, and the final precipitate was collected as the purified CSCs and CCs membrane. The protein content was analyzed using a BCA protein assay kit. The two cell membranes were then mixed at a mass ratio of 1:1, and sonicated at 100 W for 10 minutes on ice to obtain a fusion membrane of cancer stem cells and cancer cells. (2) Preparation of hydroxyapatite nanoparticles (DHAp NPs) loaded with the chemotherapeutic drug doxorubicin. 10 mg of HAPNs was weighed and added to a brown glass bottle, followed by 5 mL of 600 μg / mL DOX aqueous solution. The bottle was placed on a magnetic stirrer in the dark and reacted at room temperature for 72 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 15 min, the supernatant was removed, and the precipitate was collected. The precipitate was resuspended in ultrapure water and centrifuged multiple times to wash away the unadsorbed DOX. The washed precipitate was freeze-dried for 24 h to obtain red powdered hydroxyapatite nanoparticles (DHAp NPs) loaded with the chemotherapeutic drug doxorubicin. (3) Preparation of drug-loaded nanoparticles coated with cancer stem cell and cancer cell fusion membrane. 1 mg / mL of the above-mentioned cancer stem cell and cancer cell fusion membrane was mixed with 1 mg / mL of hydroxyapatite nanoparticles loaded with the chemotherapeutic drug doxorubicin at a membrane-to-core weight ratio of 1:1. The mixture was then sonicated at 100W for 30 s on ice (1 s of sonication each time, with a 2 s interval between every two sonications). Subsequently, the mixture was repeatedly extruded 10 times through an Avanti micro extruder (0.8 μm polycarbonate membrane) to obtain drug-loaded nanoparticles CSC / CC@DHAp NPs coated with cancer stem cell and cancer cell fusion membrane.

[0042] Example 2

[0043] (1) Preparation of fusion membrane of cancer stem cells and cancer cells. The preparation method is the same as in Example 1, except that the ultrasonic power is set to 200W and the ultrasound is performed on ice for 15 minutes. (2) Preparation of hydroxyapatite nanoparticles (DHAp NPs) loaded with the chemotherapeutic drug doxorubicin. The preparation method was the same as in Example 1, with the main difference being that the reaction was carried out at room temperature for 48 hours. (3) Preparation of drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells. Refer to the preparation method in Example 1.

[0044] Example 3

[0045] (1) Preparation of fusion membrane of cancer stem cells and cancer cells. The preparation method is the same as in Example 1, with the main difference being that the ultrasonic power is set to 200W and ultrasonication is performed on ice for 15 minutes. (2) Preparation of hydroxyapatite nanoparticles (DHAp NPs) loaded with the chemotherapeutic drug doxorubicin. The preparation method was the same as in Example 1, with the main difference being that the concentration of the DOX aqueous solution was 1000 μg / mL. (3) Preparation of drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells. The preparation method is the same as in Example 1, with the main difference being: the membrane-to-core weight ratio is 2:1; sonication at 200W on ice for 15s (1s per sonication, with a 2s interval between sonications); and the use of an Avanti micro extruder (0.4μm polycarbonate membrane).

[0046] Successful encapsulation of the fusion membrane was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy images.

[0047] Figure 1 Characterization images of CSC / CC@DHAp NPs prepared in Example 1 are shown below; (A) SDS-PAGE analysis; (BD) Transmission electron microscopy images (scale bar: 100 nm) of HAp NPs, DHAp NPs, and CSC / CC@DHAp NPs; (EF) Zeta potential and hydrated particle size distribution; (G) Cumulative DOX release of DHAp and CSC / CC@DHAp within 48 h at different pH values. pH values ​​correspond to blood pH (pH 7.4) and endosome / lysosomal pH (pH 5.0). Error bars represent standard deviations (n ​​= 3).

[0048] like Figure 1 As shown in Figure A, protein bands are clearly visible in CSC / CC@DHAp NPs, while no protein bands are observed in standalone DHAp NPs. TEM images show a visible core-shell structure in CSC / CC@DHAp NPs, while no membrane structure was found in uncoated NPs. Figure 1 These results indicate that the CSCs-CCs fusion membrane successfully encapsulated DHAp NPs. The hydration particle size of CSC / CC@DHAp NPs was slightly increased compared to DHAp NPs, and the zeta potential increased from 1.5 mV to 8.6 mV, further demonstrating the successful preparation of CSC / CC@DHAp NPs. Figure 1 E, F). Drug release from CSC / CC@DHAp NPs exhibits typical sustained and pH-responsive behavior. For example... Figure 1As shown in Figure G, both CSC / CC@DHAp NPs and DHAp NPs exhibited rapid release in the first 12 hours at different pH levels, with the release rate remaining relatively constant after approximately 24 hours. At pH 7.2, the cumulative release rate of CSC / CC@DHAp NPs reached 28.5%, and when the pH decreased to 5.0, the release rate of DOX exceeded 50%. This pH-responsive release capability reduces premature drug leakage in the bloodstream and facilitates rapid release of chemotherapeutic drugs in the acidic tumor microenvironment, thereby improving therapeutic efficacy. Furthermore, we found that cell membrane coating slightly reduced the drug release rate compared to uncoated DHAp NPs, but did not negatively impact the cytotoxicity of CSC / CC@DHAp NPs, which may be related to the homologous targeting of the fusion membrane.

[0049] After successfully confirming the CSCs-CCs fusion membrane coating on DHAp, we continued to explore the homologous targeting ability of biomimetic NPs (CSC / CC@DHApNPs) in vitro and in vivo.

[0050] Normal hepatocytes (L-02), breast cancer cells (MCF-7), and corresponding CSCs were incubated with FITC-labeled CSC / CC@DHAp NPs for 4 hours, and their targeting ability was detected by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM), respectively.

[0051] Figure 2 The image shows the targeting characterization of CSC / CC@DHAp nanoparticles prepared in Example 1 on cancer stem cells. (A) Schematic diagram of cell uptake using laser confocal microscopy after 4 hours of pretreatment with FITC-labeled DHAp, CSC / CC@DHAp on L-02, MCF-7, and cancer stem cells (scale bar: 20 μm); (B) Schematic diagram of cell uptake of FITC-labeled CSC / CC@DHAp analyzed by flow cytometry; (C) Schematic diagram of Ca2+ uptake detected by ICP-OES. 2+Intake, error bars represent standard deviation (n=3), *P<0.05, **P<0.01, ***P<0.001, bioelectron microscopy images of L-02(D), MCF-7(E), and cancer stem cells (F) after 4 hours of treatment with CSC / CC@DHAp, where arrows indicate CSC / CC@DHAp NPs (scale bar: 4μm), (G) biodistribution of DOX in MCF-7 tumor-bearing nude mice at different time points after intravenous injection of DOX, DHAP, and CSC / CC@DHAp NPs, circles indicate tumor sites (dose: 5mg / kg), mice were sacrificed after 48h to investigate the in vivo biodistribution of DOX, DHAP, and CSC / CC@DHAp NPs in tumors and major organs (heart, liver, spleen, lung, and kidney), (H) and (G) quantification of DOX fluorescence intensity in fluorescence images, (I) measurement of Ca in various organs and tumors by ICP-OES. 2+ The biodistribution of CSC / CC@DHAp NPs in mice was investigated 48 hours after injection.

[0052] After incubation with CSC / CC@DHAp NPs, MCF-7 and CSCs showed similar intensity of bright green fluorescence, while the fluorescence in normal cells was weaker. Figure 2 A). Specifically, the fluorescence intensity of MCF-7 and CSCs after incubation with NPs coated with the fusion membrane was stronger than that of DHAp NPs, indicating that the CSCs-CCs fusion membrane has a specific targeting ability for tumor cells and corresponding CSCs. Similar results were also found in the FCM results. Figure 2 B), where more fusion-membrane-encapsulated nanoeffectants accumulate in CSCs. Furthermore, cells co-cultured with CSC / CC@DHAp NPs were collected, and the targeting ability of the NPs was further examined using inductively coupled plasma optical emission spectrometer (ICP-OES) and transmission electron microscopy. The results showed that Ca in CSCs or MCF-7 cells... 2+ The total number of cells increased by nearly 0.7 times compared to L-02 cells. Figure 2 C). Simultaneously, bio-TEM images showed an increase in the abundance of NPs marked with arrows in CSCs or MCF-7 cells compared to normal cells. Figure 2D). This difference in NP internalization is attributed to the homologous targeting ability of the source cells. Subsequently, to investigate the specific tumor-targeting ability of CSC / CC@DHAp NPs in vivo, we intravenously injected free DOX, DHAp, and CSC / CC@DHAp NPs into MCF-7 tumor-bearing nude mice and performed in vivo imaging at different time points. After injection of DHAp and CSC / CC@DHAp NPs, the fluorescence signal at the tumor site gradually increased, while the fluorescence signal of free DOX was more widely distributed throughout the body and showed a rapid decreasing trend after 24 hours. Figure 2 G). Notably, mice treated with CSC / CC@DHAp NPs exhibited the highest fluorescence signal at the tumor site, reflecting the excellent tumor-targeting ability of the fusion-coated NPs in vivo. Subsequently, all mice were euthanized, and their major organs and tumor tissues were harvested for further analysis. Ex vivo fluorescence images showed relatively high drug accumulation and retention in the tumor and liver compared to other organs. Compared to DOX or DHAp treatment, tumor tissues from mice treated with CSC / CC@DHAp NPs showed the highest fluorescence intensity (G). Figure 2 H) indicates that the nanoparticles coated with the fusion membrane accumulated the most in the tumor tissue, which is consistent with the results of ICP-OES assay. Figure 2 I). These data demonstrate that CSC / CC@DHAp NPs exhibit good specificity for targeting CCs and CSCs both in vitro and in vivo, laying a solid foundation for inhibiting tumor growth, reversing the stemness of CSCs, and preventing CSC-mediated tumor progression and metastasis.

[0053] Given that CSC / CC@DHAp NPs exhibit good CSC-targeting properties both in vitro and in vivo, we will continue to test the cytotoxicity of CSC / CC@DHAp NPs against CSCs.

[0054] Figure 3Characterization of the effect of CSC / CC@DHAp nanoeffectors on the bioactivity of breast cancer CSCs; (A) Cell viability of CSCs after 24 hours of incubation with different concentrations of free DOX, DHAp and CSC / CC@DHAp NPs (n=4); (B) Effect of CSC / CC@DHAp NPs on the apoptosis level of CSCs assessed by Annexin V-FITC / PI double staining method; (C) Apoptosis ratio statistically analyzed from Figure (B); (D) Intracellular ATP levels of CSCs after 24 hours of incubation with free DOX, DHAp and CSC / CC@DHAp NPs. Relative ATP levels and fluorescence intensity are expressed as a percentage of untreated control cells. Error bars represent standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001. (E) Relative activities of caspase-9 (left) and caspase-3 (right) (n=3), with untreated cells as a control, relative caspase activities were calculated. *p<0.05, **p<0.01, ***p<0.001. (F) Western blot analysis of the expression levels of caspase 3, cleaved caspase 3, and Bcl-2 proteins. (G) Cell viability of MCF-7 breast cancer cells after incubation with different concentrations of free DOX, DHAp, and CSC / CC@DHAp NPs for 24 hours (n=4).

[0055] CCK-8 assay results showed that free DOX, DHAp, and CSC / CC@DHAp NPs all exhibited dose-dependent toxicity to CSCs. Notably, the activity of CSCs treated with CSC / CC@DHAp NPs was significantly lower than that of CSCs treated with the same concentration of DOX or DHAp NPs. This may be because the targeting of CSC / CC@DHAp NPs promotes DOX accumulation in CSCs. Annexin V-FITC / PI double staining further confirmed the toxic effect of CSC / CC@DHAp NPs on CSCs. After 24 hours of incubation with free DOX, the percentage of apoptosis and necrosis in CSCs was only 24.1%, while the percentage of apoptosis and necrosis in CSCs treated with CSC / CC@DHAp NPs reached 46.2%. Figure 3 (B, C). Subsequent mechanistic analysis revealed that the mitochondrial apoptosis pathway was activated in CSCs treated with CSC / CC@DHAp NPs. ATP production in CSCs was inhibited after exposure to CSC / CC@DHAp NPs. Figure 3D), compared with untreated cells, ATP production was reduced to 26.0%. Furthermore, CSC / CC@DHAp NPs treatment activated caspase 9 (a marker of mitochondrial apoptosis) and caspase 3 (an executor of apoptosis), with activities 1.1-fold and 4.1-fold higher, respectively, than the control group. Figure 3 E). Western blot (WB) analysis also showed that, under the action of CSC / CC@DHAp NPs, the expression level of caspase 3 decreased, while the expression level of cleaved caspase 3 increased. Figure 3 F). Meanwhile, the expression level of full-length Bid was also significantly reduced (F). Figure 3 F) indicates that the full-length Bid is cleaved into tBid, inducing mitochondrial-dependent apoptosis. Similarly, in MCF-7 breast cancer cells, the action of CSC / CC@DHAp NPs can also significantly inhibit their cell viability (F). Figure 3 G). In summary, fusion-membrane-coated NPs exhibit excellent CSC-targeting activity, enabling these nano-effectors to activate the mitochondrial apoptosis pathway, interrupt energy metabolism, and exert potent anti-tumor activity in CSCs and MCF-7 breast cancer cells.

[0056] The therapeutic effect of CSC / CC@DHAp NPs in vivo was evaluated using a subcutaneous orthotopic breast cancer nude mouse model.

[0057] MCF-7 cells were injected subcutaneously into the fat pad of the right breast of nude mice, and the tumor volume reached approximately 50 mm. 3 At that time, tumor-bearing mice were administered free DOX, DHAp or CSC / CC@DHAp NPs, and saline, respectively. On day 24, four mice from each group were sacrificed to evaluate the antitumor effect.

[0058] Figure 4This diagram illustrates the effect of the CSC / CC@DHAp nanoeffector on inhibiting stem-mediated tumor growth and deterioration. (A) Drug treatment regimen in a subcutaneous breast cancer mouse model (n=4); (B) Tumor growth curves of tumor-bearing nude mice during drug administration; tumor volume in each group was normalized to the initial volume on day 1; statistical analysis was performed using the non-parametric Kruskal-Wallis test (*P<0.05, **P<0.01, ***P<0.001); tumor weight after drug administration (C) and tumor tissue photographs (D); (E) Tumor sections from tumor-bearing nude mice after different treatments. (F) H&E, TUNEL, and Ki67 staining images (scale bar: 50 μm), (G) Changes in body weight of tumor-bearing nude mice during drug administration (n=6), (H) Representative images of metastatic nodules in lung tissue on day 62, (H) H&E staining of lung tissue, with arrows indicating metastatic nodules, (I) CD44, CD133, and ALDH1 staining images of tumor tissue collected after treatment (scale bar: 50 μm), (J) and (K) ELISA measurements of the expression levels of metastasis-related proteins CXCR4, VEGF, TGF-β, and MMP9 in tumor tissue (D) and lung tissue (E), n=4. *p<0.05, **p<0.01, ***p<0.001.

[0059] The results showed that the tumors in the control group mice treated with saline grew rapidly, increasing 4.3 times in volume after 24 days of treatment compared to the initial volume, with a final weight of 4.8g. Figure 4 (B, C) Free DOX and DHAp showed comparable tumor-suppressing effects, with final tumor volume and weight being 2.6 times and 2.4 times that of the former, respectively, at 3.0 g and 2.6 g. Notably, CSC / CC@DHAp NPs outperformed the above three groups and almost completely inhibited the growth of MCF-7 breast cancer tumors. This is attributed to the in vivo targeting of the fusion membrane, which increased drug accumulation at the tumor site. The tumor tissue images showed that the trend of tumor size change was consistent with the trend of tumor volume or tumor weight change, directly demonstrating the anti-tumor growth effect of CSC / CC@DHAp NPs in vivo.

[0060] Furthermore, H&E, TUNEL, and Ki67 staining of tumor sections further demonstrated the therapeutic effect of CSC / CC@DHAp NPs. H&E staining images showed that tumors treated with CSC / CC@DHAp NPs exhibited cavitation in tissue structure and a reduced cell number compared to the control group. Figure 4E). TUNEL staining also confirmed that CSC / CC@DHAp NPs induced tumor apoptosis more effectively than other treatments. Simultaneously, treatment with CSC / CC@DHAp NPs significantly reduced Ki67 expression and inhibited tumor cell proliferation. These results indicate that CSC / CC@DHAp NPs can effectively inhibit tumor growth. Although free DOX can inhibit tumor growth to some extent, systemic administration may produce serious adverse reactions, including a significant decrease in mouse body weight (…). Figure 4 F). In contrast, CSC / CC@DHAp NPs injections did not cause significant systemic toxicity, as evidenced by serum biochemical analysis and histological analysis of major organs. Figure 5 This indicates that the biomimetic nano-effector possesses ideal biocompatibility. Furthermore, when we evaluated the in vivo distribution of CSC / CC@DHAp NPs in tumor tissues and major organs, the NPs coated with the CSCs-CCs fusion membrane accumulated extensively at the tumor site due to their targeting specific tumor locations, while showing less accumulation in the spleen and lungs. Figure 3 G, H), this may be the reason why nano-effectors have strong therapeutic effects and low toxicity.

[0061] Tumor stemness is a key driver of tumor development and progression; further evaluation is needed to determine whether this treatment can suppress tumor stemness.

[0062] Mice treated for the above 24 days continued to receive CSC / CC@DHAp treatment to assess the progression of lung deterioration. Figure 4 A) During the experiment, different formulations were injected intravenously via the tail vein every 4 days until day 62. Photographs of whole lung tissue and H&E-stained lung sections clearly showed that, compared with saline-treated mice, mice treated with DOX, DHAp, and CSC / CC@DHAp had significantly reduced lung metastases. Figure 4 (A, B) Among them, the reduction in lung metastases was most significant in the CSC / CC@DHAp group.

[0063] Studies have shown that CD44 and CD133 receptors are overexpressed in cancer stem cells (CSCs) and are highly correlated with the degree of carcinogenesis. Furthermore, ALDH1 has been identified as an important regulator of CSC growth and differentiation. Therefore, we monitored the expression levels of CD44, CD133, and ALDH1 to investigate the potential role of parental CSCs / CCs hybrid membrane-coated NPs in cancer stem cell regulation. The results showed that CSC / CC@DHAp NPs could attenuate tumor stemness, as evidenced by a significant downregulation of the fluorescence intensity of CD44, CD133, and ALDH1 compared to saline-treated tumors. Figure 4I). The reduction in the expression levels of metastasis-related proteins (such as CXCR4, VEGF, TGF-β, and MMP9) in tumors and lung tissues further demonstrates that this CSC-targeted nanoeffector reverses tumor stemness. Specifically, compared to saline treatment, CSC / CC@DHAp treatment reduced the expression levels of CXCR4, MMP9, TGF-β, and VEGF in tumor tissues to 44.4%, 81.3%, 71.8%, and 60.3%, respectively. Figure 4 In lung tissue, the expression levels of CXCR4, MMP9, TGF-β, and VEGF decreased to 65.8%, 45.2%, 83.5%, and 73.7%, respectively. Figure 4 K).

[0064] In summary, this biomimetic CSC-targeted nanoeffector exhibits biological regulation of cancer stem cell plasticity in breast cancer cells, thereby inhibiting tumor development and making it an ideal treatment option for preventing tumor recurrence and metastasis.

[0065] Comparative Example 1

[0066] (1) Preparation of fusion membrane of cancer stem cells and cancer cells. Refer to the preparation method in Example 1. (2) Preparation of hydroxyapatite nanoparticles (DHAp NPs) loaded with the chemotherapeutic drug doxorubicin. The preparation method is as described in Example 1. (3) Preparation of drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells. The preparation method is the same as in Example 1, with the main difference being that the membrane-to-core weight ratio is 1:0.1, and the resulting product is used as a control particle.

[0067] Figure 6 The image shows a TEM image (scale bar: 200 nm) of the control particles prepared in Comparative Example 1. As can be seen from the image, the control particles have a clear membrane structure, but there are almost no DHAp particles inside the membrane, indicating that the product obtained in the comparative example did not successfully achieve sufficient loading of chemotherapeutic drugs onto the cell membrane with hydroxyapatite nanoparticles.

[0068] The technical principles of the present invention have been described above with reference to specific embodiments. These descriptions are merely for explaining the principles of the invention and should not be construed as limiting the scope of protection of the invention in any way. Based on this explanation, those skilled in the art can readily conceive of other specific embodiments of the invention without inventive effort, and these embodiments will all fall within the scope of protection of the present invention.

Claims

1. A drug-loaded nanoparticle coated with a fusion membrane of cancer stem cells and cancer cells, characterized in that, The drug-loaded nanoparticles encapsulated in the cancer stem cell and cancer cell fusion membrane comprise: a cancer stem cell and cancer cell fusion membrane, and hydroxyapatite nanoparticles loaded with chemotherapeutic drugs encapsulated within the cancer stem cell and cancer cell fusion membrane; the drug-loaded nanoparticles encapsulated in the cancer stem cell and cancer cell fusion membrane target occult cancer stem cells in the body in addition to targeting solid tumors. The mass ratio of the chemotherapy drug to the hydroxyapatite nanoparticles is (0.1-0.6):1; the mass ratio of the cancer stem cell and cancer cell fusion membrane to the hydroxyapatite nanoparticles loaded with the chemotherapy drug is 1:(0.5-1.5).

2. The drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells according to claim 1, characterized in that, The hydroxyapatite nanoparticles have a short rod-like structure with a length of 30–100 nm and a width of 10–50 nm.

3. The drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells according to claim 2, characterized in that, The hydroxyapatite nanoparticles have a length of 50 nm and a width of 30 nm.

4. The drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells according to claim 1, characterized in that, The chemotherapy drugs mentioned are doxorubicin and paclitaxel.

5. A method for preparing drug-loaded nanoparticles coated with a fusion membrane of cancer stem cells and cancer cells as described in claim 1, characterized in that, The preparation method includes the following steps: (1) The cancer stem cell membrane and the cancer cell membrane were mixed in a solution and then sonicated on an ice bath to obtain a fusion membrane of cancer stem cells and cancer cells; (2) The chemotherapy drug and hydroxyapatite nanoparticles were mixed in solution and stirred at room temperature and in the dark. After the reaction was completed, the precipitate was collected, washed and dried to obtain hydroxyapatite nanoparticles loaded with chemotherapy drug. (3) The cancer stem cell and cancer cell fusion membrane and hydroxyapatite nanoparticles loaded with chemotherapy drugs are ultrasonically mixed in an ice bath, and then the mixture is extruded using an extrusion device to obtain the cancer stem cell and cancer cell fusion membrane coated with drug-loaded nanoparticles.

6. The preparation method according to claim 5, characterized in that, In step (1), the mass ratio of the cancer stem cell membrane to the cancer cell membrane is (0.5-1.5):

1.

7. The preparation method according to claim 6, characterized in that, The mass ratio of the cancer stem cell membrane to the cancer cell membrane is 1:

1.

8. The preparation method according to claim 5, characterized in that, In step (1), the cancer stem cell membrane and cancer cell membrane are mixed and then sonicated on an ice bath with a power of 50-300 W and a sonication time of 5-15 min.

9. The preparation method according to claim 8, characterized in that, The mixture of cancer stem cell membrane and cancer cell membrane was subjected to ultrasound in an ice bath at a power of 100 W for 10 min.

10. The preparation method according to claim 5, characterized in that, In step (2), the mass ratio of chemotherapy drugs to hydroxyapatite nanoparticles is controlled to be (0.3-1):

1.

11. The preparation method according to claim 10, characterized in that, The mass ratio of chemotherapy drugs to hydroxyapatite nanoparticles was controlled to be (0.5–1):

1.

12. The preparation method according to claim 11, characterized in that, The mass ratio of chemotherapy drugs to hydroxyapatite nanoparticles was controlled at (0.6–0.8):

1.

13. The preparation method according to claim 5, characterized in that, In step (3), the power of ultrasonic mixing of the cancer stem cell and cancer cell fusion membrane with hydroxyapatite nanoparticles loaded with chemotherapeutic drugs in an ice bath is 50-300 W, and the ultrasonic time is 10-50 s.

14. The preparation method according to claim 13, characterized in that, The cancer stem cell and cancer cell fusion membrane and hydroxyapatite nanoparticles loaded with chemotherapeutic drugs were ultrasonically mixed in an ice bath at a power of 100 W for 30 s, with each ultrasonic session lasting 1 s and an interval of 2 s between each ultrasonic session.

15. The use of the drug-loaded nanoparticles encapsulated in the fusion membrane of cancer stem cells and cancer cells as described in claim 1 in the preparation of targeted tumor therapeutic drugs.

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