A CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects, and its preparation method and application

Abstract

The present invention relates to the technical field of nano drug delivery systems, and specifically to a CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects, as well as a preparation method and application thereof. The nanoparticles include a core formed by copper sulfide nanoparticles and a shell formed by a cancer cell membrane integrated with CD47, and the mesopores of the copper sulfide nanoparticles are loaded with β-lapachone. The nanoparticles of this solution mediate photothermal and chemodynamic precision treatment of breast cancer through a "don't eat me" signal. Under laser irradiation, the nanoparticles produce a photothermal therapeutic effect on tumor tissue; Lap generates H2O2 in breast cancer cells, which is then converted into OH· through the catalytic action of CuS nanoparticles. By combining photothermal and chemodynamic effects, the optimal breast cancer treatment effect is achieved. This technical solution solves the technical problem in the prior art that nano drug presentation systems are difficult to effectively avoid the phagocytic action of macrophages, and has ideal application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of nano drug delivery systems, and in particular to a CD47 signal-mediated targeted nanoparticle having both photothermal and chemodynamic therapeutic effects, as well as a preparation method and application thereof. Background Art

[0002] The rapid development of nanomedicine has brought hope and confidence to the precision treatment of tumors. However, the efficacy of nanoparticle-mediated therapeutics is severely limited by the phagocytic and clearance of macrophages. Specifically, several nanoparticles, such as Doxil and Abraxane, have been approved by the US Food and Drug Administration for the treatment of breast cancer. These nanoparticles are typically administered intravenously and reach the tumor site through the bloodstream. However, during this process, most nanoparticles are phagocytosed by the mononuclear phagocyte system (MPS), significantly reducing the number of nanoparticles circulating in the bloodstream. Ultimately, less than 1% of nanoparticles reach the tumor site, significantly diminishing the therapeutic effect. To this end, many researchers have modified or modified the nanoparticle surface to evade the MPS. The most common approach is to add hydrophilic polymer polyethylene glycol (PEG) groups to the nanoparticle surface. PEG chains enhance the hydrophilicity of the nanoparticles and, due to their charge effect, significantly reduce the adsorption of serum proteins, thereby reducing MPS phagocytosis and prolonging the nanoparticle's blood circulation time. However, PEGylation does not solve the problem of active targeting, and studies have found that the immune system can produce antibodies that specifically bind to PEG, which can lead to "accelerated blood clearance" of PEGylated nanoparticles.

[0003] Biomimetic nanotechnology, particularly cell membrane-coated nanoparticles, offers an alternative to PEGylation and has been applied to a variety of novel biomaterials and delivery systems. A variety of cell membranes have been used to coat nanoparticles, including erythrocyte membranes and cancer cell membranes. Erythrocyte membranes contain numerous CD47 molecules, which interact with SIRP-α expressed on the surface of macrophages, thereby preventing phagocytosis. Studies have shown that nanoparticles coated with erythrocyte membranes can prolong their blood circulation time, with a blood elimination half-life even longer than that of PEGylated nanoparticles (39.6 h vs. 15.8 h). Cancer cell membranes contain various tumor-homologous proteins (such as Galectin-3 and EpCAM). Nanoparticles coated with cancer cell membranes can adhere to tumor cells through homologous targeting, resulting in high accumulation of nanoparticles in tumor tissue. To achieve both prolonged blood circulation time and tumor targeting, some studies have physically mixed erythrocyte and cancer cell membranes before coating the nanoparticles. However, the number of CD47 molecules on the red blood cell membrane is limited, and the process of extracting and fusing red blood cells and cancer cells separately is cumbersome. There is an urgent need to develop a nanodrug delivery system that can effectively avoid phagocytosis and achieve cancer tissue targeting and multiple therapeutic functions. Summary of the Invention

[0004] The present invention aims to provide a CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects to solve the technical problem that the nano drug delivery system in the prior art is difficult to effectively avoid the phagocytosis and clearance of macrophages.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects, comprising a core formed by mesoporous copper sulfide nanoparticles and an outer shell formed by a cancer cell membrane integrated with CD47.

[0007] This solution also provides a method for preparing CD47 signal-mediated targeted nanoparticles with both photothermal and chemodynamic therapeutic effects, comprising the following steps in sequence:

[0008] S1: Preparation of copper sulfide nanoparticles:

[0009] S2: Preparation of cancer cell membranes with integrated CD47;

[0010] S3: Preparation of CD47@CCM-Lap-CuS nanoparticles: An ethanol solution of β-lapachone and an aqueous suspension containing copper sulfide nanoparticles were mixed and stirred in the dark, and then the ethanol was evaporated to obtain a mixture of β-lapachone and copper sulfide nanoparticles; next, an ultrasonically treated suspension of cancer cell membranes integrated with CD47 was added, and then ultrasonicated; then co-extruded through a liposome extruder to obtain CD47@CCM-Lap-CuS nanoparticles.

[0011] This solution also provides a CD47 signal-mediated application of targeted nanoparticles with both photothermal and chemodynamic therapeutic effects in the preparation of drugs for treating breast cancer.

[0012] The principles and advantages of this solution are:

[0013] This technical solution provides a new nano-drug delivery strategy for photothermal and chemodynamic precision therapy of breast cancer mediated by "don't eat me" signal ( Figure 3 ). CD47 molecules were overexpressed on the breast cancer cell membrane through genetic engineering technology, and the cell membrane was extracted and coated on CuS nanoparticles loaded with Lap. Compared with physically mixing the red blood cell membrane and cancer cell membrane and then coating, this method of preparing the cell membrane coating is simpler and easier to obtain, and has a lower error tolerance. Under the action of the "don't eat me" signal of CD47, the residence time of the nanoparticles in the blood circulation is prolonged, and they can actively target breast cancer cells through homologous targeting. Under NIRⅡ laser irradiation, a photothermal therapeutic effect is produced on the tumor tissue. At the same time, Lap is released into the breast cancer cells to generate H2O2, which is then converted into the more cytotoxic OH· through the catalytic action of CuS nanoparticles. By combining photothermal and chemokinetic effects, the best breast cancer treatment effect is achieved.

[0014] Photothermal therapy is an emerging non-invasive tumor treatment modality that utilizes photothermal materials to rapidly generate heat within the tumor region under near-infrared laser irradiation, causing irreversible damage to cancer cells through high temperatures (40-45°C) or thermal ablation (>45°C). Among the numerous photothermal materials, hollow mesoporous copper sulfide (CuS) nanoparticles are a relatively superior semiconductor, offering many unique advantages, including a strong absorption peak in the near-infrared II region (NIR II, 900-1700nm), excellent photothermal conversion performance, good photothermal stability, good biocompatibility and degradability, simple synthesis, controllable particle size, and a hollow structure suitable for drug loading. Furthermore, the inventors accidentally discovered that copper sulfide nanoparticles also possess catalytic properties similar to nanozymes, catalyzing hydrogen peroxide (H2O2) into more cytotoxic hydroxyl radicals (OH·). Combining photothermal therapy with other treatment modalities (such as chemotherapy) using nanomaterials as a medium can not only improve therapeutic efficacy but also reduce drug dosage and mitigate toxic side effects.

[0015] Furthermore, β-lapachquinone is loaded into the mesopores of the copper sulfide nanoparticles.

[0016] β-Lapachone (Lap) is a natural naphthoquinone compound that selectively inhibits topoisomerase I and cell cycle progression to induce apoptosis. It is currently undergoing a Phase II clinical trial for pancreatic cancer. Lap is also a reactive oxygen species inducer that can activate the redox reaction of NAD(P)H:quinone oxidoreductase (NQO1), which is specifically overexpressed in tumor tissues, thereby generating excess hydrogen peroxide (H2O2) that kills tumor cells.

[0017] Furthermore, the copper sulfide nanoparticles are prepared by the following method: polyvinyl pyrrolidone K30 and copper chloride solution are added to water, stirred, and then sodium hydroxide solution is added, and then hydrazine hydrate and sodium sulfide solution are added in sequence to obtain a reaction system; the reaction system is stirred and reacted in contact with air to obtain copper sulfide nanoparticles.

[0018] The preparation method used in this protocol is a template-sacrificial method. First, CuCl2 reacts with hydrazine hydrate in an alkaline environment to form solid Cu2O nanoparticles. Subsequently, Na2S is added to the Cu2O, which acts as a template for sulfurization. This process produces the Kirkendall effect, gradually forming hollow CuS NPs. The formation of CuS NPs requires oxygen. To ensure the yield and purity of CuS NPs, the reaction solution should be kept in open air and stirred at a high speed.

[0019] Furthermore, the cancer cell membrane integrated with CD47 is prepared by the following method: constructing cancer cells that overexpress CD47, and then extracting the cell membrane of the cancer cells.

[0020] Constructing an expression vector and transferring it into recipient cells through lentiviral infection to achieve overexpression of a specific gene in the recipient cells is a conventional research method in the field of molecular biology. The method is mature and easy to implement.

[0021] Furthermore, the cancer cells are breast cancer cells. By extracting the cell membranes of breast cancer cells, nanoparticles can be used to achieve targeted therapy of breast cancer tissue.

[0022] Furthermore, in S3, the amount of the ethanol solution of β-lapachone is 2 mL, and the concentration of β-lapachone is 0.5 mg / mL; the amount of the aqueous suspension containing copper sulfide nanoparticles is 2 mL, and the concentration of copper sulfide nanoparticles is 1 mg / mL.

[0023] Controlling the concentration of β-lapachone is crucial for the successful preparation of nanoparticles. Excessively high concentrations can result in the liposome extruder failing to extrude the nanoparticles, preventing the formation of the CD47@CCM-Lap-CuS NPs described in this protocol. Excessively low concentrations can lead to low drug loading on the nanoparticles.

[0024] Furthermore, in S3, the ethanol solution of β-lapachone and the aqueous suspension containing copper sulfide nanoparticles were mixed, stirred in a dark environment for 24 hours, and then continued to stir at 60° C. until the ethanol was completely evaporated.

[0025] The β-lapachone and copper sulfide nanoparticles were mixed evenly by stirring, and the ethanol was removed by evaporation to avoid its effect on the subsequently added cell membrane.

[0026] Furthermore, in S3, the CD47-integrated cancer cell membrane suspension was first sonicated for 30 seconds, then added to a mixture of β-lapachone and copper sulfide nanoparticles, and then sonicated again for 30 seconds; the amount of the CD47-integrated cancer cell membrane suspension was 1 mL, and contained 3 mg of CD47-integrated cancer cell membranes; the co-extrusion parameters of the liposome extruder were set to: pass through 0.8 μm and 0.4 μm membrane filters in sequence.

[0027] Passing through 0.8 μm and 0.4 μm membrane filters in sequence can ensure that the prepared nanoparticles have more ideal stability.

[0028] In summary, breast cancer is the most common malignancy worldwide and the leading cause of cancer-related death in women. Due to the high heterogeneity of breast cancer, single treatments often fall short of expectations. The rapid development of nanomedicine has brought hope and confidence to this field. However, circulating nanoparticles are often phagocytosed by hepatic and splenic macrophages, significantly limiting their application in cancer. To address this, the inventors developed an engineered biomimetic copper sulfide nanozyme, termed CD47@CCM-Lap-CuS NPs. These nanoparticles, through the CD47 molecule on their surface, inhibit macrophage phagocytosis, thereby increasing their return to the bloodstream. They then target tumor tissue through homologous targeting of the cancer cell membrane. Under near-infrared laser irradiation, they produce photothermal therapy on tumor tissue. Simultaneously, Lap is released into breast cancer cells to generate H2O2, which is then catalyzed by the CuS nanoparticles to convert H2O2 into the more cytotoxic OH·. By combining photothermal and chemokinetic effects, optimal breast cancer treatment is achieved.

[0029] In this technical solution, CD47@CCM-Lap-CuS NPs were prepared and their characteristic characterization was analyzed and identified. It was confirmed that the core of the nanoparticle is hollow mesoporous copper sulfide and the outer layer is the cancer cell membrane. Lap was successfully encapsulated in it, with an encapsulation efficiency and drug loading rate of 18.9% and 9.5%, respectively; the particle size potential of CCM-Lap-CuS NPs was 186.3nm and -14.07mV, respectively; in vitro verification demonstrated that CD47@CCM-Lap-CuS NPs had good photothermal and enzyme catalytic properties; it was verified that CD47 molecules were present in excess on the surface of the nanoparticles, and in vitro and in vivo experiments verified that the nanoparticles could use CD47 molecules to evade phagocytosis of macrophages and increase the aggregation of nanoparticles in tumor tissues; in vitro and in vivo experiments confirmed that CD47@CCM-Lap-CuS NPs can produce a good therapeutic effect on breast cancer cells or tissues through photothermal combined with chemokinetic effects; and CD47@CCM-Lap-CuS NPs have good biosafety.

[0030] The main innovation of this technical solution is to overexpress CD47 molecules on breast cancer cell membranes, giving them the biological functions of both red blood cell membranes and cancer cell membranes. Compared with the mixed membrane method, this method is simpler to prepare and can ensure that the majority of nanoparticles coated with cancer cell membranes can extend blood circulation retention time and target breast cancer cells. This nanoparticle preparation model provides a new approach and platform for tumor-targeted drug delivery. The key innovation of this research is that β-lapachone is loaded into copper sulfide nanoparticles, which generate and synergistically amplify reactive oxygen species in the tumor environment, thereby optimizing the chemodynamic treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart for the preparation of CuS NPs in Example 1.

[0032] Figure 2 Schematic diagram of the expression vector structure of Example 1.

[0033] Figure 3 Schematic diagram of the preparation process and mechanism of action of CD47@CCM-Lap-CuS NPs in Example 1.

[0034] Figure 4 These are images of the characterization results of CuS NPs and CCM-Lap-CuS NPs in Example 1.

[0035] Figure 5 These are the experimental results of the cellular uptake of CuS NPs and CCM-Lap-CuS NPs in Experimental Example 1.

[0036] Figure 6 These are the in vitro photothermal and catalytic activity test results of CD47@CCM-Lap-CuS NPs in Experimental Example 2.

[0037] Figure 7 These are the experimental results of the photothermal and chemodynamic therapeutic effects of CD47@CCM-Lap-CuS NPs on 4T1 cells in Experimental Example 3.

[0038] Figure 8 In vivo and ex vivo fluorescence imaging of tumor-bearing mice at different time points after injection of DiR-labeled CCM-Lap-CuS NPs or CD47@CCM-Lap-CuS NPs in Experimental Example 4.

[0039] Figure 9 Pharmacokinetic and biodistribution curves of CuS NPs, CCM-Lap NPs and CD47@CCM-Lap-CuS NPs of Experimental Example 4.

[0040] Figure 10 Photoacoustic imaging of tumor tissue at different time points after injection of CuS NPs, CCM-Lap-CuS NPs, and CD47@CCM-Lap-CuS NPs in Experimental Example 4.

[0041] Figure 11 This is the infrared thermal imaging image of the mouse and the temperature rise curve of the tumor area in Experimental Example 4.

[0042] Figure 12 These are the in vivo anti-tumor treatment experimental results of Experimental Example 5. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0044] Example 1:

[0045] A CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects is prepared by the following method:

[0046] (1) Preparation of copper sulfide nanoparticles (CuS NPs):

[0047] 100 μL 0.5 mol / L CuCl2 and 240 mg PVP-K30 (polyvinyl pyrrolidone K30) were added to 25 mL of deionized water and stirred thoroughly (about 1400 rpm) at room temperature (25 ± 5 ° C). Then 25 mL of 0.01 mM NaOH (pH 9.0) was added, and 6.4 μL N2H4·H2O was added after 2 minutes. After 5 minutes, 200 μL 320 mg / mL Na2S was added, and the reaction mixture was magnetically stirred at 60 ° C (need to be controlled to be greater than 1000 rpm, the speed used in this embodiment is 1400 rpm) for 2 hours to generate CuSNPs. The reaction mixture was centrifuged at 10000 rpm for 10 minutes, the supernatant was discarded, and the precipitated CuS NPs were washed twice with deionized water. The successfully prepared CuS NPs solution should appear dark green. See the preparation flow chart for details. Figure 1 .

[0048] The preparation method adopted in this scheme is the template sacrificial method. First, CuCl2 reacts with hydrazine hydrate in an alkaline environment to generate solid Cu2O nanoparticles. Then, Na2S is added to perform sulfurization using Cu2O as a template. During the sulfurization process, the Kirkendall effect is produced, gradually forming hollow CuS NPs. Oxygen is required to participate in the formation of CuS NPs. In order to ensure the yield and purity of CuS NPs, the reaction solution should be kept in the open air during the preparation process, and the stirring speed should be increased as much as possible. In this technical solution, the entire preparation process is carried out in an open container to ensure that the reaction system is connected to the air. If the CuS NPs need to be tested after freeze-drying, they must be washed with deionized water at least 3 times before freeze-drying to remove PKP-K30 on the surface of the CuS NPs. Otherwise, after freeze-drying, the nanoparticles will stick together, affecting subsequent experiments.

[0049] This example demonstrates a 50 mL reaction system, producing approximately 2 mg of CuS NPs (determined by ICP analysis of the copper content). Larger reaction volumes can be used, but the stirring speed and oxygen-contact area must be maintained (for example, a 500 mL beaker can be used as the reaction vessel).

[0050] (2) Lentiviral transfection:

[0051] CD47, also known as integrin-associated protein, is a transmembrane protein with a molecular weight of 50 kDa. In 2000, in the journal Science, CD47 was first identified as a "self-marker" on mouse red blood cells. It interacts with SIRP-α to prevent splenic red pulp macrophages from clearing red blood cells from the bloodstream. In 2009, the CD47-SIRP-α axis was considered a tumor phagocytic checkpoint signal that transmits the "don't eat me" signal to macrophages and is used by various tumors as a mechanism of immune escape. Taking advantage of this characteristic of the CD47-"don't eat me" signal, many studies have significantly reduced the phagocytosis of nanoparticles by macrophages and prolonged their blood circulation retention time by functionalizing CD47 (including recombinant CD47 protein or CD47 functional peptide fragments; this protocol uses mouse CD47, AB012693.1) on the surface of nanoparticles.

[0052] Shanghai GeneCare Gene was commissioned to construct, verify and purify the CD47 expression vector using conventional means of existing technologies, and to carry out lentiviral packaging of the CD47 expression vector and the control vector (the empty vector of the lentiviral vector is GV367, and the element sequence is Ubi-MCS-SV40-EGFP-IRES-puromycin. The plasmid map can be found in Figure 2 ). This technical solution directly uses a lentivirus packaged with a CD47 expression vector or a control vector to infect cells. The nucleotide sequence of CD47 is shown in SEQ ID NO.1:

[0053] (SEQID NO.1)。

[0054] The specific process is as follows: 4T1 cells (inoculation volume 1×10 5 The culture conditions are: 37°C temperature, 5% CO2, 95% relative humidity). The complete culture medium is specifically: 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100U / mL penicillin-G and 100μg / ml streptomycin). After about 50-60% confluence (after about 24 hours of culture), the culture medium is discarded, 1mL of complete culture medium is added to each well, and 40μL of infection enhancement solution P (provided by Jikai Gene) is added. The volume of the virus liquid (lentivirus packaged with CD47 vector or lentivirus packaged with empty vector) is calculated based on the MOI of the infected virus particles = 50, and the volume is 5μL. Gently shake and continue to culture. The cells are cultured routinely and fresh culture medium is replaced regularly. About 72 hours after lentiviral infection, the infection efficiency (EGFP green fluorescence) is observed under a fluorescence microscope, and 5μg / mL puromycin is used to screen successfully transfected cells.

[0055] (3) Extraction of 4T1 cell membrane:

[0056] The cell membrane extraction kit (Shanghai Biotech Co., Ltd.) was used and the operation was carried out according to the instructions. 8 Resuspend each cell in 3 mL of cell membrane extraction solution containing 1 mM PMSF, then incubate on ice for 15 minutes and freeze-thaw three times at -80°C and room temperature. Centrifuge the freeze-thawed solution at 2000 rpm for 10 minutes at 4°C. The supernatant is then centrifuged at 14000 rpm for another 30 minutes. The precipitate is the extracted cell membrane CD47@CCM. Place the CD47@CCM in PBS and freeze at -80°C until ready for use.

[0057] In this step, it is necessary to ensure that the cells are frozen and thawed repeatedly more than three times, otherwise some cells are not broken, which will affect subsequent experiments. This example uses a -80°C refrigerator for repeated freezing and thawing, and liquid nitrogen should be used for the best results. To determine the quality of the cell membrane, the quality of the cytoplasmic protein can be measured (protein content can be determined using the BCA method or the Coomassie Brilliant Blue method), and then the quality of the cell membrane can be inferred.

[0058] (4) Preparation of CD47@CCM-Lap-CuS Nanoparticles:

[0059] 2 mL of a 0.5 mg / mL Lap (β-lapachone) ethanol solution (ethanol as solvent, solute Lap) was added to 2 mL of a 1 mg / mL CuS NPs aqueous suspension. The suspension was magnetically stirred (1400 rpm) at room temperature (25 ± 5 ° C) in the dark for 24 hours, and then stirred at 60 ° C until the ethanol was completely evaporated. 1 mL of a PBS suspension containing 3 mg of cell membranes was sonicated for 30 seconds (50 W, 5 seconds on, 5 seconds off) to completely disperse the cell membranes and added to the mixture of CuS NPs and Lap. The mixture was sonicated for another 30 seconds and then co-extruded through 0.8 μm and 0.4 μm membrane filters (state-of-the-art liposome extruder, Avanti, USA). The obtained CD47@CCM-Lap-CuS NPs were then centrifuged at 8000 rpm and 4 ° C for 5 minutes and washed twice with PBS to remove excess cell membranes.

[0060] In this step, the condition for evaporating ethanol is 60°C, which does not affect the properties and activity of Lap. Subsequent experiments confirmed that CCM-Lap-CuS NPs can generate a large amount of reactive oxygen. And there are documents showing that the thermal stability of Lap is relatively good, up to 191°C. In the ethanol solution of Lap, the content of Lap cannot be too high, and it is advisable to maintain it at <1 mg / mL (optimally ≤0.5 mg / mL). This embodiment specifically uses 0.5 mg / mL. If the content of Lap is ≥1 mg / mL, the liposome extruder will not be able to extrude the nanoparticles, and the CD47@CCM-Lap-CuS NPs of this scheme cannot be formed. This shows that the increase in the content of Lap will affect the fluidity of the system formed by Lap+CuS NPs+cancer cell membrane.

[0061] The prior art uses ultrasonic vibration to prepare nanoparticles, but in this technical solution, ultrasonic vibration cannot completely coat the cell membrane with the nanoparticles. When a liposome extruder is used correctly, the coverage rate of the cell membrane is close to 100% (observed under a transmission electron microscope). This technical solution currently uses 0.8μm and 0.4μm membrane filters. The nanoparticles obtained by using only a 0.8-μm membrane filter have the same effect as those obtained by passing through a 0.4-μm membrane filter under a transmission electron microscope, but the stability is deteriorated. The stability here means that after the nanoparticles are successfully prepared and left to stand for 5-10 days, the particle size of the nanoparticles will not change or will not agglomerate. If only a 0.8μm membrane filter is used, the nanoparticles will increase in size and agglomerate after being left for 5-10 days.

[0062] Schematic diagram of the preparation process of CD47@CCM-Lap-CuS nanoparticles. Figure 3A, The mechanism of CD47@CCM-Lap-CuS NPs-mediated photothermal and chemodynamic precision therapy for breast cancer can be found in Figure 3 B. Figure 4 The properties of the nanoparticles prepared by this scheme are shown. A and B are scanning electron microscope images of CuS NPs; C is the pore size distribution of CuS NPs; D is the X-ray diffraction pattern (XPS) of CuS NPs; E and F are high-resolution Cu 2p and S 2p XPS spectra; G, transmission electron microscopy images of CuS NPs and CCM-Lap-CuS NPs (scale bar = 100 nm); H, elemental distribution map of CCM-Lap-CuS NPs (scale bar = 100 nm, red arrow indicates the cell membrane); I, UV-visible-near-infrared absorption spectra of CuS NPs, Lap, and CCM-Lap-CuS; J, hydrodynamic diameters and zeta potentials of CuS NPs, CCM-CuS NPs, and CCM-Lap-CuS NPs. For all studies in this protocol, all data are presented as mean ± SD and analyzed using GraphPad Prism 5.0. Analyses were performed using one-way analysis of variance (ANOVA) followed by a post hoc Tukey test. Ns indicates not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistically significant differences at each level.

[0063] This technical solution uses a template sacrificial method to prepare hollow mesoporous copper sulfide nanoparticles (CuS NPs). CuS NPs are regular, complete, and uniformly sized spherical particles with many fluffy small particles on the surface ( Figure 4 A, B). Nitrogen adsorption-desorption experiments confirmed that CuS NPs have a mesoporous morphology with a specific surface area of ​​22.4 m 2 / g, average pore size 3.3nm ( Figure 4 C). The mesoporous and hollow structure of CuS NPs provides favorable conditions for subsequent drug loading. The X-ray diffraction (XRD) pattern of CuS NPs is shown together with the standard peaks. Figure 4 In D, the inventors found that the peaks of the sample were highly consistent with the hexagonal copper blue structure (JCPD 06-0464), indicating that the CuS NPs were both pure and had a good crystal structure. X-ray photoelectron spectroscopy (XPS) confirmed the presence of sulfur and copper elements. High-resolution Cu2p XPS spectrum ( Figure 4 E) shows two absorption peaks at 931.88eV and 951.78eV, which are characteristic Cu2p 3 / 2 and Cu2p 1 / 2There is a 19.9 eV separation between the two peaks, indicating that Cu is in the +2 oxidation state. Figure 4 Two peaks at 162.78 eV and 161.78 eV can be seen in F, corresponding to S2p 3 / 2 and S2p 1 / 2 Transmission electron microscopy (TEM) showed that CuS NPs were hollow with a particle size of approximately 140 nm ( Figure 4 G). The anti-tumor drug β-lapachone (Lap) was loaded into CuS NPs and then coated with cancer cell membrane (CCM), termed CCM-Lap-CuS NPs. These nanoparticles had a clear core / shell structure, indicating the presence of a single layer of cell membrane coating on the nanoparticles. The shell thickness of CCM-Lap-CuS NPs was approximately 12 nm, indicating that the CCM effectively covered the nanoparticles ( Figure 4 H). In addition, elemental scanning, especially phosphorus, confirmed the successful coating of CCM on the nanoparticle surface ( Figure 4 I). The mesoporous hollow structure of CuS NPs and the coating of CCM enable Lap to be effectively loaded into the nanoparticles. Draw a Lap standard curve (measurement wavelength 257nm), and calculate the amount of Lap in the supernatant based on the standard curve. Calculate according to the following formula: Encapsulation efficiency (%) = 100×(amount of input Lap-amount of supernatant Lap) / amount of input Lap; Drug loading rate (%) = 100×(amount of input Lap-amount of supernatant Lap) / amount of input CuS NPs. After testing and calculation, the encapsulation efficiency (EE) and drug loading capacity (DLC) of Lap in CCM-Lap-CuSNPs are 18.9% and 9.5%, respectively. CuS NPs show strong light absorption at 1064nm, and the absorbance increases with the increase of CuS NP concentration. As shown Figure 4As shown in Figure J, the UV-Vis-NIR spectrum of CCM-Lap-CuS not only shows the absorption characteristics of CuS NPs in the NIR, but also the absorption characteristics of Lap in the UV. CCM-Lap-CuS NPs and Lap exhibited the same absorption peak at 257 nm, indicating that Lap was successfully encapsulated into CuS NPs. Dynamic light scattering (DLS) analysis showed that the hydrodynamic size of CCM-Lap-CuS NPs (~186 nm) increased by ~10 nm and ~36 nm compared to CCM-CuS NPs (~179 nm) and CuS NPs without cell membrane coating (~150 nm), respectively. The zeta potential of CCM-Lap-CuS NPs (~-14 mV) was restored to the original level of CuS NPs (~-14 mV) compared to CCM-CuS NPs (~-24.87 mV). The changes in the hydrodynamic size and zeta potential of the nanoparticles reflected, to some extent, the successful coating of CCM and the loading of Lap.

[0064] Comparative Example 1: Anhydrous ethanol stirring method

[0065] 2 mL of 0.5 mg / mL Lap ethanol solution was added to 2 mL of 1 mg / mL CuS NPs ethanol suspension; the suspension was magnetically stirred at room temperature in a dark environment for 24 hours; centrifuged at 11000 rpm for 10 minutes, and the precipitate was collected; the precipitate was washed three times with anhydrous ethanol and deionized water; and the precipitate was resuspended in deionized water.

[0066] The supernatant was collected, and the encapsulation efficiency and drug loading rate were calculated, both of which were close to 0%. The entire drug loading process was carried out in anhydrous ethanol solution, in which Lap is completely soluble. Furthermore, CuS NPs have mesoporous and hollow structures, allowing Lap to freely enter and exit the CuS NPs. However, Lap has difficulty precipitating and adsorbing within the mesopores of CuS NPs, resulting in encapsulation and drug loading efficiencies close to 0%.

[0067] Comparative Example 2: DMSO / H2O stirring method

[0068] Prepare 50 mg / mL Lap stock solution: dissolve 5 mg of Lap in 100 μL DMSO; take 3 mg of CuS NPs and 1 mg of Lap (50 mg / mL, 20 μL) and mix them, add deionized water to 1 mL, stir magnetically for 24 hours, collect the precipitate by centrifugation, and retain the supernatant; wash the precipitate three times with DMSO / H20 (v / v, 1 / 49) solution to remove excess Lap, and retain the supernatant; finally, resuspend the precipitate with deionized water.

[0069] This technical solution failed to achieve Lap loading on CuS NPs, resulting in both drug loading and encapsulation efficiency of 0%. With a DMSO to water ratio of 1:49, Lap precipitates from the solution. However, this precipitated Lap is too large to pass through the 3nm pore size of CuS NPs. Subsequent loading with cancer cell membranes was also difficult to achieve.

[0070] Comparative Example 3: Ethanol-water stirring method

[0071] Prepare a 5 mg / mL stock solution of Lap in ethanol. Take 400 μL of the Lap ethanol stock solution and add it to 600 μL of a 2 mg CuS NPs aqueous solution, for a total of 1 mL. Seal the tube and magnetically stir at 37°C for 24 h. After cooling to room temperature, centrifuge at 11,000 rpm for 10 min. Wash three times with deionized water and resuspend.

[0072] The supernatant was collected, and the encapsulation efficiency and drug loading rate were calculated to be close to 5%. Theoretically, Lap can be dissolved in an ethanol-water solution (v / v, 2:3) and, after mixing with CuS NPs and cooling (to room temperature), can precipitate and settle into the pores of the CuS NPs. Subsequent attempts were made to cool the solution on ice and centrifuge it in a low-temperature centrifuge. However, the final encapsulation efficiency and drug loading rate were still very low. This may be due to the low concentration of Lap entering the copper sulfide nanoparticles, and only a trace amount of Lap may have settled into the pores.

[0073] The preparation methods of Example 1 and Comparative Examples 1-3 are compared and analyzed: Due to the poor adsorption capacity of CuS NPs for Lap, there are certain difficulties in loading Lap onto CuS NPs. The process of achieving drug loading is mainly to mix Lap and CuS NPs, and Lap is precipitated and deposited into the mesopores and hollow structures of CuS NPs in a certain solvent environment. The inventors have tried to use anhydrous ethanol as a solvent environment, but Lap is completely dissolved in anhydrous ethanol. Lap can freely enter and exit the mesopores and hollow structures in CuS NPs, but it is difficult to achieve precipitation, deposition, and effective retention and adsorption in CuS NPs (Comparative Example 1). The inventors have also tried to use an aqueous solution of DMSO as a solvent environment. Although Lap can be precipitated after mixing with CuS NPs, the precipitated Lap is difficult to enter the mesoporous structure, so this method cannot achieve drug loading (Comparative Example 2). The inventors have also tried to use an aqueous solution of ethanol as a solvent environment. Lap can be dissolved in an aqueous solution of ethanol. When a larger amount of Lap is added (Lap concentration), Lap can be precipitated after mixing with CuS NPs and layered into CuS NPs in small amounts. However, the encapsulation efficiency and drug loading rate are not ideal (Comparative Example 3). The inventors also tried to carry out the cooling and placement process on ice after magnetic stirring, and to maintain the centrifugation process at a low temperature of 4°C, hoping that the low temperature can promote the precipitation of Lap. However, it is difficult to effectively improve the encapsulation efficiency and drug loading on the basis of Comparative Example 3. From Comparative Examples 1-3, it can be seen that the use of an aqueous solution of ethanol as a solvent environment for drug loading can allow CuS NPs to achieve a small amount of Lap drug loading to a certain extent, but the effect is not ideal. The use of other types of solvent environments cannot achieve the Lap drug loading of CuS NPs at all.

[0074] In order to add targeting ability and evasion of phagocytosis to the nanoparticles, the inventors tried to encapsulate the nanoparticles with breast cancer cell membranes integrated with CD47. During the above attempts, the inventors found that the loading of the cell membrane not only achieved the purpose of targeting and evasion of phagocytosis, but also that the cell membrane blocked the mesopores of CuS NPs, and when the Lap concentration was reduced, the encapsulation efficiency and drug loading capacity (DLC) of Lap were greatly improved to 18.9% and 9.5%, respectively, achieving unexpected technical effects. Moreover, in the process of using cell membranes to encapsulate nanoparticles, the concentration of Lap needs to be maintained at a relatively low level to ensure that the liposome extruder effectively extrude the nanoparticles, and successfully prepare CD47@CCM-Lap-CuS NPs nanoparticles.

[0075] Experimental Example 1: Study on Cellular Uptake of Nanoparticles

[0076] RAW264.7 and 4T1 cells were seeded in glass confocal culture dishes and cultured overnight. DiI-labeled nanoparticles (50 μg / mL) were then added and incubated for 4 hours. Subsequently, the cells were washed three times with PBS to remove nanoparticles that were not bound or taken up by the cells, and the cell nuclei were stained with DAPI for 5 minutes. Finally, the cells were observed by confocal microscopy (red fluorescence represents nanoparticles). In addition, the cell uptake of nanoparticles was quantitatively analyzed by flow cytometry and ICP-OES, respectively. After the above-mentioned PBS washing step, the cells were collected by trypsin digestion, centrifuged at 1000 rpm for 5 minutes, and then resuspended in PBS for relevant detection and analysis.

[0077] The experimental results can be found in Figure 5 , where A-C are confocal microscopy (CLSM) images, flow cytometry, and Western blot (WB) of CD47 molecules in 4T1 cells transfected with lentivirus overexpressing CD47 or negative control (scale bar = 50 μm); D is SDS-PAGE of proteins in different samples (1: CuS NPs, 2: CCM-CuS NPs, 3: CD47@CCM-CuS NPs, 4: RBCM-CuS NPs); E, F are flow cytometry and mean fluorescence intensity analysis of CD47 on the surface of CCM-CuS NPs, CD47@CCM-CuS NPs, and RBCM-CuS NPs; G, H are DiI-stained images of CCM-CuS NPs and CD47@CCM-CuS Representative CLSM images and flow cytometry profiles of RAW264.7 cells incubated with CuS NPs for 4 h (scale bar = 50 μm); I is the copper concentration of 4T1 cells and RAW264.7 cells after incubation with CuS NPs, CCM-CuS NPs, and CD47@CCM-CuS NPs for 4 h, measured by ICP-OES.

[0078] CD47 is considered a "don't eat me" signal molecule that can bind to the SIRP-α receptor on the surface of macrophages, thereby inhibiting the phagocytic function of macrophages. In order to enable nanoparticles to escape the phagocytosis of macrophages and thus prolong their blood retention time, the inventors used gene editing technology to overexpress CD47 molecules on the 4T1 cell membrane. The inventors detected the expression of CD47 mRNA by qRT-PCR and analyzed the expression of CD47 mRNA by cell immunofluorescence ( Figure 5 A) Flow cytometry ( Figure 5 B) and protein immunoblotting (Western blot, WB) ( Figure 5C) Detect the expression of CD47 protein. Flow cytometric analysis showed that the expression of CD47 protein in the transfected cell membrane was 3.5 times that in the normal 4T1 cell membrane. It is known that CD47 is present in large quantities on the red blood cell membrane, so the inventors used red blood cell membrane as a positive control and coated it on the surface of the nanoparticles, which are called RBCM-CuS NPs. The inventors analyzed the protein band pattern of each nanoparticle by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 5 The protein bands of CCM-CuS NPs and CD47@CCM-CuS were almost the same, but the protein bands of RBCM-CuS NPs were completely different. The inventors analyzed the density of CD47 on the surface of each nanoparticle by flow cytometry ( Figure 5 E), the results showed that the content of CD47 molecules on the surface of CD47@CCM-CuS NPs was 3.3 times that of CCM-CuS NPs ( Figure 5 F), which is consistent with the results of flow cytometry ( Figure 5 B). In addition, the inventors also found that the CD47 concentration on CD47@CCM-CuS NPs was even higher than that on RBCM-CuS NPs ( Figure 5 F). These results indicate that the inventors have successfully prepared nanoparticles coated with CD47-overexpressing cancer cell membranes.

[0079] Then, the inventors studied the phagocytosis of nanoparticles by RAW264.5 macrophages. The amount of CD47@CCM-CuS NPs (red signal) phagocytosed by RAW264.7 cells was significantly lower than that of CCM-CuS NPs ( Figure 5 G). However, when CD47 on the nanoparticle surface was blocked by antibodies, the phagocytosis of CD47@CCM-CuS NPs by cells increased significantly, indicating that the content and function of CD47 on the nanoparticle surface affect the phagocytosis of macrophages. Flow cytometry showed that the phagocytosis rate after 4 hours of incubation ( Figure 5 H) and confocal images ( Figure 5 G). There was no difference between the phagocytic ratios of CCM-CuS NPs and CD47@CCM-CuS NPs by 4T1 cells, indicating that CD47 overexpression does not affect the targeting of nanoparticles to 4T1 cells. The phagocytic ratio of CD47@CCM-CuS by 4T1 cells increased with the extension of incubation time, reaching 93.4% after 4h, demonstrating the good tumor targeting ability of CD47@CCM-CuS NPs. To further verify the ability of nanoparticles to evade macrophages and target tumor cells, the inventors measured the copper content in 4T1 cells and RAW264.7 cells by ICP-OES ( Figure 5I). The results showed that CCM coating of the nanoparticles significantly increased the tumor targeting ability of the nanoparticles, which was not affected by CD47 overexpression. In addition, CCM coating significantly reduced macrophage phagocytosis, which was further reduced by CD47 overexpression.

[0080] Experimental Example 2: In vitro photothermal and enzymatic catalytic activity of CD47@CCM-Lap-CuS NPs

[0081] The specific process of the in vitro photothermal experiment of CD47@CCM-Lap-CuS NPs was as follows: different concentrations of CD47@CCM-Lap-CuS NPs (100, 50, 25, and 12.5 μg / mL), CuS NPs (100 μg / mL), and PBS were placed in a 96-well plate and irradiated with a 1064-nm laser (1.0 W / cm 2 , 5 min). The plate was thermally imaged using an infrared camera (Fotric 226, Shanghai, China), and the real-time temperature was recorded. Five heating and cooling cycles were performed with 50 μg / mL CD47@CCM-Lap-CuS NPs to evaluate their photothermal stability.

[0082] The in vitro catalytic activity of CD47@CCM-Lap-CuS NPs was demonstrated as follows: The enzymatic activity of CD47@CCM-Lap-CuS NPs was verified using 3,3',5,5'-tetramethylbenzidine (TMB) as both a substrate and a chromogen. When exposed to oxidants such as hydroxyl radicals (OH·), TMB is oxidized to form a blue oxTMB product. Leveraging this characteristic, TMB was used to test whether CD47@CCM-Lap-CuS NPs could catalyze the conversion of H2O2 to OH·. Different groups of nanoparticles (CD47@CCM-Lap-CuS NPs+H2O2, CuS NPs+H2O2, CuS NPs, H2O2 and deionized water as control group) were incubated with TMB at 37°C for 30 min; or different concentrations of CD47@CCM-Lap-CuS NPs (50, 25, 12.5, 6.25, 3.13 and 1.56 μg / mL) were incubated with H2O2 (5 mM) and TMB (1 mM) for 3 min; or CD47@CCM-Lap-CuS NPs (1.56 μg / mL) were incubated with different concentrations of H2O2 (10, 7.5, 5, 2.5 and 1 mM) and TMB (1 mM) for 30 min; or CD47@CCM-Lap-CuS NPs (1.56 μg / mL) were incubated with H2O2 (10 mM) and different concentrations of TMB (0.2, 0.1, 0.08, 0.05, and 0.01 mM) for 30 minutes. Finally, the cells were centrifuged at 11,000 rpm for 10 minutes, and the supernatant was analyzed by UV / Vis spectrophotometry.

[0083] The experimental results can be found in Figure 6 , where A is the concentration of CD47@CCM-Lap-CuS NPs (100 μg / ml), CuS NPs (100 μg / ml) and PBS after 1064 nm laser irradiation (1 W / cm 2 ) of the heating curve; B is the heating curve of CD47@CCM-Lap-CuSNPs with different concentrations after 1064nm laser irradiation (1W / cm 2 ) temperature rise curve; C is the heating curve of CD47@CCM-Lap-CuS NPs (50 μg / ml) under heating (1 W / cm 2) and cooling under 5 cycles of heating and cooling; D is the UV-visible absorption spectra of TMB in different reaction systems: CD47@CCM-Lap-CuS+H2O2, CuS+H2O2, CuS only, H2O2 only and deionized water; E is the UV-visible absorption spectrum of TMB catalyzed by CD47@CCM-Lap-CuS NPs (1.56 μg / ml) in the presence of different concentrations of H2O2 (reaction time: 30 minutes); F is the UV-visible absorption spectrum of TMB catalyzed by CD47@CCM-Lap-CuS NPs at different concentrations in the presence of H2O2 (5 mM) (reaction time: 3 minutes).

[0084] In this study, CuS NPs were used not only as drug carriers but also as photothermal converters and nanozymes. This section mainly demonstrates the in vitro photothermal effect and catalytic activity of CD47@CCM-Lap-CuS NPs. Figure 6 A shows that the temperature rise curves of CD47@CCM-Lap-CuS NPs and CuS NPs are similar, indicating that neither the membrane coating nor the loading of Lap affects the photothermal effect of CuS NPs. The heating curve of CD47@CCM-Lap-CuS NPs shows concentration and time dependence ( Figure 6 B, from top to bottom are 100, 50, 25, 12.5 μg / mL, PBS). The higher the concentration, the faster the temperature rises. For example, after 5 minutes of 1064 nm laser irradiation, the temperature of the CD47@CCM-Lap-CuS NPs (100 μg / ml) aqueous dispersion increased sharply from 25°C to 76.2°C. After 5 heating and cooling cycles, the photothermal efficiency of CD47@CCM-Lap-CuS NPs did not decrease, indicating that the nanoparticles have good photothermal stability ( Figure 6 C, laser on / off cycle from left to right). 3,3',5,5'-Tetramethylbenzidine (TMB) was used to evaluate the catalytic activity of CD47@CCM-Lap-CuS NPs. In the presence of H2O2, CuSNPs effectively catalyzed the conversion of colorless TMB substrate to blue oxidized TMB (oxTMB), exhibiting a characteristic absorption peak at 652 nm ( Figure 6D, only CD47@CCM-Lap-CuS+H2O2 and CuS+H2O2 groups showed absorption peaks). CD47@CCM-Lap-CuSNPs showed almost the same level of enzymatic activity as CuS NPs, indicating that neither membrane coating nor drug loading affected the enzymatic reaction. However, when only CuS NPs or only H2O2 were present, TMB could not be converted into oxTMB, indicating that H2O2 was required for the catalytic reaction of CuS NPs. The inventors also found that the catalytic effect was H2O2 and CD47@CCM-Lap-CuS NP concentration dependent ( Figure 6 E and F; in E, from top to bottom, they are 10, 7.5, 5.0, 2.5, 1.0 mM; in F, from top to bottom, they are 50, 25, 12.5, 6.25, 3.13, and 1.56 μg / mL). In addition, even at a concentration as low as 1.56 μg / ml, CD47@CCM-Lap-CuS NPs exhibited a significant catalytic effect, indicating that CD47@CCM-Lap-CuS NPs are a highly efficient nanozyme ( Figure 6 E).

[0085] Experimental Example 3: Study on the Effects of Photothermal and Chemodynamic Therapy on 4T1 Cells

[0086] The specific process of in vitro cytotoxicity experiment is as follows: LO2 and 4T1 cells were cultured at 1×10 4 Cells were seeded in 96-well plates and grown overnight. The cells were then treated with various concentrations of CD47@CCM-Lap-CuS NPs, CCM-Lap-CuS NPs, CCM-CuS NPs, and CuS NPs for 24 hours. Cell viability was calculated by adding 10 μL of CCK-8 solution and incubating at 37°C for 30 minutes. The absorbance at 450 nm was measured using a microplate reader and compared with the control group.

[0087] The specific process of in vitro photothermal and chemodynamic therapy is as follows: 4T1 cells (10 cells per well) 4 Cells were cultured overnight in 96-well plates. Then, different concentrations of nanoparticles (CD47@CCM-Lap-CuS NPs, CCM-Lap-CuS NPs, CCM-CuS NPs, and CuS NPs) were incubated with the cells for 4 hours. After washing with PBS three times, the cells were illuminated with or without a 1064 nm laser (1 W / cm 2 4T1 cells were irradiated for 5 minutes and then incubated in complete medium for 24 hours. Cell viability was assessed using CCK-8.

[0088] The live / dead cell staining experiment was performed as follows: 4T1 cells were plated at 2×10 5Cells were seeded in a 35-mm diameter confocal culture dish and cultured overnight. The cells were then treated with different nanoparticles for 4 hours, washed three times with PBS, and illuminated with a 1064-nm laser (1 W / cm 2 ) for 5 min. The cells were then cultured for 24 h and then incubated in a normal incubator with 2 × 10 -6 M calcein-AM and 4×10 -6 MPI was treated for 30 min and then washed again with PBS and observed by confocal microscopy.

[0089] The experimental process of cell reactive oxygen species detection was as follows: DCFH-DA probe sensitive to reactive oxygen species was used to evaluate the reactive oxygen species produced by CD47@CCM-Lap-CuS NPs in 4T1 cells. 6 4T1 cells were cultured in 35-mm glass dishes and grown to 70-80% confluence. Different nanoparticles (CuS NPs, CCM-CuS NPs, CCM-Lap-CuS NPs, and CD47@CCM-Lap-CuS NPs, all at 50 μg / mL) were then added along with PBS and Lap (5 μM) and incubated for 24 hours. After washing the cells three times with PBS, they were stained with 10 mM DCFH-DA for 20 minutes, washed again, and imaged and evaluated by confocal microscopy and flow cytometry.

[0090] The cytotoxicity and therapeutic effects of various nanoparticles were evaluated by CCK-8 assay. Figure 7 As shown in A, CuS NPs are non-toxic to normal liver cells LO2. However, even at very low concentrations, they can inhibit the proliferation of tumor cells 4T1. For example, after incubation with 1.56 μg / ml of CuS NPs for 24 hours, the proliferation rate of 4T1 cells was about 91% ( Figure 7 A). The inventors speculate that the reason for this difference is that there is more H2O2 in cancer cells than in normal cells. Under the enzymatic reaction of CuS NPs, this will lead to the production of more cytotoxic OH· in cancer cells. Compared with CuS NPs, CCM-CuS NPs showed greater cytotoxicity to 4T1 cells ( Figure 7 B), which may be due to the stronger targeting of CCM-CuS NPs to 4T1 cells. CD47@CCM-Lap-CuS NPs and CCM-Lap-CuS NPs showed similar cytotoxicity to 4T1 cells and were more toxic than CCM-CuS NPs. Only 8.2% of the 4T1 cell proliferation rate was found in the presence of 100 μg / ml CD47@CCM-Lap-CuS NPs ( Figure 7B), which means that Lap in the nanoparticles has a significant contribution to the cytotoxic effect. Before conducting the cell photothermal experiment, the inventors explored different laser irradiation intensities and times to determine the optimal conditions for laser irradiation of 4T1 cells. The inventors found that "1.5W / cm 2 10min” is safe for 4T1 cells, while “2W / cm 2 5min" resulted in a proliferation rate of about 50%. Therefore, the inventors chose "1W / cm 2 5min" for cell experiments, select "1.5W / cm 2 The inventors incubated 4T1 cells with nanoparticles at different concentrations and irradiated them with near-infrared laser (1 W / cm 2 5min). Figure 7 C shows that cytotoxicity increased with increasing nanoparticle concentration and laser irradiation, and combined laser irradiation caused higher cytotoxicity compared with the nanoparticles alone. CD47@CCM-Lap-CuS NPs and CCM-Lap-CuS NPs combined with laser irradiation had the best therapeutic effect. At a concentration of 100 μg / ml, almost all 4T1 cells were killed ( Figure 7 C). Specific staining of live and dead cells further confirmed these findings ( Figure 7 D). Green fluorescence, indicating live cells, was mainly observed in the control group, laser-only, CuS NP, and CCM-CuS NP groups, whereas red fluorescence, indicating dead cells, was mainly observed in the CCM-Lap-CuS NP+laser group and CD47@CCM-Lap-CuS NPs+laser group ( Figure 7 D). CD47@CCM-Lap-CuS NPs can mediate chemodynamic therapy of tumor cells because Lap can produce H2O2 under the action of NQO1 overexpressed in tumor cells, which is then catalyzed by CuS NPs to form highly cytotoxic reactive oxygen species. 2',7'-dichlorofluorescein diacetate (DCFH-DA) can produce green fluorescence after being oxidized by reactive oxygen species. The inventors used the DCFH-DA probe to detect reactive oxygen species in 4T1 cells. Figure 7 As shown in Figure E, the green fluorescence was the least in the PBS-treated group. A small amount of fluorescence was observed in the CuS NP and CCM-CuS NP groups, which may be because the CuS NPs catalyzed H2O2 in the tumor cells to generate more active OH·. Although Lap alone can induce tumor cells to generate a certain degree of reactive oxygen species, CCM-Lap-CuS NPs and CD47@CCM-Lap-CuS NPs produced the most reactive oxygen species ( Figure 7 E). In addition, the above results were further verified by flow cytometry ( Figure 7 F).

[0091] Experimental Example 4:

[0092] The animal and tumor model establishment process is as follows: BALB / c mice (female, 6 to 8 weeks old) were purchased from Enswell Biotechnology (Chongqing, China). All protocols used in the study were approved by the ethics committee. 100 μL containing 2 × 10 6 A PBS solution of 4T1 cells was subcutaneously injected into the right dorsal region of mice to establish a xenograft model. 3 Relevant processing is performed when the size is large.

[0093] In vivo and ex vivo fluorescence imaging procedures were performed as follows: 200 μL of DiR-labeled CCM-Lap-CuS NPs and CD47@CCM-Lap-CuS NPs (2.5 mg / mL) were injected into the tail vein of tumor-bearing mice (n=3). Fluorescence (FL) images were recorded 2, 4, 8, 24, and 48 hours after injection. Mice were sacrificed 48 hours later, and tumor tissue and major organs were collected for further ex vivo fluorescence imaging.

[0094] In vivo pharmacokinetic and biodistribution studies were conducted as follows: To investigate the in vivo pharmacokinetic characteristics of CD47@CCM-Lap-CuS NPs, normal BALB / c mice were injected intravenously with 200 μL of PBS solution containing 500 μg of CD47@CCM-Lap-CuS NPs or an equivalent amount of CCM-Lap-CuS NPs or CuS NPs. Blood samples (30-50 μL) were collected from the orbital venous plexus at 1 minute, 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after injection. Copper concentrations in the blood samples were measured by ICP-OES. To assess the biodistribution of the nanoparticles, a tumor-bearing mouse model was established. After 8 days of tumor growth, the mice received an intravenous injection of 200 μL of PBS containing 500 μg of CD47@CCM-Lap-CuS NPs or an equivalent amount of CCM-Lap-CuS NPs or CuS NPs. The mice were killed 24 hours later, and the tumors and major organs, including the heart, liver, spleen, lungs, and kidneys, were removed and analyzed for copper content using ICP-OES.

[0095] The photoacoustic imaging research process is as follows:

[0096] In vitro photoacoustic imaging: Different concentrations of CD47@CCM-Lap-CuS NPs (12.5, 50, 100, 250, 500 μg mL -1) aqueous solution 100uL, added it to the agarose gel mold, and then the image was recorded by photoacoustic imaging. In vivo photoacoustic imaging, when the tumor volume of the tumor-bearing mice reached 100 to 160mm 3 In vivo imaging was performed by injecting 200 μL of 2.5 mg / mL LCD47@CCM-Lap-CuS NPs into the tail vein of mice. Photoacoustic signals in the tumor area were measured at specific time points (2 h, 4 h, 8 h, 24 h, and 48 h) after injection.

[0097] In vivo photothermal experiment: CD47@CCM-Lap-CuS NPs (2.5 mg / mL, 200 μL) and an equal amount of CD47@CCM-CuS, CCM-CuS NPs or saline were injected into tumor-bearing mice via the tail vein. After 24 hours, the tumor was photothermally stimulated by 1064-nm laser (1.5 W / cm 2 , 10 min) to irradiate the tumor, and an infrared thermal imaging recorder was used to record the temperature and infrared thermal images.

[0098] The experimental results are as follows: Figure 8 In vivo and ex vivo fluorescence imaging of tumor-bearing mice at different time points after injection of DiR-labeled CCM-Lap-CuS NPs or CD47@CCM-Lap-CuS NPs; Figure 9 A and B are the pharmacokinetics and biodistribution curves of CuS NPs, CCM-Lap NPs, and CD47@CCM-Lap-CuS NPs, evaluated by measuring the copper content; Figure 10 Photoacoustic imaging of tumor tissue at different time points after injection of CuS NPs, CCM-Lap-CuS NPs, and CD47@CCM-Lap-CuS NPs; Figure 11 After 24 h of injection of normal saline, CCM-CuS NPs, CD47@CCM-CuS NPs, and CD47@CCM-Lap-CuS NPs into tumor-bearing mice, 1064 nm laser irradiation (1.5 W / cm 2 ,10min) after the mouse infrared thermal imaging image and the temperature rise curve of the tumor area.

[0099] The in vivo biodistribution of CD47@CCM-Lap-CuS NPs in breast cancer tumor-bearing mice was studied. Figure 8As shown, after intravenous injection of DiR-labeled CD47@CCM-Lap-CuS NPs, they accumulated rapidly in the tumor area, reaching a maximum value 24 hours after injection. In contrast, most CCM-Lap-CuS NPs stagnated in the liver and spleen, and the content in the tumor was low. Fluorescence imaging of ex vivo samples showed that CD47@CCM-Lap-CuS NPs accumulated less in the liver and spleen than CCM-Lap-CuS NPs, indicating that CD47 has a significant contribution to the nanoparticles' escape from MPS in vivo. The inventors evaluated the pharmacokinetics of the nanoparticles by measuring the copper content in the blood at different time points. Compared with CCM-coated nanoparticles with a longer half-life, CuS NPs were rapidly cleared from the blood ( Figure 9 A). CD47@CCM-Lap-CuS NPs have the longest blood half-life, indicating that the increased CD47 on the surface of the nanoparticles leads to a longer blood retention time. In addition, the inventors measured the copper content in the main organs and tumor tissues of mice to clarify the tissue distribution of the nanoparticles ( Figure 9 B). The results showed that CD47@CCM-Lap-CuS NPs accumulated less in the liver than CuS NPs and CCM-Lap-CuS NPs, while accumulating most at the tumor site ( Figure 9 B), which is consistent with the fluorescence imaging results ( Figure 9 A). Since CD47@CCM-Lap-CuS NPs exhibited excellent NIRI / II absorption and photothermal conversion, the inventors subsequently studied their in vitro and in vivo photoacoustic imaging (PA) imaging performance. In vitro experiments showed that the PA signal intensity increased with increasing CD47@CCM-Lap-CuS NP concentration, indicating the potential of CD47@CCM-Lap-CuS NPs as an in vivo PA imaging contrast agent. After CD47@CCM-Lap-CuS NPs were injected into the mouse tail vein, the photoacoustic signal (red) in the mouse tumor area gradually increased, and the maximum photoacoustic signal ( Figure 10 However, CuS NPs and CCM-Lap-CuS NPs showed very weak photoacoustic signals. In addition, the inventors also evaluated the real-time temperature changes of the tumor during laser irradiation by infrared thermal imaging ( Figure 11 Left). After intravenous administration of CD47@CCM-Lap-CuS NPs and CD47@CCM-CuS NPs, the temperature of the tumor area of ​​mice increased rapidly from 33.2°C to 55.2°C within 600 seconds, while under the same irradiation conditions, the temperature change of the saline group was only 7.8°C ( Figure 11Right). Although coated with CCM, the maximum temperature of the tumor site after administration of CCM-CuS NPs could only reach 50°C ( Figure 11 These results again demonstrate that increasing the number of CD47 molecules on the nanoparticle surface can prolong the nanoparticle's blood retention time and enhance its accumulation in tumor tissue.

[0100] Experimental Example 5: In vivo anti-tumor therapy study

[0101] The tumor-bearing mouse model was established as described above. 3 At the same time, 45 mice were randomly assigned to 9 groups (n = 5 per group): saline; laser alone; Lap; CCM-CuS NP; CCM-Lap-CuS NP; CD47@CCM-Lap-CuS NP; CCM-CuS NP + laser; CD47@CCM-CuS NP + laser; CD7@CCM-Lap-CuS NP + laser. Nanoparticles (2.5 mg / mL, 200 μL) were injected only once on day 0. 24 h after injection, 1064 nm laser (1.5 W / cm 2 ) irradiated the tumor for 10 minutes. Tumor size, body weight, and images of the mice were recorded every other day. Mice were sacrificed on day 22, and tumors and organs were harvested for pathological analysis.

[0102] The experimental results can be found in Figure 12 , where A is the tumor growth curve; B is the tumor weight on day 22; C is the weight change curve of tumor-bearing mice; D is the representative images of mice in different groups at different times; E is the representative images of in vitro tumors in different groups; F is the H&E, TUNEL, and Ki-67 staining images of tumor tissues in different groups (scale bar: 200 μm).

[0103] To explore the therapeutic effect of CD7@CCM-Lap-CuS NPs in vivo, breast cancer tumor-bearing mice were randomly divided into 9 groups: ① saline control group; ② laser group; ③ Lap group; ④ CCM-CuS NP group; ⑤ CCM-Lap-CuS NP group; ⑥ CD47@CCM-Lap-CuS NP group; ⑦ CCM-CuS NP+laser group; ⑧ CD47@CCM-CuS NP+laser group; ⑨ CD47@CCM-Lap-CuS NP+laser group. The mice were examined every other day and their body weight and tumor volume were recorded. The experimental results showed that the tumor volume of mice in the laser, Lap and CCM-CuS NP groups continued to increase, which was consistent with the relative tumor volume growth curve in the control group ( Figure 12A, B, D and E), indicating that these experimental conditions mentioned above had no effect on inhibiting tumor growth. However, the tumor volume of mice in the CCM-Lap-CuS NP and CD47@CCM-Lap-CuS NP groups grew relatively slowly on day 22. Compared with the CCM-Lap-CuS NPs group, the tumors in the CD47@CCM-Lap-CuS NPs group were smaller, which may be due to the increase in CD47 molecules on the surface of the nanoparticles, resulting in reduced phagocytosis, and more nanoparticles entering the tumor tissue to exert a chemodynamic therapeutic effect. Among the 3 groups of nanoparticles + laser irradiation groups, laser irradiation resulted in almost complete eradication of the tumor. Unfortunately, however, tumor recurrence was observed in both the CCM-CuS NP + laser and CD47@CCM-CuS NP + laser groups on days 6 and 14 after treatment, respectively. There was no tumor recurrence in the CD47@CCM-Lap-CuSNPs + laser group during the 22-day observation period ( Figure 12 A, B, D, and E), indicating that CD47@CCM-Lap-CuS NPs mediated and enhanced the effect of combined photothermal and chemodynamic therapy through the "don't eat me" signal. In addition, the body weight of mice in all groups did not change significantly during the treatment period, indirectly indicating that CD47@CCM-Lap-CuS NPs have good biosafety ( Figure 12 C). To further evaluate the biosafety of the nanoparticles, the main organs of the mice were taken for H&E staining 1, 3, 7, 14 and 28 days after intravenous injection of normal saline and CD47@CCM-Lap-CuS NPs. The results showed that no histopathological abnormalities were found. In addition, routine blood tests and biochemical indicators were also normal. After the mouse tumors were treated, the inventors used H&E staining to detect changes in tumor tissue. The results showed that the number of blue-stained nuclei in the CD47@CCM-Lap-CuS NPs+laser group was significantly reduced ( Figure 12 F), indicating that the tumor tissue was significantly damaged. In addition, the inventors evaluated tumor cell proliferation and apoptosis by anti-Ki67 and TUNEL immunofluorescence, and the experimental results showed that the tumor cell proliferation in the CD47@CCM-Lap-CuS NPs+laser group was significantly reduced ( Figure 12 F), while the levels of apoptosis and necrosis were significantly increased ( Figure 12 F) H&E staining revealed no significant tissue damage to the major organs of mice in each group after treatment, demonstrating the good biosafety of this nanoparticle-mediated photothermal combined with chemodynamic therapy.

[0104] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects, characterized by: The nanostructured nanoparticles are composed of a core made of mesoporous copper sulfide nanoparticles and a shell made of a cancer cell membrane integrated with CD47. The cancer cell membrane integrated with CD47 is prepared by the following method: constructing cancer cells that overexpress CD47, and then extracting the cell membrane of the cancer cells.

2. The CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects according to claim 1, characterized in that: The mesopores of the copper sulfide nanoparticles are loaded with β-lapachone.

3. The CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects according to claim 1, characterized in that: The copper sulfide nanoparticles are prepared by the following method: polyvinyl pyrrolidone K30 and a copper chloride solution are added to water, and after stirring, a sodium hydroxide solution is added, and then hydrazine hydrate and a sodium sulfide solution are added in sequence to obtain a reaction system; the reaction system is stirred and reacted in contact with air to obtain copper sulfide nanoparticles.

4. The CD47 signal-mediated targeted nanoparticle with both photothermal and chemodynamic therapeutic effects according to claim 3, characterized in that: The cancer cells are breast cancer cells.

5. The method for preparing a CD47 signal-mediated targeted nanoparticle having both photothermal and chemodynamic therapeutic effects according to any one of claims 1 to 4, characterized in that: The method includes the following steps: S1: Preparation of copper sulfide nanoparticles: S2: Preparation of cancer cell membranes with integrated CD47; S3: Preparation of CD47@CCM-Lap-CuS nanoparticles: An ethanol solution of β-lapachone and an aqueous suspension of copper sulfide nanoparticles were mixed and stirred in the dark. The ethanol was then evaporated to obtain a mixture of β-lapachone and copper sulfide nanoparticles. Next, an ultrasonically treated suspension of CD47-integrated cancer cell membranes was added, followed by further ultrasonic treatment. Subsequently, CD47@CCM-Lap-CuS nanoparticles were obtained by co-extrusion through a liposome extruder.

6. The method for preparing a CD47 signal-mediated targeted nanoparticle having both photothermal and chemodynamic therapeutic effects according to claim 5, characterized in that: In S3, the amount of the ethanol solution of β-lapachone is 2 mL, and the concentration of β-lapachone is 0.5 mg / mL; the amount of the aqueous suspension containing copper sulfide nanoparticles is 2 mL, and the concentration of copper sulfide nanoparticles is 1 mg / mL.

7. The method for preparing a CD47 signal-mediated targeted nanoparticle having both photothermal and chemodynamic therapeutic effects according to claim 6, characterized in that: In S3, the ethanol solution of β-lapachone and the aqueous suspension containing copper sulfide nanoparticles were mixed, stirred in a dark environment for 24 hours, and then continued to stir at 60°C until the ethanol was completely evaporated.

8. The method for preparing a CD47 signal-mediated targeted nanoparticle having both photothermal and chemodynamic therapeutic effects according to claim 7, characterized in that: In S3, the CD47-integrated cancer cell membrane suspension was first sonicated for 30 seconds, then added to a mixture of β-lapachone and copper sulfide nanoparticles, and then sonicated for another 30 seconds; the amount of the CD47-integrated cancer cell membrane suspension was 1 mL, containing 3 mg of CD47-integrated cancer cell membranes; the liposome extruder co-extrusion parameters were set to: pass through 0.8 μm and 0.4 μm membrane filters in sequence.

9. Use of a CD47 signal-mediated targeted nanoparticle having both photothermal and chemodynamic therapeutic effects according to any one of claims 1 to 4 in the preparation of a drug for treating breast cancer.

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