Nano-probe, preparation method thereof and application of nano-probe in preparation of medicines for treating cancers

Abstract

The invention provides a nano-probe, a preparation method thereof and application of the nano-probe in preparation of drugs for treating cancers. The invention provides the nano probe. The nano probe comprises 131I, a cancer cell membrane, a base trapping agent and serum albumin modified copper sulfide nano particles. The nano probe provided by the invention realizes more efficient treatment on refractory thyroid cancer, the anti-tumor effect is remarkably improved, experiments possibly prove that the killing rate of cancer cells is improved compared with a single treatment mode, damage of radioactive substances to non-cancer tissues is remarkably reduced through CCM wrapping, and the application prospect is wide. Meanwhile, the local heat effect of CuS under near-infrared light irradiation also reduces damage to surrounding normal tissues, and the probe can be used for treatment and also can be used for tumor imaging, diagnosis and treatment are combined, and diagnosis and treatment integration is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical drugs, and particularly relates to a nanoprobe, a preparation method thereof, and an application thereof in the preparation of cancer therapeutic drugs. Background Art

[0002] Thyroid cancer is the most common endocrine malignancy, which is divided into differentiated thyroid cancer, undifferentiated thyroid cancer and medullary carcinoma. The vast majority of them are differentiated thyroid cancer (DTC), accounting for more than 80% of thyroid cancer, while undifferentiated thyroid carcinoma (ATC) is less common, only accounting for 1.0% - 7.5% of the incidence of thyroid cancer. The ATC tissue makes radioactive iodine therapy (RIT) ineffective due to the loss of iodine uptake ability, and at the same time, due to the strong local lesion invasiveness and high metastasis rate, the effects of conventional treatment methods such as surgery, external radiotherapy and chemotherapy are not good, and the 5-year survival rate of patients is less than 10%. As for more effective intervention measures (such as targeted drug therapy), side effects often occur due to poor selectivity. Therefore, there is currently no treatment method for curing ATC patients. Therefore, there is an urgent need to develop new treatment strategies for ATC.

[0003] Radionuclides are currently widely used in cancer treatment. Radioactive 131 I can emit β and γ rays, and the β rays are used to kill tumor cells. However, the key to the successful application of radioactive 131 I in tumor treatment is that 131 I must be taken up by the tumor site and retained for a certain period of time. Therefore, how to enable radioactive 131 I to accumulate in tumor cells while reducing its radiation damage to normal tissues has always been an urgent problem for scientific researchers to solve. It has been found that the cancer cells of ATC patients have extremely poor ability to absorb and utilize iodine, resulting in the inability to use 131 I for treatment under normal circumstances. However, the targeted therapy technology brings hope for 131 I treatment of ATC. But how to effectively target and transport 131 I into tumor cells for the treatment of ATC still needs to be studied.

[0004] Photothermal ablation therapy (PTA) is also a tumor treatment method based on nanomedicine. It utilizes the absorption of light energy by special photothermal conversion agents and converts the absorbed light energy into heat, raising the local temperature of the tumor, thereby inducing apoptosis and necrosis of tumor cells. The newly reported novel photothermal conversion agent - copper sulfide nanoparticles (CuS), with simple preparation process, low cost, excellent photothermal stability and good biocompatibility, has become a research hotspot in the field of nanomedicine today. The research on photothermal nanomaterials has always been a hotspot in the field of materials science. In recent years, CuS has been widely used in the targeted treatment of cancer and achieved definite anti-tumor efficacy. However, the inventors' previous research confirmed that direct intratumoral injection of 131 I-BSA@CuS can achieve the combined therapy of the photothermal effect of CuS and 131 I, but its targeting and therapeutic efficacy via tail vein injection are poor.

[0005] During normal human life activities, DNA, as the genetic material, often suffers various damages, and one very important form of damage is base deletion damage. Apurinic / apyrimidinic (AP) sites can form spontaneously under normal physiological conditions in the body, or can be induced by exogenous factors and oxidative damage. Under normal physiological conditions, apurinic sites are more common than apyrimidinic sites. About 10,000 - 20,000 apurinic sites and about 500 apyrimidinic sites are generated in each cell every day. The number of AP sites generated varies according to cell types. In the base excision repair (BER) process of AP sites, apurinic / apyrimidinic endonuclease (APE) plays an important role. APE1 is a very important member of the APE family. Research shows that the excision of AP sites in mammalian cells is mainly achieved under the action of APE1 (accounting for more than 95% of the entire BER pathway). It can specifically cleave the DNA strand at the AP site and act successively with other enzymes, such as DNA repair enzyme (PARP), DNA polymerase β, etc., to achieve the excision repair of base deletion damage. Blocking the APE1 repair of AP sites will lead to cell death, which is currently a research hotspot in cancer treatment. However, in different cells, non-selective AP site capture will produce side effects. Targeted anti-tumor drugs utilize the unique chemical environment of cancer cells to selectively target tumor cells, greatly reducing side effects on normal cells. How to obtain an AP capture agent for ATC tissue is of great significance for the treatment of ATC. Therefore, the limitations of existing technologies for treating anaplastic thyroid cancer are that current radiotherapy methods (such as131 I) There are problems with limited efficacy in undifferentiated thyroid cancer. Especially when the cancer develops resistance to traditional radiotherapy, single radiotherapy cannot meet the treatment needs. At the same time, although existing photothermal therapies (such as CuS) have certain potential in tumors, their treatment efficiency and safety are limited due to the lack of an effective targeted delivery mechanism. Existing nanoprobe technologies are also mostly single-functional and do not fully combine multiple treatment mechanisms. Summary of the Invention

[0006] An object of the present invention is to provide a nanoprobe, a preparation method thereof, and an application thereof in the preparation of cancer therapeutic drugs.

[0007] To achieve the above object, the following technical solutions are adopted in the present invention:

[0008] A nanoprobe, which comprises 131 I. Cancer cell membrane, base capturer, and bovine serum albumin-modified copper sulfide nanoparticles.

[0009] For the nanoprobe as described above, preferably, the cancer cell membrane coats the base capturer and bovine serum albumin-modified copper sulfide nanomaterial, and the 131 I is labeled on the cancer cell membrane.

[0010] For the nanoprobe as described above, preferably, the cancer cell membrane is selected from one or more of lung cancer cell membrane, esophageal cancer cell membrane, gastric cancer cell membrane, duodenal cancer cell membrane, colorectal cancer cell membrane, renal cancer cell membrane, liver cancer cell membrane, pancreatic cancer cell membrane, gallbladder cancer cell membrane, cervical cancer cell membrane, endometrial cancer cell membrane, vulvar cancer cell membrane, ovarian cancer cell membrane, breast cancer cell membrane, thyroid cancer cell membrane, prostate cancer cell membrane, bladder cancer cell membrane, and testicular cancer cell membrane.

[0011] For the nanoprobe as described above, preferably, the cancer cell membrane is ATC cancer cell membrane.

[0012] For the nanoprobe as described above, preferably, the base capturer is selected from 2,4-dinitrobenzenesulfonamide caged coumarin-based alkoxylamine; the bovine serum albumin-modified copper sulfide nanomaterial is bovine serum albumin-modified copper sulfide nanomaterial or human serum albumin-modified copper sulfide nanomaterial.

[0013] For the preparation method of the nanoprobe as described above, it comprises the following steps:

[0014] S1. Mix the bovine serum albumin-modified copper sulfide nanoparticles and the base capturer solution thoroughly under ultrasonic waves to obtain the AP-BSA@CuS nanoprobe;

[0015] S2, fully mixing the cancer cell membrane with the AP-BSA@CuS nanoprobe under ultrasound, and then extruding it with a liposome extruder to coat it on the surface of the AP-BSA@CuS nanoprobe, and then centrifuging the solution to remove excess cell membrane to obtain CCM@AP-BSA@CuS;

[0016] S3. Chloramine T labeling method was used to label CCM@AP-BSA@CuS 131 I labeled; centrifuged and purified by ultrafiltration centrifuge tube 131 I-CCM@AP-BSA@CuS.

[0017] In the preparation method as described above, preferably, the mass ratio of serum albumin-modified copper sulfide nanoparticles to base capture agent and cancer cell membrane protein is 1-2:0.0000625-1:3-5.

[0018] In the preparation method as described above, preferably, in step S1, the ultrasonic time is 5 to 10 minutes, and the base capture agent is dissolved in the DMSO solution; in step S2, the ultrasonic time is 1 to 3 minutes.

[0019] In the preparation method described above, preferably, in step S3, the chloramine T labeling method is to mix the CCM@AP-BSA@CuS solution with Na 131 After the I solution is mixed, chloramine T solution is added, and after the reaction, sodium metabisulfite solution is added to terminate the reaction. The radioactive mixture solution is transferred to an ultrafiltration centrifuge tube and centrifuged, and the radioactivity count of the supernatant is measured. Ultrapure water is added to the supernatant and centrifugation is continued until the radioactivity of the filtrate no longer increases.

[0020] Furthermore, CCM@AP-BSA@CuS solution, Na 131 The concentrations of I solution and chloramine T solution are 2-50 mg / mL, and the added amounts are in a volume ratio of 5:2:3; the reaction time is 2-5 min; the molecular weight cutoff of the ultrafiltration centrifuge tube is 30,000; and the centrifugation conditions are 6,000 r / min centrifugation speed for 30 min.

[0021] The preparation method as described above also includes the step of preparing serum albumin modified copper sulfide nanoparticles: mixing a BSA-containing solution with a Cu(NO3)2 solution to produce a blue mixture, then adding a NaOH solution to turn it purple, then adding a Na2S solution to turn it brick red, and then heating to turn it dark green to obtain serum albumin modified copper sulfide nanoparticles.

[0022] Further, the concentration of the BSA solution is 0.01 - 0.5 g / mL, the concentration of the Cu(NO3)2 solution is 0.1 - 0.5 M, the concentration of the NaOH solution is 0.5 - 2 M, and the concentration of the Na2S solution is 0.05 - 1 M. The volume ratio of the above solutions is 7.5:1:0.5:2; the conditions for heat treatment are 80 - 100 °C for 15 - 60 min.

[0023] Another object of the present invention is to provide a nanosensor and a drug for treating anaplastic thyroid cancer, and the nanosensor is mainly used for treating anaplastic thyroid cancer.

[0024] In one embodiment of the present invention, a nanosensor for treating anaplastic thyroid cancer is further provided, which includes 131 I (radioactive iodine), CCM (cancer cell membrane), AP (base capturer), and BSA-CuS (bovine serum albumin modified copper sulfide nanomaterial).

[0025] Further, the base capturer is 2,4-dinitrobenzenesulfonamide cage coumarin-based alkoxylamine; CCM is ATC cancer cell membrane.

[0026] A drug for treating anaplastic thyroid cancer includes the above-mentioned nanosensor for treating anaplastic thyroid cancer.

[0027] When using radioactive therapy alone 131 The efficacy of I or photothermal therapy with CuS in the treatment of thyroid cancer is limited; radioactive therapy may lead to treatment resistance, and photothermal therapy may be difficult to effectively control the growth of cancer cells alone. However, by combining the two and with the assistance of CCM and AP, the nanosensor can achieve precise targeting and efficiently attack tumors through a triple mechanism (radiotherapy + photothermal therapy + blocking DNA repair).

[0028] The nano-probe for treating undifferentiated thyroid cancer provided by the present invention uses CCM (cancer cell membrane encapsulation), which is isolated and purified from undifferentiated thyroid cancer tissue, has good biocompatibility, and can achieve tumor-targeted delivery by mimicking the natural membrane properties of cells. This not only improves the stability of the probe in vivo, but also reduces the immune response, enabling the radioactive drug to better accumulate in the tumor area; the used AP (base) capturer utilizes the high concentration of glutathione in ATC cells to activate the base (AP) capturer, capture the AP site, and block DNA damage repair. BSA-CuS (bovine serum albumin modified copper sulfide nanomaterial) BSA can increase the water solubility and stability of the probe. As a biological macromolecule, BSA can effectively reduce non-specific adsorption and enhance the targeting effect of the probe by binding to the targeting ligand. CuS is a material with photothermal therapy function, which can generate a thermal effect under near-infrared light irradiation, combine with radiotherapy, block DNA repair, and achieve the synergistic effect of a triple treatment mode. The present invention combines radiotherapy ( 131 I), blocking DNA repair (AP), and photothermal therapy (CuS), and the probe can achieve more efficient treatment for refractory thyroid cancer. In the prior art, these four materials are rarely combined, especially in the treatment of undifferentiated thyroid cancer.

[0029] The beneficial effects of the present invention are as follows:

[0030] When the nano-probe provided by the present invention is used to treat tumors, the treatment efficiency is significantly improved, and the anti-tumor effect is significantly enhanced. The experimental results prove that the novel nano-probe has a higher killing rate of cancer cells than a single treatment method, effectively reduces side effects, and at the same time, the local thermal effect of CuS under near-infrared light irradiation also reduces the damage to surrounding normal tissues; moreover, the probe can also be used for tumor imaging to observe the tumor morphology.

[0031] The novel nano-probe ( 131 I-CCm@AP-BSA@CuS) provided by the present invention for treating undifferentiated thyroid cancer has the following effects: the treatment efficiency is significantly improved, and the anti-tumor effect is significantly enhanced. The experimental results prove that the novel nano-probe has a higher killing rate of cancer cells than a single treatment method (the shrinkage rate of the tumor mass in combination treatment is statistically significant compared with single treatment, P < 0.05 in both cases, see Figure 9 ); side effects are reduced: mainly by the encapsulation of CCM, the damage of radioactive substances to non-cancerous tissues is significantly reduced, and at the same time, the local thermal effect of CuS under near-infrared light irradiation also reduces the damage to surrounding normal tissues; multifunctionality: the probe can not only be used for treatment, but also for tumor imaging, combining diagnosis and treatment to achieve "diagnosis and treatment integration", which is an effect difficult to achieve in the prior art.

[0032] The novel nanoprobe ([ 131 131 I-CCm@APBSA@CuS) provided by the present invention can actively target ATC cells, where the AP capturer is selectively activated by glutathione to capture the AP site and block the activity of APE1, blocking the DNA damage repair of ATC cells. At the same time, 131 the ionization radiation of [[ 131 131 I and near-infrared laser irradiation can jointly promote the action of the AP capturer, thereby leading to cell senescence and ultimately inhibiting the progression of ATC. For patients with undifferentiated thyroid cancer and those who experience dedifferentiation during diagnosis and treatment, resulting in

[0033] poor efficacy of [[ 131 131 I or even disease progression, it will produce good social and economic benefits. 131 The novel actively targeted nanoprobe provided by the present invention has the following characteristics: ① It has an active targeting function: on the one hand, the specific targeting effect of glutathione (GSH): the base (AP) capturer can be targeted and activated by the high concentration of GSH in ATC cells to capture the AP site and block the activity of the endonuclease 1 (APE1), blocking DNA damage repair and causing the death of ATC cells. On the other hand, the homologous aggregation targeting effect: the thyroid cancer cell membrane coated on the surface of the nanoprobe can efficiently recognize thyroid cancer cells. According to the homologous aggregation characteristics and combined with the enhanced permeability and retention effect of the nanoprobe, its active targeting function can be realized, effectively reducing tissue toxicity and side effects. ② Triple treatment improves efficacy: It realizes the purpose of triple treatment of thyroid cancer by photothermal therapy, iodine therapy and blocking DNA damage repair. ③ Multifunctionality and multimodal imaging: Since the novel nanoprobe provided by the present invention carries

[0034] 131 131 I-CCM@APBSA@CuS) for the treatment of undifferentiated thyroid cancer, ① applying the triple combination of photothermal therapy, radionuclide therapy and blocking DNA damage repair to improve efficacy is a bold exploration and practice of the current comprehensive treatment strategy for thyroid cancer. ② Combining the advanced actively targeted nanotechnology with the classic radioactive 131 I therapy expands the new ideas for the treatment of undifferentiated thyroid cancer and is a new application of nanotechnology in the field of radionuclide therapy; at the same time, it explores the possibility of its further application in the radionuclide therapy of other non-thyroid-derived tumors. Brief Description of the Drawings

[0035] Figure 1 For 131Schematic diagram and mechanism diagram of the construction of I-CCm@AP-BSA@CuS.

[0036] Figure 2 From left to right are the TEM images of ATC cell membrane vesicles, AP-BSA@CuS, and CCM@AP-BSA@CuS.

[0037] Figure 3 Hydrodynamic size and Zeta potential of CCM@AP-BSA@-CuS and I-CCM@AP-BSA@CuS.

[0038] Figure 4 a) Heating-time curves of aqueous solutions with the same material concentration of water, BSA, BSA@CuS, and CCM@AP-BSA@CuS irradiated by 1.5 W / cm 2 NIR laser for 10 min; b) Photothermal heating curve images at different light intensities with a material concentration of 2 mg / mL; c) Photothermal heating images at different material concentrations with a laser intensity of 1.5 W / cm 2 for 808 nm laser; d) Real-time temperature change images of the photothermal cycle of CCM@AP-BSA@CuS aqueous solution.

[0039] Figure 5 For 131 SPECT / CT imaging of mice before injection (day 0) and 2 h, 1st, 3rd, and 7th days after injection of I-CCm@AP-BSA@CuS.

[0040] Figure 6 For the apoptosis analysis of CAL-62 cells after treatment with 3 groups of protocols ( 131 I-BSA@CuS, 131 I-CCm@AP-BSA@CuS and the control group) for 24 h.

[0041] Figure 7 For the cell cycle analysis of CAL-62 cells treated with 3 groups of protocols ( 131 I-BSA@CuS, 131 I-CCm@AP-BSA@CuS and the control group).

[0042] Figure 8 For the DNA damage results of CAL-62 cells treated with 3 groups of protocols ( 131 I-BSA@CuS, 131 I-CCm@AP-BSA@CuS and the control group).

[0043] Figure 9 For 131In vivo study of I-CCm@AP-BSA@CuS against ATC, where (a): photos of mice before treatment (day 0) and 3, 6, 12, and 24 days after treatment; (b): statistical analysis of excised tumor weights; *P<0.05. (c): statistical analysis of the body weights of mice in each subgroup.

[0044] Figure 10 To 131 The toxicity of I-CCM@APBSA@CuS to organisms. Detailed implementation manners

[0045] The inventors of the present invention developed a glutathione-responsive AP trapping agent with targeted anti-tumor activity, namely 2,4-dinitrobenzenesulfonamide-caged coumarin-based alkoxylamine. At the same time, the previous research of the inventors has confirmed that the AP trapping agent (selectively releases sulfur dioxide (SO2) and fluorescent coumarin-based alkoxylamine by high-concentration GSH in ATC cells to trap AP sites and block the activity of apurinic / apyrimidinic endonuclease 1 (APE1), block DNA damage repair, and induce the death of ATC cells; in vitro experiments show that the AP trapping agent has a good therapeutic effect on subcutaneous implanted tumors of ATC cells Chai J, Su M, Zhang R, Li N, Jia Y, Zheng W, Tan J, Jia Q, Sun H, Meng Z. Selective anti-tumor activity of glutathione-responsive abasic site trapping agent in anaplastic thyroid carcinoma. BMC Cancer. 2024 Jul 8;24(1):816.). However, there are certain limitations in single treatment, and the treatment effect is not as good as that of combination treatment.

[0046] The inventors found in previous research that the photothermal therapy of CuS nanoprobes is combined with 131 I combination therapy (Zhang C#, Chai J#, Jia Q, Tan J, Meng Z*, Li N*, Yuan M*. Evaluating the therapeutic efficacy of radiolabeled BSA@CuS nanoparticle-induced radio-photothermal therapy against anaplastic thyroid cancer. IUBMB Life, 2022, 74(5):433-445.), which can produce a synergistic anti-tumor effect, and it is confirmed that directly injecting 131 I-BSA@CuS into the ATC tumor can achieve the photothermal effect of CuS and131 I co - therapy, but its tail - vein injection targeting and curative effect are poor. On this basis, the inventor extracted thyroid cancer cell membrane proteins and coated them on the surface of the nano - probe. Utilizing the homologous aggregation of tumor cells, it has active targeting, improves the curative effect while reducing tissue toxicity. Thus, 131 I - CCM@AP - BSA@CuS nano - probe was constructed.

[0047] A novel cancer cell membrane - active - targeting nano - probe constructed by the present invention ( 131 I - CCM@APBSA@CuS) is based on bovine serum albumin (BSA) to construct BSA@CuS nano - probe. After fully mixing it with AP capturer, AP - BSA@CuS is formed. Its surface is further coated with the extracted ATC cancer cell membrane proteins and labeled with 131 I. Among them, by coating the cancer cell membranes (CCM) on the surface of the nano - probe and utilizing the homologous targeting ability of the cancer cell membrane, the diffusion of the nano - probe in the tumor is promoted, thereby improving the treatment effect. The present invention also comprehensively analyzes through a series of in vitro and in vivo studies 131 the feasibility and safety of the triple - drug administration regimen of I - CCM@AP - BSA@CuS nano - probe to achieve photothermal therapy, 131 I therapy combined with blocking DNA damage repair, and verifies its inhibitory effect and mechanism on ATC.

[0048] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, the modifications or replacements made to the present invention all belong to the scope of the present invention.

[0049] Unless otherwise specified, the technical means used in the examples are conventional means well - known to those skilled in the art. Except as otherwise specified, the reagents used in the methods of the present invention are all of analytical purity or above specifications, and commercially available products can be used. The sources of the reagents and instruments used in the examples are shown as follows:

[0050] (1) Main reagents

[0051] Name QS2510101 Source

[0052] DMEM medium VivaCell

[0053] Trypsin 0.25%, EDTA 0.02% VivaCell

[0054] Fetal bovine serum (FBS) Procell

[0055] Sterile PBS buffer Servicebio

[0056] Dimethyl sulfoxide (DMSO) Solarbio

[0057] 1% Penicillin / Streptomycin Antibody Mixture Procell

[0058] MTT Beyotime Biotechnology

[0059] Bax Abcam

[0060] Marker Thermo

[0061] Bovine Serum Albumin (BSA) Dingguo Biotechnology Co., Ltd.

[0062] Copper nitrate (Cu(NO3)2·3H2O) Tianjin Chemical Reagent Supply and Marketing Company Sodium Hydroxide (NaOH) Aladdin Reagent Company Sodium sulfide (Na2S·9H2O) Aladdin Reagent Co., Ltd. (Shanghai, China Na 131 I SolutionBeijing Atom High-Tech Co., Ltd. Chloramine T Tianjin CNS Biochemical Technology Co., Ltd. The cell lines of ATC cells (CAL-62) and normal thyroid cells (Nthy-ori 3-1) were kindly provided by Professor Junichi Yamashita of Nagasaki University, Japan.

[0063] (2) Main instruments

[0064] Origin of the name

[0065] WaterJacketed CO2 Incubator Thermo Forma USA RB-200 Sub-Room Temperature Water Bath GIBCO BRL USA TGL-16M centrifuge Cence Xiangyi Company

[0066] J2-21 low temperature high speed centrifuge Beckman

[0067] Liquid nitrogen tank Aviation Industry Ministry Yuxin Machinery Factory Microbalance Sartorious

[0068] Adjustable pipetting gun Dragon

[0069] Optical Microscope Olympus TH4-200

[0070] Analytical Flow Cytometer BD BD, USA

[0071] FACSVerse

[0072] Laser scanning confocal (Zeiss LSM800), Carl Zeiss AG, Germany

[0073] Vertical electrophoresis apparatus, bio-rad

[0074] Drying heater - Dry bath incubator HGT-2(HA)

[0075] Ice maker, XUEKE

[0076] Dehydrator, DIAPATH

[0077] Embedding machine, Wuhan Junjie Electronics Co., Ltd. Microtome, Leica Microsystems Shanghai Co., Ltd. Freezing table, Wuhan Junjie Electronics Co., Ltd. Tissue spreading machine, Jinhua Cody Instrument & Equipment Co., Ltd., Zhejiang Province Oven, Tianjin Laiborui Instrument & Equipment Co., Ltd. Cryostat microtome, Thermo Fisher Scientific (China) Co., Ltd. Glass slide, Wuhan Sevier Biotechnology Co., Ltd. Coverslip, Jiangsu Shitai Experimental Equipment Co., Ltd. Decolorizing shaker (pendulum type), Wuhan Sevier Biotechnology Co., Ltd. Vortex mixer, Wuhan Sevier Biotechnology Co., Ltd. Pipette, Wuhan Sevier Biotechnology Co., Ltd. Animal gas anesthesia machine, PATTERSON VETERINARY

[0078] (3) Animals: Male BALB / C nude mice were purchased from Spf (Beijing) Biotechnology Co., Ltd.

[0079] Example 1 Construction of stable cancer cell membrane actively targeted 131 I-CCM@AP-BSA@CuS nanoprobe and its characterization

[0080] I. Preparation method of actively targeted nanoprobe coated with thyroid cancer cell membrane protein, the process schematic diagram is as Figure 1 , which includes the following steps:

[0081] (1) Preparation of Bovine Serum Albumin Modified Copper Sulfide Nanoparticles (BSA@CuS): Using bovine serum albumin (BSA) as a template, BSA@CuS nanoparticles were synthesized by the biomineralization method. The specific preparation method is as follows: Dissolve 250 mg of BSA in 7.5 mL of ultrapure water. Under magnetic stirring, add 1 mL of 0.2 M Cu(NO3)2 solution to the above solution to produce a blue mixture. After quickly adding 0.5 mL of 1 M NaOH solution, the mixture turns purple. Then add 2 mL of 0.2 M Na2S solution, and the color of the solution immediately turns brick red. The solution was further treated at 90 °C for 0.5 h, and then the color turned dark green, indicating the formation of BSA@CuS nanoparticles. The obtained BSA@CuS nanoparticles were purified by dialysis (MW 8KDa) for 24 hours, then freeze-dried, and stored at 4 °C for later use.

[0082] (2) Preparation of CCM@AP-BSA@CuS:

[0083] Cancer cell membranes were obtained through separation and purification. The specific method is as follows: A large number of ATC cells (kindly provided by Professor Junichi Yamashita of Nagasaki University, Japan) were cultured. The cells were digested with ice-cold 0.1% EDTA-PBS, and the collected cells were resuspended and collected in 1.5 mL tubes, washed twice with PBS, centrifuged at 700 g at 4 °C for 5 min, and the precipitate was resuspended in ice-cold PBS (pH = 7.4). Then, it was centrifuged again at 700 g at 4 °C for 5 min. Subsequently, the supernatant was discarded, and the cell precipitate was suspended in 1 mL of hypotonic lysis buffer [(20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, and 1% phenylmethylsulfonyl fluoride (PMSF), 10 μL), also containing a membrane protein extraction reagent (commercially available membrane protein extraction kit) (990 μL)], and lysed in an ice bath for 10 - 15 min. It was snap-frozen at -80 °C for 20 min and quickly thawed at 37 °C for 20 min, repeated 3 - 5 times. The cell suspension was treated with a cell disruptor in an ice-water bath: ultrasonicated for 5 s each time, with a 5 s interval and an amplitude of 30%, for a total of 3 min. Centrifuged at 700 g at 4 °C for 10 min, the supernatant was collected, and then centrifuged at 14000 g for 30 min. The supernatant was discarded, and the precipitate was the obtained cell membrane fragments, namely cancer cell membrane proteins. Furthermore, the extracted cancer cell membrane proteins were measured for protein content by protein quantification (BCA method) (purchased from Wuhan Boster Biological Engineering Co., Ltd., China) and then identified by SDS-PAGE gel electrophoresis.

[0084] First, mix the BSA@CuS nanoprobe (1 mg) with 100 μL of the AP capturer (2,4-dinitrobenzenesulfonamide cage coumarin-based alkoxylamine) solution. (The preparation of the AP capturer solution is as follows: first, dissolve 0.5 mg of the AP capturer in 101.5 μL of DMSO to prepare a 10 mM / L stock solution, and then add physiological saline to dilute it to the required AP drug concentration. Since the AP concentrations used in cell experiments and animal experiments are different, during cell experiments, physiological saline is added according to the volume ratio of the stock solution to physiological saline of 1:100 - 8000, and during animal experiments, physiological saline is added according to the volume ratio of the stock solution to physiological saline of 1:1 - 20.) Sonicate for 10 minutes and mix well to obtain the AP-BSA@CuS nanoprobe. Then, mix the extracted cancer cell membrane protein (2 mg) after identification with the AP-BSA@CuS nanoprobe under sonication for 1 minute, and then extrude it 10 times through a 200 nm polycarbonate porous membrane using a liposome extruder Avanti Mini-Extruder (Beckman Coulter, USA) to coat it on the surface of the AP-BSA@CuS nanoprobe, namely CCM@AP-BSA@CuS, and store it at 4°C.

[0085] Among them, the structural formula of the AP capturer 2,4-dinitrobenzenesulfonamide cage coumarin-based alkoxylamine can be seen in paragraph

[0054] of the patent specification with the publication number CN 113979981 A.

[0086] (3) 131 Preparation, purification and identification of I-CCM@AP-BSA@CuS:

[0087] Use the chloramine-T labeling method to perform 131 I labeling on CCM@AP-BSA@CuS. The specific method is as follows: 1) Weigh the powders of chloramine-T and sodium metabisulfite and dissolve them in PB to prepare a 5 mg / mL chloramine-T solution and a sodium metabisulfite solution respectively. Prepare them freshly each time and filter and sterilize them through a membrane before use. Weigh the lyophilized CCM@AP-BSA@CuS and prepare it into a 5 mg / mL aqueous solution. Take 0.5 mL and add it to a glass vacuum bottle for subsequent reactions. Add a fresh radioactive Na 131 I solution (10 mCi / 200 μL), mix well, then add 300 μL of the chloramine-T solution, and after reacting at room temperature for 2 min, add 400 μL of the sodium metabisulfite solution to terminate the reaction. 2) Transfer the above radioactive mixture solution to an ultrafiltration centrifugal tube (MW 30000) and centrifuge at a speed of 6000 r / min for 30 min. After centrifugation, measure the radioactive count of the supernatant, and add ultrapure water to the supernatant and continue centrifugation until the radioactivity of the filtrate no longer increases. Determine its binding rate by ultraviolet spectrophotometry (the labeling rate is about 76% ± 6%, and the radiochemical purity can reach 97.2 ± 2%).

[0088] II. Characterization of Actively Targeted Nanoprobe Coated with Thyroid Cancer Cell Membrane Protein

[0089] The ATC cell membrane can be successfully extracted by the above method and coated on the surface of the AP-BSA@CuS nanocarrier to synthesize CCM@AP-BSA@CuS, which is labeled 131 with I, the labeling rate is about 70-82%, and the radiochemical purity can reach 95.2-99.2%. A new type of nanoprobe ( 131 I-CCM@AP-BSA@CuS) is successfully constructed. Transmission electron microscopy reveals the morphology of the nanoprobe (see Figure 2 ).

[0090] A series of characterization analyses were initially carried out on the 131 I-CCM@AP-BSA@CuS nanoprobe: hydrodynamic size, zata potential, stability, preliminary exploration of photothermal conditions, etc. (see Figure 3 , Figure 4 ). The Zeta potential proves the stability of the nanoparticles in the solution. The specific operations are as follows:

[0091] 1) Transmission electron microscopy morphology analysis

[0092] Dissolve the above CCM@AP-BSA@CuS nanomaterial in an aqueous solution to form a slightly colored solution, and then sonicate it in an ultrasonic cleaner until no visible particles are present (i.e., completely dissolved). Take a clean filter paper, place two brand-new carbon support films on top of the filter paper, and then take an appropriate amount of the sonicated solution and drop it onto the copper grid. After waiting for natural drying, drop 1 drop of 1% phosphotungstic acid solution (pH 6.4) on one of the carbon films and stain for 3 min. Use the filter paper to absorb the excess liquid on the copper grid. After waiting for natural drying, observe the material morphology at different magnifications of the TEM. It is observed through the electron microscope that the ATC cell membrane is vesicular (see the first figure in Figure 2 ), with a particle size range of approximately 20-30 nm; the particle size range of AP-BSA@CuS is approximately 9-22 nm (see the second figure in Figure 2 ); the particle size range of CCM@AP-BSA@CuS is approximately 30-40 nm (see the third figure in Figure 2 ). From the transmission electron microscope, the size and morphology of the nanoparticles are confirmed by dynamic light scattering.

[0093] 2) Colloidal stability of the material solution

[0094] Dissolve the above CCM@AP-BSA@CuS nanoparticles in ultrapure water and PBS solution (10 mM, pH = 7.4) respectively to prepare solutions with concentrations of 0.1 mg / mL and 0.5 mg / mL. After complete dissolution, measure the particle size and zeta potential of the nanoparticles on a Malvern particle size analyzer. Measure the hydrodynamic radius of CCM@AP-BSA@CuS at the same time point every day within the next week. The results are shown in Figure 3 , and the zeta potential results show that the materials are all negatively charged in the solution and there is no obvious change in the measured results within 1 week, thus ensuring that the nanoparticles have good water solubility and stability.

[0095] 3) Evaluation of the photothermal stability of the nanomaterials

[0096] When irradiated with near-infrared laser, use an infrared thermal imager to monitor the real-time change of temperature and evaluate the photothermal conversion performance of the nanoparticles. The results are shown in Figure 4 , and from the heating-time curves of aqueous solutions of water, BSA, BSA@CuS and CCM@AP-BSA@CuS with the same material concentration under 1.5 W / cm 2 NIR laser irradiation for 10 min, it can be seen that the CCM@AP-BSA@CuS nanoparticles show excellent photothermal heating ability in vitro. When the material concentration is 2 mg / mL, from the photothermal heating curves at different light intensities, it can be seen that the CCM@AP-BSA@CuS nanoparticles heat up the fastest under 3.0 W / cm 2 NIR laser irradiation. The photothermal heating images at different material concentrations show that the laser intensity at 808 nm is 1.5 W / cm 2 . After the CCM@AP-BSA@CuS aqueous solution has undergone five consecutive laser on-off cycles, no obvious decrease in temperature is monitored in each cycle. This result is sufficient to show that the CCM@AP-BSA@CuS nanomaterial still has excellent photothermal heating ability after laser irradiation, and the photothermal conversion ability is not affected by laser irradiation.

[0097] Finally, 131 I-CCM@AP-BSA@CuS nanoprobes were successfully constructed.

[0098] III. Targeted imaging after tail vein injection of the novel nanoprobe

[0099] Perform SPECT-CT imaging by tail vein injection of 131 I-CCm@AP-BSA@CuS to preliminarily verify its targeting, which is better than 131 I-BSA@CuS, specifically manifested in: achieving tail vein injection instead of local tumor injection, with a long and stable imaging time (see Figure 5 ). 131The I-BSA@CuS group 131 could concentrate in the tumor area for a longer time than the 131 I group, accumulating at the tumor site until the 5th day. However, 131 the I-CCM@AP-BSA@CuS nanoprobe group accumulated at the tumor site until the 7th day. In contrast, 131 the radioactive accumulation in the I group almost disappeared within 24 hours after injection. Therefore, 131 the long-term retention of I-CCM@AP-BSA@CuS at the tumor site will contribute to 131 I better exerting the therapeutic effect of radionuclides. Among them,

[0100] the specific experimental method is as follows: 1) Tumor model construction: After establishing an ATC mouse model with the CAL-62 cell line, when the tumor volume reached approximately 100 mm 3 , the retention of the nanomaterial in the tumor was investigated by collecting the γ-rays emitted by 131 I through SPECT / CT imaging on NM 670 (GE Healthcare, USA).

[0101] 2) Grouping: The mice were randomly divided into two groups, and then the mice were anesthetized (isoflurane gas anesthesia, concentration 1 - 4%), and the tumor-bearing mice were respectively injected via the tail vein with 131 I-CCm@AP-BSA (5 mg / mL, 30 MBq) or 131 I (30 MBq), with a volume of 50 μL for both.

[0102] 3) Image acquisition: Image acquisition was performed at multiple time points before injection (day 0), 2 h after injection, and on the 1st, 3rd, and 7th days until the radioactivity in the mice disappeared. The obtained images were post-processed by reconstruction to obtain fused tomographic images and three-dimensional stereoscopic images of SPECT and CT.

[0103] Example 2 In vitro investigation 131 of the mechanism of action of I-CCM@AP-BSA@CuS nanoparticles on ATC

[0104] The ATC cell line (CAL-62) was cultured, and normal thyroid cells (NTHY ORI 3-1) were cultured as a control. Different protocols ( 131 I-BSA@CuS, 131 I-CCM@AP-BSA@CuS and the control group) were used to treat the ATC cell line respectively. The cells were irradiated with 808 nm NIR laser for 5 min. Among them, 131The preparation of I-CCM@AP-BSA@CuS is described in Example 1. The AP capturer solutions (100, 75, 50, 25, 10, 5, 2.5, 1.25 μM) used in the MTT assay were solutions diluted at a volume ratio of the stock solution to normal saline of 1:100 - 8000; the AP capturer solutions used in the remaining cell experiments were solutions diluted at a volume ratio of the stock solution to normal saline of 1:500.

[0105] (1) MTT assay: 131 The IC50 values of I-CCM@AP-BSA@CuS after 24-hour and 48-hour treatments in ATC cells (CAL-62) and normal thyroid cells (Nthy-ori 3-1) are shown in Table 1 (see details below).

[0106] Table 1 shows 131 the IC50 values of I-CCM@AP-BSA@CuS after 24-hour and 48-hour treatments in ATC cells (CAL-62) and normal thyroid cells (Nthy-ori 3-1)

[0107] Nthy-ori 3-1 CAL-62 24h-IC50 (mg / L) 625±34 378±22 48h-IC50 (mg / L) 328±38 138±26

[0108] (2) Cell apoptosis: Collect the cell suspension, centrifuge and discard the supernatant, add 300 μL of PBS, and resuspend the precipitate. Add 1 ml of ice-cold 70% ethanol to fix for 2 h. Then add the prepared propidium iodide staining solution and mix gently. Place in the dark and incubate on ice for 30 min. Immediately detect using a flow cytometer at an excitation wavelength of 488 nm. Analyze cell DNA content analysis and light scattering analysis. Detection using an apoptosis detection kit (KGA107) showed that 131 I-CCM@AP-BSA@CuS significantly promoted cell apoptosis at a later stage (late apoptosis rate Q2 was 39.2% vs 72.9%, and the results are as Figure 6 shown).

[0109] (3) Cell cycle: Analysis showed that 131 I-CCM@AP-BSA@CuS arrested the cell cycle at the G2 / M phase in CAL-62 cells (see Figure 7 ). The results indicate that 131 I-CCM@AP-BSA@CuS promoted ATC cell apoptosis and arrested the ATC cell cycle more effectively than 131 I-BSA@CuS (23% vs 53%).

[0110] (4) DNA damage detection: Use a DNA damage detection kit (AP site) to detect the formation of AP sites in DNA damage; use a flow cytometer to detect γ-H2AX to evaluate DNA damage. Seed the cells in confocal dishes and use131 I-CCM@AP-BSA@CuS, 131 treated with I-BSA@CuS for 24 h. 131 Compared with 131 I-BSA@CuS, the γ-H2AX fluorescence in cells was significantly enhanced after treatment with I-CCM@AP-BSA@CuS. These results indicate that 131 Compared with 131 I-BSA@CuS, I-CCM@AP-BSA@CuS induced an increase in DNA damage in CAL-62 cells (see Figure 8 ).

[0111] Example 3 In Vivo Verification 131 Inhibitory Effect of I-CCM@AP-BSA@CuS Nanoprobe on ATC

[0112] (1) Establish a subcutaneous implantation model of ATC (CAL-62 cell line) in nude mice: Use SPF-grade male BALB / C nude mice, with a required age of 4-5 weeks, healthy and weighing within the normal range (15-18 g). Animal experiments were approved by the Animal Ethics Committee of Tianjin Medical University and complied with the ethical guidelines of the National Institutes of Health. The specific operations are as follows:

[0113] ① Select CAL-62 cells in the logarithmic growth phase with a density of about 90% in the culture dish. Digest them with 0.25% trypsin, terminate the digestion with complete medium after detaching from the wall, gently pipette to make a relatively uniform single-cell suspension, and collect it into a centrifuge tube. Centrifuge at 4°C (1500 rpm, 5 min), collect the cell pellet, wash it once with PBS, count the cells, and prepare a cell suspension with a concentration of 2×10 7 cells / mL with normal saline, and store it on ice.

[0114] ② Grasp the nude mouse, wipe and disinfect the area near the hind limb on the back with an iodophor cotton ball. Use a 1 mL PD needle to aspirate about 100 μL of the above CAL-62 cell suspension, insert the needle subcutaneously on the dorsal side of the right hind limb of the mouse, and slowly inject to form a small round dermal papule. Observe whether there are any reactions such as bleeding, leakage, or subcutaneous emphysema after withdrawing the needle;

[0115] ③ Number the nude mice. Approximately 10 days after inoculation, the formation of subcutaneous tumor masses can be observed. Observe and photograph the changes in the tumor mass size. Starting from the first day of tumor mass formation, measure the length (L) and width (W) of the tumor with a vernier caliper every 2 days, and calculate the tumor size. The tumor volume calculation formula is as follows: V (mm 3 ) = (L × W 2 ) / 2.

[0116] (2) Thyroid gland blockade: The thyroid gland tissue was blocked by adding 1% NaI to the drinking water of nude mice to reduce the iodine uptake of the thyroid gland tissue, maximize the iodine uptake rate of transplanted tumors, and protect normal thyroid gland tissue from radiation damage by iodine. 131 The iodine uptake 131 rate, and protect normal thyroid tissue from 131 radiation damage by iodine.

[0117] (3) Physiological index monitoring: The experimental animals were randomly divided into 3 groups and intravenously injected with normal saline + 1% DMSO, 131 I-BSA@CuS, 131 I-CCM@AP-BSA@CuS. Among them, 131 the preparation of I-CCM@AP-BSA@CuS is shown in Example 1, and the used AP capture agent solution is a solution diluted at a volume ratio of stock solution to normal saline of 1:20.

[0118] Six or seven weeks after injecting cancer cells (when the diameter of cancer nodules generally reaches about 0.6 - 0.8 cm), start injecting the nanoprobe via the tail vein, and observe the general conditions of the nude mice such as spirit, activity, diet, body weight, and urination and defecation.

[0119] (4) Tumor weight and survival time of nude mice: Measure the tumor volume, calculate the tumor growth inhibition rate of animals in each group; record the survival time of animals in each group, draw the survival curve of nude mice, and analyze the median survival period.

[0120] (6) Acute toxicity reaction of animals: After 30 days of treatment in different treatment groups, perform H&E staining on different organs (heart, liver, spleen, lung, kidney) of nude mice to observe the long-term safety of the nanoprobe.

[0121] (7) Observe the tumor tissue morphology by H&E staining of tumor tissue and evaluate the effect of the nanoprobe by apoptosis.

[0122] HE staining: Take the tissue paraffin sections of nude mice in each group, dewax them with xylene and gradient alcohol, and then stain the sections with Weigert iron hematoxylin staining solution for 7 minutes; after washing thoroughly with water, differentiate with hydrochloric acid alcohol for 5 s; use Masson blueing solution for blueing for 5 minutes, then wash with water; use ponceau fuchsin staining solution for staining for 5 minutes; dehydrate in alcohol with different concentrations in turn, and finally make it transparent with xylene; drop neutral gum for mounting, and observe the histological morphological changes under an optical microscope.

[0123] The results are as Figure 9 and Figure 10 shown, among which, Figure 9(a): Photos of the mice before treatment (day 0) and 3, 6, 12, and 24 days after treatment; (b): Statistical analysis of the excised tumor weights; *P<0.05. (c): Statistical analysis of the body weights of the mice in each subgroup. Note 131 The tumors excised from the I-CCM@AP-BSA@CuS treatment group were smaller in volume and lighter in mass, indicating that it effectively destroyed the tumors and effectively inhibited tumor growth, and had the potential for synergistic cancer treatment. There were no differences in the body weights of the mice in the two treatment groups during the treatment process, indicating that the nanotherapy did not cause any obvious systemic toxicity or side effects to the mice.

[0124] The results of HE staining were as Figure 10 shown. It can be seen from the figure that compared with the control group, there were no obvious differences in the morphology and structure of the main organs of the mice in the experimental group. Normal cell and cell nucleus morphologies could be observed, and no pathological changes such as necrosis, apoptosis, and degeneration occurred, confirming that the novel nanoprobe had no obvious biosafety problems at the organ level.

[0125] As can be seen from the above, 131 the targeting of the I-CCM@AP-BSA@CuS nanoprobe was better than that of 131 I-BSA@CuS, specifically manifested as: realizing tail vein injection instead of local tumor injection. 131 The effect of I-BSA@CuS has been mentioned in a previously published article (Zhang C, Chai J, Jia Q, Tan J, Meng Z, Li N, Yuan M. Evaluating the therapeutic efficacy of radiolabeled BSA@CuS nanoparticle-induced radio-photothermal therapy against anaplastic thyroid cancer. IUBMB Life. 2022 May;74(5):433-445. doi:10.1002 / iub.2601.) 131 The I-BSA@CuS group could concentrate in the tumor area for a longer time than the 131 I group, accumulating at the tumor site until day 5. However, 131 the I-CCM@AP-BSA@CuS nanoprobe group accumulated at the tumor site until day 7. In contrast, 131 the radioactive accumulation in the I group almost disappeared within 24 hours after injection. A longer residence time means a longer irradiation and action time, resulting in a greater killing effect. Therefore, 131 the long-term retention of I-CCM@AP-BSA@CuS at the tumor site will contribute to 131 I better exerting the effect of radionuclide therapy.

Claims

1. A nanoprobe, characterized in that: It includes 131 I. Cancer cell membrane, base-trapping agent, and serum albumin modified copper sulfide nanoparticles.

2. The nanoprobe according to claim 1, characterized in that The cancer cell membrane is coated on the outside of the base capture agent and the serum albumin modified copper sulfide nanomaterial. 131 I is labeled on the cancer cell membrane.

3. The nanoprobe according to claim 1, characterized in that The cancer cell membrane is selected from one or more of lung cancer cell membrane, esophageal cancer cell membrane, gastric cancer cell membrane, duodenal cancer cell membrane, colorectal cancer cell membrane, kidney cancer cell membrane, liver cancer cell membrane, pancreatic cancer cell membrane, gallbladder cancer cell membrane, cervical cancer cell membrane, endometrial cancer cell membrane, vulvar cancer cell membrane, ovarian cancer cell membrane, breast cancer cell membrane, thyroid cancer cell membrane, prostate cancer cell membrane, bladder cancer cell membrane and testicular cancer cell membrane.

4. The nanoprobe according to claim 1, characterized in that The cancer cell membrane is an ATC cancer cell membrane.

5. The nanoprobe according to claim 1, characterized in that The base capture agent is 2,4-dinitrobenzenesulfonamide caged coumarin-based alkoxyamine; the serum albumin-modified copper sulfide nanomaterial is bovine serum albumin-modified copper sulfide nanomaterial or human serum albumin-modified copper sulfide nanomaterial.

6. The method for preparing a nanoprobe according to any one of claims 1 to 5, characterized in that: It includes the following steps: S1. The serum albumin-modified copper sulfide nanoparticles and the base capture agent solution were fully mixed under ultrasound to obtain AP-BSA@CuS nanoprobes; S2, fully mix the cancer cell membrane and AP-BSA@CuS nanoprobe under ultrasound, and then extrude it with a liposome extruder to coat it on the surface of the AP-BSA@CuS nanoprobe, and then centrifuge the solution to remove excess cell membrane to obtain CCM@AP-BSA@CuS; S3. Chloramine T labeling method was used to label CCM@AP-BSA@CuS 131 I labeling; centrifugation and purification 131 I-CCM@AP-BSA@CuS.

7. The preparation method according to claim 6, characterized in that: The amount of serum albumin modified copper sulfide nanoparticles, base capture agent and cancer cell membrane protein is in a mass ratio of 1-2:0.0000625-1:3-5.

8. The preparation method according to claim 6, characterized in that: In step S3, the chloramine T labeling method is to mix the CCM@AP-BSA@CuS solution with Na 131 I solution is mixed and chloramine T solution is added, and after the reaction, sodium metabisulfite solution is added to terminate the reaction, the radioactive mixture solution is transferred to an ultrafiltration centrifuge tube and centrifuged, the radioactivity count of the supernatant is measured, and ultrapure water is added to the supernatant and centrifuged continuously until the radioactivity of the filtrate no longer increases; Among them, CCM@AP-BSA@CuS solution, Na 131 The concentrations of I solution and chloramine T solution are 2-50 mg / mL, and the added amounts are in a volume ratio of 5:2:3; the reaction time is 2-5 min; the molecular weight cutoff of the ultrafiltration centrifuge tube is 30,000; and the centrifugation conditions are 6,000 r / min centrifugation speed for 30 min.

9. The preparation method according to claim 7, characterized in that: The method further comprises the step of preparing serum albumin modified copper sulfide nanoparticles: mixing a BSA solution with a Cu(NO3)2 solution to produce a blue mixture, then adding a NaOH solution to turn it into purple, then adding a Na2S solution to turn it into brick red, and then heating to turn it into dark green to obtain serum albumin modified copper sulfide nanoparticles; Among them, the concentration of the BSA solution is 0.01-0.5 g / mL, the concentration of the Cu(NO3)2 solution is 0.1-0.5 M, the concentration of the NaOH solution is 0.5-2 M, the concentration of the Na2S solution is 0.05-1 M, and the volume ratio of the above solutions is 7.5:1:0.5:2; the heating treatment conditions are 80-100°C and the treatment time is 15-60 min.

10. Use of the nanoprobe according to any one of claims 1 to 5 in the preparation of drugs for treating cancer.

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