A contrast agent for breast cancer diagnosis and its preparation method and application

Abstract

The present invention discloses a method for preparing a contrast agent for breast cancer diagnosis, which comprises the following steps: (1) encapsulating a quantum dot mixture with Ti3C2MXenes, wherein the quantum dot complex is composed of carbon quantum dots and black phosphorus quantum dots in a weight ratio of 1:1-2; (2) preparing a suspension from the result of step (1), adding a hybrid cell membrane and performing homogenization to obtain a contrast agent; the hybrid cell membrane is composed of red blood cell membrane, cancer cell membrane and macrophage membrane in a membrane protein weight ratio of 1:1:1, and the weight ratio of the result of step (1) to the hybrid cell membrane is 3:1:1.

Description

Technical Field

[0001] The present invention belongs to the technical field of contrast agents, and in particular relates to a contrast agent for breast cancer diagnosis, a preparation method thereof and an application thereof. Background Art

[0002] Photoacoustic imaging is a novel imaging technique. When endogenous chromophores or exogenous contrast agents absorb short laser pulses and then expand due to heat, they generate ultrasound waves. Ultrasonic probes on the tissue surface detect, analyze, and process these waves, creating a distribution map of the differences in light energy absorption in the tissue, often referred to as an image. Photoacoustic imaging, characterized by strong acoustic penetration and high resolution, combined with high optical sensitivity and resolution, has become a research hotspot in the medical field and clinical applications in recent years.

[0003] Common photoacoustic imaging contrast agents are mainly divided into two categories: inorganic materials and organic materials. Organic materials are mainly various organic near-infrared dyes and various polymers. [1-2] , the most common one is indocyanine green. Inorganic materials mainly include metal nanomaterials such as gold nanorods, gold nanostars, silver nanohorns [3] etc.; Carbon nanomaterials such as carbon nanotubes, graphene [4] etc., as well as some other related materials such as quantum dots, palladium blue, germanium nanocrystals, etc. [5-6] .

[0004] Among inorganic photoacoustic imaging contrast agents, some two-dimensional inorganic nanomaterials have the characteristics of strong near-infrared light absorption and high photothermal conversion efficiency due to their optoelectronic properties, large band gap and band gap tunability. Among them, Ti3C2MXenes and black phosphorus are research hotspots. [7-8] .

[0005] However, inorganic nanomaterials are prone to agglomeration and poor dispersion, which means that when they are prepared into contrast agents, they can only be used immediately after preparation, and their industrial application value is low. Although the agglomeration problem can be solved by multiple dispersion methods, the particle size distribution of some inorganic nanomaterials is difficult to restore after multiple dispersion, making them unusable. In order to solve the dispersion problem, some scholars combined the sulfonate of titanium ligand with black phosphorus quantum dots to improve the water dispersibility. [9] However, since there is currently no universal method to improve the dispersibility of different inorganic contrast agents, the dispersibility of inorganic contrast agents remains a major challenge in the preparation of contrast agents.

[0006] In addition, in addition to being highly dispersible, the contrast agent must also be able to penetrate the circulatory system into tissue cells and have the ability to specifically bind to tumor cells, and be metabolized by the body and cleared from the circulatory system.

[10] , in addition, it is also necessary to have higher targeting to the desired imaging site. Specifically, although existing contrast agents can achieve tumor targeting, the targeting is still low, resulting in a lot of contrast agents being enriched in non-tumor sites. Therefore, in actual use, a higher dose is required to obtain an acceptable photoacoustic signal (PA) value, which makes it take longer to clear from the body. For example, XiaoxiaHan

[11] The contrast agent studied by et al. could only achieve a PA above 1 at high concentrations (50 ppm), and within 24 hours after tail vein injection, the PA did not reach 1. Furthermore, ZL202210940520.5 prepared a contrast agent by loading the inorganic material silica nanoparticles (MSNs) with the organic dye indocyanine green and coating them with the corresponding cell membrane. However, this contrast agent was unable to produce the photoacoustic effect on its own, requiring the addition of a high dose of indocyanine green. Furthermore, its PA value still failed to reach 1 within 48 hours.

[0007] In summary, it can be seen that the art needs a tumor contrast agent that has high dispersion stability, a lower usage dosage, and a higher photoacoustic signal value.

[0008] [1]Wang K, Yao H, Meng Y, et al.Specific aptamer-conjugated mesoporoussilica-carbon nanoparticles for HER2-targeted chemo-photothermal combined therapy[J]. Acta Biomater, 2015, 16(1): 196-205.

[0009] [2] Beziere N, Lozano N, Nunes A, et al. Dynamic imaging of PEGylatedindocyanine green (ICG) liposomes within the tumor microenvironment using multi-spectral optoacoustic tomography (MSOT) [J]. Biomaterials, 2015, 37(37C): 415-424.

[0010] [3]Nie L,Chen X.Structural and functional photoacoustic moleculartomography aided by emerging contrast agents[J].Chem Soc Rev,2014,43(20):7132-7170.

[0011] [4]Chen D,Dougherty CA,Zhu K,et al.Theranostic applications of carbonnanomaterials in cancer:focus on imaging and cargo delivery[J].J ControlRelease,2015,210(1):230-245.

[0012] [5]Homan K,Kim S,Chen YS,et al.Prospects of molecular photoacousticimaging at 1064 nm wavelength[J].Opt Lett,2010,35(15):2663-2665.

[0013] [6]Yu J,Yin W,Zheng X,et al.Smart MoS2 / Fe3O4 Nanotheranostic formagnetically targeted photothermal therapy guided by magnetic resonance / photoacoustic imaging[J].Theranostics,2015,5(9):931-945.

[0014] [7]Lin H,Wang X,Yu L,et al.Two-dimensional ultrathin MXene ceramicnanosheets for photothermal conversion.Nano Lett,2017,17:384.

[0015] [8]Shao J, Xie H, Huang H, et al. Biodegradable black phosphorus-basednanospheres for in vivo photothermal cancer therapy. Nat Commun, 2016,7:12967.

[0016] [9]Sun ZB, Zhao YT, Li ZB, et al. TiL4-coordinated black phosphorusquantum dots as an efficient contrast agent for in vivo photoacoustic imaging of cancer. Small, 2017,13:1602896.

[0017]

[10] Tang Hewen, Yang Meng, Jiang Yuxin. Molecular contrast agents for photoacoustic imaging[J]. Journal of Peking Union Medical College, 2018, 9(04): 358-363.

[0018]

[11] Han Summary of the Invention

[0019] In response to the shortcomings of the prior art, the present invention aims to provide a method for preparing a contrast agent for breast cancer diagnosis. The contrast agent obtained by this preparation method can overcome the defects of the prior art such as the high dosage and poor dispersion stability required, and has the characteristics of having a higher photoacoustic signal value at a lower dosage.

[0020] In order to achieve the above object, the scheme adopted by the present invention is as follows:

[0021] A method for preparing a contrast agent for breast cancer diagnosis, comprising the following steps:

[0022] (1) encapsulating a quantum dot mixture with Ti3C2 MXenes, wherein the quantum dot composite is composed of carbon quantum dots and black phosphorus quantum dots in a weight ratio of 1:1-2;

[0023] (2) preparing a suspension of the product obtained in step (1), adding a hybrid cell membrane and homogenizing the suspension to obtain a hybrid cell membrane; the hybrid cell membrane is composed of erythrocyte membrane, cancer cell membrane and macrophage membrane in a membrane protein weight ratio of 1:1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1:1;

[0024] in,

[0025] The preparation method of Ti3C2 MXenes is:

[0026] A mixture of titanium powder, aluminum powder and graphite powder is sintered at high temperature, and then the sintered product is reacted with hydrofluoric acid. The reactant is then added to a tetrapropylammonium hydroxide aqueous solution for reaction, and then the reactant is centrifuged and washed to obtain Ti3C2MXenes.

[0027] The preparation method of carbon quantum dots is as follows:

[0028] Glucose, ornithine, and arginine were dissolved in water in a weight ratio of 3:1:1, followed by carbonization reaction. The reactants were then dissolved in water, centrifuged to remove the precipitate, and the supernatant was dialyzed. The dialyzed solution was then freeze-dried to obtain a powder, which was the carbon quantum dots.

[0029] The preparation method of the black phosphorus quantum dots is as follows:

[0030] The N-methylpyrrolidone solution of black phosphorus is ultrasonically treated, the treated solution is centrifuged to obtain the supernatant, and the supernatant is rotary evaporated to obtain a powder that is black phosphorus quantum dots.

[0031] Preferably, in step (1), when the quantum dot mixture is encapsulated with Ti3C2 MXenes, the quantum dot mixture is placed in a Ti3C2 MXenes ethanol solution with a concentration of 1 to 2 mg / mL, mixed, and then centrifuged to remove the supernatant, and then dried.

[0032] Preferably, the weight ratio of the quantum dot mixture to Ti3C2 MXenes is 1:60-70.

[0033] Preferably, in step (2), when the product obtained in step (1) is prepared into a suspension, PBS is used as the solution.

[0034] Preferably, in step (2), during the homogenization process, an ultrasonic homogenizer is used for ultrasonic dispersion.

[0035] Preferably, when preparing Ti3C2 MXenes, the weight ratio of titanium powder, aluminum powder and graphite powder is 7:3:1; and the high-temperature sintering is carried out in an argon atmosphere.

[0036] Preferably, when using hydrofluoric acid for reaction, the weight concentration of hydrofluoric acid is 40%, and the reaction time of hydrofluoric acid reaction is 50 hours; the solute weight concentration of tetrapropylammonium hydroxide aqueous solution is 20%, and the reaction time in tetrapropylammonium hydroxide aqueous solution is 50 hours.

[0037] Preferably, when preparing carbon quantum dots, the carbonization temperature is 190-200°C, and the carbonization time is 8-10 hours; when preparing black phosphorus quantum dots, water bath ultrasound is used for ultrasonic treatment; when performing water bath ultrasound, the temperature is controlled at 1-4 degrees, the power of the water bath ultrasound is 300-500W, and the ultrasound time is 24 hours.

[0038] Another object of the present invention is to provide a contrast agent prepared by the above method for breast cancer diagnosis.

[0039] Another object of the present invention is to provide a use of the above-mentioned contrast agent for breast cancer diagnosis in the preparation of a contrast agent for breast cancer diagnosis.

[0040] Beneficial effects of the present invention:

[0041] The contrast agent for breast cancer diagnosis obtained by the present invention has high dispersion stability and can be stored in an aqueous solution for more than 60 days. At the same time, at a low dosage of 3 μg / mL, the photoacoustic signal PA value can reach more than 1 within 24 hours. Moreover, the contrast agent obtained by the present invention also has excellent targeting, and the enrichment level in breast cancer tumors is much higher than that in other parts. DETAILED DESCRIPTION

[0042] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0043] Example 1

[0044] 1. Preparation of Ti3C2 MXenes

[0045] Titanium and aluminum powders, both with a purity of at least 99% w / w, were ground to -325 mesh. Graphite powder, also with a purity of 99% w / w, was ground to -300 mesh. The mixture was mixed in a weight ratio of 7:3:1 and ball-milled for 10 hours. The mixture was then pressed into a round cake under a pressure of 30 MPa. The cake was then fired in a furnace at 1500°C for 2 hours under argon gas to produce the Ti3AlC2 ceramic material.

[0046] After grinding the obtained Ti3AlC2 ceramic material, 10g of powder was collected and placed in 60ml of a 40% hydrofluoric acid aqueous solution for etching reaction at room temperature for 50 hours. The powder was then collected by centrifugation and washed with water and ethanol. It was then dispersed in 50ml of a 20% w / w tetrapropylammonium hydroxide aqueous solution and stirred at room temperature for 50 hours. The powder was then centrifuged and washed with water and ethanol to remove residual tetrapropylammonium hydroxide to obtain Ti3AlC2 MXenes.

[0047] 2. Preparation of Carbon Quantum Dots

[0048] 1.5 g of glucose, 0.5 g of ornithine, and 0.5 g of arginine were dissolved in 20 mL of water, and then carbonized at 190-200 ° C for 10 hours. The reactants were then dissolved in water, centrifuged to remove the precipitate, and the supernatant was dialyzed (MWCO = 100000) for 3 days. The dialyzed solution was then freeze-dried (1.00 KPa, -80 ° C). The resulting powder was carbon quantum dots.

[0049] 3. Preparation of Black Phosphorus Quantum Dots

[0050] A black phosphorus N-methylpyrrolidone solution (black phosphorus concentration is 3 mg / mL) is ultrasonically treated (300W) in a water bath (1-4°C) for 24 hours. The treated solution is centrifuged to obtain the supernatant, which is then rotary evaporated to obtain a powder, namely black phosphorus quantum dots.

[0051] 4. Contrast Agent Preparation

[0052] (1) Carbon quantum dots and black phosphorus quantum dots were mixed in a weight ratio of 1:1 to obtain a quantum dot mixture. The quantum dot mixture was placed in a Ti3C2 MXenes ethanol solution (Ti3C2 MXenes concentration was 1 mg / mL) and then centrifuged to remove the supernatant. The mixture was then dried at 60°C. The weight ratio of the quantum dot mixture to the Ti3C2 MXenes was 1:70.

[0053] (2) The product obtained in step (1) is prepared into a PBS suspension, and the hybrid cell membrane is added and dispersed by ultrasonic homogenizer (500W) to obtain the hybrid cell membrane; the hybrid cell membrane is composed of red blood cell membrane, cancer cell membrane and macrophage membrane in a membrane protein weight ratio of 1:1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1:1.

[0054] The red blood cell membrane was prepared according to the literature (Gao Changyong. Bionic design of natural cell membrane camouflaged micro-nanocarriers and their biomedical applications [D]. Harbin Institute of Technology, 2017.), and the cancer cell (HepG2 cell) membrane and macrophage membrane were prepared according to this method.

[0055] Example 2

[0056] The preparation methods of Ti3C2 MXenes, carbon quantum dots and black phosphorus quantum dots are the same as those in Example 1.

[0057] Contrast agent preparation

[0058] (1) Carbon quantum dots and black phosphorus quantum dots were mixed in a weight ratio of 1:2 to obtain a quantum dot mixture. The quantum dot mixture was placed in a Ti3C2 MXenes ethanol solution (Ti3C2 MXenes concentration was 1 mg / mL) and then centrifuged to remove the supernatant. The mixture was then dried at 60°C. The weight ratio of the quantum dot mixture to the Ti3C2 MXenes was 1:60.

[0059] (2) The product obtained in step (1) is prepared into a PBS suspension, and the hybrid cell membrane is added and dispersed by ultrasonic homogenizer (500W) to obtain the hybrid cell membrane; the hybrid cell membrane is composed of red blood cell membrane, cancer cell membrane and macrophage membrane in a membrane protein weight ratio of 1:1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1:1.

[0060] Example 3

[0061] The preparation methods of Ti3C2 MXenes, carbon quantum dots and black phosphorus quantum dots are the same as those in Example 1.

[0062] Contrast agent preparation

[0063] (1) Carbon quantum dots and black phosphorus quantum dots were mixed in a weight ratio of 1:1.5 to obtain a quantum dot mixture. The quantum dot mixture was placed in a Ti3C2 MXenes ethanol solution (Ti3C2 MXenes concentration was 1 mg / mL) and then centrifuged to remove the supernatant. The mixture was then dried at 60°C. The weight ratio of the quantum dot mixture to the Ti3C2 MXenes was 1:65.

[0064] (2) The product obtained in step (1) is prepared into a PBS suspension, and the hybrid cell membrane is added and dispersed by ultrasonic homogenizer (500W) to obtain the hybrid cell membrane; the hybrid cell membrane is composed of red blood cell membrane, cancer cell membrane and macrophage membrane in a membrane protein weight ratio of 1:1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1:1.

[0065] Comparative Example 1

[0066] On the basis of Example 1, carbon quantum dots were used to replace the quantum dot mixture, and the rest were the same as in Example 1.

[0067] Comparative Example 2

[0068] On the basis of Example 1, black phosphorus quantum dots were used to replace the quantum dot mixture, and the rest were the same as in Example 1.

[0069] Comparative Example 3

[0070] On the basis of Example 1, the hybrid cell membrane was replaced by cancer cell membrane and macrophage membrane, and the rest was the same as Example 1.

[0071] Comparative Example 4

[0072] Based on Example 1, except that only glucose and arginine were used as raw materials in the preparation of carbon quantum dots, the rest were the same as Example 1.

[0073] Experimental methods and results

[0074] 1. Dispersion experiment

[0075] The dispersion was characterized by dynamic light scattering method, and the average particle size and particle size distribution after standing for different times were examined. The results are shown in Table 1.

[0076] Table 1

[0077]

[0078] As shown in Table 1, the choice of quantum dots has a certain effect on dispersibility, and the choice of cell membrane has a greater effect on dispersibility. The possible reason is that different cell membrane combinations have an effect on the adsorption of inorganic materials. At the same time, different quantum dots are prone to microscopic separation due to weak binding force when they are in aqueous solution for a long time, resulting in poor dispersibility. As shown in the dispersibility results of Example 1 of the present invention, the present invention can still maintain very good dispersibility after standing for 60 days, and the standing time has little effect on the average particle size. The inventors also measured the particle size distribution of the contrast agent of Example 1 after standing for 60 days, and the particle size distribution PDI was 0.031, which shows that the resulting contrast agent is basically not agglomerated.

[0079] 2. In vivo experiments

[0080] A breast tumor mouse model was constructed according to the reference (Gu Xingju. Establishment of a breast tumor mouse model and analysis of its bone marrow MDSC cell subtypes [D]. Shenyang Agricultural University, 2017). The aqueous solutions of the contrast agents obtained in Examples 1-3 and Comparative Examples 1-4 were then injected into the tail vein to investigate targeting and imaging properties.

[0081] Four hours after injection, the distribution ratio of the contrast agent in different body parts (tumor, heart, liver, spleen, lung, and kidney) was examined. The tumor distribution ratio (i.e., targeting efficiency) of the contrast agents obtained in Examples 1-3 ranged from 26% to 30%, compared to the targeting efficiencies of 11.6%, 12.3%, 16.4%, and 13.5% for the contrast agents in Comparative Examples 1-4, respectively.

[0082] The photoacoustic signal (PA) values of Examples 1-3 of the present invention and Comparative Examples 1-4 within 24 hours after tail vein injection (10 μg / mL, calculated as Ti element) were recorded, as shown in Table 2.

[0083] Table 2

[0084]

[0085]

[0086] Based on the above experiments, the inventors also investigated photoacoustic imaging at even lower injection concentrations. The contrast agents obtained in Examples 1-3 exhibited photoacoustic signal (PA) values exceeding 1 within 24 hours of injection at an injection concentration of 3 μg / mL. At an injection concentration of 1 μg / mL, the PA values also exceeded 0.7 within 24 hours of injection. This demonstrates that the contrast agents obtained in the present invention can be used at extremely low injection concentrations.

Claims

1. A method for preparing a contrast agent for breast cancer diagnosis, characterized in that: The preparation method of the contrast agent comprises the following steps: (1) encapsulating a quantum dot mixture with Ti3C2 MXenes, wherein the quantum dot composite is composed of carbon quantum dots and black phosphorus quantum dots in a weight ratio of 1:1-2; (2) preparing a suspension of the product obtained in step (1), adding a hybrid cell membrane and homogenizing the suspension to obtain a hybrid cell membrane; the hybrid cell membrane is composed of erythrocyte membrane, cancer cell membrane and macrophage membrane in a membrane protein weight ratio of 1:1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1:1; in, The preparation method of Ti3C2 MXenes is: A mixture of titanium powder, aluminum powder and graphite powder is sintered at high temperature, and then the sintered product is reacted with hydrofluoric acid. The reactant is then added to a tetrapropylammonium hydroxide aqueous solution for reaction, and then the reactant is centrifuged and washed to obtain Ti3C2MXenes. The preparation method of carbon quantum dots is as follows: Glucose, ornithine, and arginine were dissolved in water in a weight ratio of 3:1:1, followed by carbonization reaction. The reactants were then dissolved in water, centrifuged to remove the precipitate, and the supernatant was dialyzed. The dialyzed solution was then freeze-dried to obtain a powder, which was the carbon quantum dots. The preparation method of black phosphorus quantum dots is as follows: The N-methylpyrrolidone solution of black phosphorus is ultrasonically treated, the treated solution is centrifuged to obtain the supernatant, and the supernatant is rotary evaporated to obtain a powder that is black phosphorus quantum dots.

2. The preparation method according to claim 1, characterized in that In step (1), when the quantum dot mixture is encapsulated with Ti3C2MXenes, the quantum dot mixture is placed in a Ti3C2MXenes ethanol solution with a concentration of 1 to 2 mg / mL and mixed, and then centrifuged to remove the supernatant, and then dried.

3. The preparation method according to claim 2, characterized in that The weight ratio of the quantum dot mixture to Ti3C2 MXenes is 1:60-70.

4. The preparation method according to claim 1, characterized in that In step (2), when the product obtained in step (1) is prepared into a suspension, PBS is used as the solution.

5. The preparation method according to claim 1, characterized in that In step (2), during the homogenization process, ultrasonic dispersion is performed using an ultrasonic homogenizer.

6. The preparation method according to claim 1, characterized in that When preparing Ti3C2 MXenes, the weight ratio of titanium powder, aluminum powder and graphite powder is 7:3:1; high-temperature sintering is carried out in an argon atmosphere.

7. The preparation method according to claim 6, characterized in that When hydrofluoric acid is used for the reaction, the weight concentration of hydrofluoric acid is 40%, and the reaction time is 50 hours; the solute weight concentration of the tetrapropylammonium hydroxide aqueous solution is 20%, and the reaction time in the tetrapropylammonium hydroxide aqueous solution is 50 hours.

8. The preparation method according to claim 1, characterized in that When preparing carbon quantum dots, the carbonization temperature is 190-200°C, and the carbonization time is 8-10 hours; when preparing black phosphorus quantum dots, water bath ultrasound is used for ultrasonic treatment; when performing water bath ultrasound, the temperature is controlled at 1-4 degrees, the power of the water bath ultrasound is 300-500W, and the ultrasound time is 24 hours.

9. A contrast agent for breast cancer diagnosis prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the contrast agent for breast cancer diagnosis according to claim 9 in the preparation of a contrast agent for breast cancer diagnosis.

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