A highly targeted liver cancer contrast agent and its preparation method

Abstract

The present invention discloses a method for preparing a highly targeted liver cancer contrast agent, comprising the following steps: (1) encapsulating a quantum dot mixture with Ti3C2MXenes, wherein the quantum dot complex is composed of carbon quantum dots and graphyne quantum dots in a weight ratio of 1:1-2; (2) preparing a suspension from the product obtained in step (1), adding a hybrid cell membrane and homogenizing the suspension to obtain a highly targeted liver cancer contrast agent; the hybrid cell membrane is composed of erythrocyte membrane and macrophage membrane in a membrane protein weight ratio of 1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1. The liver cancer contrast agent obtained by the 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 0.5 μg / mL, the photoacoustic signal PA value can reach more than 1 within 24 hours. Moreover, the contrast agent obtained by the present invention has excellent targeting, with a targeting efficiency of up to 32% for liver cancer tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials and contrast agents, and particularly relates to a highly targeted liver cancer contrast agent and a preparation method thereof. Background Art

[0002] In the preparation of contrast agents, inorganic nanomaterials can often only be used immediately after preparation, and their industrial application value is low. Although the agglomeration problem can be solved by means of multiple dispersion, the particle size distribution of some inorganic nanomaterials is difficult to restore after multiple dispersion, resulting in their unusability. In order to solve the dispersion problem, some scholars have combined the sulfonate of titanium ligands 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.

[0003] 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.

[0004] 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.

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[0014]

[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.

[0015]

[11] Han Summary of the Invention

[0016] To address the shortcomings of existing technologies, the present invention provides a method for preparing a highly targeted liver cancer contrast agent. This method overcomes the drawbacks of existing technologies, such as the high dosage and poor dispersion stability, resulting in a contrast agent with high photoacoustic signal values ​​at lower dosages. Specifically, the present invention achieves a contrast agent that exhibits high photoacoustic signal values ​​even at an extremely low injection volume of 0.5 μg / mL.

[0017] In order to achieve the above object, the present invention adopts the following technical solutions:

[0018] A highly targeted liver cancer contrast agent and a preparation method thereof, the preparation method of the contrast agent comprising the following steps:

[0019] (1) encapsulating a quantum dot mixture with Ti3C2 MXenes, wherein the quantum dot composite is composed of carbon quantum dots and graphyne quantum dots in a weight ratio of 1:1-2;

[0020] (2) preparing a suspension of the product obtained in step (1), adding a hybrid cell membrane and homogenizing the suspension to obtain a suspension; the hybrid cell membrane is composed of erythrocyte membrane and macrophage membrane in a membrane protein weight ratio of 1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1;

[0021] in,

[0022] The preparation method of Ti3C2 MXenes is:

[0023] 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.

[0024] The preparation method of carbon quantum dots is as follows:

[0025] 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.

[0026] The preparation method of the graphene quantum dots is as follows:

[0027] The graphyne aqueous solution is hydrothermally treated, the treated solution is dialyzed and then freeze-dried, and the obtained powder is graphyne quantum dots.

[0028] 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.

[0029] Preferably, the weight ratio of the quantum dot mixture to Ti3C2 MXenes is 1:80-100.

[0030] Preferably, in step (2), when the product obtained in step (1) is prepared into a suspension, PBS is used as the solution.

[0031] Preferably, in step (2), during the homogenization process, ultrasonic dispersion is performed using an ultrasonic homogenizer.

[0032] 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.

[0033] 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.

[0034] Preferably, when preparing carbon quantum dots, the carbonization temperature is 190-200° C., and the carbonization time is 8-10 hours; when preparing graphyne quantum dots, the hydrothermal temperature is 165-170° C., and the hydrothermal treatment time is 2.5-3 hours.

[0035] Another object of the present invention is to provide a highly targeted liver cancer contrast agent prepared by the above method.

[0036] Another object of the present invention is to provide the use of the above-mentioned highly targeted liver cancer contrast agent in the preparation of liver cancer contrast agents.

[0037] The present invention is developed based on another patented technology applied by the inventor, "A contrast agent for breast cancer diagnosis, its preparation method and application". During the exploration process, it was found that the contrast agent used for breast cancer diagnosis is not good for targeted imaging of liver cancer (such as comparative example 3 of the present invention), the tumor enrichment is not good, and the required injection concentration is high. In order to solve this problem, the inventor obtained a highly targeted liver cancer contrast agent by adjusting the quantum dot mixture and the hybrid cell membrane. It is worth noting that, on the basis of removing the cancer cell membrane in the hybrid cell membrane, graphyne quantum dots are selected to replace black phosphorus quantum dots, which not only does not affect the dispersion stability, but improves the imaging effect. The reason may be that the hybrid cell membrane of the present invention has a good binding property for the combination of carbon quantum dots and graphyne quantum dots, and is not easy to fall off during circulation in the body, thereby having higher targeting and imaging properties.

[0038] Beneficial effects of the present invention:

[0039] The liver cancer contrast agent 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 0.5 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, with a targeting efficiency of up to 32% for liver cancer tumors. DETAILED DESCRIPTION

[0040] 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.

[0041] Example 1

[0042] 1. Preparation of Ti3C2 MXenes

[0043] 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.

[0044] After grinding the obtained Ti3AlC2 ceramic material, 10g of the powder was collected and placed in 60ml of a 40% hydrofluoric acid aqueous solution, and an etching reaction was carried out at room temperature for 50 hours. The powder was then collected by centrifugation and washed with water and ethanol, and 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 Ti3AlC2MXenes.

[0045] 2. Preparation of Carbon Quantum Dots

[0046] 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.

[0047] 3. Preparation of Graphdine Quantum Dots

[0048] The graphyne aqueous solution (graphyne concentration is 2 mg / mL) is hydrothermally treated (165-170°C) for 3 hours, the treated solution is dialyzed (MWCO=100000) for 3 days and then freeze-dried (1.00 KPa, -80°C). The resulting powder is graphyne quantum dots.

[0049] 4. Contrast Agent Preparation

[0050] (1) Carbon quantum dots and graphyne 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 1:100.

[0051] (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 erythrocyte membrane and macrophage membrane in a membrane protein weight ratio of 1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1.

[0052] 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 macrophage membrane was prepared according to this method.

[0053] Example 2

[0054] The preparation methods of Ti3C2 MXenes, carbon quantum dots and graphyne quantum dots are the same as those in Example 1.

[0055] Contrast agent preparation

[0056] (1) Carbon quantum dots and graphyne quantum dots were mixed in a weight ratio of 1:1 to obtain a quantum dot mixture, and the quantum dot mixture was placed in a Ti3C2 MXenes ethanol solution (Ti3C2 MXenes concentration was 1 mg / mL) for mixing, and then the supernatant was removed by centrifugation, and then dried at 60°C; the weight ratio of the quantum dot mixture to Ti3C2 MXenes was 1:80;

[0057] (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 erythrocyte membrane and macrophage membrane in a membrane protein weight ratio of 1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1.

[0058] Example 3

[0059] The preparation methods of Ti3C2 MXenes, carbon quantum dots and graphyne quantum dots are the same as those in Example 1.

[0060] Contrast agent preparation

[0061] (1) Carbon quantum dots and graphyne 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 Ti3C2 MXenes was 1:65.

[0062] (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 erythrocyte membrane and macrophage membrane in a membrane protein weight ratio of 1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1.

[0063] Comparative Example 1

[0064] 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.

[0065] Comparative Example 2

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

[0067] Comparative Example 3

[0068] 1. Preparation of Ti3C2 MXenes

[0069] 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.

[0070] After grinding the obtained Ti3AlC2 ceramic material, 10g of the powder was collected and placed in 60ml of a 40% hydrofluoric acid aqueous solution, and an etching reaction was carried out at room temperature for 50 hours. The powder was then collected by centrifugation and washed with water and ethanol, and 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 Ti3AlC2MXenes.

[0071] 2. Preparation of Carbon Quantum Dots

[0072] 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.

[0073] 3. Preparation of Black Phosphorus Quantum Dots

[0074] 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.

[0075] 4. Contrast Agent Preparation

[0076] (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.

[0077] (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.

[0078] 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.

[0079] Comparative Example 4

[0080] 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.

[0081] Experimental methods and results

[0082] 1. Dispersion experiment

[0083] 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.

[0084] Table 1

[0085]

[0086]

[0087] As shown in Table 1, the selection of quantum dots has a certain impact on dispersibility. The possible reason is that when different quantum dots are in aqueous solution for a long time, they are prone to microscopic separation due to weak binding force, resulting in poor dispersibility. As shown in the dispersibility results of Examples 1-3 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 in Example 1 after standing for 60 days. The particle size distribution PDI was 0.034, which indicates that the obtained contrast agent has basically not agglomerated.

[0088] In addition, Comparative Example 3 (an invention patent applied for separately by the inventor) also has good dispersion stability.

[0089] 2. In vivo experiments

[0090] Reference (Wang Jinyin, Li Xueying, Yi Jinke, et al. Establishment of a primary liver cancer model in mice [J]. Chinese Journal of Applied Physiology, 2022, 38(06): 820-823.) A liver cancer tumor mouse model was constructed, and then the aqueous solution of the contrast agent obtained in Examples 1-3 and Comparative Examples 1-4 was injected into the tail vein to investigate the targeting and imaging properties.

[0091] Four hours after injection, the distribution ratio of the contrast agent in different body parts (tumor, heart, spleen, lung, and kidney) was examined. The tumor distribution ratio (i.e., targeting efficiency) of the contrast agents obtained in Examples 1-3 was 32%, while the targeting efficiencies of the contrast agents in Comparative Examples 1-4 were 12.1%, 13.9%, 19.6%, and 11.7%, respectively.

[0092] 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 (1 μg / mL, calculated as Ti element) were recorded, as shown in Table 2.

[0093] Table 2

[0094]

[0095] Based on the above experiments, the inventors also investigated photoacoustic imaging at even lower injection concentrations. At an injection concentration of 0.5 μg / mL, the contrast agents obtained in Examples 1-3 all exhibited photoacoustic signal (PA) values ​​exceeding 1 within 24 hours after injection. In contrast, the contrast agent in Comparative Example 3 exhibited a photoacoustic signal (PA) value of no more than 0.7 within 24 hours after injection at a concentration of 0.5 μg / mL. This demonstrates that the contrast agents obtained in the present invention can be used at extremely low injection concentrations and are particularly suitable for imaging liver cancer.

Claims

1. A method for preparing a liver cancer contrast agent, 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 graphyne 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 suspension; the hybrid cell membrane is composed of erythrocyte membrane and macrophage membrane in a membrane protein weight ratio of 1:1, and the weight ratio of the product obtained in step (1) to the hybrid cell membrane is 3:1; 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 the graphene quantum dots is as follows: The graphyne aqueous solution is hydrothermally treated, the treated solution is dialyzed and then freeze-dried, and the obtained powder is graphyne 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 1:80-100.

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 homogenizer is used for ultrasonic dispersion.

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 graphyne quantum dots, the hydrothermal temperature is 165-170° C. and the hydrothermal treatment time is 2.5-3 hours.

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

10. Use of the liver cancer contrast agent according to claim 9 in the preparation of a liver cancer contrast agent.

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