Positive and negative CT nanometer contrast agent for detecting liver cancer and preparation method thereof

Abstract

The application discloses a positive-negative CT nanometer contrast agent for detecting liver cancer, which is a CT imaging nano probe with Hf-MOF nanoparticles as an inner core, channels carrying ammonia borane and an outer layer modified with polyvinylpyrrolidone; the Hf-MOF nanoparticles are prepared from hafnium tetrachloride, benzoic acid and 2-amino terephthalic acid. The positive-negative CT nanometer contrast agent for detecting liver cancer is cheap and easy to obtain, has mild reaction conditions, few synthesis steps and simple product processing.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedical technology, specifically, it relates to a positive and negative CT nanocontrast agent for detecting liver cancer and its preparation method. Background Technology

[0002] Liver cancer is a common primary malignant tumor of the digestive system, with a high incidence and poor prognosis, seriously threatening human life and health. Early imaging diagnosis of liver cancer is of paramount importance for its effective treatment. CT imaging has advantages such as simple operation and high spatial and temporal resolution, playing a crucial role in clinical disease screening, including tumors. However, CT's soft tissue resolution is not high, hindering its sensitive detection of early-stage liver cancer. This is mainly because X-ray attenuation depends on the density of the material itself. Soft tissue lesions such as liver cancer have similar densities to surrounding normal tissue, making precise differentiation difficult in CT imaging. Therefore, there is an urgent need to develop suitable CT contrast agents to improve the sensitivity of early liver cancer detection.

[0003] Currently, most CT contrast agents (including iodine-based contrast agents commonly used in clinical practice) exhibit high density and are generally defined as positive CT contrast agents. Among them, positive CT nanocontrast agents have many advantages in tumor detection, such as multifunctional design and enhanced penetration and retention (EPR) effects. However, positive CT nanocontrast agents still cannot sensitively detect liver cancer. This is mainly because CT nanocontrast agents are simultaneously phagocytosed by Kupffer cells and tumor cells in the liver, resulting in similar high CT values ​​for both normal liver tissue and tumor tissue, making sensitive differentiation difficult. Recently, Meng et al. innovatively developed a negative CT nanocontrast agent for detecting osteosarcoma. This contrast agent can specifically respond to the acidic microenvironment of osteosarcoma, generating hydrogen gas. The negative CT signal generated by hydrogen gas contrasts strongly with the positive CT signal generated by the surrounding bone, thereby achieving accurate CT diagnosis of osteosarcoma. Therefore, if positive CT nanocontrast agents and negative nanocontrast agents can be combined and specifically enriched at the liver tumor site, specifically forming a positive and negative CT signal contrast, it is hoped that sensitive CT diagnosis of early liver cancer can be achieved. Summary of the Invention

[0004] The purpose of this invention is to provide a positive and negative CT nanocontrast agent for detecting liver cancer, thereby achieving accurate CT imaging detection of liver cancer using clinical CT imaging technology.

[0005] Another object of the present invention is to provide a method for preparing the positive and negative CT nanocontrast agent for detecting liver cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a positive and negative CT nanocontrast agent for detecting liver cancer, which is a CT imaging nanoprobe with Hf-MOF nanoparticles as the core, pores carrying ammonia borane (AB) and an outer layer modified with polyvinylpyrrolidone (PVP); the Hf-MOF nanoparticles are prepared from hafnium tetrachloride (HfCl4), benzoic acid, and 2-aminoterephthalic acid.

[0008] A second aspect of the present invention provides a method for preparing the positive and negative CT nanocontrast agents for detecting liver cancer, comprising the following steps:

[0009] The first step involves dispersing hafnium tetrachloride and benzoic acid in DMF at a molar ratio of 1:8 to 20, adding 2-aminoterephthalic acid to the above system, dissolving by sonication, with a molar ratio of 2-aminoterephthalic acid to hafnium tetrachloride of 1:1 to 3, reacting for 1 to 10 minutes (preferably 3 minutes) at a temperature of 60 to 200°C (preferably 120°C) for 24 to 72 hours (preferably 24 hours) to obtain Hf-MOF nanoparticles, which are then dispersed in water to a concentration of 0.1 to 10 mg / mL (preferably 0.5 mg / mL).

[0010] In the second step, ammonia borane is added to the above Hf-MOF solution, with a molar ratio of ammonia borane to hafnium tetrachloride of 7–15:1. The mixture is stirred at room temperature and evacuated for 0.1–2 hours (preferably 1 hour), and then the reaction is continued for 24–72 hours (preferably 24 hours). After the reaction is complete, polyvinylpyrrolidone is added to the solution, with a molar ratio of polyvinylpyrrolidone to hafnium tetrachloride of 7–20:1. The mixture is stirred for another 24–72 hours (preferably 24 hours) to obtain a positive and negative CT nanocontrast agent for detecting liver cancer.

[0011] The molar ratio of hafnium tetrachloride to benzoic acid is 1:10.

[0012] The molar ratio of 2-aminoterephthalic acid to hafnium tetrachloride is 1:1.

[0013] The molar ratio of ammonia borane to hafnium tetrachloride is 9.4:1.

[0014] The molar ratio of polyvinylpyrrolidone to hafnium tetrachloride is 14.6:1.

[0015] A third aspect of the present invention provides the application of the positive and negative CT nanocontrast agents for detecting liver cancer in the preparation of liver cancer detection reagents.

[0016] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0017] The positive and negative CT nanocontrast agents for detecting liver cancer provided by this invention are made from inexpensive and readily available raw materials, have mild reaction conditions, require few synthesis steps, and are simple to process. The novel positive and negative CT nanocontrast agents can achieve rapid and sensitive CT image detection of liver cancer, with stable detection signals that are not affected by external factors. They can also achieve accurate CT image detection in subcutaneous liver cancer model mice and in situ liver cancer model mice.

[0018] This invention provides a positive and negative CT nanocontrast agent for detecting liver cancer, comprising an Hf-MOF core, pores carrying ammonia borane (AB), and an outer layer modified with polyvinylpyrrolidone (PVP) as a CT imaging nanoprobe. This nanoprobe primarily concentrates in the liver in vivo, initially exhibiting a positive CT signal. Over time, the contrast agent at the tumor site specifically responds to the acidic tumor microenvironment, releasing hydrogen gas, significantly reducing the CT signal value in the tumor area, thus creating a strong contrast between negative and positive CT signals. More importantly, the relative position between the negative and positive CT contrast areas can sensitively distinguish the relative position of liver cancer from surrounding normal tissue. By recording changes in the density values ​​of the tumor area, sensitive CT detection of liver cancer is expected to be achieved. Attached Figure Description

[0019] Figure 1 This is a transmission electron microscope (TEM) schematic diagram of the novel positive and negative CT nanocontrast agent in Example 1.

[0020] Figure 2 This is a schematic diagram of the hydrated particle size of the novel positive and negative CT nanocontrast agent in Example 1 at pH=7.4.

[0021] Figure 3 This is a schematic diagram illustrating the hydrogen release capacity of Hf-MOF@AB@PVP nanoparticles in aqueous solutions at different pH values ​​(pH = 7.4, 6.5, and 5.5) in Example 2, using gas chromatography.

[0022] Figure 4 This is a schematic diagram of CT images of the novel positive and negative CT nanocontrast agent in aqueous solutions at different pH values ​​(pH = 7.4, 6.5 and 5.5) in Example 3.

[0023] Figure 5 This is a schematic diagram of a CT image of a mouse with a subcutaneous tumor, obtained using the novel positive and negative CT nanocontrast agent described in Example 4.

[0024] Figure 6 This is a schematic diagram of CT images of mice with in situ liver cancer detected by the novel positive and negative CT nanocontrast agent in Example 4. Detailed Implementation

[0025] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] Preparation and characterization of Hf-MOF@AB@PVP, a positive and negative CT nanocontrast agent for detecting liver cancer:

[0028] Step 1, Preparation of Hf-MOF nanoparticles:

[0029] Hafnium tetrachloride (HfCl4) (0.343 mmol, 110 mg) and benzoic acid (3.43 mmol, 418 mg) were dispersed in 20 mL of DMF. 2-Aminoterephthalic acid (0.343 mmol, 62.0 mg) was added to the system, and the mixture was sonicated for 3 minutes. The reaction was carried out at 120 °C for 24 h. After the reaction was complete, the solution was cooled to room temperature, and the precipitate was separated by centrifugation. The precipitate was washed twice with DMF and water, respectively. 5 mg of Hf-MOF nanoparticles were dispersed in 10 mL of water.

[0030] In the second step, ammonia borane (AB) (3.24 mmol, 100 mg) was added to the above Hf-MOF solution, stirred at room temperature, and vacuumed for 1 hour, then the reaction was continued for 24 hours to obtain the product Hf-MOF@AB nanoparticles. After the reaction was complete, polyvinylpyrrolidone (PVP) (5.0 mmol, 200 mg) was added to the solution, and the mixture was stirred for another 24 hours. The precipitate was separated by centrifugation and washed with water to obtain Hf-MOF@AB@PVP nanoparticles, a positive and negative CT nanocontrast agent for detecting liver cancer. These nanoparticles were then freeze-dried into powder under vacuum conditions.

[0031] The positive and negative CT nanocontrast agents Hf-MOF@AB@PVP nanoparticles for detecting liver cancer are CT imaging nanoprobes with Hf-MOF nanoparticles as the core, pores carrying ammonia borane (AB) and an outer layer modified with polyvinylpyrrolidone (PVP). The Hf-MOF nanoparticles are prepared from hafnium tetrachloride (HfCl4), benzoic acid, and 2-aminoterephthalic acid.

[0032] TEM characterization was performed on Hf-MOF@AB@PVP nanoparticles, a positive and negative CT nanocontrast agent used for the detection of liver cancer. Dynamic light scattering (DLS) (GB / T 29022-2021) was used to characterize the hydration kinetic diameter of the Hf-MOF@AB@PVP nanoparticles at pH 7.4. Specific results are shown below. Figure 1 As shown, Figure 2As shown. Figure 1 This is a transmission electron microscope (TEM) schematic diagram of the novel positive and negative CT nanocontrast agent in Example 1. The figure shows that the diameter of the Hf-MOF@AB@PVP nanoparticles is approximately 45.2 nm, indicating that the synthesized Hf-MOF@AB@PVP probe is a nanoparticle. Figure 2 This is a schematic diagram of the hydrated particle size of the novel positive and negative CT nanocontrast agent in Example 1 at pH = 7.4. As can be seen from the figure, the hydrodynamic diameter of Hf-MOF@AB@PVP is 91.3 nanometers, indicating that the molecular probe in this invention is at the nanoscale and is a nanoparticle.

[0033] The hydrogen release capacity of Hf-MOF@AB@PVP nanoparticles in aqueous solutions at different pH values ​​(pH = 7.4, 6.5, and 5.5) was analyzed using gas chromatography. The prepared Hf-MOF@AB@PVP nanoparticles were redispersed in 50 mL phosphate buffer solutions at different pH values ​​(pH = 5.5, 6.5, 6.5, or 7.4). The final concentration of Hf-MOF@AB@PVP was 0.5 mg / mL. The volume of hydrogen produced at different time points was measured quantitatively using gas chromatography. The hydrogen release was calculated using the ideal gas equation (pV = nRT). The results are as follows: Figure 3 As shown, Figure 3 This is a schematic diagram illustrating the hydrogen release capacity of Hf-MOF@AB@PVP nanoparticles in aqueous solutions at different pH values ​​(pH = 7.4, 6.5, and 5.5) in Example 2, analyzed using gas chromatography. The diagram shows that the Hf-MOF@AB@PVP nanoparticles released a large amount of hydrogen in acidic solutions; the stronger the acidity, the more hydrogen was released. In contrast, very little hydrogen was produced in the phosphate buffer at pH 7.4. Specifically, hydrogen was produced rapidly in phosphate buffers at pH 5.5 and pH 6.5, but almost no hydrogen was released in the phosphate buffer at pH 7.4. After 4 hours, the ammonia borane was almost completely decomposed, and the amount of hydrogen released did not increase further in the phosphate buffers at pH 5.5 and pH 6.5. The hydrogen generation rate was fastest in the phosphate buffer at pH 5.5, followed by the phosphate buffer at pH 6.5, but almost no hydrogen was generated in the phosphate buffer at pH 7.4. This indicates that Hf-MOF@AB@PVP nanoparticles can generate hydrogen gas in an acidic environment, and the more acidic the solution, the more hydrogen gas is released. Therefore, the results show that Hf-MOF@AB@PVP nanoparticles have the potential to release hydrogen gas in response to the acidic tumor microenvironment.

[0034] CT images of Hf-MOF@AB@PVP nanoparticles, used for detecting liver cancer, in phosphate buffer at different pH values ​​(pH = 7.4, 6.5, and 5.5):

[0035] Hf-MOF@AB@PVP nanoparticles were redispersed in phosphate buffer (2 mL) at different pH values ​​(pH = 5.5, 6.5, 6.5, or 7.4) to achieve a final concentration of 0.5 mg / mL. CT imaging was performed at different time points using a dual-energy CT scanner (100 mAs, 70 / Sn 150 kV). The results are shown in [Figure number missing]. Figure 4 As shown, Figure 4 This is a schematic diagram of CT images of the novel positive and negative CT nanocontrast agent in Example 3 in aqueous solutions at different pH values ​​(pH = 7.4, 6.5, and 5.5). As can be seen from the figures, the Hf-MOF@AB@PVP nanoparticles exhibit significant positive CT enhancement performance. Over time, at pH 5.5 and 6.5, the negative CT signal gradually increases, and the CT value decreases significantly. At pH 5.5 and 6.5, positive CT contrast images gradually disappear, but there is little change at pH 7.4. The results indicate that the Hf-MOF@AB@PVP nanocontrast agent has the ability to sensitively respond to acidic environments and generate positive and negative CT signal contrast.

[0036] Application of Hf-MOF@AB@PVP nanoparticles, a positive and negative CT contrast agent for detecting liver cancer, in mouse in vivo liver cancer: Huh7 cells were cultured in DEME medium (9% fetal bovine serum, 1% penicillin-dextrose antibody) for 48 h, then digested with trypsin, centrifuged, and the supernatant was discarded before being uniformly dispersed in the culture medium. Balb / c nude mice (6 weeks old, male, 10 mice) were subcutaneously injected with approximately 200 μL of Hf-MOF@AB@PVP nanoparticles. 6 The Huh 7 cells were obtained using the above method.

[0037] Approximately 7-10 days later, the tumor volume exceeds 100 mm. 3 In vivo CT imaging experiments were conducted on a subcutaneous liver cancer tumor model: 100 μL of an aqueous solution containing Hf-MOF@AB@PVP (10000 ppm) was injected directly into the tumor of tumor-bearing mice, and CT images were performed before and after injection; CT images at different time points (Pre, 10 min, 15 min, 30 min, 60 min, 120 min) were compared, and the results are shown in [Figure number missing]. Figure 5 As shown, Figure 5This is a schematic diagram of CT images of mice with subcutaneous tumors, using the novel positive and negative CT nanocontrast agents described in Example 4. As can be seen from the image, after injecting Hf-MOF@AB@PVP nanoparticles into the tumor, a positive CT enhancement signal was immediately observed in the tumor tissue within the region of interest (the gray dashed circle). Over time (0-120 min), a black area appeared in the tumor tissue and gradually increased in size; this was caused by hydrogen gas released from the acidic tumor tissue by the Hf-MOF@AB@PVP nanoparticles. More importantly, the significant contrast and relative position of the positive and negative CT signal areas facilitate sensitive tumor detection. This demonstrates that Hf-MOF@AB@PVP nanoparticles can accurately detect subcutaneous hepatocellular carcinoma tumors in mice.

[0038] Precise detection of an orthotopic hepatocellular carcinoma model in mice: 200 μL (approximately 1 million cells) of the prepared Huh7 cells were injected into the livers of 10 male mice (6 weeks old). Approximately 7-10 days later, in vivo CT imaging of the orthotopic hepatocellular carcinoma model was performed: 100 μL of an aqueous solution containing Hf-MOF@AB@PVP (25000 ppm) was injected into the tail vein of tumor-bearing mice, and CT images were taken before and after injection; CT images at different time points (Pre, 120 min, 180 min, 240 min, 300 min, 360 min) were compared. Results are shown below. Figure 6 As shown, Figure 6 This is a schematic diagram of CT images of mice bearing hepatocellular carcinoma using the novel positive and negative CT nanocontrast agent in Example 4. The transverse CT images of the mice show an increase in CT values ​​of the liver 120 minutes after injection. Over time, negative CT signals begin to appear and increase around the positive CT contrast signals. This is because Hf-MOF@AB@PVP encounters the acidic environment of hepatocellular carcinoma and releases H2, generating negative CT contrast in the process. The difference between the positive and negative CT signals is as high as 216.3 HU. More importantly, the relative positions of the positive and negative CT signals are more helpful in determining the relative positions of hepatocellular carcinoma and normal tissue. This demonstrates that Hf-MOF@AB@PVP nanoparticles can accurately detect orthotopic hepatocellular carcinoma in mice.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing positive and negative CT nanocontrast agents for detecting liver cancer, characterized in that, Includes the following steps: The first step involves dispersing hafnium tetrachloride and benzoic acid in DMF at a molar ratio of 1:8~20, adding 2-aminoterephthalic acid to the above system, dissolving by sonication, with a molar ratio of 2-aminoterephthalic acid to hafnium tetrachloride of 1:1~3, reacting for 1~10 minutes at a temperature of 60~200℃ for 24~72 h to obtain Hf-MOF nanoparticles, which are then dispersed in water to a concentration of 0.1~10 mg / mL. The second step involves adding ammonia borane to the above Hf-MOF solution, with a molar ratio of ammonia borane to hafnium tetrachloride of 7~15:

1. The mixture is stirred at room temperature and evacuated for 0.1~2 hours, and then the reaction continues for 24~72 hours. After the reaction is complete, polyvinylpyrrolidone is added to the solution, with a molar ratio of polyvinylpyrrolidone to hafnium tetrachloride of 7~20:

1. The mixture is stirred for another 24~72 hours to obtain positive and negative CT nanocontrast agents for detecting liver cancer. The positive and negative CT nanocontrast agents used for detecting liver cancer are CT imaging nanoprobes with Hf-MOF nanoparticles as the core, pores carrying ammonia borane and an outer layer modified with polyvinylpyrrolidone; the Hf-MOF nanoparticles are prepared from hafnium tetrachloride, benzoic acid, and 2-aminoterephthalic acid.

2. The method for preparing positive and negative CT nanocontrast agents for detecting liver cancer according to claim 1, characterized in that, The molar ratio of hafnium tetrachloride to benzoic acid is 1:

10.

3. The method for preparing positive and negative CT nanocontrast agents for detecting liver cancer according to claim 1, characterized in that, The molar ratio of 2-aminoterephthalic acid to hafnium tetrachloride is 1:

1.

4. The method for preparing positive and negative CT nanocontrast agents for detecting liver cancer according to claim 1, characterized in that, The molar ratio of ammonia borane to hafnium tetrachloride is 9.4:

1.

5. The method for preparing positive and negative CT nanocontrast agents for detecting liver cancer according to claim 1, characterized in that, The molar ratio of polyvinylpyrrolidone to hafnium tetrachloride is 14.6:

1.

6. The use of a positive and negative CT nanocontrast agent for detecting liver cancer prepared by any one of claims 1 to 5 in the preparation of liver cancer detection reagents.

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