Phospholipid-coated silver / manganese dioxide coprecipitation particle MRI contrast agent and preparation method and application thereof

Abstract

The invention provides a phospholipid coated silver / manganese dioxide coprecipitation particle MRI contrast agent and a preparation method and application thereof. The prepared contrast agent has efficient MRI contrast performance, and compared with a traditional gadolinium-based contrast agent, the biological safety is higher, and the risk of heavy metal ion leakage is avoided; the contrast agent has the advantages that the contrast agent is high in targeting ability and accurate in targeting ability, phospholipid layer modified cRGD targeting molecules can specifically recognize high-expression integrin alpha v beta 3 on the surfaces of tumor cells, enrichment of the contrast agent in tumor tissues is realized, and imaging resolution and focus positioning accuracy are improved. The nano-silver / manganese dioxide composite material has good biocompatibility and multifunctional diagnosis and treatment integrated potential, creatively combines a silver / manganese dioxide coprecipitation core and a phospholipid wrapping structure, has good magnetic resonance imaging (MRI) contrast performance and biocompatibility, and shows remarkable application potential in the medical imaging fields of tumor targeted imaging, disease diagnosis and the like. And a new direction is provided for the development of MRI contrast agents.

Description

Technical Field

[0001] This invention relates to the field of medical imaging technology, specifically to a phospholipid-encapsulated silver / manganese dioxide coprecipitated MRI contrast agent, its preparation method, and its application. Background Technology

[0002] Magnetic resonance imaging (MRI) is a commonly used non-invasive imaging technique in clinical practice, relying on contrast agents to enhance tissue contrast. Traditional gadolinium (Gd)-based contrast agents have limited application in patients with renal insufficiency due to the potential toxicity of gadolinium ions (such as the risk of renal systemic fibrosis). Manganese-containing nanomaterials (such as MnO2) serve as T1 contrast agents, enhancing imaging signals through the Mn²⁺ ion relaxation effect and exhibiting higher biocompatibility; however, single MnO2 nanoparticles suffer from poor targeting and insufficient in vivo stability. The surface plasmon resonance properties of silver (Ag) nanoparticles can synergistically enhance MRI signals, but their biocompatibility requires further optimization. Combining Ag and MnO2 to form co-precipitated particles, combining the advantages of both, while simultaneously utilizing phospholipid encapsulation technology to improve particle dispersibility, biocompatibility, and targeting modification potential, has become a research hotspot for novel MRI contrast agents. Summary of the Invention

[0003] To address the above problems, this invention proposes an MRI contrast agent containing phospholipid-encapsulated silver / manganese dioxide coprecipitates and its preparation method.

[0004] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing an MRI contrast agent with phospholipid-encapsulated silver / manganese dioxide co-precipitated particles includes the following steps: (1) Synthesis of Ag@MnO2 hybrid core: Prepare 0.3M polyallylamine hydrochloride (PAH) solution and dissolve it in a volumetric flask; prepare 0.04M AgNO3 and 0.02M KMnO4 solution respectively, dissolve them, make up to volume, mix them well and set aside. Add the mixture dropwise to the PAH solution (v:v=8:1), react at 900 r / min for 15 min under ice bath conditions, and dialyze through a 20KD dialysis bag for 4 h to remove unreacted substances, thus obtaining the Ag@MnO2 core; (2) Synthesis of Ag@MnO2-sis-L: Take 1 mL of the dialyzed Ag@MnO2 solution, add 20 μL of 0.01M citric acid solution to adjust the Ag@MnO2 solution to acidity; add an appropriate volume of siRNA and incubate for 10 min; mix SM-102, CHO-HP, HSPC and DMG-PEG2000 (molar ratio = 18:16:4:1) and dissolve in an appropriate amount of anhydrous ethanol. Inject the ethanol solution of lipids into the Ag@MnO2 solution, control the temperature at 55℃, rotate at 600 r / min, react for 30 min, dialyze in a dialysis bag with a molecular weight cutoff of 20 KD for 4 h to obtain Ag@MnO2-sis-L; (3) Synthesis of Ag@MnO2-sis-cL: Add DSPE-PEG2000-cRGD to the solution obtained in step (2), and dialyze after reaction to obtain Ag@MnO2-sis-cL.

[0005] Furthermore, the solvent for the PAH solution in step (1) is ultrapure water, and the final volume is 10 mL.

[0006] Furthermore, in step (1), the amount of PAH solution added is 500 μL, and the amount of AgNO3 and KMnO4 mixed solution added is 4 mL.

[0007] Furthermore, in step (2), the concentration of the citric acid solution is 0.01M, and the pH is adjusted to 4.0-6.0.

[0008] Furthermore, in step (2), the volume of ethanol in the lipid ethanol solution is 100 μL.

[0009] Furthermore, in step (3), the amount of DSPE-PEG2000-cRGD added is 1 / 10 of the molar amount of DMG-PEG2000, and the reaction time is 30 min.

[0010] Furthermore, the prepared phospholipid encapsulates silver / manganese dioxide coprecipitated particles of MRI contrast agent (Ag@MnO2-sis-cL). The contrast agent has a core-shell structure, with the core being Ag@MnO2 coprecipitated particles and the outer shell being a phospholipid layer containing siRNA and SM-102, CHO-HP, HSPC, DMG-PEG2000, and DSPE-PEG2000-cRGD.

[0011] Furthermore, the prepared phospholipid-encapsulated silver / manganese dioxide coprecipitated particles MRI contrast agent Ag@MnO2-sis-cL are used in the preparation of tumor-targeted magnetic resonance imaging diagnostic reagents and in the application of therapeutic formulations that combine imaging and drug delivery functions.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention has high-efficiency MRI contrast performance. By utilizing the Mn²⁺ ion relaxation effect of MnO2 and the surface plasmon resonance characteristics of Ag through the Ag@MnO2 core, it significantly improves T1-weighted imaging contrast. Compared with traditional gadolinium-based contrast agents, it has higher biosafety and no risk of heavy metal ion leakage. 2. This invention has precise targeting capabilities. The cRGD targeting molecules modified with phospholipid layers can specifically recognize integrin αvβ3, which is highly expressed on the surface of tumor cells, thereby enriching the contrast agent in the tumor tissue and improving imaging resolution and lesion localization accuracy. 3. This invention has good biocompatibility. Both the phospholipid material (HSPC) and the PEGylated lipids (DMG-PEG2000, DSPE-PEG2000-cRGD) are FDA-approved pharmaceutical excipients, which reduce immunogenicity and toxic side effects, and ensure in vivo circulation stability. The phospholipid layer coating improves particle dispersibility, and the PDI is maintained at 0.23-0.25 to ensure in vivo stability. 4. This invention has the potential to integrate multiple functions for diagnosis and treatment. The core is further loaded with siRNA, and the shell is targeted modified and has phospholipid encapsulation properties, which endow it with drug delivery function, realizing integrated diagnosis and treatment and providing a new platform for precision medicine. Attached Figure Description

[0013] Figure 1 SEM images of Ag@MnO2, Ag@MnO2-sis-L, and Ag@MnO2-sis-cL prepared in this invention; Figure 2 TEM images of Ag@MnO2, Ag@MnO2-sis-L, and Ag@MnO2-sis-cL prepared in this invention; Figure 3 Nuclear magnetic resonance imaging (NMR) images of Ag@MnO2-sis-cL prepared in this invention at different time points in mice. Detailed Implementation Example

[0014] (1) Place 0.3 M PAH solution (500 μL) in an ice bath, add AgNO3 / KMnO4 mixture (4 mL) dropwise, stir for 15 min, and after dialysis, obtain Ag@MnO2 core (particle size 65.4 nm, PDI=0.089, potential -45.57 mV). (2) Take 1 mL of dialyzed Ag@MnO2 solution, add 0.01 M citric acid to adjust the pH to acidic, add siRNA, and incubate for 10 min; (3) Dissolve SM-102 (18 μmol), CHO-HP (16 μmol), HSPC (4 μmol), and DMG-PEG2000 (1 μmol) in 100 μL of ethanol, inject into Ag@MnO2 solution, react at 55℃ for 30 min, and dialyze to obtain Ag@MnO2-sis-L (particle size 80.7 nm, PDI=0.247, potential 6.54mV); (4) Add DSPE-PEG2000-cRGD (0.1 μmol), and after reaction, dialyze to obtain Ag@MnO2-sis-cL (particle size 84.4 nm, PDI=0.234, potential 4.73 mV). 1. Characterization analysis: The characterization was performed using scanning electron microscopy (SEM). Figure 1 ) and transmission electron microscopy (TEM) Figure 2 Observation showed that the Ag@MnO2 core was spherical with uniform particle size; after being coated with phospholipid, a smooth shell was formed on the surface of the particles; after targeted modification, the particle morphology did not change significantly, confirming the structural stability. 2. In vivo imaging results: In tumor-bearing mouse models, Ag@MnO2-sis-cl was injected via tail vein. T1-weighted imaging was performed using a SKYSCAN1276 small animal MRI scanner. Figure 3 The results showed that clear imaging of blood vessels throughout the body was visible 0.167 h after drug administration, and the signal intensity at the tumor site reached its peak at 0.5 h, then gradually decreased, indicating that the contrast agent rapidly accumulated in the tumor tissue and had good targeted imaging capabilities.

Claims

1. A method for preparing an MRI contrast agent with phospholipid-encapsulated silver / manganese dioxide co-precipitated particles, characterized in that: Includes the following steps: (1) Synthesis of Ag@MnO2 hybrid core: Prepare 0.3M polyallylamine hydrochloride (PAH) solution and dissolve it in a volumetric flask; prepare 0.04M AgNO3 and 0.02M KMnO4 solution respectively, dissolve them, make up to volume, mix them well and set aside. Add the mixture dropwise to the PAH solution (v:v=8:1), react at 900 r / min for 15 min under ice bath conditions, and dialyze through a 20KD dialysis bag for 4 h to remove unreacted substances, thus obtaining the Ag@MnO2 core; (2) Synthesis of Ag@MnO2-sis-L: Take 1 mL of the dialyzed Ag@MnO2 solution, add 20 μL of 0.01M citric acid solution to adjust the Ag@MnO2 solution to acidity; add an appropriate volume of siRNA and incubate for 10 min; mix SM-102, CHO-HP, HSPC and DMG-PEG2000 (molar ratio = 18:16:4:1) and dissolve in an appropriate amount of anhydrous ethanol. Inject the ethanol solution of lipids into the Ag@MnO2 solution, control the temperature at 55℃, rotate at 600 r / min, react for 30 min, dialyze in a dialysis bag with a molecular weight cutoff of 20 KD for 4 h to obtain Ag@MnO2-sis-L; (3) Synthesis of Ag@MnO2-sis-cL: Add DSPE-PEG2000-cRGD to the solution obtained in step (2), and dialyze after reaction to obtain Ag@MnO2-sis-cL.

2. The method for preparing an MRI contrast agent with phospholipid-encapsulated silver / manganese dioxide co-precipitated particles according to claim 1, characterized in that: The solvent for the PAH solution in step (1) is ultrapure water, and the final volume is 10 mL.

3. The method for preparing an MRI contrast agent with phospholipid-encapsulated silver / manganese dioxide co-precipitated particles according to claim 1, characterized in that: In step (1), the amount of PAH solution added is 500 μL, and the amount of AgNO3 and KMnO4 mixed solution added is 4 mL.

4. The method for preparing an MRI contrast agent with phospholipid-encapsulated silver / manganese dioxide co-precipitated particles according to claim 1, characterized in that: In step (2), the concentration of the citric acid solution is 0.01M, and the pH is adjusted to 4.0-6.

0.

5. The method for preparing an MRI contrast agent with phospholipid-encapsulated silver / manganese dioxide co-precipitated particles according to claim 1, characterized in that: In step (2), the volume of ethanol in the lipid ethanol solution is 100 μL.

6. The method for preparing an MRI contrast agent with phospholipid-encapsulated silver / manganese dioxide co-precipitated particles according to claim 1, characterized in that: In step (3), the amount of DSPE-PEG2000-cRGD added is 1 / 10 of the molar amount of DMG-PEG2000, and the reaction time is 30 min.

7. The phospholipid-encapsulated silver / manganese dioxide co-precipitated MRI contrast agent Ag@MnO2-sis-cl prepared according to any one of claims 1-6, characterized in that: The contrast agent has a core-shell structure, with the core being Ag@MnO2 co-precipitated particles and the outer shell being a phospholipid layer containing siRNA, SM-102, CHO-HP, HSPC, DMG-PEG2000, and DSPE-PEG2000-cRGD.

8. The application of the phospholipid-encapsulated silver / manganese dioxide coprecipitated particle MRI contrast agent Ag@MnO2-sis-cL prepared according to any one of claims 1-6 in the preparation of tumor-targeted magnetic resonance imaging diagnostic reagents and in the application of therapeutic formulations that combine imaging and drug delivery functions.

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