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Multi-mode ferritin nanometer contrast agent as well as preparation method and application thereof

A nano-contrast agent and ferritin technology, which is applied in the field of biological and medical nano-materials, can solve the problems of incomplete detection information and missed detection, and achieve the effects of comprehensive and accurate diagnosis, simple process and strong repeatability

Active Publication Date: 2018-09-04
JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to address the deficiencies in the sentinel lymph node detection technology, mainly the limitations of a single imaging diagnosis, which may cause missed detection due to incomplete detection information, and propose a multi-modal ferritin nano-contrast agent and its preparation method, the multimodal ferritin nano-contrast agent can be used for sentinel lymph node blue dyeing and living non-invasive imaging imaging of patients through subcutaneous injection, so as to realize comprehensive and accurate diagnosis, thereby promoting the development of clinical diagnostics

Method used

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  • Multi-mode ferritin nanometer contrast agent as well as preparation method and application thereof
  • Multi-mode ferritin nanometer contrast agent as well as preparation method and application thereof
  • Multi-mode ferritin nanometer contrast agent as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Horse spleen ferritin was selected as the biological nano-template, Prussian blue nanoparticles were used as the contrast agent for MRI and photoacoustic imaging, and near-infrared fluorescent dye (IR-782) was coupled on the ferritin shell as the fluorescent probe part to prepare multimodal iron Protein nano-contrast agent, the specific method is as follows:

[0050] (1) 2.22g PVP (K-30) was ultrasonically dissolved in 50mL pure water, 200mg ferrous chloride tetrahydrate (FeCl 2 4H 2 O) be dissolved in 30mL pure water, weigh 0.33g potassium ferricyanide (K 3 [Fe(CN) 6 ]) was dissolved in 20 mL of pure water. FeCl 2 4H 2 The O solution was dropped into the PVP aqueous solution dropwise; under magnetic stirring at 0°C, the K 3 [Fe(CN) 6 ] solution, after the dropwise addition, continue to react with magnetic stirring at 0°C for 20 minutes. Acetone and the reaction solution were mixed at a volume ratio of 2:1, centrifuged and washed three times, and washed with pur...

Embodiment 2

[0055] Characterization of Multimodal Ferritin Nanocontrast Agents:

[0056] (1) Take 10 μL of the multimodal ferritin nano-contrast agent prepared in Example 1 and drop it on the copper grid. Stain for 1 minute, and finally observe the sample morphology under a transmission electron microscope (TEM, JEM-2100, Japan JEOL), the results are as attached figure 2 As shown, it can be seen from the right half of the figure that the SPB is a nearly spherical shape with a size of 2-8nm, and the distribution is relatively regular. figure 2 It can be seen in the left half of the figure that the ferritin negatively stained with phosphotungstic acid has a well-shaped shell structure.

[0057] (2) The ferritin aqueous solution with a ferritin content of 1 μg / mL and the multimodal ferritin nanocontrast agent aqueous solution were used to detect the absorption spectrum in the range of 300-800 nm with a UV-visible spectrophotometer (UV-3600, Shimadzu, Japan). The results are attached im...

Embodiment 3

[0060] Validation of macrophage targeting of multimodal ferritin nanocontrast agents:

[0061] (1) Prepare phosphate buffered saline (PBS) with pH 7.4; methanol and acetone 1:1 and store at 4°C for later use; prepare 95% ethanol; prepare 1% commercially available bovine serum albumin (BSA) and store at 4°C for later use;

[0062] (2) Place the sterilized cover glass in a 6-well plate, and inoculate the mouse macrophage RAW264.7 (1×10 5 well), after the cell fusion rate reaches 50%, wash the cells with PBS, fix with pre-cooled methanol-acetone mixture for 15 minutes, and dry in the fume hood for 5 minutes;

[0063] (3) Add 1 mL of commercially available peroxidase blocker, incubate at 37°C for 30 minutes, and wash 3 times with PBS;

[0064] (4) Add 1% BSA, block at 37°C for 30 minutes, wash with PBS 3 times;

[0065] (5) Add 1 mL of multimodal ferritin nanocontrast agent, incubate at 37°C for 1 hour, and wash with PBS 3 times;

[0066] (6) After adding commercially available...

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Abstract

The invention discloses a multi-mode ferritin nanometer contrast agent based on ferritin and a preparation method thereof. Horse spleen ferritin with the targeted performance on macrophage surface scavenger receptors 5 is used as a biological nanometer template (shell); prussian blue nanometer particles are used as cores to realize MRI and photoacoustic imaging; next, fluorescent dye IR-782 is coupled to be used as a fluorescence probe part; the multi-mode ferritin nanometer contrast agent of a shell core structure is prepared. The multi-mode ferritin nanometer contrast agent combines the photoacoustic imaging, fluorescence imaging and MRI trimodal imaging, can be used for sentinel lymph node photoacoustic / MRI / fluorescence trimodal radiography before breast cancer, can also be used for performing blue dyeing on the sentinel lymph node before breast cancer by using the dyeing effect of the prussian blue, realizes the comprehensive and precise diagnosis of the breast cancer lymphatic metastasis, and promotes the development of clinic medicine and image diagnostics.

Description

technical field [0001] The invention relates to a multi-modal ferritin nano-contrast agent for lymph node staining and imaging diagnosis and its preparation method and application, belonging to the technical field of biological and medical nano-materials. Background technique [0002] From a clinical point of view, the sentinel lymph node (SLN) refers to the first lymph node where the primary tumor occurs in a specific part of an organ, and the tumor cells first invade. Sentinel lymph node biopsy is a reliable method to assess the metastatic status of axillary lymph nodes in early breast cancer. Whether to perform further lymph node dissection is determined according to whether there is lymph node metastasis. In theory, positive sentinel lymph node metastasis can indicate tumor lymph node metastasis in this region. The currently commonly used sentinel lymph node detection methods include staining method, radionuclide method and fluorescent dye method. [0003] The tracers...

Claims

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Application Information

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IPC IPC(8): A61K49/00A61K49/14A61K49/18A61K49/22B82Y5/00B82Y15/00
CPCA61K49/0002A61K49/0056A61K49/006A61K49/0093A61K49/143A61K49/1818A61K49/225B82Y5/00B82Y15/00
Inventor 唐金海张薇丁莉吴旸张建何蕴藉朱溢智陈秀
Owner JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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