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Dual-targeting ultrasonic contrast agent and preparation method thereof

An ultrasonic contrast agent and dual-targeting technology, applied in echo/ultrasound imaging agents, pharmaceutical formulations, preparations for in vivo tests, etc., can solve problems such as low targeting ability, affecting gene therapy, and affecting the concentration of carried genes. Achieve the effects of improved targeting efficiency, enhanced enrichment degree, and strong adsorption

Active Publication Date: 2016-06-01
SHENZHEN PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can achieve the specific binding of microvesicles and target cells through the use of specific ligands such as monoclonal antibodies, due to the design of a single target, it is difficult to achieve an ideal concentration of microvesicles in the target tissue, thus affecting It carries the concentration of the gene in the target tissue, which in turn affects the subsequent gene therapy
[0006] In summary, the current targeted contrast agents can achieve specific binding to target cells, but single-target designed microbubbles can only bind to a single target, and are affected by the shear force of blood flow. The vesicles and the target tissue are often not tightly adhered, and the targeting ability is low
This defect is evident in contrast agents targeting breast cancer

Method used

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  • Dual-targeting ultrasonic contrast agent and preparation method thereof
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  • Dual-targeting ultrasonic contrast agent and preparation method thereof

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preparation example Construction

[0047] A preferred method of preparing the above-mentioned dual-targeted ultrasound contrast agent of the present invention comprises the following steps:

[0048] (1) Dissolve DSPC, Stearic-PEI600, DSPE-PEG2000-Biotin in chloroform;

[0049] (2) remove above-mentioned chloroform under the effect of inert gas flow, make above-mentioned DSPC, Stearic-PEI600, DSPE-PEG2000-Biotin form uniform film on container wall;

[0050] (3) Shake until transparent after adding the aqueous solution, and then pack in sealed bottles;

[0051] (4) replace the air in the bottle with perfluoropropane, and make biotinylated microbubbles after shaking;

[0052] (5) Add avidin to the biotinylated microbubbles and incubate at room temperature;

[0053] (6) Add Integrinα v beta 3 and MCP-1 biotinylated monoclonal antibody, incubated at room temperature to prepare the above dual-targeted ultrasound contrast agent.

[0054] Wherein, in step (1), the molar ratio of DSPC, Stearic-PEI600, DSPE-PEG2000-...

Embodiment 1

[0063] Embodiment 1 prepares Integrin α v beta 3 / MCP-1 dual targeting microbubbles

[0064] 1. Dissolve DSPC, Stearic-PEI600, DSPE-PEG2000-Biotin with a molar ratio of 8.5:0.5:1 in chloroform.

[0065] 2. In N 2 The chloroform is removed under the action of air flow, so that the phospholipids form a uniform film on the test tube wall.

[0066] 3. After adding an appropriate amount of 0.1M Tris buffer solution, place it in a water-bath ultrasonic oscillator and vibrate until it is transparent, then pack it into a clean vial and seal it.

[0067] 4. The air in the bottle was replaced with perfluoropropane, and placed on a mechanical shaker for 30 seconds to prepare biotinylated microbubbles.

[0068] 5. After adding it to PBS and centrifuging to wash it, take 10ml (1×10 8 / ml) microbubbles were added with 50 μg avidin and incubated at room temperature for 20 min.

[0069] 6. After centrifuging again, add excess biotinylated Integrinα at a molar ratio of 1:1 v beta 3 and M...

Embodiment 2

[0071] Example 2 Detection of Integrinα v beta 3 / MCP-1 dual-targeting microbubble targeting efficiency in vivo and in vitro

[0072] 1. Cultivate breast cancer cells (MCF-7): use DMEM medium containing 10% fetal calf serum at 37°C, 5% CO 2 , routine subculture under 100% saturated humidity conditions.

[0073] 2. Test the in vitro targeting efficiency of dual-targeted microbubbles: Integrinα v beta 3 The monoclonal antibodies of MCP-1 and MCP-1 were labeled with different fluorescent lights to prepare Integrinα respectively v beta 3 Single targeting, MCP-1 single targeting, Integrinα v beta 3 / MCP-1 dual-targeting and non-targeting control nano-microbubbles, the method is the same as in Example 1. The prepared microbubbles were added to the breast cancer cell (MCF-7) culture dish cultured in vitro, reacted at room temperature for 30min, rinsed twice with PBS, observed the target combination of microbubbles and cancer cells in each group under a fluorescence microscope...

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Abstract

The invention discloses a dual-targeting ultrasonic contrast agent and a preparation method thereof. The contrast agent is an Integrin alpha v beta 3 / MCP-1 dual-targeting micro-vesicle, the dual-targeting micro-vesicle comprises a lipid shell and a gas inner core, and the external surface of the lipid shell is connected with an Integrin alpha v beta 3 monoclonal antibody and an MCP-1 monoclonal antibody. Through the combined application of different target spots, the targeting efficiency of the contrast agent can be effectively increased, and the degree of enrichment of the carrying genes in the target tissue can be greatly increased. In addition, the contrast agent disclosed by the invention is an ultrasonic contrast agent with double functions of development and treatment, and tumor images can be accurately observed in real time through an ultrasonic imaging system; and after gene transfection is realized through high-strength ultrasonic irradiation, the tumor gene treatment effect can be achieved.

Description

technical field [0001] The invention relates to the technical field of ultrasound contrast agents, in particular to a dual-target ultrasound contrast agent and a preparation method thereof. Background technique [0002] Traditional ultrasound contrast agents are microbubbles prepared by wrapping gas with membrane materials such as phospholipids and albumin. The particle size of these gas-containing microbubbles is about several microns. They have similar characteristics to red blood cells in the body and can pass through the systemic circulation and Pulmonary circulation is mostly used clinically to show the distribution and integrity of the entire blood pool system. The most significant difference between targeted ultrasound contrast agents and ordinary contrast agents is that they can recognize specific antigenic epitopes of target tissues at the molecular level and bind firmly to them, finally enabling specific visualization of target tissues. In order to achieve this go...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K49/22A61K48/00
CPCA61K48/0008A61K49/221A61K49/223
Inventor 徐金锋刘莹莹严飞郑海荣周玉丽曾欣欣
Owner SHENZHEN PEOPLES HOSPITAL
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