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Targeted micro-bubble contrast agent and preparation method thereof

A contrast agent, targeting technology, applied in pharmaceutical formulations, echo/ultrasonic imaging agents, gene therapy, etc., can solve the problem of short contact time between targeted microbubbles and receptors, and limited number of targeted microbubble ligands-receptors , the gene transfection rate cannot be reached, etc., to achieve the effect of reducing recognition and phagocytosis, high activity, and good water solubility

Inactive Publication Date: 2013-08-07
THE THIRD XIANGYA HOSPITAL OF CENT SOUTH UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the in vivo environment, due to the fluidity of the blood, the contact time between the targeting microvesicles and the receptors is short, and the number of ligand-receptor pairs formed on the targeting microvesicles is in

Method used

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  • Targeted micro-bubble contrast agent and preparation method thereof
  • Targeted micro-bubble contrast agent and preparation method thereof
  • Targeted micro-bubble contrast agent and preparation method thereof

Examples

Experimental program
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Example Embodiment

[0029] Example 1: Synthesis of DSPE-PEG-c (RGDyK) targeting microbubble contrast agent of the present invention

[0030] The DSPE-PEG-c (RGDyK) targeting microbubble contrast agent of the present invention is such as figure 2 As shown, it was prepared by the following steps:

[0031] (1) Synthesis of DSPE-PEG-c (RGDyK): Add 10 mmoles of c(RGDyK) and 10 mmoles of DSPE-PEG2000, 30 mmoles of DIEA (N'N-diisopropylethylamine) to 15mL of acetonitrile Then add 20 millimoles of HBTU and react at room temperature for 30 minutes. The cyclic pentapeptide c (RGDyK) -NH 2 The product DSPE-PEG-c(RGDyK) is obtained by covalent coupling between the sulfonate group and the -OH group of DSPE-PEG, the solvent is evaporated under reduced pressure, column chromatography is analyzed by high performance liquid chromatography, and the purity is> 95%, the molecular structure identified by mass spectrometry is correct;

[0032] (2) Suspend 40 mg DSPE, 4 mg DPPA, 5 mg poloxamer in 5 ml double-distilled water ...

Example Embodiment

[0047] Example 4: Comparative detection of in vitro targeting ability

[0048] 1) Cell culture: human hepatoma cell line HepG2 and normal hepatocyte L-O2 are cultured in DMEM medium containing 10% newborn fetal calf serum, 100U penicillin and streptomycin, at 37℃, 5% CO 2 Culture in a cell incubator. Take 1×10 5 Cells in logarithmic growth phase are plated on a 24-well plate for culture.

[0049] 2) Comparative experiment of targeting binding ability between targeted microbubbles and HepG2 cells

[0050] ① Rosette formation experiment: Taking normal hepatocytes L-O2 cells as the control group, take 100μL of HepG2 cell suspension and add 20μL of two kinds of microbubble contrast agents respectively. After reacting at room temperature for 10 minutes, observe the combination of cells and microbubbles under light microscope . Take the number of microbubbles in the wreath (referred to as the number of microbubbles)> Five wreaths are standard wreaths. Counting the average number of stand...

Example Embodiment

[0060] Example 5: In vivo gene transfection experiment of carrying CD / TK double suicide gene plasmid combined with ultrasound for subcutaneous transplantation tumor in liver cancer mice

[0061] (1) Plasmid extraction

[0062] The plasmid pEGFP-KDRP-CD / TK (constructed in the previous stage) containing the enhanced green fluorescent protein sequence was transformed into Escherichia coli DH5α, and ampicillin-resistant single clones were selected, amplified by Escherichia coli, and purified plasmids were extracted and digested. 0.8% agarose gel electrophoresis for identification.

[0063] (2) Establishment of subcutaneous transplantation tumor model of mouse liver cancer

[0064] Each mouse was injected with 0.1 mL subcutaneously on the back of the lumbar and ribs, and then observed the growth of the mouse. The subcutaneous nodule diameter was 0.5-1.0 cm as the standard for tumor formation.

[0065] (3) Targeted gene transfection experiment of mouse liver cancer model

[0066] 1) Detection...

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Abstract

The invention relates to a targeted micro-bubble contrast agent. Cyclic peptide ligands c (RGDyK) and DSPE-PEG are coupled through covalent bonds to synthesize DSPE-PEG-C (RGDyK), so that PEG derivative (PEG-DSPE) connecting arms are inserted between the ligands and micro-bubbles; and then, the targeted micro-bubble contrast agent with the connecting arms is prepared by ultrasonic oscillation homogenization and in a high-speed shearing method. The targeted micro-bubble contrast agent with the PEG derivative connecting arms inserted between the ligands and the micro-bubbles is advantageous for enhancing adhesion between the ligands and receptors, and capable of effectively increasing formed targeted micro-bubble ligand-receptor pairs, so that bonding between the micro-bubbles and target organs is closer and the transfection efficiency of carrier genes, namely the micro-bubbles, can be further improved.

Description

technical field [0001] The invention relates to a targeting microbubble contrast agent and a preparation method thereof. Background technique [0002] As a safe carrier in gene therapy, microbubble contrast agent can effectively carry out targeted gene transfection under ultrasound irradiation, and has become a research hotspot in the field of tumor gene therapy. After the microbubble contrast agent carrying the target gene is injected intravenously, the target tissue is given certain conditions of ultrasound irradiation, which can cause the microbubble in the blood vessel to burst and release the gene, and simultaneously produce "cavitation effect" and "sonic hole effect". It can increase capillary permeability and cell membrane permeability, thereby promoting the release of a large number of genes into target cells. It is safe, non-invasive, and can act on deep tissues and organs. It is a new type of gene targeted delivery technology. Has a broad space for development. ...

Claims

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Application Information

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IPC IPC(8): A61K49/22A61K48/00A61P35/00
Inventor 周平李家乐高峰
Owner THE THIRD XIANGYA HOSPITAL OF CENT SOUTH UNIV
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