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Preparation method of DNA nano flowers drug for targeted regulation of browning of white fat

A nanoflower, targeted technology, applied in nanomedicine, nanotechnology for materials and surface science, nanotechnology, etc., can solve problems such as life-threatening patient safety, poor obesity effect, and treatment failure, and achieve simple operation. , The effect of improving solubility and targeting, and high safety

Active Publication Date: 2021-04-02
安徽凯博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, current obesity-fighting strategies are ineffective and show significant inter-individual variability
At the same time, most anti-obesity drugs often cause serious side effects, which lead to treatment failure, and even threaten the life safety of patients in severe cases.

Method used

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  • Preparation method of DNA nano flowers drug for targeted regulation of browning of white fat
  • Preparation method of DNA nano flowers drug for targeted regulation of browning of white fat
  • Preparation method of DNA nano flowers drug for targeted regulation of browning of white fat

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Preparation process of DNA nanomedicine NFA.

[0040] The preparation process figure of DNA nano-medicine of the present invention, as figure 1 As shown, the details are as follows:

[0041] 1. Ligation: 5' phosphorylated DNA template (10 μM, 4.5 μL) (shown in SEQ ID NO:2) containing adipocyte aptamer targeting sequence and primer (10 μM, 4.5 μL) (SEQ ID NO :4) mixed in a final volume of 27 μL DNA ligase buffer (5 mM Tris-HCl, 1 mM MgCl 2 , 0.1 mM ATP and 1 mM dithiothreitol), annealing (keep at 95°C for 5 min, then slowly cool down to room temperature within 2 h). The annealed product was ligated with T4 DNA ligase (40 U / μL, 3 μL) at 25 °C for 2 h to form a circularized DNA template.

[0042] 2. Polymerization: the concentration of circularized DNA template is 0.6-0.8 μM, the concentration of phi29 DNA polymerase is 8-12U / μL, the concentration of BSA is (0.04-0.06)%, and the polymerase buffer is composed of 50 mM Tris- HCl, 10 mM (NH 4 ) 2 SO 4 , 10mM ...

Embodiment 2

[0047] The condition optimization of embodiment 2 DNA nano flower preparation

[0048] The DNA nanoflowers constructed in this example use adipocyte-specific nucleic acid aptamers as RCA amplification templates, successfully synthesized DNA nanoflowers with a dense layered structure, and have the effect of targeting adipocytes. Optimization of reaction conditions during the preparation of DNA nanoflowers such as figure 2 As shown, the optimal ratio (1:0; 0:1; 2:1; 1:1; 1:2) of the combination of primers and circular templates for rolling circle amplification reactions was studied by Page, and the results showed that the primers When the concentration ratio of cyclic template and cyclic template is 1:1, the binding condition is good. At the same time, the concentration of dNTP (0.05-0.30mmol / L) and phi29 enzyme (0.10-0.35U / μL) and the reaction system were optimized, such as figure 2 B. figure 2 C agarose electrophoresis results showed that the amplification was good when ...

Embodiment 3

[0049] Formation and characterization of embodiment 3DNA nanoflowers

[0050] image 3 The results show that with the progress of the rolling circle amplification reaction, the particle size of the DNA nanoflowers gradually increases and has a monodisperse sheet structure, which proves that the particle size of the DNA nanoflowers is adjustable and has a certain correlation with the rolling circle time , also provides a good basis for allicin loading and targeting. For the first time, we used nuclear magnetic resonance technology to track and verify the formation process of DNA nanoflowers, because as the rolling circle amplification proceeds, the molecular weight of the rolling circle amplification products gradually increases and stays in the pores, so it cannot be detected in the later stage of agarose gel electrophoresis. To characterize the progress of this reaction. Figure 4 The results showed that as the rolling circle amplification proceeded, the two signal peaks a...

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Abstract

The invention discloses a DNA nano flowers drug for targeted regulation of energy metabolism as well as a preparation method and application thereof. The drug is composed of fat cell targeted DNA NF (Nano Flowers) and Allicin A (Allicin). The Allicin acts with nucleic acid through multiple interaction forces and is combined on the surface of the DNA NF; and the generated DNA nanoflower has fat cell targeting property and efficient Allicin loading capacity. The DNA NF drug disclosed by the invention has excellent targeting property on fat cells, and compared with a single-stranded DNA aptamer,the formed DNA NF has higher targeting ability and higher stability on fat cells and can specifically enter the fat cells; and the Allicin has excellent capacity of regulating energy metabolism and can regulate browning of white fat, and after the two components are combined, the Allicin can be specifically delivered into the fat cells. Therefore, the constructed DNA NF drug can achieve the aim oftargeting fat tissues to accurately regulate energy metabolism and has high biosecurity.

Description

technical field [0001] The invention belongs to the technical field of biological nano-medicines, and in particular relates to a DNA nano-medicine for targeting and regulating the browning of white fat, a preparation method and an application thereof. Background technique [0002] Obesity has been recognized by the World Health Organization as the most serious public health disease in the world, which seriously affects people's quality of life and induces various major diseases and complications. At present, the clinical treatment strategy and clinical research for obesity are mainly carried out by controlling the energy intake, absorption and metabolism of the body. However, current anti-obesity strategies are not effective and there are significant individual differences. At the same time, most anti-obesity drugs often cause serious side effects, which lead to treatment failure, and even threaten the life safety of patients in severe cases. Therefore, the development of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/54A61K31/255A61P3/04B82Y5/00B82Y30/00B82Y40/00
CPCA61K31/255A61K47/549A61P3/04B82Y5/00B82Y30/00B82Y40/00
Inventor 许文涛贺晓云黄昆仑陈旭兰欣悦
Owner 安徽凯博生物科技有限公司
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