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Preparation and evaluation of novel RGDF-targeting ruthenium polypyridine coordination compound vector

A bipyridine and targeting technology, which is applied in the field of preparation and evaluation of new ruthenium polypyridine complex carriers targeted by RGDF, can solve the problems of cells that cannot pass through the cell membrane

Inactive Publication Date: 2017-11-07
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, siRNA has many disadvantages, such as being negatively charged so that it cannot pass through the cell membrane and enter the cell. The solution to such problems becomes the key to the use of RNAi to achieve gene therapy.

Method used

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  • Preparation and evaluation of novel RGDF-targeting ruthenium polypyridine coordination compound vector
  • Preparation and evaluation of novel RGDF-targeting ruthenium polypyridine coordination compound vector
  • Preparation and evaluation of novel RGDF-targeting ruthenium polypyridine coordination compound vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] The preparation of embodiment 1.Ru-CO-RGDF

[0103] Ru-CO-RGDF preparation route such as figure 1

[0104] Synthesis of Boc-Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl

[0105] The main chain of arginine and aspartic acid is protected by Boc at the amino terminus, the carboxyl terminus of glycine and benzoic acid is protected by OBzl, and the side chain is protected by a suitable protecting group (tert-butoxycarbonyl) L-amino acid, liquid phase method The peptide was connected to obtain two fully protected dipeptides: Boc-Arg(Tos)-Gly-OBzl and Boc-Asp(OBzl)-Phe-OBzl. After removing the protection of OBzl by hydrogenolysis and the protection of Boc by acidolysis, respectively, the peptide was connected with the liquid phase method to obtain the fully protected tetrapeptide. Under the condition of ice bath, dissolve 170 mg of Boc-Arg(Tos)-Gly-Asp(OBzl)-Phe-OBzl with 5 ml of HCl-dissolved ethyl acetate solution, stir the reaction, and monitor the reaction with a TLC plate. After t...

example 2

[0116] Example 2 Determination of the loading capacity of survivin-siRNA in Ru-CO-RGDF / survivin-siRNA

[0117] Prepare samples

[0118] Use RNA dilution buffer to prepare different concentrations of Ru-CO-RGDF 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg / mL, and different ratios of protamine mixed solutions, mix well, and stand at room temperature for 10 minutes , were mixed with 2 μL of survivin-siRNA, beat repeatedly, and stood for 10 minutes. Then add an appropriate volume of 5×Loading Buffer, vortex and mix well, and add 20 μL of the above solution to the prepared agarose gel sample well with a protein sample pipette tip.

[0119] Experimental procedure

[0120] Weigh 0.5g of agarose into a 100mL beaker, add 5mL of TBE (10×) and 45mL of deionized water, heat in a microwave oven on medium-high heat for 2min to dissolve the agarose. After cooling down slightly, add 8 μL of ethidium bromide solution (10 mg / mL), stir well, pour into the mold (washed in advance and rinse with ...

Embodiment 2

[0123] Example 2 Determination of the nanostructure of Ru-CO-RGDF and Ru-CO-RGDF / survivin-siRNA

[0124] experimental method

[0125] Take 10 μL of Ru-CO-RGDF aqueous dispersion with a concentration of 625 μg / mL, prepare Ru-CO-RGDF / protamine aqueous dispersion according to the volume ratio of 1:8, then mix 2 μL of siRNA and appropriate calf thymus DNA, take 20 μL of loaded survivin-siRNA and unloaded pure carrier were dropped onto the carbon support membrane (copper grid), and dried in an oven at 37°C. Each sample was observed under a transmission electron microscope (TEM, JEM-1230, JEOL, Japan) and photographs were taken. Then 20 μL of each was dropped onto a glass slide, and left to air dry at room temperature. Each sample was observed under a scanning electron microscope (JSM-6360LV, JEOL, JAPAN) and photographs were taken.

[0126] Experimental results

[0127] from Figure 11 From the scanning electron microscope and transmission electron microscope images, we can se...

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Abstract

The invention discloses preparation methods of a gene nanometer vector (4'-methyl-2,2'-bipyridine-4-amide-RGDDF)bis(2,2'-bipyridine)ruthenium dichloride(Ru-CO-RGDF) and a Ru-CO-RGDF / survivin-siRNA gene delivery system, wherein a concentration ratio of a gene nanometer vector Ru-CO-RGDF to protamine in the Ru-CO-RGDF / survivin-siRNA gene delivery system is 1:8 ,the concentration of Ru-CO-RGDF is 0.21875 [mu]g / [mu]L, a ratio of surviving-siRNA to a DNA complex is 2:1, and the Ru-CO-RGDF / survivin-siRNA gene delivery system has effects of slow release and targeting. According to the present invention, the antitumor activity of the complex and the gene silencing efficiencies at the mRNA level and the protein level are evaluated by using the breast cancer cell line MCF-7 as the model, wherein the results show that the Ru-CO-RGDF / surviving-siRNA gene delivery system has excellent antitumor activity and excellent gene silencing efficiency compared to various control groups; and by using the mice bearing S180 ascitic tumor as the model to evaluate the antitumor activity of the Ru-CO-RGDF / surviving-siRNA gene delivery system, the results show that the Ru-CO-RGDF / surviving-siRNA group has the antitumor effect.

Description

technical field [0001] The present invention relates to ruthenium polypyridine complex (4'-methyl-2,2'-bipyridine-4-amide-RGDF) bis(2,2'-bipyridine) ruthenium dichloride (Ru) modified by sequence peptide RGDF -CO-RGDF) and the Ru-CO-RGDF / survivin-siRNA gene delivery system were constructed, and the anti-tumor activity and gene silencing efficiency were evaluated in vivo and in vitro. This patent mainly uses RGDF modification to enhance the targeting of ruthenium polypyridine complexes, increasing the concentration at the target site, thereby reducing side effects and improving the therapeutic index; Background technique [0002] At present, cancer has become the world's first fatal disease, and cancer treatment is one of the main goals of current scientific researchers. With the development of medicine and nanotechnology, gene therapy for cancer becomes possible. Gene therapy is a method of introducing exogenous target genes into the body through vectors or other means and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/42A61K48/00C07F15/00C12Q1/68C12Q1/02
CPCY02P20/55A61K47/42A61K48/005C07F15/0053C12Q1/6886C12Q2600/158
Inventor 崔纯莹钟荣荣
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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