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Method for preparing antitumor drug carrier by using rolling circle amplification technology

A rolling circle amplification technology, anti-tumor drug technology, applied in pharmaceutical formulations, medical preparations with inactive ingredients, etc., can solve problems such as affecting the efficiency of carriers entering cells and the biosafety of metal nanoparticles

Inactive Publication Date: 2014-01-15
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the anti-tumor research of using nanoparticles or nanostructures as anti-tumor drug carriers, polymer nanomaterials, liposomes and metal nanoparticles are used more, and there are many precedents of successful application of nano-drug delivery systems. , but it is undeniable that there are still many problems in the research of high-efficiency drug loading, targeted delivery, and multifunctional drug carriers that integrate treatment and detection. For example, the particle size distribution of liposomes may affect the carrier’s entry into cells. The efficiency of other metal nanoparticles has biosafety controversies, etc.

Method used

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  • Method for preparing antitumor drug carrier by using rolling circle amplification technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Add 20 μL (100μM) SH-DNA to 1mL 15nm gold colloid solution, shake at room temperature overnight, add 100mM PB buffer (pH7.4), the final concentration is 10mM, add aging solution (10 mM PB, 2M NaCl, pH 7.4) The final concentration of salt in the solution is 0.15M. After overnight at room temperature, the mixture solution was centrifuged at 12000 rpm / min for 15 min at 4°C and washed three times. The precipitate was resuspended in 200 μL, pH7.4 PB (10 mM, 0.15 M NaCl) solution and stored at 4°C.

[0029] Add 5 μL of 100 μM pre-circular DNA and 10 μL of 100 μM complementary sequence into a 0.5 mL centrifuge tube and mix evenly, at 90°C for 1 min, when the solution in the tube is naturally cooled to room temperature, add 3 μL of LT4 ligase and 2 μL of LT4 ligase buffer, at 25°C The ligation was carried out for more than 16 hours under ambient conditions. After the end, the enzyme was denatured at 65°C for 10 minutes, then 3 μL T4 polymerase and 6 μL T4 polymerase buffer were...

Embodiment 2

[0032] Add 20 μL (100μM) SH-DNA to 1mL 30nm gold colloid solution, shake overnight at room temperature, add 100mM PBbuffer (pH7.4), the final concentration is 10mM, add aging solution (10 mM PB, 2M NaCl, pH 7.4) after overnight at room temperature ) The final concentration of salt in the solution is 0.15M. After overnight at room temperature, the mixture solution was centrifuged at 10,000 rpm / min for 15 min at 4°C and washed three times. The precipitate was resuspended in 200 μL, pH 7.4 PB (10 mM, 0.15 M NaCl) solution, and stored at 4°C.

[0033]Add 5 μL of 100 μM pre-circular DNA and 10 μL of 100 μM complementary sequence into a 0.5 mL centrifuge tube and mix evenly, at 90°C for 1 min, when the solution in the tube is naturally cooled to room temperature, add 3 μL of LT4 ligase and 2 μL of LT4 ligase buffer, and store at 25°C The ligation was carried out for more than 16 hours. After the end, the enzyme was denatured at 65°C for 10 minutes, then 3 μL T4 polymerase and 6 μL T...

Embodiment 3

[0036] 1 mg of single-walled carbon nanotubes was dissolved in 1 mL of PBS solution at pH 7, 0.4 mL of EDC (400 mM) and NHS (100 mM) solutions were added after ultrasonication for 10 min, and shaken at room temperature for 15 min. The above solution was centrifuged at 15,000 rpm / min at 4°C for 5 min, and repeated twice to remove excess EDC and NHS. Then 20 μL of 1 mg / mL streptavidin solution was added, and the assembly was shaken overnight at room temperature. Then centrifuge at 15,000 rpm / min at 4°C for 10 min, repeat three times to remove unlinked streptavidin, then add 20 μL of biotin-amplification primer DNA (100 μM), shake at room temperature for 1 hour and then centrifuge (4°C, 15,000 rpm / min, 10min) three times. The precipitate was resuspended in 200 μL, pH7.4 PB (10 mM, 0.15 M NaCl) solution, and stored at 4 °C.

[0037] Add 5 μL of 100 μM pre-circular DNA and 10 μL of 100 μM complementary sequence into a 0.5 mL centrifuge tube and mix evenly, at 90°C for 1 min, when...

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Abstract

The invention relates to a method for preparing an antitumor drug carrier by using a rolling circle amplification technology. An amplification primer DNA is assembled on the surface of a nano-mateiral, a circular DNA amplification template and other amplification mixtures are added and then rolling circle amplification is performed, and the prepared product is a long single-stranded DNA antitumor drug carrier obtained by extension from the surface of nano-gold. The primer DNA is extended to a microsize scale by means of room temperature amplification and can be in comparison with a non-amplified gold-DNA control group by means of agarose electrophoresis; after amplification, the DNA becomes longer, resulting in delay in the electrophoresis experiment. Besides, in an atomic force microscopy image, the amplified long-chain DNA extending from the surface of the nano-material can be intuitively observed. The nano-scale biological composite structure constructed by the rolling circle amplification technology can be used for the researches on novel antitumor drug carriers.

Description

technical field [0001] The invention belongs to the field of functionalization and application of nanomaterials, and relates to a rolling circle amplification signal amplification technology performed on the surface of nanomaterials. The biological composite structure constructed can be used for the development and research of new anti-tumor drug carriers. Background technique [0002] Malignant tumors are one of the important diseases that seriously endanger human health. In many countries, the mortality rate of malignant tumors ranks among the top. The rapid development of nano-biotechnology in recent years provides a powerful means to solve major problems in the field of biomedicine, and forms a new research field of nano-biomedicine. The research on nanomaterials as a new generation of drug carriers is booming. It can improve the water solubility of drugs, improve the stability of drugs, and realize slow and controlled release of drugs, targeted positioning, and multi-ta...

Claims

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Application Information

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IPC IPC(8): A61K47/46
Inventor 颜娟樊春海金彩虹何丹农
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH
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