Adriamycin-loaded aptamer-modified DNA nanocage and preparation method thereof

A nanocage and aptamer technology, applied in the field of medicine, can solve the problems of limiting tumor treatment effect, toxic and side effects, etc.

Inactive Publication Date: 2017-08-18
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, after direct intravenous administration of Dox, it is widely distributed in the blood and various tissues of the whole body, which will have a wide range of biochemic

Method used

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  • Adriamycin-loaded aptamer-modified DNA nanocage and preparation method thereof
  • Adriamycin-loaded aptamer-modified DNA nanocage and preparation method thereof
  • Adriamycin-loaded aptamer-modified DNA nanocage and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0025] step 1:

[0026] Send the following 7 DNA oligonucleotide strands to the company for synthesis:

[0027] A: 5′-CGT ATC ACC AGG CAG TTG AGA CGA ACA TTC CTA AGT CTG AA A TTT ATCACC C GC CAT AGT AG-3′

[0028] B: 5′-CGA TTA CAG CTT GCT ACA CGA TTC AGA CTT AGG AAT GTT CGA CAT GCGAGG GTC CAA TAC CG-3′

[0029] C: 5′-CGT GTA GCA AGC TGT AAT CGA CGG GAA GAG CAT GCC CAT CCA CTA CTATGG CGG GTG ATA AA-3′

[0030] D: 5′-CTC AAC TGC CTG GTG ATA CGA GGA TGG GCA TGC TCT TCC CGACGG TATTGG ACC CTC GCA TG-3′

[0031] A′: 5′-CGT ATC ACC AGG CAG TTG AGA CGA ACA TTC CTA AGT CTG AA A TTTATC ACC C GC CAT AGT AGTTTTTTTT GCAGTTGATCCTTTGGATACCCTGG-3′

[0032] B′: 5′-CGA TTA CAGCTT GCT ACA CGA TTC AGA CTT AGG AAT GTT CGA CAT GCGAGG GTC CAA TAC CGTTTTTTTT GCAGTTGATCCTTTGGATACCCTGG-3′

[0033]C′: 5′-CGT GTA GCA AGC TGT AAT CGA CGG GAA GAG CAT GCC CAT CCA CTA CTATGG CGG GTG ATA AATTTTTTT GCAGTTGATCCTTTGGATACCCTGG-3′

[0034] Use TM buffer (10mM Tris-HCl, 5mMMgCl 2 , pH 8.0) was dissolved for ...

specific Embodiment 2

[0038] Human liver cancer Hep G2 cells with low MUC1 expression were used as a negative control to conduct an in vitro cell uptake test to investigate the cell-targeted uptake capabilities of Td, apt-Td, 2apt-Td, and 3apt-Td obtained in Example 1.

[0039] Human breast cancer MCF-7 cells and human liver cancer HepG2 cells were mixed at a density of 1×10 5 Cells / well were seeded in 24-well plates and cultured for 24 hours. Then, the cells were treated with serum-free medium containing samples (Td, apt-Td, 2apt-Td and 3apt-Td obtained in Example 1, the same final concentration was 1000 nM) for 5 h (37° C.). After the cells were incubated for 4 hours, they were washed 3 times with fresh PBS, and then the cells were digested with trypsin. After the digestion was completed, PBS was added to make a cell suspension. After passing through a microporous membrane, the uptake ability of the cells was observed by flow cytometry. The results are attached Figure 4 shown. The results sho...

specific Embodiment 3

[0041] Dox@Td, Dox@apt-Td, Dox@2apt-Td, Dox@3apt-Td obtained in Example 3 of the present invention were subjected to in vitro cell uptake experiments.

[0042] Human breast cancer MCF-7 cells were cultured at a density of 1×10 5 Cells / well were seeded in 24-well plates and cultured for 24 hours. Then, cells were treated with samples containing (Dox@Td obtained in Example 3, Dox@apt-Td, Dox@2apt-Td, Dox@3apt-Td, and free Dox, the same final concentration of doxorubicin was 11.6 μg / mL) The serum-free medium was treated for 1, 2 and 3 hours (37°C) respectively. After the cells were incubated for a fixed time, they were washed 3 times with fresh PBS, and the uptake ability of the cells was observed by an inverted fluorescence microscope. The results are attached Figure 5 shown. The results showed that the cell uptake of free doxorubicin was the strongest at 1 h, and the cell entry ability of Dox@Td, Dox@apt-Td, Dox@2apt-Td, Dox@3apt-Td increased with the increase of the numbe...

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Abstract

The invention discloses an adriamycin-loaded aptamer-modified DNA nanocage and a preparation method thereof, and belongs to the field of medicine science and technology. Oligonucleotide sequences of MUC1 mucoprotein aptamers are modified onto four oligonucleotide sequences of a DNA regular tetrahedron structure through nucleotide sequence synthesis; and on the basis of a base complementation pairing rule, the stable DNA regular tetrahedron structure is formed through self-assembling under certain conditions, so that the DNA nanocage which is modified with different numbers of the MUC1 mucoprotein aptamers at various vertexes, and adriamycin is loaded through physical trapping so as to achieve a purpose of conducting targeting therapy of tumors. Experiments prove that the drug-loaded DNA nanocage can be formed through self-assembling of a nanocage nucleic acid skeleton, without the participation of complex preparation steps; efficient targeted intake of human breast cancer MCF-7 cells is achieved on the basis of a targeting effect of the MUC1 mucoprotein aptamers and a transmembrane transport property of the DNA nanocage; and meanwhile, the aptamers are protected from being degraded by nuclease in vivo; therefore, a novel drug-delivery system is provided for tumor-targeted delivery of chemotherapeutics.

Description

[0001] Technical field: [0002] The invention relates to a tumor-targeting aptamer-modified DNA nanocage loaded with doxorubicin and a preparation method thereof, belonging to the technical field of medicine. [0003] Background technique: [0004] DNA nanocages are nanoscale three-dimensional polyhedral structures formed by the self-assembly of multiple DNA oligonucleotide chains through complementary base pairing. Common ones include tetrahedron, octahedron, triangular bipyramid and dodecahedron. The self-assembly of this DNA nanostructure has high controllability and precision, and its application in the fields of biosensors, molecular imaging and nanocomputers has attracted more and more attention. Among them, the regular tetrahedral DNA nanostructure has good mechanical rigidity and structural stability, and can also be called a regular tetrahedral DNA nanocage, which can be obtained through rapid self-assembly of four synthetic DNA oligonucleotide chains. And it is easy...

Claims

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Application Information

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IPC IPC(8): A61K47/61A61K31/704A61P35/00
CPCA61K31/704
Inventor 吴正红姜妤婕祁小乐许辰辰李姝宜杨金龙
Owner CHINA PHARM UNIV
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