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Anti-tumor targeting drug delivery system as well as preparation method and application thereof

A targeted drug delivery and anti-tumor technology, applied in anti-tumor drugs, pharmaceutical formulations, drug combinations, etc., can solve the problem of long experimental period of exosome method, poor cell targeting ability of nucleic acid drugs, poor endocytosis and escape of endosomes, etc. To achieve the effect of inhibiting breast cancer tumor proliferation, inhibiting tumor proliferation, and weakening the inhibitory effect

Active Publication Date: 2018-06-19
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, naked nucleic acid drugs have the following disadvantages: (1) nucleic acid drugs are easily degraded by ribozymes in tissues or cells; (2) the cell targeting ability of nucleic acid drugs is poor; Repulsion between negatively charged cell membranes results in poor endocytosis and escape from endosomes
[0003] Traditional gene medicines mainly rely on viral vectors for transportation. However, as a drug carrier, viruses may cause immune responses, hidden dangers of integration into the genome, and the possibility of virus resurrection toxicity, etc., have been controversial.
However, this method of constructing a targeted exosome has a long experimental cycle, so how to more effectively endow the exosome with targeting is a key direction of research

Method used

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  • Anti-tumor targeting drug delivery system as well as preparation method and application thereof
  • Anti-tumor targeting drug delivery system as well as preparation method and application thereof
  • Anti-tumor targeting drug delivery system as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Collection of Exosomes

[0049] The culture supernatant of immature dendritic cells (iDC) derived from cultured mice was collected and centrifuged at 300×g for 10 min. The supernatant was concentrated by the filter tube (5000×g, 30min) to obtain a concentrated solution, which was then diluted to the original volume with PBS, and concentrated again by centrifugation at 5000×g for 30min in a 100kDa ultrafiltration tube. Then add exosome extract according to the volume ratio of concentrate: exosome extract reagent = 2:1, mix thoroughly, and incubate overnight at 4°C. Finally, centrifuge at 12000×g for 2 hours, remove the supernatant, collect exosome, and resuspend with sterilized PBS. After testing the protein concentration with a Nandrop 2000 spectrophotometer, store at -20°C, and store at 4°C within one month.

[0050] With about 10 μg of purified exosome, add an equal volume of 4% glutaraldehyde, and fix at room temperature for 30 min. Then put the sample ...

Embodiment 2

[0052] Example 2: Preparation of AS1411-exosome

[0053] The specific steps are:

[0054](1) Stock solution A and B configuration: Weigh 3 mg of cholesterol-labeled polypeptide (cholesterol-IIASTIGGIFGSSTTQSGGGG) and dissolve it in 1.5 mL of sterile water to prepare 2 mg / mL stock solution A; 5OD labeled CY3 nucleic acid aptamer AS1411 (5′-TTGGTGGTGGTGGTTGTGGTGGTGGT GG-CY3-3′,) was dissolved in 500 μL sterile water to prepare stock solution B;

[0055] (2) Take three glass bottles and add 500 μL of stock solution A to each bottle; then add equimolar EDC and NHS aqueous solutions respectively, and stir at room temperature for 2 hours to activate the polypeptide;

[0056] (3) Add 500 μL of stock solution B to the solution in step 2 above, and stir at room temperature for 4 days.

[0057] (4) After the reaction is finished, dialyze overnight with a dialysis bag with a molecular weight of 3 kDa to obtain a polypeptide modified by the nucleic acid aptamer.

[0058] (5) 150 μg of ...

Embodiment 3

[0061] Example 3: Preparation of AS1411-exosome-let-7

[0062] Mix 150 μg AS1411-exosome and 150 μg miRNA let-7 with OPTI-MEM serum-free medium, add to a 0.4 cm electric shock cup, ice bath, and then use Eppendorf’s Eppendorf Co. Multiporator click to load.

[0063] In order to further judge whether the loading was successful or not, Cy3-labeled let-7 was used for loading and fluorescence microscopy was used to analyze the fluorescence intensity of AS1411-exosome-let-7-Cy3 adsorbed by magnetic beads. From image 3 It can be seen that the magnetic beads have a strong fluorescent signal after adsorbing AS1411-exosome-let-7-Cy3, indicating that let-7-Cy3 was successfully loaded into the AS1411-exosome by electric shock, and endowed AS1411-exosome with a strong fluorescent signal ( Scale bar is 100 μm).

[0064] The loaded AS1411-exosome-let-7 was observed under a transmission electron microscope to determine the effect of electric shock on the morphology of the complex. resul...

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Abstract

The invention discloses an anti-tumor targeting drug delivery system as well as a preparation method and an application thereof, and belongs to the technical field of biology. The system comprises a small nucleic acid drug, a lipid-coated saclike object wrapping the small nucleic acid drug as well as a target head targeting tumor cells, and a let-7-delivering drug delivery system formed by the target head based on aptamer AS1411 and the lipid-coated saclike object adopting an exosome is particularly provided. Besides the preparation method of the system, the invention further provides the application of AS1411-exosome-let-7 to targeting detection results and tumor resistance on cellular level and mouse in-vivo level. Results prove that compared with individual exosome, the AS1411-exosome has higher targeting performances on the cellular level and in a mouse body, let-7 can be delivered more effectively to tumor tissue to play a role in inhibiting tumor proliferation and migration.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a drug delivery system, in particular to an anti-tumor targeted drug delivery system and its preparation method and application. Background technique [0002] In recent years, nucleic acid drugs have shown great potential in the research of major human diseases, especially tumor treatment, and opened up a new direction for the modern biomedical industry. However, naked nucleic acid drugs have the following disadvantages: (1) nucleic acid drugs are easily degraded by ribozymes in tissues or cells; (2) the cell targeting ability of nucleic acid drugs is poor; There is repulsion between negatively charged cell membranes, resulting in their poor ability to endocytose and escape endosomes. The above shortcomings lead to poor therapeutic effect of nucleic acid drugs. Therefore, it is necessary to find a suitable carrier to load nucleic acid drugs, deliver them to target cells, effectively e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/51A61K47/46A61K9/127A61K47/26A61K31/7105A61K31/713A61K48/00A61P35/00
CPCA61K9/127A61K9/5176A61K31/7105A61K31/713A61K47/26
Inventor 陈小佳王晓刚洪岸
Owner JINAN UNIVERSITY
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