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Exosome carrier of target integrin alpha v beta 3 and preparation method and application of exosome carrier

A technology of integrins and exosomes, applied in the field of medicine, to achieve the effects of low immunogenicity, promotion of apoptosis, and mild reaction conditions

Active Publication Date: 2019-04-23
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no exosome targeting integrin αvβ3 overexpressing tumor cells, and it can realize the targeted delivery of chemotherapy drugs and gene drugs

Method used

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  • Exosome carrier of target integrin alpha v beta 3 and preparation method and application of exosome carrier
  • Exosome carrier of target integrin alpha v beta 3 and preparation method and application of exosome carrier
  • Exosome carrier of target integrin alpha v beta 3 and preparation method and application of exosome carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Extraction and transmission electron microscope observation of exosomes secreted by undifferentiated and differentiated THP-1 cells

[0056] Cultivate THP-1 mononuclear suspension cells at a culture density of 5×10 5 -10 6 / ml. Adjust the cell density to 6 x 10 5 / ml, add PMA at a concentration of 50ng / ml, stimulate the culture for 24 hours, wash the cells with PBS, replace with a medium that does not contain PMA and continue to culture for 1-5 days. In order to quickly obtain sufficient yield and relatively pure exosomes from a large volume of culture medium, it is proposed to combine ultrafiltration and ultracentrifugation to separate and extract exosomes. First, obtain cell-free medium by centrifugation, and then use gradient centrifugation: centrifuge at 300×g for 10 minutes, centrifuge at 1200×g for 20 minutes, and centrifuge at 10,000×g for 30 minutes to remove cell debris and microbubbles, etc., and then pass through a 0.22 μm microporous membrane ...

Embodiment 2

[0057] Example 2: Nanoparticle tracking analysis technology (NTA) of exosomes secreted by undifferentiated and differentiated THP-1 cells to investigate the concentration and particle size of the collected exosomes

[0058] Take the exosomes isolated in Example 1 to analyze their concentration and particle size by NTA method, use a 1ml syringe to take 200ul and dilute to 1ml, and analyze the sample concentration and particle size on the machine. The result is as image 3 , a) is the NTA result of the exosomes isolated from the THP-1 cell culture medium without PMA stimulation, b) is the NTA knot of the exosomes isolated from the THP-1 cell culture medium stimulated with PMA, the result It shows that the average particle size of exosomes secreted by undifferentiated THP-1 cells is 179.4nm, and the particle size of exosomes secreted by differentiated THP-1 cells is 94.1nm.

Embodiment 3

[0059] Example 3: Nanoparticle Tracking Analysis Technology (NTA) of exosomes secreted by undifferentiated and differentiated THP-1 cells investigates the protein identification of the collected exosomes

[0060] The two kinds of exosomes collected at the bottom of the centrifuge tube in Example 1 are the exosomes (Exo) secreted by undifferentiated THP-1 cells and the exosomes (A15-Exo) secreted by differentiated THP-1 cells . Add an appropriate amount of RIPA cell lysate, lyse on ice for 30 min, centrifuge at 13,000 g for 15 min, and take the supernatant. Protein quantification was performed according to the protein quantification kit (BCA method for quantifying protein), and an equal amount of protein was mixed with 5X denaturing buffer and boiled for 5 min. Prepare 10% separating gel, and separate the protein by SDS-PAGE electrophoresis, first at constant voltage 60V, electrophoresis for 50min, then at constant voltage 120V, electrophoresis for 2h. Cut the PVDF membrane a...

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Abstract

The invention relates to the technical field of medicine, in particular to an exosome carrier of a target integrin alpha v beta 3 and a preparation method and application of the exosome carrier. Mononuclear suspension cells THP-1 are adopted and stimulated by PMA of the certain concentration to obtain the exosome carrier, the surface of an exosome after separating contains an RGD target molecule metalloprotease disintegrin family 15, and the target integrin receptor alpha v beta 3 can be obtained. The in-vitro releasing experiment shows that the prepared exosome can slowly release loaded chemotherapeutic medicine, and the better capacity of jointly loading genes and the chemotherapeutic medicine is achieved. In the tumor treatment process, the exosome can enhance the effects of cells of aspecificity target overexpression integrin alpha v beta 3 on tumor cells through the chemotherapeutic medicine or gene medicine, apoptosis of tumor cells is promoted, and therefore the exosome carrierforms a targeted and efficient low-toxicity bionic nanoscale delivery system of tumor treatment.

Description

technical field [0001] The invention relates to the field of medical technology, in particular, an exosome carrier targeting integrin αvβ3, its preparation method and application. Background technique [0002] With the development of molecular biology, immunology and related disciplines, as well as in the study of the molecular mechanism of resistance to androgen withdrawal and chemotherapeutic drug resistance in the treatment of prostate cancer, gene therapy has gradually shown its great advantages. It has the characteristics of good toxicity, low toxicity and side effects, and can specifically kill tumor cells (Yap, T.A., et al., Drug discovery in advanced prostate cancer: translating biology into therapy. NatRev Drug Discov, 2016.15(10): p.699-718 .). However, exogenous genes are prone to degradation in vivo, so gene therapy needs a safe, efficient and stable delivery carrier for clinical application (Milcovich, G., et al., Recent advances in smart biotechnology: Hydroge...

Claims

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Application Information

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IPC IPC(8): C12N15/85A61K47/46A61K48/00A61K31/704A61P35/00
CPCA61K47/46A61K48/005A61P35/00A61K31/704C12N15/85C07K14/47A61K45/06C12N15/88C12N15/111C12N15/113C12N2310/141C12N2310/3515C12N2320/32C12N2320/31A61K2039/53A61K2039/5156A61K39/461A61K39/001102
Inventor 原永芳宫春爱韩璐俞晓艳王彧杰王蓉
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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