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Preparation method and application of engineering bionic exosome for delivering specific protein

An engineering, exosome technology, applied in the field of engineering biomimetic exosome preparation, can solve problems such as unstable exosome yield, achieve improved biosafety and bioavailability, low immunogenicity, high Practical value and effect of clinical prospects

Pending Publication Date: 2021-03-09
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects of instability of existing protein drugs in vivo and low yield of exosomes, and provide a preparation method of engineered biomimetic exosomes for delivery of specific proteins

Method used

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  • Preparation method and application of engineering bionic exosome for delivering specific protein
  • Preparation method and application of engineering bionic exosome for delivering specific protein
  • Preparation method and application of engineering bionic exosome for delivering specific protein

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Embodiment 1

[0036] 1. Preparation of engineered biomimetic exosomes

[0037] Culture HEK-293T cells in a 6-well plate until the density reaches about 70%, then replace with Opti-MEM for culture. Add 1 μg of target plasmid, 1.2 μg of packaging plasmid and 4 μL of lipo2000 mixed into each well, transfect and incubate. Change the medium after 6-8 hours, and culture with DMEM medium containing 10% FBS. The treated HEK-293T cells were observed under a fluorescence microscope at a specific time. The result is as figure 1 As shown, HEK-293T cells can efficiently produce specific lentiviruses. Virus supernatants were collected at 24h, 48h, and 72h, respectively, and stored at 4°C. Then the collected virus supernatant was centrifuged at 500 g for 10 min, and the collected supernatant was filtered with a 0.45 μm filter to remove cell debris in the supernatant. Then the filtrate was mixed with the virus purification reagent (LentiConcentrator) at a ratio of 1:5, and kept on ice overnight. Afte...

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Abstract

The invention discloses a preparation method and application of an engineering bionic exosome for delivering specific protein. The preparation method comprises the following steps: S1, packaging viruses by using engineering cells to prepare specific lentiviral particles; S2, infecting target cells by using the obtained lentivirus, and adding a resistant drug for screening, so as to prepare a stable cell line for expressing a specific gene with high purity; and S3, culturing the obtained stable cell line, extracting cell membranes of the stable cell line, preparing the stable cell line into membrane nano-vesicles, and finally performing centrifugal purification to obtain the engineering bionic exosome. The invention provides a synthetic method for preparing a large number of specific protein drug delivery systems, the method can effectively enhance the stability of protein biomacromolecule drugs in vivo, prolong the drug effect duration and realize effective delivery of the drugs, a newthought is provided for the administration route of the protein biomacromolecule drugs, the engineering bionic exosome can be used for immunotherapy for treating tumors, is good in safety and strongin action effect, and has very high application value and clinical transformation prospect.

Description

technical field [0001] The present invention relates to the technical field of biomedical materials, more specifically, to a preparation method and application of engineered biomimetic exosomes for delivering specific proteins. Background technique [0002] In recent years, immunotherapy, including immune checkpoints, has revolutionized the way tumors are treated and brought hope to the cure of cancer. [NATURE REVIEWS CANCER, 2015, 12, 4, 252-264]. There are two types of immune checkpoints. One is the well-known negative immune checkpoints represented by PD-1 / PDL1 and CTLA4, which can inhibit the body’s immune system. Currently, most immune checkpoint monoclonal antibodies Both are based on negative immune checkpoints; the other type is positive immune checkpoints, which activate the body's immune system. [NATURE BIOMEDICAL ENGINEERING, 2017, 1, 0011]. Although immune checkpoint monoclonal antibody drugs have achieved great success, some patients did not benefit from it a...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/12C12N5/10A61K47/46A61K39/00A61P35/00
CPCC12N15/86C07K14/70575C12N5/0645A61K47/46A61K39/001117A61P35/00C12N2740/15043C12N2800/107C12N2510/00
Inventor 黄宇星梅林辛卓张旭东程琴珍余永康
Owner NANCHANG UNIV
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