Chemotherapy drug loaded and TIGIT overexpressed engineered drug-loaded cell membrane vesicle as well as preparation method and application thereof

A chemotherapeutic drug and cell membrane technology, applied in botany equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of short circulation time in the body, easy to be degraded and cleared, unstable properties, etc. problems, to achieve the effect of improving bioavailability, effective enrichment, and high biosafety

Pending Publication Date: 2022-04-29
SUN YAT SEN UNIV SHENZHEN
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] At present, chemotherapy and immunotherapy have shown great therapeutic potential, but only direct injection of these drugs, such as small molecule drugs, monoclonal antibodies, etc., still have certain shortcomings, including unstable properties, easy to be degraded and eliminated, short circulation time in the body, poor targeting ability

Method used

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  • Chemotherapy drug loaded and TIGIT overexpressed engineered drug-loaded cell membrane vesicle as well as preparation method and application thereof
  • Chemotherapy drug loaded and TIGIT overexpressed engineered drug-loaded cell membrane vesicle as well as preparation method and application thereof
  • Chemotherapy drug loaded and TIGIT overexpressed engineered drug-loaded cell membrane vesicle as well as preparation method and application thereof

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Embodiment 1

[0049] 1. Preparation of engineered drug-loaded cell membrane vesicles

[0050] 1. Packaging lentivirus. Culture HEK-293T cells (purchased) in a cell incubator at 37°C, add the mixed target plasmid (plasmid encoding TIGIT gene), packaging plasmid and lipo2000 (liposome) in a ratio of 1:1.2~1.5:2~4 2000). Change the medium after 5-9 hours, continue to culture with normal complete medium, and observe the transfected HEK-293T cells under a fluorescence microscope at different times. The result is as figure 1 As shown, it shows that HEK-293T cells are efficiently packaging and producing lentivirus.

[0051] The culture supernatant was collected and centrifuged to remove cells. Continue to collect the supernatant and filter through a 0.45 μm filter to remove cell debris in the supernatant. Then mix the filtrate with the virus purification reagent (Lenti Concentrator) at a volume ratio of 1:4, centrifuge at 3000g~4000g for 20-30min after incubation, remove the supernatant, and ...

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Abstract

The invention discloses a preparation method and application of engineered drug-loaded cell membrane vesicles loaded with small-molecule chemotherapeutic drugs and over-expressed in TIGIT. The preparation method comprises the following steps: S1, packaging viruses, and preparing and collecting a specific lentivirus solution for coding a TIGIT gene; s2, infecting target cells by using the obtained lentivirus solution, and screening by using a resistant drug so as to construct a stable cell line of an overexpressed TIGIT gene; s3, culturing the obtained stable cell line, extracting cell membranes of the stable cell line, preparing the cell membranes into membrane nano-vesicles, and centrifugally purifying the membrane nano-vesicles to obtain engineered cell membrane vesicles; and S4, loading a small molecule chemotherapeutic drug by using the obtained cell membrane vesicle, and carrying out centrifugal purification to obtain the engineered drug-loaded cell membrane vesicle. The engineered drug-loaded cell membrane vesicle prepared by the invention is good in biological safety, shows an excellent effect in immunotherapy of tumors, and has relatively good clinical transformation value and application prospect.

Description

technical field [0001] The invention relates to the technical field of biomedical materials, and more specifically, relates to an engineered drug-loaded cell membrane vesicle loaded with chemotherapy drugs and overexpressed TIGIT, as well as its preparation method and application. Background technique [0002] With the maturity of immunotherapy, human beings seem to see the dawn of victory over cancer. However, although related therapies targeting immune checkpoints such as PD-1 and CTLA-4 have achieved unprecedented success, clinical studies have shown that only some patients achieve objective remission. At the same time, the existing immune checkpoints are difficult to meet the diversity and complexity of clinical tumor patients, and there will also be treatment-related toxicity in clinical use, which is called immune-related adverse events. For example, these immune-related adverse events occurred in 90% of patients treated with CTLA-4 antibodies and 70% of patients trea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K9/51A61K47/46A61P35/00C12N5/10C12N15/12A61K31/555
CPCA61K31/555A61K38/1774A61K47/46A61K9/5176A61P35/00C12N5/0656C12N15/86C07K14/70521C12N2510/00C12N2740/15043A61K2300/00
Inventor 梅林余永康张帆程琴珍张锦勰李倩倩
Owner SUN YAT SEN UNIV SHENZHEN
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