Composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small-molecule antitumor drug

A tumor necrosis factor and apoptosis-inducing ligand technology, which is applied in the fields of biomedicine and oncology, can solve the problems of enhanced targeting and low bioavailability, and achieves reduced side effects, good reproducibility, and improved therapeutic effect. Effect

Pending Publication Date: 2021-11-12
FUDAN UNIV SHANGHAI CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The simultaneous delivery of small molecule anti-tumor drug TPL by TRAIL-modified exosomes can not only overcome the drug resistance, in vivo instability and low bioavailability of TRAIL and TPL, but also produce synergistic anti-tumor effects through the combination of the two. Enhance targeting and promote tumor apoptosis, reduce the toxic and side effects of TPL, so it has great potential for clinical application

Method used

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  • Composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small-molecule antitumor drug
  • Composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small-molecule antitumor drug
  • Composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small-molecule antitumor drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Construction of overexpression TRAIL lentiviral vector

[0055] The TRAIL (Homo sapiens TNF superfamily member 10, TNFSF10) nucleotide sequence (NM_003810) was synthesized into the pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO lentiviral expression vector. The specific process is as follows: select the vector enzyme digestion system → enzyme digestion of the carrier at 37°C, gel recovery → fragment PCR recovery → put the nucleotide sequence of TRAIL and the lentiviral expression vector connection reaction system in a 50°C warm bath for 20 minutes → transform (competent cells DH 5a; resistance: ampicillin, 37°C, 230rpm, 24h) → pick bacteria from the transformed plate, shake the bacteria at 37°C, 250rpm for 14h → perform PCR identification on the bacterial liquid, and perform overexpression sequencing on the positive clone liquid.

[0056] figure 1 It is the TRAIL lentiviral expression vector map constructed in Example 1.

Embodiment 2

[0057] Example 2: Lentiviral packaging

[0058] Use a plasmid DNA extraction kit to extract a large number of constructed lentiviral expression vectors and helper plasmids. The plasmid concentration must be greater than 1 μg / μL, and the A260 / 280 between 1.7 and 1.8 can be used for virus packaging. The 293T cells were digested, centrifuged, resuspended with complete culture medium, subcultured into petri dishes at a ratio of 1:10, and placed in an incubator to continue culturing. Transfection can be carried out when the density of 293T cells reaches 70-80%. Replace the culture medium with serum-free medium, and perform lipotransfer complex: 10 μg pSPAX2, 5 μg pMD2G, 10 μg shuttle plasmid containing the target gene, 75 μL Lipofiter TM reagent. Six hours after transfection, the cells were replaced with complete culture medium containing 10% FBS. Virus supernatants were collected twice at 48h and 72h after transfection. When the virus was harvested at 48 hours, the culture med...

Embodiment 3

[0059] Embodiment 3: detection of virus titer

[0060] After digesting and counting 293T cells, dilute to 1~3×10 5 / mL, add to 96-well plate, 100 μL / well, prepare 6 wells for each virus. On the next day, prepare six 1.5mL EP tubes, add 10 μL of virus solution to the first EP tube, and then make a 3-fold serial dilution, a total of 6 dilutions. On the third day, if there are wells that need to be screened with puromycin, first aspirate 100 μL of medium containing lentiviral particles, and then add 100 μL of complete medium containing puromycin. On the fifth day, observe under a fluorescent microscope. Six hours before the observation, fresh complete medium needs to be replaced. Aspirate 80 μL of medium from the well, then add 80 μL of fresh complete medium, and put it into the incubator for cultivation. After 6 hours, the results were observed under a fluorescence microscope, and the wells with a fluorescence percentage of 10-50% were selected to calculate the virus titer. A...

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Abstract

The invention relates to the technical field of biological medicine and oncology, in particular to a composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small-molecule antitumor drug as well as a preparation method and application of the composite exosome. The membrane surface of the composite exosome carries TRAIL protein, and the small-molecule antitumor drug is entrapped in a membrane. A preparation process of the composite exosome is mature, efficient, good in reproducibility and low in cost. The composite exosome has the advantages of obvious in-vitro and in-vivo anti-tumor effects, low drug dosage, no toxic or side effect and good biological safety, and provides a new strategy for clinical treatment of tumors.

Description

technical field [0001] The invention relates to the technical fields of biomedicine and oncology, specifically, a composite exosome loaded with a membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and a small-molecule anti-tumor drug, and a preparation method and application thereof. Background technique [0002] Cutaneous malignant melanoma is a malignant tumor produced by melanocytes of the skin and other organs, accounting for about 3% of all tumors, with strong invasiveness and metastasis; high incidence, poor prognosis, and high mortality in advanced melanoma 70-90%, ranking first in skin malignancies. According to the statistics of the American Cancer Society in 2021, in 2020, there will be 106,110 newly diagnosed cases and 7,180 deaths of skin melanoma in situ globally in 2020, and it will grow at a rate of 3-5% per year, becoming the fastest growing malignant tumor in the world One, the five-year survival rate for advanced melanoma is only 25%. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/19A61K31/585A61K47/46A61P35/00
CPCA61K38/191A61K31/585A61K47/46A61P35/00A61K2300/00
Inventor 刘继勇姜良弟顾永卫武鑫杜月李爱雪赵语南唐晓萌
Owner FUDAN UNIV SHANGHAI CANCER CENT
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