Stomach-targeted drug-loaded exosome and application thereof, and drug for treating stomach diseases
An exosome and targeting technology, applied in the field of biomedicine, can solve the problems of insufficient curative effect and strong side effects, so as to improve the therapeutic effect, reduce drug toxicity and side effects, and have good application prospects
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Embodiment 1
[0027] The gastric-targeted drug-loaded exosomes are obtained by introducing drugs for treating gastric diseases into gastric tissue-targeted exosomes, and the gastric tissue-targeted exosomes are derived from pancreatic cells .
[0028] Collect normal human pancreatic cells HPC-Y5 (this example takes human pancreatic cells HPC-Y5 as an example), and the HPC-Y5 cell culture medium is cultured with exosome-free serum and DMEM medium; centrifuge at 2000g for 10 minutes, and take Supernatant; then centrifuge at 10000g for 30min, take the supernatant, discard the cell debris and sediment; centrifuge the centrifuged cell culture solution at 100000g for 2 h, resuspend the precipitate with sterile PBS and collect in Stomach-targeted exosomes can be obtained by short-term storage at 4°C; the surface marker proteins and particle size distribution of the stomach-targeted exosomes obtained by extraction and separation are as follows: figure 1 , figure 2 As shown; the expression level ...
Embodiment 2
[0030] The exosomes secreted by HPC-Y5 pancreatic cells were collected and stained with PKH67 to trace the distribution of exosomes secreted by pancreatic cells in the body. The specific steps are as follows:
[0031] Take 100 μg of exosomes secreted by HPC-Y5 pancreatic cells, incubate with 1 μL of PKH67 overnight at 4°C in the dark, centrifuge at 100,000 g for 2 h, discard the supernatant, wash twice with PBS, and resuspend the pancreas in sterile PBS The exosomes secreted by the cells were injected into C57bl / 6 mice through the tail vein (in this example, male C57bl / 6 mice (4-6 weeks) were purchased from Beijing Huafukang Biotechnology Co., Ltd. All mice were bred in SPF facilities); 24 hours later, the mice were anesthetized, and the hearts, livers, spleens, lungs, kidneys, stomachs and intestines of the mice were taken to make frozen sections. Biodistribution of exosomes in various organs of mice; the results showed that exosomes secreted by pancreatic cells HPC-Y5 in thi...
Embodiment 3
[0033] In this example, Sunitinib, a drug used to treat gastric cancer, was selected.
[0034] In this example, drugs for the treatment of gastric cancer are introduced into gastric-targeted exosomes to prepare gastric tumor-targeted therapeutic drugs. The specific operations are as follows:
[0035] Step 1: Take 150 μg of stomach-targeted exosomes and mix them with sunitinib. The electrotransfer buffer can be (PBS, DMEM, Cytomix, Tris-HCl) to make up to 150 μL, and transfer to different specifications (0.2cm, 0.4cm ) in the electroporation cup; different waveforms (exponential wave, square wave) and different voltages (100V, 200V, 300V, 400V, 450V) were used for electroporation of gastric-targeted exosomes and sunitinib respectively;
[0036] Step 2: The electroporation product was ultracentrifuged at 100,000 g for 120 min, and the supernatant was collected to measure the drug loading.
[0037] Results: For sunitinib, 150 μg gastric-targeted exosomes were electroporated with...
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