Metabolic drug loading of EVs
A drug, pharmacological technique, applied in the field of therapeutic purposes, can solve the problem of little loading and practical therapeutic application
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Embodiment 1
[0041] Example 1: Loading of immune cell-derived EVs with cytarabine by fatty acid conjugation
[0042] Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood and plated at appropriate densities in cell culture medium. After 24 hours the cell culture medium was removed and the plate was washed 3 times with PBS. Fresh EV-depleted medium or serum-free medium containing C18 fatty acid-conjugated cytarabine (C18-AraC) was added to preload EV-producing cells with the drug for 1 h. Thereafter, cells were washed and the medium was replaced with fresh EV-depleted medium or serum-free medium and incubated for 24 h to allow drug incorporation into the membrane of secreted EVs through endogenous metabolic pathways. EVs were purified from conditioned media. Alternatively, EVs from untreated cells were first isolated, then loaded with C18-AraC by co-incubation, and unloaded drug was removed by a washing step. C18-AraC loading capacity in secreted EVs was quantified b...
Embodiment 2
[0046] Example 2: Loading of MSC-EVs with NSAIDs by Phospholipid Conjugation
[0047] Seed mesenchymal stem cells (MSCs) in cell culture medium at an appropriate density. After 24 hours the cell culture medium was removed and the plate was washed 3 times with PBS. Add fresh EV-depleted fresh medium or serum-free medium containing a phospholipid conjugated to isobutylphenylpropionate through its phosphor head group (PhLip-IBU) to generate EVs preloaded with drugs cells for 1 hr. Thereafter, cells were washed and the medium was replaced with fresh EV-depleted medium or serum-free medium and incubated for 24 h to allow drug incorporation into the membrane of secreted EVs via endogenous metabolic pathways. EVs were purified from conditioned media (PhLip-IBU endogenous EVs). Alternatively, EVs derived from untreated cells were first isolated, then PhLip-IBUs were loaded by co-incubation, and unloaded drug (PhLip-IBU exogenous EVs) were removed by a washing step. PhLip-IBU loadi...
Embodiment 3
[0049] Example 3: Loading of MSC-EVs with NSAIDs via vitamin B7 or B9 conjugation
[0050] MSCs stably expressing streptavidin, receptor peptide and / or folate receptor alpha (FRα) were cultured and prepared as in Example 2, but with isobutylphenylpropionic acid and biotin (IBU-B7 ) or a heterotrimeric conjugate of isobutylphenylpropionic acid and folic acid (IBU-B9) was fed. Alternatively, isolated wild-type exosomes were incubated exogenously with IBU-B7 or IBU-B9. IBU-B7 and IBU-B9 loading capacities in secreted EVs were quantified by HPLC.
[0051] EVs were obtained and processed as in Example 1. COX-2 inhibition of EV-loaded and free IBU-B7 and IBU-B9 was measured in RAW264.7 cells seeded in 24-well plates. The next day, cells were pre-incubated with free PhLip-IBU, EV PhLlp-IBU or CTRL EV and then stimulated with 100 ng / ml LPS for 24 hours. Cell culture supernatants were collected, centrifuged at 1000 x g for 15 min, and COX-2 inhibition was assessed by measuring the ...
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