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We demonstrated that phospholipid-free small unilamellar vesicles (PFSUVs) composed of TWEEN 80 and cholesterol (25/75, mol%) could be fabricated using a staggered herringbone micromixer with precise controlling of their mean size between 54 nm and 147 nm. Increasing the temperature or decreasing the flow rate led to an increase in the resulting particle diameter. In zebrafish embryos, 120-nm PFSUVs showed 3-fold higher macrophage clearance compared to the 60-nm particles, which exhibited prolonged blood circulation. In mice, the 60-nm particles showed dominant accumulation in the liver hepatocytes (66% hepatocytes positive), while the 120-nm particles were delivered equally to the liver and spleen macrophages. Accordingly, in a murine model of acetaminophen-induced hepatotoxicity the 60-nm particles loaded with chlorpromazine reduced the serum alanine aminotransferase level and liver necrosis 2- to 4-fold more efficiently than their 120-nm counterparts and the free drug, respectively. This work showed that the intra-liver distribution of PFSUVs was largely determined by the size. Most other nanoparticles published to date are predominantly cleared by the liver Kupffer cells. The 60-nm PFSUVs, on the other hand, focused the delivery to the hepatocytes with significant advantages for the therapy of liver diseases.
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