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Cell-penetrating mechanism of intracellular targeting albumin: Contribution of macropinocytosis induction and endosomal escape

Ichimizu, Shota, Watanabe, Hiroshi, Maeda, Hitoshi, Hamasaki, Keisuke, Ikegami, Komei, Chuang, Victor Tuan Giam, Kinoshita, Ryo, Nishida, Kento, Shimizu, Taro, Ishima, Yu, Ishida, Tatsuhiro, Seki, Takahiro, Katsuki, Hiroshi, Futaki, Shiroh, Otagiri, Masaki, Maruyama, Toru
Journal of controlled release 2019 v.304 pp. 156-163
CXCR4 receptor, Western blotting, cytosol, dextran, drug carriers, electrostatic interactions, endosomes, fluorescent antibody technique, heparan sulfate, heparin, human serum albumin, lysosomes, proteoglycans, signal transduction, staining
We recently developed a cell-penetrating drug carrier composed of albumin (HSA) combined with palmitoyl-cyclic-(D-Arg)12. While it is possible that the palmitoyl-cyclic-(D-Arg)12/HSA enters the cell mainly via macropinocytosis, the mechanism responsible for the induction of macropinocytosis and endosomal escape remain unknown. We report herein that palmitoyl-cyclic-(D-Arg)12/HSA might interact with heparan sulfate proteoglycan and the chemokine receptor CXCR4 followed by multiple activations of the PKC/PI3K/JNK/mTOR signaling pathways to induce macropinocytosis. This result was further confirmed by a co-treatment with 70 kDa dextran, a macropinocytosis marker. Using liposomes that mimic endosomes, the leakage of 5,6-carboxyfluorescein from liposome was observed in the presence of palmitoyl-cyclic-(D-Arg)12/HSA only in the case of the anionic late endosome-like liposomes but not the neutral early endosome-like liposomes. Heparin largely inhibited this leakage, suggesting the importance of electrostatic interactions between palmitoyl-cyclic-(D-Arg)12/HSA and the late-endosomal membrane. Immunofluorescence staining and Western blotting data indicated that the intact HSA could be transferred from endosomes to the cytosol. These collective data suggest that the palmitoyl-cyclic-(D-Arg)12/HSA is internalized via macropinocytosis and intact HSA is released from the late endosomes to the cytoplasm before the endosomes fuse with lysosomes. Palmitoyl-cyclic-(D-Arg)12/HSA not only functions as an intracellular drug delivery carrier but also as an inducer of macropinocytosis.