title: Systematic investigation on the intracellular trafficking network of polymeric nanoparticles.
abstract: Polymeric nanoparticles such as PLGA-based nanoparticles are emerging as promising carriers for controlled drug delivery. However, little is known about the intracellular trafficking network of polymeric nanoparticles. Here, more than 30 Rab proteins were used as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy to investigate in detail the intracellular trafficking pathways of PLGA nanoparticles. We observed that coumarin-6-loaded PLGA nanoparticles were internalized by the cells mainly through caveolin and clathrin-dependent endocytosis and Rab34-mediated macropinocytosis. Then the PLGA nanoparticles were transported to early endosomes (EEs), late endosomes (LEs), and finally to lysosomes. Two novel transport pathways were identified in our research: the macropinocytosis (Rab34 positive)-LE (Rab7 positive)-lysosome pathway and the EE-liposome (Rab18)-lysosome pathway. Moreover, the slow (Rab11 and Rab35 positive), fast (Rab4 positive) and apical (Rab20 and Rab25 positive) endocytic recycling endosome pathways could transport the PLGA nanoparticles to lysosomes. The PLGA nanoparticles were transported out of the cells by GLUT4 transport vesicles (Rab8, Rab10 positive), classic secretory vesicles (Rab3, Rab27 positive vesicles) and melanosomes (Rab32, Rab38 positive vesicles). Besides, the PLGA nanoparticles were observed in autophagosomes (LC3 positive), which means that the nanoparticles can be delivered by the autophagy pathway. Multiple cross-talk pathways were identified connecting autophagy and endocytosis or exocytosis by screening the co-localization of the Rab proteins with the LC3 protein. Degradation of nanoparticles through lysosomes can be blocked by autophagy inhibitors (3 MA and CQ). A better understanding of intracellular trafficking mechanisms involved in polymeric nanoparticle-based drug delivery is a prerequisite to clinical application.
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China. mei.lin@sz.tsinghua.edu.cn huanglq@mail.tsinghua.edu.cn and The Shenzhen Key Lab of Gene and Antibody Therapy, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China.
authors.forename: Jinxie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China. mei.lin@sz.tsinghua.edu.cn huanglq@mail.tsinghua.edu.cn and The Shenzhen Key Lab of Gene and Antibody Therapy, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China.
authors.forename: Danfeng
authors.initials: D
authors.lastname: Chang
authors.affiliation: College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, People's Republic of China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Yang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China. mei.lin@sz.tsinghua.edu.cn huanglq@mail.tsinghua.edu.cn and The Shenzhen Key Lab of Gene and Antibody Therapy, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China and Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, USA. x.d.zhang@foxmail.com.
authors.forename: Xudong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China. mei.lin@sz.tsinghua.edu.cn huanglq@mail.tsinghua.edu.cn and The Shenzhen Key Lab of Gene and Antibody Therapy, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Tao
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China. mei.lin@sz.tsinghua.edu.cn huanglq@mail.tsinghua.edu.cn and The Shenzhen Key Lab of Gene and Antibody Therapy, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Jiang
authors.affiliation: College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, People's Republic of China and School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, Guangzhou, 510006, People's Republic of China.
authors.forename: Xin
authors.initials: X
authors.lastname: Liang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China. mei.lin@sz.tsinghua.edu.cn huanglq@mail.tsinghua.edu.cn and The Shenzhen Key Lab of Gene and Antibody Therapy, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China.
authors.forename: Hsiangi
authors.initials: H
authors.lastname: Tsai
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China. mei.lin@sz.tsinghua.edu.cn huanglq@mail.tsinghua.edu.cn and The Shenzhen Key Lab of Gene and Antibody Therapy, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China.
authors.forename: Laiqiang
authors.initials: L
authors.lastname: Huang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China. mei.lin@sz.tsinghua.edu.cn huanglq@mail.tsinghua.edu.cn and The Shenzhen Key Lab of Gene and Antibody Therapy, Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China and School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, Guangzhou, 510006, People's Republic of China.
authors.forename: Lin
authors.identifier: http://orcid.org/0000-0001-6503-5149
authors.initials: L
authors.lastname: Mei