title: Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.
abstract: Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization.
authors.affiliation: Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China; Laboratory for Drug Delivery and Biomaterials, Faculty of Pharmacy, University of Manitoba, 750 McDermot Ave, Winnipeg, Manitoba R3E 0T5, Canada.
authors.forename: Jijin
authors.initials: J
authors.lastname: Gu
authors.affiliation: Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China.
authors.forename: Junguo
authors.initials: J
authors.lastname: Hao
authors.affiliation: Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Fang
authors.affiliation: Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China. Electronic address: shaxy@fudan.edu.cn.
authors.forename: Xianyi
authors.initials: X
authors.lastname: Sha
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002470:Cell Survival / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D061986:MCF-7 Cells; D018613:Microscopy, Confocal; D010957:Plasmids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011060:Poloxalene / Q000737:chemistry*; D011126:Polypropylenes / Q000737:chemistry*; D014162:Transfection / Q000379:methods*
chemical_list: D011126:Polypropylenes; C443641:poly(propyleneimine); C464484:pluronic block copolymer P123; D011060:Poloxalene; D004247:DNA
keywords: Colocalization; Endocytosis Mechanism; Non-viral vector; P123-PPI; Polyplexes