title: pH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells.
abstract: This paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0±0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6-2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL.
authors.affiliation: School of Pharmacy, The University of Auckland, Auckland 1142, New Zealand.
authors.forename: Mimi M
authors.initials: MM
authors.lastname: Yang
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, New Zealand.
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
authors.affiliation: School of Pharmacy, The University of Auckland, Auckland 1142, New Zealand. Electronic address: z.wu@auckland.ac.nz.
authors.forename: Zimei
authors.initials: Z
authors.lastname: Wu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D001549:Benzamides / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D016503:Drug Delivery Systems; D011992:Endosomes / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D015674:Mammary Neoplasms, Animal / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D018613:Microscopy, Confocal; D009588:Nitrogen Mustard Compounds / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry; D011355:Prodrugs; D012995:Solubility; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D008081:Liposomes; D009588:Nitrogen Mustard Compounds; D011355:Prodrugs; D011092:Polyethylene Glycols
keywords: (CID 406952); (CID 44134771); (CID 5997); 1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]; 1,2-dioleoy-sn-glycero-3-phosphoethanolamine; 1,2-distearoyl-sn-glycero-3-phosphocholine; Active loading; CHEMS (CID 65082); Cholesterol; Cholesteryl hemisuccinate; DOPE (CID 9546744); DSPC (CID 94190); Endosome escape; Gene-directed enzyme-prodrug therapy; Hydroxypropyl-β-cyclodextrin; pH-sensitive liposomes