title: Uptake mechanism of furosemide-loaded pegylated nanoparticles by cochlear cell lines.
abstract: This study tests the hypothesis that pegylated nanoparticles (NPs) could be taken up by the cochlear cells [House Ear Institute-organ of Corti 1 (HEI-OC1) and Stria vascularis K-1 (SVK-1)], through endocytic pathways. Furthermore, the in vitro drug release and the cytotoxicity of Furosemide (FUR)-loaded NPs on these two cochlear cells are investigated. FUR-loaded pegylated NPs are prepared by the emulsion-solvent diffusion method without surfactant. The NPs are characterized for particle mean diameter, polydispersity index (PDI), morphology, percent drug encapsulation efficiency (EE%), and FUR release kinetics. The methyl tetrazolium salt (MTS) and lactate dehydrogenase (LDH) bioassays are used to evaluate in vitro, the cytotoxicity of FUR-loaded NPs and native FUR. The NPs uptake is investigated using confocal microscopy, microplate reader/fluorimetry, and flow cytometry. Spherical NPs with a mean diameter range of 133-210 nm and PDI values varying from 0.037 to 0.41 are produced. The FUR EE% is 86% and the drug is released from the NPs according to the zero-order and Higuchi models. After treatment with blank NPs, the percentage of cell viability and cell death are 95.96% and 8.95%, in HEI-OC1 cells, respectively. The NPs are internalized by HEI-OC1 cells through a clathrin-dependent pathway. In addition, results show that NPs can be taken up via clathrin and cytoskeleton mediated pathways in SVK-1 cells. The internalization of the pegylated NPs can enhance the drug toxicity by necrosis in a dose-dependent and sustained release manner. The formulated NPs provide a promising template for a targeted drug delivery system to the inner ear.
authors.affiliation: Laboratory of Future Nanomedicines and Theoretical Chronopharmaceutics, Division of Pharmaceutical Sciences, University of Missouri Kansas City, MO 64108, USA.
authors.forename: Ibrahima
authors.initials: I
authors.lastname: Youm
authors.forename: Bi-Botti C
authors.initials: BB
authors.lastname: Youan
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003051:Cochlea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004337:Drug Carriers / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000633:toxicity; D016503:Drug Delivery Systems; D004705:Endocytosis; D005665:Furosemide / Q000008:administration & dosage* / Q000493:pharmacokinetics* / Q000633:toxicity; D034381:Hearing Loss / Q000139:chemically induced / Q000188:drug therapy / Q000517:prevention & control; D006801:Humans; D051379:Mice; D053769:Nanocapsules / Q000008:administration & dosage / Q000633:toxicity / Q000648:ultrastructure; D053758:Nanoparticles / Q000008:administration & dosage / Q000633:toxicity / Q000648:ultrastructure; D036103:Nanotechnology; D009925:Organ of Corti / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D013316:Stria Vascularis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
chemical_list: D004337:Drug Carriers; D053769:Nanocapsules; D011092:Polyethylene Glycols; D005665:Furosemide
keywords: ANOVA; CCD; CLSM; CPZ; DCM; DLS; EE%; FBS; FITC; FUR; Furosemide; GNT; HEI-OC1; HPLC; House Ear Institute-Organ of Corti 1; I.V; LDH; MTS; NPs; P; P-value; PCL-PEG; PDI; PEG; PI; PLGA; PMA; PS; R(2); SD; SEM; SVK-1; Stria vascularis K-1; TEM; analysis of variance; chlorpromazine; confocal laser scanning microscope; cytochalasin D; dichloromethane; dynamic light scattering; fetal bovine serum; fluorescein isothiocyanate; genistein; high performance liquid chromatography; inherent viscosity; lactate dehydrogenase; methyl tetrazolium salt; nanoparticles; percent drug encapsulation efficiency; phorbol myristate acetate; phosphatidylserine; poly(lactic acid co-glycolic acid); poly-ε-caprolactone-polyethylene glycol diblock; polydispersity index; polyethylene glycol; propidium iodide; scanning electron microscopy; square of the correlation coefficient; standard deviation; transmission electron microscopy