RESUMEN
BACKGROUND: This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. METHODS: SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS. RESULTS: The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH3 and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells. CONCLUSIONS: The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis. GENERAL SIGNIFICANCE: Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Neoplasias de la Mama/metabolismo , Clatrina/metabolismo , Doxorrubicina/metabolismo , Portadores de Fármacos , Endocitosis , Ácido Fólico/metabolismo , Magnetismo/métodos , Nanopartículas de Magnetita , Nanomedicina/métodos , Polietilenglicoles/química , Neoplasias del Cuello Uterino/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Caveolas/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Doxorrubicina/química , Doxorrubicina/farmacología , Endosomas/metabolismo , Femenino , Ácido Fólico/química , Células HeLa , Humanos , Lisosomas/metabolismo , Células MCF-7 , Nanopartículas de Magnetita/química , Microscopía Confocal , Microscopía Electrónica de Transmisión de Rastreo , Microscopía de Fluorescencia por Excitación Multifotónica , Espectroscopía de Pérdida de Energía de Electrones , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.