RESUMEN
PURPOSE: This work focused on the characterization and in vitro/in vivo evaluation of an alginate/chitosan microsphere (ACMS) formulation of glucose oxidase (GOX) for the locoregional delivery of reactive oxygen species for the treatment of solid tumors. METHODS: The GOX distribution and ACMS composition were determined by confocal laser scanning microscopy and X-ray photoelectron spectroscopy. The mechanism of GOX loading and GOX-polymer interactions were examined with Fourier transform infrared spectroscopy and differential scanning calorimetry. In vitro cytotoxicity and in vivo efficacy of GOX-encapsulated ACMS (ACMS-GOX) were evaluated in EMT6 breast cancer cells and solid tumors. RESULTS: GOX was loaded into calcium alginate (CaAlg) gel beads via electrostatic interaction and the CaAlg-GOX-chitosan complexation likely stabilized GOX. Higher concentrations of GOX near the surface of ACMS were detected. GOX retained its integrity upon adsorption to CaAlg gel beads during the coating and after release from ACMS. ACMS-GOX exhibited cytotoxicity to the breast cancer cells in vitro and their efficacy increased with increasing incubation time. Intratumorally delivered ACMS-GOX significantly delayed tumor growth with much lower general toxicity than free GOX. CONCLUSION: The results suggest that the ACMS-GOX formulation has the potential for the intratumoral delivery of therapeutic proteins to treat solid tumors.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Modelos Animales de Enfermedad , Glucosa Oxidasa/administración & dosificación , Glucosa Oxidasa/farmacocinética , Microesferas , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Química Farmacéutica/métodos , Femenino , Glucosa Oxidasa/química , Humanos , Ratones , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
P-glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Microglía/metabolismo , Membrana Nuclear/metabolismo , Animales , Western Blotting , Línea Celular , Células Endoteliales/citología , Células Endoteliales/ultraestructura , Técnica del Anticuerpo Fluorescente , Microglía/citología , Microglía/ultraestructura , Membrana Nuclear/ultraestructura , Transporte de Proteínas , Ratas , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
PURPOSE: The function of ABCG2 (BCRP), a member of the ATP-binding cassette (ABC) superfamily of membrane-associated drug transporters, at the blood-brain barrier remains highly controversial. This project investigates the functional expression of endogenous ABCG2 in cultures of human and rodent brain cellular compartments. MATERIALS AND METHODS: RT-PCR, western blot and fluorescent immunocytochemical analyses were performed on ABCG2-overexpressing human breast cancer (MCF-MX100) cells, human and rat brain microvessel endothelial (HBEC and RBE4, respectively), and rat glial cells. RESULTS: RT-PCR analysis detected ABCG2 mRNA in all the cell culture systems. Western blot analysis with anti-ABCG2 monoclonal BXP-21 antibody detected a robust band at approximately 72 kDa in the ABCG2-overexpressing MCF-MX100 cell line, whereas low expression was found in human and rat brain cell systems. Immunofluorescence microscopy detected predominant plasma membrane localization of ABCG2 in MCF-MX100 cells but weak signal in all brain cellular compartments. In the presence of ABCG2 inhibitors, the accumulation of (3)H-mitoxantrone and pheophorbide A, two established ABCG2 substrates, was significantly increased in MCF-MX100 cells but not in the human and rodent brain cell culture systems. CONCLUSIONS: Our data show low endogenous ABCG2 protein expression, localization and activity in cultures of human and rat brain microvessel endothelial and glial cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Astrocitos/metabolismo , Encéfalo/irrigación sanguínea , Neoplasias de la Mama/metabolismo , Células Endoteliales/metabolismo , Microglía/metabolismo , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Clorofila/análogos & derivados , Clorofila/metabolismo , Células Endoteliales/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Indoles/farmacología , Microcirculación/citología , Microcirculación/metabolismo , Microglía/efectos de los fármacos , Mitoxantrona/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transfección , TritioRESUMEN
The objectives of this study were to evaluate the potential of a polymer-lipid hybrid nanoparticle (PLN) system to enhance cellular accumulation and retention of doxorubicin (Dox), a widely used anticancer drug and an established P-glycoprotein (Pgp) substrate, in Pgp-overexpressing cancer cell lines and to explore the underlying mechanisms. Nanoparticles containing Dox complexed with a novel anionic polymer (Dox-PLN) were prepared using an ultrasound method. Two Pgp-overexpressing breast cancer cell lines (a human cell line, MDA435/LCC6/MDR1, and a mouse cell line, EMT6/AR1) were used to investigate the effect of nanoparticles on cellular uptake and retention of Dox. Endocytosis inhibition studies and fluorescence microscopic imaging were performed to elucidate the mechanisms of cellular drug uptake. Treatment of Pgp-overexpressing cell lines with Dox-PLNs resulted in significantly enhanced Dox uptake and more substantial increases in drug retention after the end of treatment compared with free Dox solutions (p < 0.05). Fluorescence microscopic images showed improved nuclear localization of Dox and uptake of lipid when the drug was delivered in the Dox-PLN form to MDA435/LCC6/MDR1 cells. Endocytosis inhibition studies revealed that phagocytosis is an important pathway in the membrane permeability of the nanoparticles. These findings suggest that some of the Dox physically associated with the nanoparticles bypass the membrane-associated Pgp when delivered as Dox-PLNs, and in this form, the drug is better retained within the Pgp-overexpressing cells than the free drug. The present study suggests a new mechanism for overcoming drug resistance in Pgp-overexpressing tumor cells using lipid-based nanoparticle formulations.