RESUMEN
A strategy for the light-activated release of bioactive compounds (BODIPY, colchicine, paclitaxel, and methotrexate) from membrane-enclosed depots is described. We have found that membrane-permeable bioagents can be rendered membrane impermeable by covalent attachment to cobalamin (Cbl) through a photocleavable linker. These Cbl-bioagent conjugates are imprisoned within lipid-enclosed compartments in the dark, as exemplified by their retention in the interior of erythrocytes. Subsequent illumination drives the secretion of the bioactive species from red blood cells. Photorelease is triggered by wavelengths in the red, far-red, and near-IR regions, which can be pre-assigned by affixing a fluorophore with the desired excitation wavelength to the Cbl-bioagent conjugate. Pre-assigned wavelengths allow different biologically active compounds to be specifically and unambiguously photoreleased from common carriers.
Asunto(s)
Compuestos de Boro/química , Colchicina/química , Metotrexato/química , Paclitaxel/química , Vitamina B 12/química , Compuestos de Boro/metabolismo , Colchicina/metabolismo , Eritrocitos/química , Células HeLa , Humanos , Metotrexato/metabolismo , Estructura Molecular , Paclitaxel/metabolismo , Procesos Fotoquímicos , Vitamina B 12/metabolismoRESUMEN
Light-activatable drugs offer the promise of controlled release with exquisite temporal and spatial resolution. However, light-sensitive prodrugs are typically converted to their active forms using short-wavelength irradiation, which displays poor tissue penetrance. We report herein erythrocyte-mediated assembly of long-wavelength-sensitive phototherapeutics. The activating wavelength of the constructs is readily preassigned by using fluorophores with the desired excitation wavelength λ(ex). Drug release from the erythrocyte carrier was confirmed by standard analytical tools and by the expected biological consequences of the liberated drugs in cell culture: methotrexate, binding to intracellular dihydrofolate reductase; colchicine, inhibition of microtubule polymerization; dexamethasone, induced nuclear migration of the glucocorticoid receptor.
Asunto(s)
Portadores de Fármacos/química , Eritrocitos/química , Profármacos/química , Supervivencia Celular/efectos de los fármacos , Colchicina/química , Colchicina/toxicidad , Dexametasona/química , Dexametasona/toxicidad , Fluoresceína/química , Células HeLa , Humanos , Luz , Metotrexato/química , Metotrexato/toxicidad , Profármacos/toxicidad , Receptores de Glucocorticoides/metabolismo , Rodaminas/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Vitamina B 12/químicaRESUMEN
Inâ vivo optical imaging must contend with the limitations imposed by the optical window of tissue (600-1000â nm). Although a wide array of fluorophores are available that are visualized in the red and near-IR region of the spectrum, with the exception of proteases, there are few long wavelength probes for enzymes. This situation poses a particular challenge for studying the intracellular biochemistry of erythrocytes, the high hemoglobin content of which optically obscures subcellular monitoring at wavelengths less than 600â nm. To address this, tunable fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far-red and near-IR region. These agents were used to monitor endogenous cAMP-dependent protein kinase activity in erythrocyte lysates and in intact erythrocytes when using a light-activatable reporter.
Asunto(s)
Colorantes Fluorescentes/química , Péptidos/química , Proteínas Quinasas/química , Fosforilación , Transducción de SeñalRESUMEN
Inactive in the membrane: Lipidated light-responsive constructs that sequester bioagents (R, see scheme) to the membranes of organelles and cells have been constructed. When membrane-bound, the bioagent is not susceptible to processing by its biological target. Photolysis releases the bioagent from its membrane anchor and thereby renders it biologically active.