Abstract
Despite the widely accepted assumption that most endosomal compartments are acidic, evaluation of the efficiency of pH-dependent drug release from a ligand-targeted drug conjugate during receptor-mediated endocytosis is lacking. Therefore, we have characterized the kinetics of pH-dependent drug release from a model folate-drug conjugate during folate receptor (FR)-mediated endosomal trafficking. For this purpose, we synthesized an acid-labile folate-fluorescence resonance energy transfer reporter (ALFR) that emits green fluorescence (BODIPY FL, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-propionyl)amino)hexanoic acid) only after acid-catalyzed hydrolysis of the acyl hydrazone linker. In a cell-free system, cleavage of ALFR was found to be efficient only at acidic pH values (t1/2 = 1.95, 4.63, and 75 h at pH 4, 5, and 6, respectively) and essentially resistant to hydrolysis at pH 7. Curiously, when applied to folate receptor-expressing cancer cells, the acid-labile folate-linked probe exhibited little or no recovery of BODIPY FL fluorescence (green), even after 55 h of incubation, arguing very inefficient cleavage within the FR endocytic pathway. To understand this unanticipated observation, we measured the pH of FR-containing endosomes using ratiometric fluorescence microscopy and observed that most FR+ endosomes are only mildly acidic (average ∼pH 6.5). Taken together, these data argue that the FR-trafficking pathway does not involve acidic compartments and that acyl hydrazone linkers may constitute a poor option for FR-mediated drug delivery.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.117648.
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ABBREVIATIONS: FR, folate receptor; FRET, fluorescence resonance energy transfer; BODIPY FL, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl)amino)hexanoic acid, fluorescein; GPI, glycophosphoinositol; FITC, fluorescein isothiocyanate; ALFR, acid-labile folate-FRET reporter; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry.
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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↵1 Current affiliation: St. Jude Children's Research Hospital, Memphis, Tennessee.
- Received November 26, 2006.
- Accepted February 6, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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