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Characterization of the pH of folate receptor-containing endosomes and the rate of hydrolysis of internalized acid-labile folate-drug conjugates.

Yang J, Chen H, Vlahov IR, Cheng JX, Low PS

Department of Chemistry, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.

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 approximately 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.

Published 17 April 2007 in J Pharmacol Exp Ther, 321(2): 462-8.
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