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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Characterization of the pH of Folate Receptor-Containing Endosomes and the Rate of Hydrolysis of Internalized Acid-Labile Folate-Drug Conjugates

Department of Chemistry (J.Y., H.C., J.-X.C., P.S.L.), Weldon School of Biomedical Engineering (H.C., J.-X.C.), Purdue University, West Lafayette, Indiana; and Endocyte, Inc., West Lafayette, Indiana (I.R.V.)

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 (t_1/2 = 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.

Address correspondence to: Dr. Philip S. Low, Department of Chemistry, Purdue University, West Lafayette, IN 47907. E-mail: plow{at}purdue.edu

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