PT  - JOURNAL ARTICLE
AU  - Jun Yang
AU  - Hongtao Chen
AU  - Iontcho R. Vlahov
AU  - Ji-Xin Cheng
AU  - Philip S. Low
TI  - Characterization of the pH of Folate Receptor-Containing Endosomes and the Rate of Hydrolysis of Internalized Acid-Labile Folate-Drug Conjugates
AID  - 10.1124/jpet.106.117648
DP  - 2007 May 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 462--468
VI  - 321
IP  - 2
4099  - http://jpet.aspetjournals.org/content/321/2/462.short
4100  - http://jpet.aspetjournals.org/content/321/2/462.full
SO  - J Pharmacol Exp Ther2007 May 01; 321
AB  - 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. The American Society for Pharmacology and Experimental Therapeutics