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

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 folatelinked 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 similar to 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.