title: Characterization of the pH of folate receptor-containing endosomes and the rate of hydrolysis of internalized acid-labile folate-drug conjugates.
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 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.
authors.affiliation: Department of Chemistry, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.forename: Hongtao
authors.initials: H
authors.lastname: Chen
authors.forename: Iontcho R
authors.initials: IR
authors.lastname: Vlahov
authors.forename: Ji-Xin
authors.initials: JX
authors.lastname: Cheng
authors.forename: Philip S
authors.initials: PS
authors.lastname: Low
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007624:KB Cells; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D004364:Pharmaceutical Preparations; D011956:Receptors, Cell Surface; D005492:Folic Acid