QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: jpet.106.117648v1 321/2/462    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yang, J. Articles by Low, P. S. Search for Related Content PubMed PubMed Citation Articles by Yang, J. Articles by Low, P. S.

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

This article has been cited by other articles:

				H. Chen, J. Yang, P. S. Low, and J.-X. Cheng Cholesterol Level Regulates Endosome Motility via Rab Proteins Biophys. J., February 15, 2008; 94(4): 1508 - 1520. [Abstract] [Full Text] [PDF]

				A. Qiu, S. H. Min, M. Jansen, U. Malhotra, E. Tsai, D. C. Cabelof, L. H. Matherly, R. Zhao, M. H. Akabas, and I. D. Goldman Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1669 - C1678. [Abstract] [Full Text] [PDF]