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Research ArticleMETABOLISM, TRANSPORT, AND PHARMACOGENOMICS

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

Jun Yang, Hongtao Chen, Iontcho R. Vlahov, Ji-Xin Cheng and Philip S. Low
Journal of Pharmacology and Experimental Therapeutics May 2007, 321 (2) 462-468; DOI: https://doi.org/10.1124/jpet.106.117648
Jun Yang
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Hongtao Chen
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Iontcho R. Vlahov
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Ji-Xin Cheng
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Philip S. Low
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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 ∼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.

Footnotes

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

  • doi:10.1124/jpet.106.117648.

  • ABBREVIATIONS: FR, folate receptor; FRET, fluorescence resonance energy transfer; BODIPY FL, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl)amino)hexanoic acid, fluorescein; GPI, glycophosphoinositol; FITC, fluorescein isothiocyanate; ALFR, acid-labile folate-FRET reporter; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry.

  • ↵ Embedded Image The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

  • ↵1 Current affiliation: St. Jude Children's Research Hospital, Memphis, Tennessee.

    • Received November 26, 2006.
    • Accepted February 6, 2007.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 385 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 385, Issue 1
1 Apr 2023
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Research ArticleMETABOLISM, TRANSPORT, AND PHARMACOGENOMICS

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

Jun Yang, Hongtao Chen, Iontcho R. Vlahov, Ji-Xin Cheng and Philip S. Low
Journal of Pharmacology and Experimental Therapeutics May 1, 2007, 321 (2) 462-468; DOI: https://doi.org/10.1124/jpet.106.117648

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Research ArticleMETABOLISM, TRANSPORT, AND PHARMACOGENOMICS

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

Jun Yang, Hongtao Chen, Iontcho R. Vlahov, Ji-Xin Cheng and Philip S. Low
Journal of Pharmacology and Experimental Therapeutics May 1, 2007, 321 (2) 462-468; DOI: https://doi.org/10.1124/jpet.106.117648
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