Selective disruption of early/recycling endosomes: Release of disulfide-linked cargo mediated by a N-alkyl-3β-cholesterylamine-capped peptide

Article abstract of DOI:10.1021/ja803380a

The use of endocytic uptake pathways to deliver poorly permeable molecules into mammalian cells is often plagued by entrapment and degradation of material in late endosomes and lysosomes. As a strategy to prevent the exposure of cargo to these highly hydrolytic membrane-sealed compartments, we synthesized derivatives of the membrane anchor N-alkyl-3β-cholesterylamine that selectively target linked compounds to less hydrolytic early/recycling endosomes. By targeting a pH-dependent membrane-lytic dodecapeptide and a disulfide-linked fluorophore to these compartments in Chinese hamster ovary cells or Jurkat lymphocytes, membranes of early/recycling endosomes were selectively disrupted, resulting in cleavage of the disulfide and escape of the fluorophore into the cytosol and nucleus with low toxicity. The ability of appropriately designed N-alkyl-3β-cholesterylamines to deliver cargo into and release disulfide-linked cargo from relatively nonhydrolytic early/recycling endosomes may be useful for the delivery of a variety of sensitive molecules into living mammalian cells. Copyright

Full text of DOI:10.1021/ja803380a

Published on Web 07/10/2008
Selective Disruption of Early/Recycling Endosomes: Release of
Disulfide-Linked Cargo Mediated by a N-Alkyl-3ꢀ-Cholesterylamine-Capped
Peptide
Qi Sun,† Sutang Cai,‡ and Blake R. Peterson*,§
IntegratiVe Biosciences and Chemistry Graduate Programs, The PennsylVania State UniVersity, UniVersity Park,
PennsylVania 16802, and Department of Medicinal Chemistry, The UniVersity of Kansas, Lawrence, Kansas 66045
Received May 6, 2008; E-mail: brpeters@ku.edu
Receptor-mediated endocytosis (RME) is a major mechanism of
uptake of impermeant molecules by mammalian cells.1 In this process,
extracellular ligands bind cell surface receptors that cluster in dynamic
regions of cellular plasma membranes. By actively pinching off to
form intracellular vesicles, these membrane regions are internalized,
encapsulating ligand-receptor complexes in the cytoplasm. These
vesicles fuse and form early (primary/sorting) endosomes that are
acidified (pH ≈ 6) by the activation of proton pumps, conditions that
generally promote the dissociation of receptors from bound ligands.
Free receptors often cycle back to the cell surface, generally via sub-
sequent trafficking through related recycling endosomes (also termed
the endocytic recycling compartment).2 In contrast, free ligands are
typically directed to more acidic late endosomes and lysosomes (pH
≈ 5), where hydrolases and other enzymes promote their degradation.
Some viruses and other intracellular pathogens exploit RME to enter
cells, but these organisms avoid degradation in lysosomes by expressing
pH-dependent fusogenic proteins that disrupt endosomal membranes.3
To escape entrapment within these membranes and gain access to the
cytosol, Semliki Forest virus disrupts early endosomes whereas
influenza virus disrupts late endosomes during the course of infection.3
We report here the synthesis of novel compounds designed to
enable membrane-bound disulfide-linked cargo to selectively escape
from early/recycling endosomes of living mammalian cells. Because
these endosomes are less acidic and less hydrolytically active than
late endosomes/lysosomes, this approach may be advantageous
when compared to delivery methods that penetrate deeper into the
endosomal system. To selectively deliver compounds into early/
recycling endosomes, we synthesized four derivatives of the
dynamic membrane anchor N-alkyl-3ꢀ-cholesterylamine4,5 (1-4).
Two of these compounds (1, and red fluorescent 2) incorporate PC4,
a pH-dependent membrane-lytic dodecapeptide previously reported6
by Weber to disrupt membranes of liposomes. Two others comprise
the green fluorophore 5-carboxyfluorescein linked through disulfide
(3) and amide (4) bonds. The unmodified PC4 peptide with the
amino acid sequence AcNH(SSAWWSYWPPVA)CONH2 (5) was
additionally prepared as a control.
in endosomes compared to the plasma membrane. Because early/
recycling endosomes are thought to be oxidizing,8 we hypothesized
that the disulfide of 3 should be relatively stable in these compartments.
However, if 3 were exposed to reduced glutathione (GSH), a thiol
present at mM concentrations in the cytosol, this functional group
would be cleaved (Figure 1).9 Correspondingly, disruption of early/
recycling endosomes loaded with 3 by compounds 1 or 2 was proposed
as a mechanism to enable GSH to access these compartments, reduce
the disulfide of 3, and release the soluble fluorophore 6 into the
cytoplasm and nucleus of cells (Figure 1).
Confocal laser scanning microscopy was employed to examine the
subcellular localization of fluorescent compounds added to living
mammalian cells. In Chinese hamster ovary (CHO) cells, compound
3 was found to become localized in defined intracellular compartments
that reside outside of the cell nucleus (Figure 2). These compartments
were identified as early/recycling endosomes by essentially complete
intracellular colocalization with red fluorescent transferrin protein, a
highly selective marker.10 As a control, cells were similarly treated
with red fluorescent DiI-labeled low density lipoprotein (LDL), a
protein that selectively accumulates in late endosomes and lysosomes.11
When added to mammalian cells, derivatives of N-alkyl-3ꢀ-
cholesterylamine have been shown to become avidly incorporated in
cellular plasma membranes and engage a membrane trafficking
pathway that involves rapid cycling between the cell surface and
intracellular endosomes, similar to many natural cell surface receptors.4
The partitioning of these compounds between the plasma membrane
and endosomes is affected by the structure of the linker region proximal
to the membrane anchor.7 In 1-4, the glutamic acid residue(s) in this
region were installed to enhance the localization of these compounds
† Integrative Biosciences Graduate Program, Penn State University.
‡ Chemistry Graduate Program, Penn State University.
§ The University of Kansas.
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10064 J. AM. CHEM. SOC. 2008, 130, 10064–10065
10.1021/ja803380a CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. Strategy for the selective release of disulfide-tethered cargo from
membranes of early/recycling endosomes mediated by 1 or 2: (A) products
of cleavage of 3 by glutathione; (B) mechanism of release of fluorophore
6 into the cytosol and nucleus of mammalian cells.
Figure 3. (A-J) Confocal fluorescence and DIC micrographs of living
cells treated with fluorescent probes. (A-F) CHO cells were treated with
3 or 4 (5 µM) and 1, 2, or 5 (8 µM) for 24 h. In panel F, [chloroquine] )
5 µM. In panels G-J, Jurkat lymphocytes were treated with 3 or 4 (2.5
µM) and 1 or 5 (2 µM) for 12 h. Scale bars ) 10 µm. (K) Toxicity to CHO
and Jurkat cells after incubation with 1 for 48 h at 37 °C.
Figure 2. Confocal laser scanning and differential interference contrast
(DIC) micrographs of living CHO cells treated with green fluorescent 3 (5
µM) for 12 h followed by (A) red fluorescent Texas Red transferrin (500
nM) or (B) DiI-LDL (8 nM) for 5 min. Colocalization of red and green
fluorescence is shown as yellow pixels in the DIC overlay images. Arrows
point to distinct red fluorescence. Scale bar ) 10 µm.
that disrupt early/recycling endosomes in CHO cells (8 µM) or
Jurkat lymphocytes (2 µM, see the Supporting Information).
Although other synthetic vehicles that disrupt endosomes have been
reported,14 the ability of N-alkyl-3ꢀ-cholesterylamines to specifically
target a subset of relatively nonhydrolytic early/recycling endosomes
and release disulfide-linked cargo from these compartments may be
advantageous for a variety of cellular delivery applications.
Treatment with DiI-LDL revealed distinct red fluorescence, establishing
that in this cell line the N-alkyl-3ꢀ-cholesterylamine membrane anchor
promotes delivery of the fluorophore of 3 to early/recycling endosomes
with a high level of specificity.
Acknowledgment. We thank the NIH (R01-CA83831) for
financial support. We thank Mrs. Ewa Maddox for technical
assistance.
Compared to 3 alone, living cells treated with both 3 and 1 (or
2) showed a strikingly different pattern of intracellular fluorescence
(Figure 3). When combined with 1 or 2, the green fluorescence of
3 was released from entrapment in early/recycling endosomes and
fluorescence was observed in the cytosol and nucleus. As shown
in Figure 3, this release of fluorescent cargo from endosomal
membranes was effective in both adherent cells (CHO) and
suspension cells (human Jurkat lymphocytes). Consistent with the
model shown in Figure 1, replacement of the disulfide of 3 with
the amide bond of 4 blocked release of the fluorophore (Figure
3C,I). The red fluorescence of 2 allowed visualization of the linked
PC4 peptide in early/recycling endosomes (see the Supporting
Information and Figure 3E,F). Colocalization of 1 or 2 with 3 in
these compartments was required to promote efficient cargo release;
little effect was observed with the unmodified PC4 peptide (5). To
investigate the importance of endosomal acidity on the function of
the PC4 peptide,6 we increased endosomal pH by adding chloro-
quine12 and bafilomycin A113 (Supporting Information). These
compounds blocked release of the fluorophore (Figure 3, compare
panels E and F), consistent with the pH-dependent membrane-lytic
activity of PC4. Because the acidity of endosomes is required for
efficient membrane disruption, deleterious effects of 1 and 2 on
the plasma membrane, which is surrounded by media of pH 7.4,
should be limited. Consistent with this idea, assays of cellular
viability (Figure 3K) revealed that 1 is nontoxic under conditions
Supporting Information Available: Supporting figures, experi-
mental procedures, and compound characterization data. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Conner, S. D.; Schmid, S. L. Nature 2003, 422, 37–44.
(2) Maxfield, F. R.; McGraw, T. E. Nat. ReV. Mol. Cell. Biol. 2004, 5, 121–
132.
(3) Lakadamyali, M.; Rust, M. J.; Zhuang, X. Microbes Infect. 2004, 6, 929–
936.
(4) Peterson, B. R. Org. Biomol. Chem. 2005, 3, 3607–3612.
(5) Boonyarattanakalin, S.; Hu, J.; Dykstra-Rummel, S. A.; August, A.;
Peterson, B. R. J. Am. Chem. Soc. 2007, 129, 268–269.
(6) Hirosue, S.; Weber, T. Biochemistry 2006, 45, 6476–6487.
(7) Boonyarattanakalin, S.; Martin, S. E.; Dykstra, S. A.; Peterson, B. R. J. Am.
Chem. Soc. 2004, 126, 16379–16386.
(8) Austin, C. D.; Wen, X.; Gazzard, L.; Nelson, C.; Scheller, R. H.; Scales,
S. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17987–17992.
(9) Saito, G.; Swanson, J. A.; Lee, K. D. AdV. Drug DeliV. ReV. 2003, 55,
199–215.
(10) Sheff, D.; Pelletier, L.; O’Connell, C. B.; Warren, G.; Mellman, I. J. Cell.
Biol. 2002, 156, 797–804.
(11) Ghosh, R. N.; Gelman, D. L.; Maxfield, F. R. J. Cell Sci. 1994, 107, 2177–
2189.
(12) Adachi, K.; Ichinose, T.; Takizawa, N.; Watanabe, K.; Kitazato, K.;
Kobayashi, N. Arch. Virol. 2007, 152, 2217–2224.
(13) Yoshimori, T.; Yamamoto, A.; Moriyama, Y.; Futai, M.; Tashiro, Y. J. Biol.
Chem. 1991, 266, 17707–17712.
(14) Wolff, J. A.; Rozema, D. B. Mol. Ther. 2008, 16, 8–15.
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