Cellular uptake of aminoglycosides, guanidinoglycosides, and poly-arginine

Article abstract of DOI:10.1021/ja0360135

Aminoglycosides (including neomycin B and tobramycin) exhibit poor uptake by eukaryotic cell lines. When the amines of these natural products are converted into guanidine groups, their cellular uptake is dramatically enhanced. We have synthesized BODIPY-c

Full text of DOI:10.1021/ja0360135

Published on Web 09/20/2003
Cellular Uptake of Aminoglycosides, Guanidinoglycosides, and Poly-arginine
Nathan W. Luedtke,† Peter Carmichael, and Yitzhak Tor*
Department of Chemistry and Biochemistry, UniVersity of California, San Diego, La Jolla, California 92093-0358
Received May 7, 2003; E-mail: ytor@ucsd.edu
Charged molecules over 500 amu typically exhibit poor bio-
availability.1 This limits the delivery of many therapeutically active
molecules to their intended targets. Polycationic molecules provide
important exceptions to this generalization. Modification of bovine
serum albumin (BSA) with ethylenediamine produces “cationion-
ized BSA”, a highly effective antigen carrier.2 Despite its size (over
66 000 amu), cationized BSA efficiently enters cells via an unknown
path involving adsorptive uptake.3 More recently, a number of poly-
arginine peptides,4 peptoids,5 and peptidomimetics6 have been found
to exhibit highly efficient uptake into a wide range of mammalian
cell types. The conjugation of such poly-Arg peptides to large
molecules can facilitate the transduction of peptide, protein, and
nucleic acid conjugates into cells.4a The mechanism responsible
for poly-Arg-mediated transport is still unclear, but may involve a
receptor-mediated, nonendocytotic route.4a,7 In this report, we
present two guanidine-modified natural products that exhibit
exceptional cellular membrane translocation efficiencies and share
an uptake mechanism similar to that of poly-Arg peptides.
Aminoglycoside antibiotics are a therapeutically important family
of polycations that selectively inhibit prokaryotic protein biosyn-
thesis.8 We recently reported that converting the amines on amino-
glycosides into guanidine groups increases both the RNA affinity
and the anti-HIV activities of the resulting compounds, termed
“guanidinoglycosides”.9 To examine how the cellular uptake of
these compounds is affected by guanidinylation, we have synthe-
sized a series of BODIPY-tagged aminoglycosides and guanidi-
noglycosides based upon tobramycin and neomycin B (Figure 1).
BODIPY is an excellent fluorescent probe for cellular uptake studies
because its fluorescence is relatively insensitive to changes in the
local environment. By using fluorescein as a reference (φ ) 0.93
at pH 9.0), we determined that the emission quantum efficiency
(φ) of all five BODIPY conjugates 1-5 is equal to 1.0 at pH 7.5.
The uptake of BODIPY-containing glycosides by two different
eukaryotic cell lines was studied using both fluorescence activated
cell sorting (FACS) and fluorescence microscopy. Examples of
FACS histograms are presented in Figure 2, and selected micros-
copy images are shown in Figure 3. In a typical experiment, cell
cultures were treated with 0.5-5 µΜ of each compound for 0.5-1
h, washed twice with buffer, cleaved with trypsin, and quantified
for fluorescence at 530 nm.10 Both fluorescent aminoglycosides (1
and 3) display poor cellular uptakes (slightly above the autofluo-
rescence of the cell itself) (Table 1).11,12 Upon guanidinylation, the
cellular uptake of tobramycin is enhanced by approximately 10-
fold (relative to autofluorescence), and the enhancement for neo-
mycin B is approximately 20-fold (Figure 2A,B and Table 1).12,13
Because fluorescent poly-Arg peptides are known to exhibit better
uptake than poly-Lys conjugates,5,14,15 it appears that poly-guanidino
compounds exhibit better transport properties relative to the
corresponding polyamines, regardless of the molecular scaffold.16
Figure 1. Aminoglycosides and guanidinoglycosides evaluated for cellular
uptake. See Supporting Information for the synthesis and characterization
of 1-5. Compound 6 has already been reported.9b
As compared to a common poly-Arg transduction peptide,4a,5,7,14,15
the guanidinoglycosides show the same, or even better, cellular
uptake efficiencies. Guanidino-tobra-BODIPY (2) has four fewer
guanidinium groups than BODIPY-Cys(Arg)9 (5), but shows
approximately the same transport efficiency (Table 1). Importantly,
guanidino-neo-BODIPY (4) consistently has a better cellular uptake
as compared to the poly-Arg peptide BODIPY-Cys(Arg)9 (5)
(Figure 2C, Table 1). This suggests that the semirigid preorgani-
zation of the guanidinium groups on the glycoside core may better
facilitate translocation across the cell membrane.17 In contrast to
the results obtained for a family of poly-Arg peptoids,5 the amphi-
pathic properties provided by the flexible methylene chains of poly-
(Arg)9 residues do not appear essential for membrane transport of
guanidinoglycosides. To address the possibility that guanidino-
neomycin B enters cells through a different mechanism than poly-
Arg, a competition experiment was conducted between BODIPY-
Cys(Arg)9 (5) and the unlabeled guanidino-neomycin B (6). FACS
analysis shows that guanidino-neomycin B (6) effectively inhibits
the transport of BODIPY-Cys(Arg)9 into cells (Figure 2D and Table
1), suggesting a common pathway responsible for the uptake of
both compounds.
Microscopy experiments have been conducted using both
fluorescein-labeled and BODIPY-labeled guanidinoglycosides. The
relative intensities of individual cells, following treatment with either
† Present address: Department of Chemistry and Biochemistry, Yale University,
New Haven, CT 06520-8107.
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J. AM. CHEM. SOC. 2003, 125, 12374-12375
10.1021/ja0360135 CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
labeled conjugates,10 as well as 10T1/2 cells (Figure 3C). Taken
together, this suggests that the relative uptake efficiencies and
cellular localization of these compounds are not highly dependent
on the cell type or dye molecule used.19
In summary, we have found that, unlike aminoglycosides,
guanidinoglycosides exhibit highly efficient uptake by eukaryotic
cell cultures via a mechanism similar to that of a poly-arginine
peptide.20 Guanidine-containing modified natural products, including
guanidino-neomycin, may facilitate the efficient cellular transport
of pharmacologically important cargo molecules.21
Acknowledgment. We thank Dr. Roger Tsien for his comments,
and W. Coyt Jackson for technical assistance with the FACS
experiments. Financial support was provided by the NIH (AI 47673)
and the DOE (DE-FG03-01ER63276).
Supporting Information Available: Synthesis and characterization
of compounds 1-5, conditions for the cellular uptake studies, and
microscopy images of 10T1/2 cells treated with aminoglycoside- and
guanidinoglycoside-fluorescein conjugates (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 2. FACS histograms showing the fluorescence intensity versus cell
count for 10 000 individual 10T1/2 cells following a 1 h incubation with
0.5 µM of the following: (A) tobra-BODIPY (red) and guanidino-tobra-
BODIPY (white); (B) neo-BODIPY (red) and guanidino-neo-BODIPY
(white); (C) BODIPY-Cys(Arg)9 (red) or guanidino-neo-BODIPY (white);
(D) uptake of BODIPY-Cys(Arg)9 inhibited by guanidino-neomycin B (6),
at 0 µM (red), 10 µM (black), and 200 µM (green).
References
(1) Lipinski, C. A.; Lombardo, F.; Dominsy, B. W.; Feeney, P. J. AdV. Drug
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(4) For a review, see: (a) Futaki, S. Int. J. Pharm. 2002, 245, 1-7. For
â-peptides, see: (b) Umezawa, N.; Gelman, M. A.; Haigis, M. C.; Raines,
R. T.; Gellman, S. H. J. Am. Chem. Soc. 2001, 124, 360-369.
(5) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman,
L.; Rothbard, J. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003-13008.
(6) Litovchick, A.; Lapidot, A.; Eisenstein, M.; Kalinkovich, A.; Borkow,
G. Biochemistry 2001, 40, 15612-15623.
Figure 3. (A and B) Cross-sectional images of two individual HeLa cells
in solution following a 30 min treatment with 5 µM of guanidino-neo-
BODIPY and cleavage by trypsin (see Supporting Information for full cross-
sectioning of each cell).10 (C) Two neighboring 10T1/2 cells growing on a
culture plate following a 1 h exposure to 1 µM of 4. See Supporting
Information for uptake experiments using fluorescein-containing molecules.
(7) Suzuki, T.; Futaki, S.; Niwa, M.; Tanaka, S.; Ueda, K.; Sugiura, Y. J.
Biol. Chem. 2002, 277, 2437-2443.
(8) (a) Davies, J.; Davis, B. D. J. Biol. Chem. 1968, 243, 3312-3316. (b)
Moazed, D.; Noller, H. F. Nature 1987, 327, 389-394.
(9) (a) Luedtke, N. W.; Baker, T. J.; Goodman, M.; Tor, Y. J. Am. Chem.
Soc. 2000, 122, 12035-12036. (b) Baker, T. J.; Luedtke, N. W.; Tor, Y.;
Goodman, M. J. Org. Chem. 2000, 65, 9054-9058.
Table 1. Mean Fluorescence Intensities of Treated Cellsa
(10) See Supporting Information for experimental details.
c
compound
10T /2b
1
HeLa
(11) Preliminary experiments indicate that for prokaryotic cell cultures (E. coli),
compounds 1-4 all show highly efficient cellular uptake.
none (autofluorescence)
∼40d
830
1000
2100
∼1400e
7900
2000
n.d.f
(12) Similar results are observed for fluorescein-labeled conjugates.10
(13) A recent report describes the incorporation of aminoglycoside-cholesterol
conjugates into liposomal phospholipid complexes capable of delivering
DNA into cells. Guanidinylation of a kanamycin A-cholesterol conjugate
actually decreases its efficiency as a delivery agent. See: Belmont, P.;
Aissaoui, A.; Hauchecorne, M.; Oudrhiri, N.; Petit, L.; Vigneron, J. P.;
Lehn, J. M.; Lehn, P. J. Gene Med. 2002, 4, 517-526. Our preliminary
results indicate that a minimum of five guanidinium groups is needed to
stimulate efficient cellular uptake (kanamycin A has four amines).
(14) Mitchell, D. J.; Kim, D. T.; Steinman, L.; Fathman, C. G.; Rothbard, J.
B. J. Pept. Res. 2000, 56, 318-325.
tobra-BODIPY (1)
60
guanidino-tobra-BODIPY (2)
neo-BODIPY (3)
guanidino-neo-BODIPY (4)
BODIPY-Cys(Arg)9 (5)
BODIPY-Cys(Arg)9 (5) + 10 µM (6)
BODIPY-Cys(Arg)9 (5) + 50 µM (6)
BODIPY-Cys(Arg)9 (5) + 200 µM (6)
240
60
430
280
110
90
n.d.
70
n.d.
a The FACS data are not directly comparable between cell types, as a
higher instrumental gain (about 10-fold) was used for the HeLa experiments.
b Average intensity of 10 000 individual cells treated with 0.5 µM of each
compound for 1 h. c Average intensity of 2000 individual cells treated with
1 µM of each compound for 0.5 h. Under these conditions, a “free” BODIPY
dye molecule Tris-BODIPY shows poor uptake into HeLa cells (similar to
Tobra-BODIPY).10,12 d Estimate based upon data set collected at a higher
instrumental gain. e Estimate based upon data set collected at a lower
instrumental gain. f n.d. ) not determined.
(15) Uemura, S.; Rothbard, J. B.; Matsushita, H.; Tsao, P. S.; Fathman, C. G.;
Cooke, J. P. Circ. J. 2002, 66, 1155-1160.
(16) Contrary to experiments conducted with peptide conjugates of small
organic fluorophores,5,14,15 a recent paper reports that poly-Lys protein
conjugates mediate transduction at the same or even higher levels than
poly-Arg. See: Mai, J. C.; Hongmei, S.; Watkins, S. C.; Cheng, T.;
Robbins, P. D. J. Biol. Chem. 2002, 277, 30208-30218.
(17) For peptides, secondary structure formation can increase transport
efficiencies. See: Ho, A.; Schwarze, S. R.; Mermelstein, S. J.; Waksman,
G.; Dowdy, S. F. Cancer Res. 2001, 61, 474-477.
(18) Treatment of guanidinoglycoside-containing cells with propidium iodide
(a common viability test) indicates that the cells used for these experiments
have intact membranes.
(19) Microscopy experiments suggest that trypsin-mediated cleavage prior to
FACS analysis does not affect the cellular localization, or the relative
uptake efficiencies of these compounds (Figure 3 and Figure S1).10
(20) Previous studies have shown that peptidomimetics based upon neomycin-
arginine conjugates also show efficient cellular uptake.6 This type of
modification doubles the total number of basic groups and results in
compounds possessing an equal number of amide, amine, and guanidine
functionalities. The results presented here represent a direct comparison
of purely amino- versus guanidino-containing glycosides.
fluorescent aminoglycosides or guanidinoglycosides, are consistent
with the trends from FACS experiments.10 Optical cross-sectioning
using scanning confocal fluorescence microscopy indicates that
guanidinoglycosides are found inside of living cells (Figure 3).10,18
Interestingly, two distinct types of cellular localization of guanidino-
neo-BODIPY are observed (Figure 3). Approximately one-half of
the cells exhibit a highly diffuse, cytoplasmic, and nuclear distribu-
tion (Figure 3A), while the other one-half exhibit more localized
nucleolar staining, similar to that reported for poly-Arg peptides
(Figure 3B).4a,7 We have observed similar results with fluorescein-
(21) For potential commercial applications, see: Bonetta, L. The Scientist 2002,
16, 38-40.
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