Báo cáo khoa học: Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides pdf

Cellular uptake of antisense oligonucleotides after complexing
or conjugation with cell-penetrating model peptides
J. Oehlke, P. Birth, E. Klauschenz, B. Wiesner, M. Beyermann, A. Oksche and M. Bienert
Institute of Molecular Pharmacology, Berlin, Germany
The uptake by mammalian cells of phosphorothioate oli-
gonucleotides was compared with that of their respective
complexes or conjugates with cationic, cell-penetrating
model peptides of varying helix-forming propensity and
amphipathicity. An HPLC-based protocol for the synthesis
and purification of disulfide bridged conjugates in the 10–
100 nmol range was developed. Confocal laser scanning
microscopy (CLSM) in combination with gel-capillary
electrophoresis and laser induced fluorescence detection
(GCE-LIF) revealed cytoplasmic and nuclear accumula-
tion in all cases. The uptake differences between naked
oligonucleotides and their respective peptide complexes or
conjugates were generally confined to one order of magni-
tude. No significant influence of the structural properties of
the peptide components upon cellular uptake was found.
Our results question the common belief that the increased
biological activity of oligonucleotides after derivatization
with membrane permeable peptides may be primarily due to
improved membrane translocation.
Keywords: oligonucleotide-peptide conjugates; cellular
uptake; cell-penetrating peptides.
The effectiveness of antisense oligonucleotides and peptide
nucleic acids in modifying mammalian cell function can be
improved substantially by covalent attachment or complex-
ing with natural cell-penetrating peptide sequences [1–4].
This increase in biological activity has been commonly
attributed to an enhanced cellular uptake of the conjugates

[5–7]. The peptide components used to date have been
protein-derived sequences that exhibit very different struc-
tural properties, ranging from lipophilic to unstructured and
highly positively charged sequences [5,7–11] as well as to
strongly structured amphipathic ones [12–15]. The structural
requirements for the peptide moiety and the necessity for
covalent attachment remain controversial.
In the present study we investigated the influence of the
complexing or covalent tagging of phosphorothioate oligo-
nucleotides with cationic model peptides of different
structure forming properties (Table 1, Fig. 1) upon the
cellular uptake. The a-helical amphipathic 18-mer model
peptide used here (I) and its derivatives (exhibiting reduced
helicity or amphipathicity) were previously shown to possess
analogous cell penetrating properties to the above men-
tioned natural sequences [16–18]. We observed extensive
cellular uptake of naked oligonucleotides as well as of their
peptide derivatives. The uptake rates were all within an
order of magnitude for a given cell type and oligonucleotide
length irrespective of the mode of peptide binding or peptide
structural properties. Conjugation or complexing of the
oligonucleotides with the most widely used natural vector
peptide, derived from the homeodomain of Antennapedia
[19], led to comparable results. Our results therefore imply
other aspects than an improved translocation across mam-
malian plasma membranes such as increased affinity to
target structures or interactions with oligonucleotide bind-
ing proteins to be also responsible for the enhanced
biological activity of peptide-oligonucleotide derivatives.
EXPERIMENTAL PROCEDURES

General
Peptides were synthesized by the solid phase method using
standard Fmoc chemistry as described previously [17].
Phosphorothioate oligonucleotides were synthesized using
an automated DNA synthesizer model ABI-394 (Applied
Biosystems, Inc., Foster City, CA, USA). 5¢-fluorescein- and
3¢-propyldisulfide modifications were performed using flu-
orescein phosphoramidite and the 3¢ thiol-modifier C3 S–S
CPG, respectively (both from Glen Research, Sterling, VA,
USA).
Chemicals and reagents were purchased from Sigma
(Deisenhofen, Germany) or Bachem (Heidelberg, Germany)
unless specified otherwise. Release of lactate dehydro-
genase was assessed by means of LDH-L reagent from
Sigma.
HPLC analysis
HPLC was performed using a Bischoff HPLC-gradient
system (Leonberg, Germany) with a UV-detector and a
Fluorescence HPLC Monitor RF-551 (Shimadzu).
Analysis of peptides activated with Ellman’s reagent was
carried out using a Polyencap A 300, 5 lm column
(250 · 4 mm internal diameter, Bischoff, Leonberg,
Germany) and 0.01
M
trifluoroacetic acid (trifluoroacetic
acid; A) and acetonitrile/water 9 : 1 (B) at a flow rate of
Correspondence to J. Oehlke, Institute of Molecular Pharmacology,
Robert-Ro
¨
ssle-Str. 10, D-13125 Berlin, Germany.

Fax/Tel.: + 49 30 94793 159/275, E-mail:
Abbreviations: CLSM, confocal laser scanning microscopy;
GCE-LIF, gel-capillary electrophoresis and laser induced
fluorescence detection; AEC, calf aortic endothelial cells;
MEM, minimal essential medium; ROI, regions of interest; DPBSG,
Dulbecco’s phosphate buffered saline/glucose.
(Received 7 March 2002, revised 27 June 2002, accepted 4 July 2002)
Eur. J. Biochem. 269, 4025–4032 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03093.x
1.0 mL min
)1
with gradients from 35 to 60% B (0–15 min).
The detection was performed at 320 nm; dansyl fluores-
cence was measured simultaneously at 540 nm after excita-
tion at 340 nm.
APLRP-S300A,8lm column (150 · 4.6 mm internal
diameter; Polymer Laboratories Ltd, Waltrop, Germany)
with a precolumn containing 60 mg of the same adsorbent
were used for the purification and analysis of the oligonu-
cleotides and peptide-oligonucleotide conjugates. Elution
was carried out using 0.01
M
triethylammonium acetate
pH 9.0 (A) and acetonitrile/water 9 : 1 (B) at a flow rate of
1.0 mLÆmin
)1
with gradients from 8 to 30% B (0–15 min)
for the oligonucleotides and 8–70% B (0–15 min) for the
oligonucleotide peptide conjugates. Before HPLC purifica-
tion, the respective oligonucleotide–dithiothreitol or –pep-
tide reaction mixtures were loaded onto the precolumn

preequilibrated with buffer A. The precolumn was subse-
quently washed with 250 mL of buffer A, 500 lL 0.01
M
trifluoroacetic acid, 1500 lL trifluoroacetic acid/acetonitrile
1 : 1 v/v, 500 lL 0.01
M
trifluoroacetic acid, 250 lL water
and 250 lL buffer A and was then connected to the HPLC
column. Detection was at 260 nm with simultaneous
fluorescence measurement at 540 nm (dansyl) and 520 nm
(fluorescein) after excitation at 340 nm and 488 nm,
respectively.
Peptide activation with Ellman’s reagent
Three volumes of a 1-m
M
aqueous solution of the respective
cysteine containing peptide were mixed with two volumes
of a 100-m
M
aqueous solution of di-Na-5,5¢-dithiobis
(2-nitrobenzoic acid) (Ellman’s reagent) and maintained at
60 °C for 2 h. Subsequently the precipitates were centri-
fuged off and washed four times with one volume of water,
to which 0.1
M
NaOH was added until the solution became
slightly yellow. Finally three volumes of a 1 : 1 mixture
of 0.01
M
trifluoroacetic acid and ethanol were added to

the washed precipitate, resulting in a 1-m
M
solution or
suspension. Irrespective of residual impurities (dithiobis-
nitrobenzoic acid and thio-nitrobenzoic acid) these products
gave comparable conjugate yields in subsequent syntheses
of peptide-oligonucleotide conjugates (30–50%, relative to
the oligonucleotide) to those obtained with commonly used
thiopyridine-activated peptides, which did not precipitate
and therefore required a more laborious HPLC purification.
Peptide–oligonucleotide conjugates
The 3¢ SH-oligonucleotides were obtained by reaction of
3¢ propyldisulfide tagged derivatives with a 1000-fold excess
of dithiothreitol over night at room temperature, followed
by HPLC purification. After evaporation under reduced
pressure of the HPLC fraction to 0.5–1 mL, 1 m
M
Ellman
activated peptide suspension was added (12 lL
2 lLÆnmol
)1
oligonucleotide). Subsequently ethanol was
added to 50% v/v and the reaction mixture was maintained
at 60 °C for 1 h. Thereafter sodium dodecylsulfate was
added to 0.02% and the mixture stored until processing by
HPLC. Prior to HPLC purification an equal volume of
triethylammonium acetate buffer pH 9 (0.01
M
) was added
and this final mixture was sonicated for 5 min at 60 °Cand

immediately loaded on the HPLC precolumn. Aggregation
phenomena which normally prevent the HPLC purification
of the conjugates could be overcome simply by an acidic
wash procedure (see HPLC analysis) which removed the
excess of noncovalently bound peptide while leaving
oligonucleotide conjugate fixed on the polymer support.
The HPLC fractions containing the conjugates, indicated by
simultaneous absorption at 260 nm and dansylfluorescence
at 540 nm (retention times of the residual oligonucleotide,
the conjugates with peptide I and peptides II–V were 6, 13
and 8–11 min, respectively), were lyophilized and the
resulting residues were dissolved in 0.01
M
ammonium
bicarbonate/ethanol 2 : 1 (to at least 10 l
M
). Approximate-
ly 10% of noncovalently bound oligonucleotide resisted the
HPLC purification and therefore these impurities were
tolerated in the uptake experiments. Addition of dithiothre-
itol led to the cleavage of the obtained conjugates combined
with the reappearance of the parent compunds, thus
confirming the disulfide bridged structure. MALDI-MS of
the conjugates [performed using a Voyager-DE STR
BioSpectrometry Workstation MALDI-TOF mass spec-
trometer (Perseptive Biosystems, Inc.) and a 2,4,6-trihydr-
oxyacetophenone/ammonium citrate matrix 0.5)0.1
M
(Aldrich-Chemie, Steinheim, Germany)] posed serious
problems and yielded only small signals exceeding only

slightly the background noise in the expected mass range.
ESI-MS according to Antopolsky et al. [9] failed fully to
detect molecul ions, probably because of the higher number
of positive charges in our peptides.
Table 1. Sequence and structural properties of the peptides studied.
Peptide Sequence Structural properties
I Dansyl-GC-KLALK LALKA LKAAL KLA-NH2 a Helical, amphipathic
II Dansyl-GC-KLGLKLGLKGLKGGLKLG-NH2 Reduced amphipathicity due to strongly impaired helicity
III Dansyl-GC-KALKLKAALALLAKLKLA-NH2 a Helical, nonamphipathic
IV Dansyl-GC-KGLKLKGGLGLLGKLKLG-NH2 Unstructured, nonamphipathic
V Dansyl-GC-RQIKI WFQNR RMKWK K-NH2 a Helical, reduced amphipathicity
Fig. 1. Helical wheel projections of the amphipathic/nonamphipathic
peptide pair I and III.
4026 J. Oehlke et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Cell culture
Calf aortic endothelial cells (AEC), 12th)20th subculture of
a cell line (LKB Ez 7), established and characterized by
Halle et al. [20], were cultured in 24-well plates (10
5
cells
perwell) or for CLSM on 22 · 22 mm coverslips (2 · 10
4
)
at 37 °C in a humidified 5% CO
2
containing air environ-
ment in minimal essential medium (MEM) supplemented
with 290 mgÆmL
)1
glutamine and 10% fetal bovine serum

and used for the uptake experiments after 4 days. CHO-
cells were cultured analogously (Ham’s F-12; 5 · 10
4
cells
per well).
Assessment of cellular uptake by confocal laser
scanning microscopy (CLSM)
The CLSM measurements were performed using a LSM
410 invert confocal laser scanning microscope (Carl Zeiss,
Jena GmbH, Jena, Germany) as described previously by
Lorenz et al. [21]. In brief: the fluorescent oligonucleotide
derivatives were dissolved in 1 mL prewarmed (37 °C)
Dulbecco’s phosphate buffered saline supplemented with
1gÆL
)1
D
-glucose (DPBSG) and the cells were overlayed
with this solution within 5 min. After 30 min observation,
the viability of the cells was assessed by the addition of
Trypan Blue. Excitation was performed at 365 nm (dansyl),
488 nm (Fluos) or 543 nm (Trypan Blue) and emission was
measured at 420, 515 nm or 570 nm, respectively. Three
regions of interest (ROIs, 16 · 16 pixel; 30 scans with a scan
time of 2 s with double averaging) in the cytosol and one in
the nucleus of three selected cells were chosen such that the
intensity of the diffuse fluorescence could be recorded
without substantial interference from vesicular fluorescence.
The intracellular fluorescence signal was corrected for the
contribution of the extracellular fluorescence, arising from
nonideal confocal properties of the CLSM, by estimating

the distribution function of sensitivity in the z direction of
the microscope.
Assessment of cellular uptake by gel-capillary
electrophoresis with laser-induced fluorescence
detection (GCE-LIF)
The cells were overlayed with 0.2 mL of a prewarmed
(37 °C) 0.5 l
M
solution of the fluorescent oligonucleotide
derivative in DPBSG immediately after addition of the
respective aliquot of the sonicated oligonucleotide-stock
solution to the DPBSG. After 30 min incubation at 37 °C,
the incubation solutions were checked for released lactate
dehydrogenase in order to ascertain the integrity of the cells
and the cells were washed four times with ice-cold NaCl/P
i
and lysed for 2 h at 0 °C with 0.2 mL 0.1% Triton X-100
containing 10 mmolÆL
)1
trifluoroacetic acid. The lysate,
which contained only negligible amounts of fluorescent
oligonucleotide derivatives was used for protein determina-
tion according to Bradford [22]. The wells containing
attached cell debris and nuclei virtually quantitatively along
with the bound or precipitated oligonucleotide derivatives,
were washed twice with 0.01
M
trifluoroacetic acid. Subse-
quently 0.2 mL per well of triethylammonium acetate buffer
pH 9 (0.01

M
)/ethanol 2 : 1 (v/v) containing 0.3% SDS and
1n
M
fluorescein as an internal standard were added. After
standing over night at room temperature the samples were
finally sonicated for 5 min at 60 °C. The resulting extracts
were centrifuged for 3 min at 3000 g andstoredat)20 °C;
immediately prior to the GCE-LIF analysis the extracts
were sonicated for 5 min at 60 °C.
GCE-LIF was performed using a P/ACE MDQ system
with a P/ACE MDQ Laser-Induced Fluorescence Detector
(Beckman Coulter, Fullerton, CA, USA) and an eCAP ss
DNA 100-R Kit from the same manufacturer. The LIF
detector used an argon ion laser for excitation at 488 nm
and emission was measured at 520 nm. In slight modifica-
tion of the manufacturer’s recommendations SDS was
added to 0.3% to the polyacrylamide gel and the running
buffer of the eCAP ssDNA 100-R Kit. The cell extracts
were injected into the neutral coated capillary (40 cm/
100 lm internal diameter) at 50 PSI for 0.2 min and the
separations were performed at 500 VÆcm
)1
and 15 °C. As
the exact volume of the sample injected into the capillary
remained unknown, the references used as calibration
standards were injected under essentially the same condi-
tions, so that this factor was eliminated by itself in the
subsequent calculations.
The migration times of the 15-mer and the 24-mer

phosphorothioate oligonucleotides were 25 and 31 min,
those of the corresponding peptide conjugates 29 and
36 min, respectively, related to the normal appearance of
the internal standard (fluorescein) at 19 min The quantita-
tion limits (signal-to-noise ratio > 3) were about 0.1, 1 and
10 pmolÆmL
)1
for the free oligonucleotides, the 15-mer
PTO-peptide conjugates and the 24-mer PTO-peptide
conjugates, respectively. The peaks were integrated using
the
P
/
ACE
-
SYSTEM MDQ
software (Beckman Coulter, Fuller-
ton, CA, USA), and were normalized to the area of the
internal standard fluorescein in order to eliminate irregu-
larities of injection, gel- and buffer status. Quantitation was
performed on the basis of the CE-LIF peak areas and the
concentrations determined at 260 nm of purified calibration
standards, which exhibited linear peak area to concentration
ratios in the range between the quantitation limits and
500 n
M
.
That the values obtained are not biased to more than
20% by adsorption onto the surface of cells or culture plate
was ascertained in exploratory experiments using conditions

with comparable adsorption but different uptake [e.g.
incubation of the cells for 60 min additionally to the
generally used 30-min period (not shown) or influencing the
uptake by energy depletion (see below)].
RESULTS AND DISCUSSION
Components and solubility of the oligonucleotide-
peptide conjugates and complexes
The oligonucleotides used in the present study were a
24-mer phosphorothioate oligonucleotide (acgaacactgatcgtc
ttcggcat; 24-mer PTO) directed against the mRNA of the
ERM-protein moesin [23] and a 15-mer phosphorothioate
oligonucleotide directed against base positions 16–30 (rel-
ative to the translation initiation site) of the vasopressin-
2-receptor mRNA (aggcacagc ggaagt, 15-mer PTO); both
carried a 5¢-fluorescein label and a 3¢-SH tag. The 3¢-SH tag
was either disulfide bridged with the cystein-SH of the
peptide in the conjugates or blocked with propylsulfide in
the cases of the naked oligonucleotide or the peptide
Ó FEBS 2002 Oligonucleotide–CPP constructs (Eur. J. Biochem. 269) 4027
complex, respectively (Fluo-5¢-PTO-3¢-S-S-X; X ¼ peptide
or -C
3
H
7
).
The helical amphipathic 18-mer model peptide I (Table 1;
Fig. 1) served as the parent peptide component of the
conjugates or complexes with these phosphorothioate
oligonucleotides. This synthetic peptide has previously been
shown to enter mammalian cells nonendocytically [16],

comparable to various protein derived peptide sequences
used for improving the effectivity of antisense oligonucleo-
tides [5–7,9,11,14].
Additionally, derivatives of I with graduately impaired
helix forming propensity and amphipathicity (Table 1) from
alanine-glycine replacement (II), uniform distribution
around the helix of the lysines (III, Fig. 1) or both (IV)
were included in the investigations in order to obtain
information about the role of these parameters upon the
cellular uptake of peptide-oligonucleotide complexes and
conjugates. For comparison the natural vector peptide
sequence V (Table 1) derived from the homeodomain of
Antennapedia [19] was used.
All oligonucleotide-peptide complexes (mol PTO/mol
peptide ¼ 1 : 1) and conjugates proved soluble (at least up
to 100 l
M
)in10 m
M
phosphate buffer at pH 7. However, in
the presence of physiological salt concentrations the conju-
gates containing the amphipathic parent peptide I exhibited
extensive precipitation whereas those with the other peptides
remained soluble under physiological conditions (Fig. 2).
The negative influence of the enhanced salt concentration
only upon the solubility of the conjugates containing the
strong amphipathic peptide I suggests that this effect is
accounted for primarily by nonpolar, not by charge
interactions. This notion is supported by the observation
that disturbance of the nonpolar face of the amphipathic

helix of I (after replacement of one leucine by a more polar
glutamine) significantly improved its solubility in physio-
logical buffer (Figs 1 and 2). With a view to practical aspects
this would imply that peptide amphipathicity restricts the
applicability of oligonucleotide-peptide conjugates.
Cellular uptake of the 24- and the 15-mer PTO
complexed or conjugated with I
After exposing bovine aortic endothelial cells to the 24- and
15-mer PTOs and their complexes and conjugates with I a
diffuse cytosolic and nuclear fluorescence of the same order
as that of the external oligonucleotide solution was indicated
by the fluorescence detector in all cases. The measured
fluorescence intensities reveal a higher rate of uptake for the
smaller PTO and, for reasons unclear as yet, a reduced
internalization of its peptide conjugate but an enhanced one
of that of the longer PTO (Fig. 3). In both cases, however,
the nuclear fluorescence measured after exposure of the cells
to the oligonucleotide-peptide conjugates was significantly
lower than the cytosolic fluorescence, whereas no such
difference was observed after incubation with the naked
PTOs or their peptide complexes (Fig. 3). This observation
suggests an inhibition of oligonucleotide translocation
across the nuclear envelope by the covalently attached
peptide for both PTOs.
The fluorescence intensities of the dansyl-moiety attached
to the peptide moiety exhibited an analogous pattern (not
shown) to that observed for 5¢-bound fluorescein of the
oligonucleotide, indicating uptake of the intact complex and
conjugate, respectively. In accordance with these observa-
tions, no noticeable cleavage of the conjugates could be

detected in the incubation solutions in all cases and also in
the lysates of the CHO-cells. In the lysates of the LKB-Ez7
cells on the other hand partial splitting of the disulfide bond
in the cell interior throughout the 30 min incubation period
was indicated by the presence of naked oligonucleotide.
Significant amounts of fluorescent oligonucleotide metabo-
lites, however, indicative of nuclease cleavage, could not be
detected in the lysates of both cell types, very likely due to
the fluorescein- and SH modification, respectively, at both
ends of the oligonucleotide chain.
The relatively high intensity of the cytosolic and nuclear
fluorescence, comparable to that of the external medium,
suggested equilibration between the external oligonucleotide
concentration and that within the cell. This is difficult to
reconcile, however, with the commonly anticipated endo-
cytic mechanism of oligonucleotide uptake. Hence, the
predominant portion of the incorporated oligonucleotide
Fig. 2. HPLC quantitation of the soluble portion of 0.5 l
M
solutions in
NaCl/P
i
of 24-mer PTO–peptide conjugates after various periods of
storage at 37 °C.
Fig. 3. CLSM fluorescence intensity in cytosol and nucleus of LKB-Ez7
cellsafterexposureto0.5l
M
24-mer- and 15-mer PTO alone and
complexed (1 : 1, mol/mol) or conjugated to I for 30 min at 37 °C,
normalized to the fluorescence intensity of the external oligonucleotide

solution. Each bar represents the mean from three cells ± SEM. The
differences between the respective cytosolic fluorescence intensities and
the asterisk-marked bars are statistically significant at P £ 0.05
(Student’s t-test).
4028 J. Oehlke et al. (Eur. J. Biochem. 269) Ó FEBS 2002
appears to have been internalized nonendocytically. The
same had already been indicated by the observation of the
extensive nuclear fluorescence described above, which
presupposes the presence of the internalized oligonucleotide
in a freely diffusible form in the cytosol rather than within
vesicles.
Further support for a nonendocytic mode of uptake
was provided by the high values of 67 ± 8and
140 ± 26 pmolÆmg
)1
protein ± SD determined by GCE-
LIF for the internalized naked 24-mer and 15-mer PTOs,
respectively. These values correspond, respectively, to about
10- and 2 fold enrichments within the cell interior (taking
into account a ratio of 110 lgproteinper10
6
cells in
conjunction with a cell volume of 1.4 pL) [16]. Such high
intracellular oligonucleotide concentrations as outlined
above, however, strongly contradict an endocytic mode of
entry and are in accord with numerous previous reports of
ananlogously high oligonucleotide enrichments in various
cell types [24–27].
The quantities of the internalized naked 24- and 15-mer
PTOs determined by GCE-LIF correlate well with the

corresponding fluorescence intensities measured by CLSM
(Fig. 3), suggesting that the CLSM values resemble actual
concentration profiles, irrespective of environmental influ-
ences which might prohibit quantitative deductions on the
basis of CLSM measurements alone. The analogous parallel
assessment of the cellular uptake by CLSM and GCE-LIF
of the peptide conjugates with the 24-mer PTO, however,
proved problematical because of poor recovery and exten-
sive GCE-LIF peak broadening. Therefore, further inves-
tigations were performed only with the 15-mer
oligonucleotide and its peptide derivatives, as in this case
these shortcomings did not seriously impede the GCE-LIF
analysis.
Cellular uptake of the 15-mer PTO complexed
or conjugated with peptides I-IV
Figure 4 summarizes the CLSM results after exposing
LKB-Ez7 cells to the 15-mer PTO and its complexes and
conjugates with the peptides I–IV. Normalization of the
measured cytosolic and nuclear fluorescences to the external
oligonucleotide fluorescence was omitted here because the
directly measured values correlated better with the GCE-
LIF-results (Fig. 5) than the relative ones. No significant
differences were apparent between the cellular uptakes of
oligonucleotide conjugates or complexes with the helical
amphipathic parent peptide I and those of its derivatives II–
IV exhibiting impaired amphipathicity and helicity (Figs 4
and 5). This finding suggests that peptide amphipathicity
and helicity are not essential for the cellular uptake of
oligonucleotide-peptide conjugates. Analogously complex-
ationwithpeptidesII–IValsoledtoanenhancedinternal-

ization relative to that of the naked oligonucleotide and
covalent binding rather inhibited oligonucleotide transloca-
tion through both the plasma membrane and the nuclear
envelope (Figs 4 and 5). The latter finding contradicts the
currently accepted opinion, that cell penetrating peptides
would mediate an enhanced oligonucleotide uptake directly
into the cytosol by circumventing the endosomal route [2],
but supports recent reports of an impairment of cellular
uptake of antisense oligonucleotides after covalent attach-
ment of peptides [9,11,28]. These authors nevertheless found
an enhanced biological activity of the conjugates, suggesting
that other aspects, such as impaired efflux and influences on
the affinity to the target molecule or upon interactions with
nucleic acid binding proteins, might have more importance
in this context than the translocation across the plasma
membrane.
Additional uptake experiments were performed with
CHO-cells stably transfected with the V2-receptor, which
were used in concomitant antisense experiments. These
data, principally supported the conclusions drawn from the
studies with LKB Ez 7 cells concerning the nonendocytic
mode of uptake, the limited influence of complexing or
covalently tagging with cell penetrating peptides and the
negligible role of structure forming properties of the peptide
upon the entry of oligonucleotides into the cell interior
(Figs 6 and 7). Conjugation of the 15-mer PTO to the
Antennapedia-peptide V, one of the most widely used
vectorpeptides [2,19] led to analogous results (Fig. 6) in
accord with our previous findings [17,18], confirming that
the synthetic model peptides used here, and natural cell

penetrating peptides behave similarly.
Fig. 4. CLSM fluorescence intensity in cytosol and nucleus of LKB-Ez7
cells after exposure to 0.5 l
M
15-mer PTO alone and complexed (1 : 1,
mol/mol) or conjugated to peptides I–IV for 30 min at 37 °C. Each bar
represents the mean from three cells ± SEM.
Fig. 5. Quantities of internalized oligonucleotide after exposure of LKB-
Ez7 cells to 0.5 l
M
15-mer PTO alone and complexed (1 : 1, mol/mol)
or conjugated to peptides I–IV for 30 min at 37 °CdeterminedbyGCE-
LIF. Each bar represents the mean from three wells ± SEM.
Ó FEBS 2002 Oligonucleotide–CPP constructs (Eur. J. Biochem. 269) 4029
Generally, however, the uptake values found with CHO-
cells were considerably lower than that observed after
treating LKB-Ez7 cells (Figs 5–7), consistent with the
repeatedly reported variability of oligonucleotide uptake
between different cell types [24,25,29–31]. Even here,
however, the relatively poor uptake of the naked oligonuc-
leotide into CHO-cells corresponds to an equilibration
between extra- and intracellular oligonucleotide concentra-
tions, which in accord with the above results contradict an
endocytic mode of uptake. This notion is further supported
by the observation that lowering of the temperature to 0 °C
did not significantly affect the uptake, and energy depletion
even enhanced the internalization of naked oligonucleotide
and, to a lower extent, of its complex with I (Fig. 7).
Likewise the latter finding provides an explanation for the
relatively low oligonucleotide levels found in CHO cells as

this behavior appears reconcilable with the action of energy
dependent export pumps which, under normal conditions
maintain a low intracellular oligonucleotide level by coun-
teracting the influx. Such an assumption is supported by
repeated reports of a rapid, energy-dependent export of
oligonucleotides by various cell types [24,25,29]. With
respect to the uptake of the oligonucleotide-peptide conju-
gate, which proved unaffected by energy depletion (Fig. 7),
this would imply that covalent tagging with peptides renders
the oligonucleotide less accessible to such a putative export
pump.
Toxicity of phosphorothioate oligonucleotides and their
complexes and conjugates with peptides
As both phosphorothioate oligonucleotides [32] and amphi-
pathic peptides [33–36] are known to induce biological
effects by binding to cellular proteins, we investigated the
unspecific cell toxicity of the individual components and of
the oligonucleotide-peptide complexes and conjugates by
the MTT method [37] (LDH-liberation and Trypan blue
exclusion led to comparable results, not shown). During
CLSM and GCE-LIF uptake experiments, which lasted not
more than 60 min, no significant toxicity was detected in
any instance. After twofold administration within 18 h to
CHO-cells stably transfected with the V2-receptor, as
required for antisense experiments, the oligonucleotides
and peptides alone, up to 5 l
M
and 1 l
M
, respectively, also

exhibited no toxicity. However, even 0.5 l
M
doses of both
the conjugates and the complexes of the 15-mer PTO with
all peptides, including the Antennapedia sequence V,
administered in this manner led to 20–50% reduced viability
after this treatment. Sixfold administration of 0.1 l
M
doses
over 4 days remained without effect upon viability for all
peptide complexes, but the conjugates, including that with
the Antennapedia sequence, elicited 20–50% reduction in
MTT-activity even at this low concentration. Comparable
effects were observed using analogous peptide derivatives of
a reference oligonucleotide with the same base composition
but a scrambled sequence, indicating that the found toxic
effects were not sequence specific.
Generally these findings suggest a potentiation of the
known unspecific toxicity of phosphorothioate oligonucleo-
tides [32] by complexation, and more markedly, by covalent
binding to cell penetrating peptides.
In parallel antisense experiments this unspecific toxicity,
however, superimposed the antisense effects so that incon-
sistent results were obtained. In total these results (not
shown) provided indication of the down-regulation of the
ERM-protein moesin and the V2-receptor, respectively,
already at 0.5 l
M
concentrations of the PTO-peptide
complexes and conjugates (20–50% relative to cells treated

with the respective scrambled PTO-peptide derivative)
whereas more than 5 l
M
of the naked PTOs were required
to elicit comparable effects.
Taken together the present study provides evidence that
the complexing or conjugation of phosphorothioate oligo-
nucleotides to cationic, cell-penetrating peptides, irrespective
of peptide structural properties, does not substantially alter
the ability of oligonucleotides to cross mammalian plasma
membranes. Our results support reports implying that even
Fig. 6. Quantities of internalized oligonucleotide after exposure of CHO
cells to 0.5 l
M
15-mer PTO alone and conjugated to peptides I, IV and V
for 30 min at 37 °CdeterminedbyGCE-LIF.Each bar represents the
mean from three wells ± SEM.
Fig. 7. Quantities of internalized oligonucleotide after exposure of CHO
cells to 0.5 l
M
15-mer PTO alone and complexed (1 : 1, mol/mol) or
conjugated to peptide I for 30 min at 37 °C in the absence or presence of
25 m
M
2-deoxyglucose/10 m
M
sodium azide and at 0 °C, determined by
GCE-LIF. Before exposure to the oligonucleotide derivative the cells
used for the 2-deoxyglucose/sodium azide and 0 °C experiments were
incubatedfor30mininDPBScontaining25m

M
2-deoxyglucose/
10 m
M
sodium azide at 37 °CorinDPBSGat0 °C, respectively. Each
bar represents the mean from three wells ± SEM. The differences
between the uptake of the naked PTO-15 under normal conditions
and the asterisk-marked bars are statistically significant at P £ 0.05
(Student’s t-test).
4030 J. Oehlke et al. (Eur. J. Biochem. 269) Ó FEBS 2002
naked oligonucleotides are extensively taken up across
mammlian plasma membranes in a nonendocytic manner.
Likewise our findings question the belief that enhanced
bioactivity of complexes and conjugates of cell-penetrating
peptides and oligonucleotides derives solely from an
increased delivery into the cytosol and nucleus, mediated
by the peptide. Therefore, future attempts to optimize the
peptide components of oligonucleotide-peptide derivatives
should focus on aspects other than translocation across the
plasma membrane, e.g. influences upon the binding affinity
to the target nucleic acid or interactions with oligonucleotide
binding, metabolizing or, as suggested by the present results,
exporting proteins.
ACKNOWLEDGEMENTS
We thank J. Dickson for discussion and helpful advice and
W. Schumacher, B. Mohs, A. Loose, B. Dekowski, K. Marsch and
G. Vogelreiter for excellent technical assistance. This work was
supported by the Deutsche Forschungsgemeinschaft (Oe 170/5-2).
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