Cellular uptake of antisense oligonucleotides after complexingor conjugation with cell-penetrating model peptides J.. Bienert Institute of Molecular Pharmacology, Berlin, Germany The upt
Trang 1Cellular 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-penetratcomplex-ing 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
E X P E R I M E N T A L P R O C E D U R E S
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: oehlke@fmp-berlin.de
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)
Trang 21.0 mLmin)1with 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
A PL RP-S 300 A, 8 lm 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 0.01M triethylammonium acetate
pH 9.0 (A) and acetonitrile/water 9 : 1 (B) at a flow rate of
1.0 mLÆmin)1with 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 mLof buffer A, 500 lL0.01M
trifluoroacetic acid, 1500 lLtrifluoroacetic acid/acetonitrile
1 : 1 v/v, 500 lL0.01Mtrifluoroacetic acid, 250 lLwater
and 250 lLbuffer 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-mMaqueous solution of the respective
cysteine containing peptide were mixed with two volumes
of a 100-mM aqueous solution of di-Na-5,5¢-dithiobis
(2-nitrobenzoic acid) (Ellman’s reagent) and maintained at
60C for 2 h Subsequently the precipitates were
centri-fuged off and washed four times with one volume of water,
to which 0.1MNaOH was added until the solution became
slightly yellow Finally three volumes of a 1 : 1 mixture
of 0.01M trifluoroacetic acid and ethanol were added to
the washed precipitate, resulting in a 1-m solution or
suspension Irrespective of residual impurities (dithiobis-nitrobenzoic acid and thio-(dithiobis-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 mMEllman 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 60C 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 (0.01M) was added and this final mixture was sonicated for 5 min at 60C and 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 0.01M ammonium bicarbonate/ethanol 2 : 1 (to at least 10 lM)
Approximate-ly 10% of noncovalentApproximate-ly 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)0.1M
(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.
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.
Trang 3Cell 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 (105 cells
perwell) or for CLSM on 22· 22 mm coverslips (2 · 104)
at 37C in a humidified 5% CO2 containing air
environ-ment in minimal essential medium (MEM) suppleenviron-mented
with 290 mgÆmL)1glutamine and 10% fetal bovine serum
and used for the uptake experiments after 4 days
CHO-cells were cultured analogously (Ham’s F-12; 5· 104cells
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 mLprewarmed (37C)
Dulbecco’s phosphate buffered saline supplemented with
1 gÆL)1D-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 mLof a prewarmed
(37C) 0.5 lM 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 37C,
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/Pi
and lysed for 2 h at 0C with 0.2 mL0.1% 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 0.01Mtrifluoroacetic acid
Subse-quently 0.2 mLper well of triethylammonium acetate buffer
pH 9 (0.01M)/ethanol 2 : 1 (v/v) containing 0.3% SDS and
1 nMfluorescein as an internal standard were added After
standing over night at room temperature the samples were
finally sonicated for 5 min at 60C The resulting extracts were centrifuged for 3 min at 3000 g and stored at)20 C; immediately prior to the GCE-LIF analysis the extracts were sonicated for 5 min at 60C
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 recommendamodifica-tions 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)1and 15C 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 theP/ACE-SYSTEM MDQsoftware (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 nM 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)]
R E S U L T S A N D D I S C U S S I O N
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
Trang 4complex, respectively (Fluo-5¢-PTO-3¢-S-S-X; X ¼ peptide
or -C3H7)
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
peptide¼ 1 : 1) and conjugates proved soluble (at least up
to 100 lM) in 10 mMphosphate 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 cells after exposure to 0.5 l M 24-mer- and 15-mer PTO alone and complexed (1 : 1, mol/mol) or conjugated to Ifor 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).
Trang 5appears 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 ± 8 and
140 ± 26 pmolÆmg)1protein ± 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 lg protein per 106 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-ation with peptides II–IV also led to an enhanced internal-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 °C determined by GCE-LIF Each bar represents the mean from three wells ± SEM.
Trang 6Generally, 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 0C
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 lMand 1 lM, respectively, also exhibited no toxicity However, even 0.5 lMdoses 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 lMdoses 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 lM 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 lMof 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 °C determined by GCE-LI F 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 Ifor 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
incubated for 30 min in DPBS containing 25 m M 2-deoxyglucose/
10 m M sodium azide at 37 C or in DPBSG at 0 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).
Trang 7naked 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
A C K N O W L E D G E M E N T S
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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4 Tung, C.H & Stein, S (2000) Preparation and applications of
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