Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide Johannes Oehlke1, Gerd Wallukat2, Yvonne Wolf1
Trang 1Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide
Johannes Oehlke1, Gerd Wallukat2, Yvonne Wolf1, Angelika Ehrlich1, Burkhard Wiesner1, Hartmut Berger1 and Michael Bienert1
1
Institute of Molecular Pharmacology, Berlin, Germany;2Max Delbru¨ck Center for Molecular Medicine, Berlin, Germany
In order to evaluate the ability of the cell-penetrating
a-helical amphipathic model peptide KLALKLALKALK
AALKLA-NH2 (MAP) to deliver peptide nucleic acids
(PNAs) into mammalian cells, MAP was covalently linked
to the 12-mer PNA 5¢-GGAGCAGGAAAG-3¢ directed
against the mRNA of the nociceptin/orphanin FQ receptor
The cellular uptake of both the naked PNA and its
MAP-conjugate was studied by means of capillary electrophoresis
combined with laser-induced fluorescence detection,
confo-cal laser scanning microscopy and fluorescence-activated cell
sorting Incubation with the fluorescein-labelled
PNA–pep-tide conjugate led to three- and eightfold higher intracellular
concentrations in neonatal rat cardiomyocytes and CHO
cells, respectively, than found after exposure of the cells to the naked PNA Correspondingly, pretreatment of sponta-neously-beating neonatal rat cardiomyocytes with the PNA–peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively The main rea-sons for the higher bioavailability of the PNA–peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum
Keywords: cell-penetrating peptides; cellular uptake; PNA– peptide conjugates
The wider application of peptide nucleic acids (PNAs) [1] as
antisense agents appears to be limited mainly by poor
cellular uptake [2,3] Improved delivery into mammalian
cells and enhanced antisense activity have been achieved
after covalent coupling of PNAs to cell-penetrating peptides
(CPPs), which are able to enter cells in a nonendocytic but
as yet unknown mode [3–8] The structural requirements for
the delivery activity of peptides have been unclear until now
In order to contribute to an elucidation of structure–
delivery activity relationships we have previously
investi-gated the cellular uptake and biological activity of
CPP–phosphorothioate oligonucleotide conjugates using
the cell-penetrating amphipathic model peptide MAP
(KLALKLALKALKAALKLA-NH2) [9, 10] as the lead
compound [11] The value of the results of this study was
limited, however, by a high cell toxicity of the
phosphoro-thioate oligonucleotide–peptide conjugates Therefore, in
the present study we evaluated the suitability of PNA to
serve as the cargo molecule in MAP-based structure– delivery activity investigations To this end we investigated cellular uptake and biological activity of a 12-mer peptide nucleic acid (5¢-GGAGCAGGAAAG-Lys-3¢; compound I; Table 1) complementary to bases 12–23 of the translated region of the nociceptin/orphanin FQ receptor, proven previously to be sensitive to antisense attacks [12,13], and of its conjugate with MAP (compound II; Table 1) For assessing the cellular uptake, we developed a protocol based
on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) providing absolute quantities of inter-nalized PNA which was used supplementally with confocal laser scanning microscopy (CLSM) and fluorescence-acti-vated cell sorting (FACS)
Material and methods General
Chemicals and reagents were purchased from Sigma (Deisenhofen, Germany), Bachem (Heidelberg, Germany)
or PE Biosystems unless specified otherwise Release of lactate dehydrogenase was assessed by means of LDH-L reagent from Sigma
Synthesis of PNA and PNA–MAP conjugates PNA oligomers were synthesized manually using the t-Boc strategy [14] The peptide segments of the conjugates were synthesized by the solid phase method using standard Boc chemistry [15], after which the PNA moiety was extended from the N-terminus of the peptide by manual Boc coupling according to Christensen et al [14] To introduce the
Correspondence to J Oehlke, Institute of Molecular Pharmacology,
Robert-Ro¨ssle-Str 10, D-13125 Berlin, Germany.
Fax: + 49 30 94793 159, Tel.: + 49 30 94793 267,
E-mail: oehlke@fmp-berlin.des
Abbreviations: CE-LIF, capillary electrophoresis with laser-induced
fluorescence detection; CLSM, confocal laser scanning microscopy;
CM, spontaneously-beating neonatal rat cardiomyocytes; CPP,
cell-penetrating peptide; DPBSG, Dulbecco’s phosphate buffered
saline/glucose; FACS, fluorescence-activated cell sorting;
Fluos, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; MAP,
model amphipathic peptide; PNA, peptide nucleic acid.
(Received 16 March 2004, revised 21 May 2004,
accepted 28 May 2004)
Trang 2fluorescent label, the unprotected N-termini of the PNAs or
the PNA–MAP conjugates were reacted in
dimethylform-amide for 3 days at room temperature with 10 equivalents
of 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester
(Fluos; Boehringer, Mannheim, Germany) Purification
was carried out by semipreparative HPLC on Vydac C18
using a 250· 8 mm column MALDI-MS (Voyager-DE
STR BioSpectrometry Workstation MALDI-TOF;
Persep-tive Biosystems, Inc., Framingham, MA, USA) provided
the expected [M + H]+ peaks (3878 and 5609 Da for I
and II, III, respectively)
Cell culture
CHO cells were cultured in 24-well plates (5· 104cells per
well) or for CLSM on 22· 22 mm coverslips (2 · 104cells)
at 37C in a humidified air environment containing 5%
CO2 in Ham’s F-12 medium supplemented with
290 mgÆmL)1glutamine and 10% (w/v) fetal bovine serum
Spontaneously-beating, neonatal rat cardiomyocytes (CM)
were obtained from ventricles of 1–2 day-old Sprague–
Dawley rats and cultured as described earlier [16]
Experi-ments conformed with the Guide for the Care and Use of
Laboratory Animals (NIH) and were approved by the local
government
The chronotropic response of the CM was measured as
described previously [16] on day 4 after seeding, every 5 min
after cumulative addition of nociceptin/orphanin FQ
(FGGFTGARKSARKLANQ) [17,18] at 37C Antisense
pretreatment of the heart cells was performed on days 1 and
2 after seeding by administration of either 0.2 lMPNA or
PNA–MAP conjugate
Assessment of cellular uptake by CLSM
The CLSM measurements were performed using a LSM
410 inverted confocal laser scanning microscope (Carl Zeiss,
Jena GmbH, Jena, Germany) as described previously
[19, 20] In brief, the fluorescent oligonucleotide derivatives
were dissolved in 1 mL prewarmed (37C) Dulbecco’s
phosphate buffered saline supplemented with 1 gÆL)1
D-glucose (DPBSG) and the cells were overlaid 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 488 nm (Fluos) and
543 nm (trypan blue) and emission was measured at 515 nm
and 570 nm, respectively Three regions of interest (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 FACS
The cells (105 per well) were washed three times with prewarmed DPBSG and then overlaid with 0.2 mL of a freshly prepared prewarmed (37C) solution of the fluor-escent PNA derivative in DPBSG (2 lM I or 0.1 lM II; Table 1) After 30 min incubation at 37C, the cells were washed two times with NaCl/Piand detached by 15 min trypsination at 37C using 0.5 mL 0.05% Trypsin/0.02% EDTA (v/v) per well Then 1 mL culture medium was added and the cell suspension was centrifuged at 1000 g for
8 min Subsequently the cells were resuspended in 1 mL NaCl/Pi and stored on ice until the measurement The accumulation of fluorescence was determined at 525 nm after excitation at 488 nm using a Becton Dickinson (Franklin Lakes, NJ, USA) FACS Calibur flow cytometer with CELLQUESTsoftware Cytograms were acquired with
104cells
Assessment of cellular uptake by CE-LIF The cells were overlaid with 0.2 mL of a prewarmed (37C) solution of the fluorescent oligonucleotide derivative in DPBSG (0.5 lM I; 0.2 lM II; Table 1) immediately after addition of the respective aliquot of the sonicated PNA stock solution to the DPBSG After 30 min incubation at
37C (if not indicated otherwise), the cells were washed four times with ice-cold NaCl/Piand lysed for 2 h at 0C with 0.2 mL 0.1% (v/v) Triton X-100 containing 10 mmolÆL)1 trifluoroacetic acid The lysate, which contained only negligible amounts of fluorescent PNA derivatives (below 10% of total cell-associated PNA) was used for protein determination according to the method of Bradford [21] The wells containing attached cell debris and nuclei along with bound or precipitated PNA derivative were extracted
by sonication for 5 min at 60C with 0.2 mLÆwell)1Tris/ borate buffer (20 mM, pH 7.5) supplemented with 5Murea, 0.1% (w/v) SDS and, as an internal standard, 10 nM e-fluoresceinyl lysine The resulting extracts were centri-fuged for 3 min at 3000 g and stored at)20 C; immedi-ately prior to the CE-LIF analysis the extracts were sonicated for 5 min at 60C
CE-LIF was performed using a P/ACE MDQ system with a P/ACE MDQ Laser-Induced Fluorescence Detector (Beckman Coulter, Fullerton, CA, USA) and a
CZESep-600 neutral coated capillary (31 cm, 50 lM i.d.; Pheno-menex, Aschaffenburg, Germany) Tris/borate (200 mM,
pH 7.5) with 5Murea and 0.1% (w/v) SDS was used as the running buffer The cell extracts were injected into the capillary for 5 s at 0.5 p.s.i and the separations were performed at 650 VÆcm)1 and 25C The peaks of the references appeared after 1.8 min (I), 2.1 min (e-fluorescei-nyl lysine) and 4.2 min (II) Apart from free I and II, the cell extracts contained the largest quantity of compound I or II
in a complex bound form appearing in both cases at
Table 1 Sequences of the PNA derivatives studied.
Compound Sequence
MAP KLALKLALKALKAALKLA-NH 2
(antisense)
(antisense) III Fluos-AGGAGCAGGGAA-MAP
(scrambled)
Trang 33.9 min The assignment for this peak is confirmed by its
complete disappearance after addition of an excess of 2 lM
unlabelled I and the concomitant generation of equally
intensive fluorescent peaks at the positions of the pure
reference compounds I and II, respectively
Quantitation was performed by fluorescence
measure-ment at 520 nm after excitation at 488 nm using an argon
ion laser The peaks were integrated using theP/ACE-SYSTEM
MDQ software (Beckman Coulter, Fullerton, CA, USA),
and were normalized to the area of the internal standard
e-fluoresceinyl lysine in order to eliminate irregularities of
injection and buffer status Because the exact volume of the
sample injected into the capillary remained unknown, the
references used as calibration standards were injected under
essentially the same conditions in order to eliminate this
factor in the subsequent calculations The concentrations of
the references were determined on the basis of the optical
density at 260 nm and proved linearly correlated to the peak
areas in the range between the quantitation limits and
500 nM The quantitation limits (signal-to-noise ratio > 3)
were about 0.5 pmolÆmL)1and 1.5 pmolÆmL)1for I and II,
respectively
Results
Conjugation with MAP leads to an increased intracellular
availability of PNA
In order to examine the ability of MAP to deliver PNA into
intact cells, we investigated the cellular uptake of the
conjugate of I with MAP in comparison with that of naked
I, by means of FACS, CLSM and CE-LIF The former two
protocols have been most widely used so far in such context
The results obtained in this way, however, were suspected
recently to be biased by surface adsorption or fixation
artefacts [22, 23] Moreover, due to the environmental
dependence of the fluorescence intensity, these approaches
only enable relative quantitative conclusions Therefore, we
have developed the third, a CE-LIF based protocol, which
appears capable of supplementing FACS and CLSM by
providing absolute quantitation of internalized PNA
CLSM revealed extensive fluorescence in the cytosol and
nucleus of CM and CHO cells after exposure to both the
naked PNA I and its MAP-conjugate (compound II)
(Fig 1) The intensity of this fluorescence (outside of
vesicles) in all cases was of the same order as that of the
extracellular PNA solution, indicating extensive
nonendo-cytic uptake for both I and II No differentiation between
the permeation behavior of I and II appears possible on the
basis of the CLSM data, except that a lower rate of
re-export became apparent for the conjugate (Fig 1)
FACS, on the other hand, revealed clearly higher
cell-associated fluorescence even after exposure to 100 nM
conjugate, than found after incubation with 2 lM of the
naked PNA (Fig 2) However, in this case, surface
adsorption of the conjugate, combined with washout of
the naked PNA due to the peptide tag and the relatively
long time required for the wash and trypsination processes,
respectively, might have biased the results
In addition to the information provided by CLSM and
FACS, the results obtained by the CE-LIF approach
enab-led a quantitative differentiation between the intracellular
concentrations of I and II If related to the external concentrations, the intracellular concentration of the con-jugate measured by CE-LIF in CM exceeded that of the naked PNA by about eightfold (Fig 3), which correlates well with the respective bioactivities (see below) Complementary uptake experiments performed with more conveniently available CHO cells analogously revealed a significantly higher uptake of the conjugate (Fig 3) Washout effects should influence the CE-LIF results only
to a negligible extent, considering that 3 min at 0C and
15 min at 37C are required for the wash process and for the efflux of about 50% of the internalized naked PNA (Fig 1), respectively That surface adsorption should also
Fig 1 Fluorescence intensity measured by CLSM according to [19] in cytosol and nucleus of CHO cells and cardiomyocytes Exposure to 0.5 l M I or 0.2 l M II for 30 min at 37 C and subsequent re-exchange (RE) into empty buffer for 15 min at 37 C, normalized to the fluor-escence intensity of the external PNA solution Each bar represents the mean of three samples ± SEM.
Fig 2 Cell-associated fluorescence measured by flow cytometry after exposure of CHO cells for 30 min at 37 °C to empty buffer, com-pound I and comcom-pound II Empty buffer (thin line; mean fluorescence intensity 2.6), to 2 l M I (broken line; mean fluorescence intensity 5.7)
or to 0.1 l M II (bold line; mean fluorescence intensity 7.3) Cell number
is plotted on the ordinate as a function of the fluorescence intensity on the abscissa.
Trang 4not interfere decisively with the quantification of the
internalized PNA by CE-LIF is implied by a comparison
of the uptake results depicted in Fig 3 As the extent of
surface adsorption should be comparable in all cases, its
contribution to the uptake results should be confined to the
lowest values found, which were assessed for the uptake into
CHO cells at 0C (Fig 3) Thus, the bias by surface
adsorption of the other results should amount at maximum
to about 20% and 50% for the naked PNA and the
conjugate, respectively The real bias, however, should
clearly be lower than these values because the values found
at 0C, at least in part, should also reflect
energy-independent uptake Consistent with these notions in the
extracts of cells exposed to an analogous disulfide bridged
PNA–MAP-conjugate (Y Wolf, M Bienert & J Oehlke,
unpublished observation) no surface bound conjugate
above the quantification limit of CE-LIF could be detected
In this case exclusively the naked PNA generated by
cleavage of the disulfide bond in the reducing environment
of the cell interior was found
Energy-dependent and energy-independent mechanisms
are involved in the cellular uptake of both naked PNA
and its MAP conjugate
The cellular uptake of both the naked PNA and the
conjugate proved only partially sensitive to lowered
temperature and energy depletion, implying the
involve-ment of nonendocytic mechanisms (Fig 3) On the other
hand, energy-dependent and -independent mechanisms
contributed differently to the cellular uptake of the naked
PNA and of the conjugate (Fig 3), suggesting that distinct
modes were functioning in the two cases The different
sensitivity to the presence of serum observed for the
internalization of the naked PNA and its conjugate,
respectively, probably also suggests distinct modes of uptake (Fig 3), although different association with serum components appear more likely to be the reason here
Intracellular PNA concentration increases more rapidly after exposure of cells to PNA–MAP-conjugate than to naked PNA
The quantity of cell-associated PNA increased significantly faster after exposing CHO cells to II than after incubation with I ( Figs 4 and 5) This finding unravels a further reason for the enhanced bioavailability of the PNA–MAP conju-gate, besides reduced re-export and resistance to serum influences mentioned above After 60 min the cell-associ-ated concentration of the conjugate reached a level at which apparently the efflux equalled the influx, whereas that of the naked PNA increased linearly further (Fig 4) Possible alternative reasons for the observed arrest of the uptake of
Fig 3 Amount of cell-associated PNA determined by CE-LIF in the
extracts of CHO cells and cardiomyocytes Results following exposure
to 0.5 l M I or 0.2 l M II for 30 min at 37 C without (control) and with
energy depletion or in the presence of 10% (v/v) fetal bovine serum
(FBS) and at 0 C For energy depletion, the cells were incubated in
DPBS containing 25 m M 2-deoxyglucose/10 m M sodium azide (DOG/
NaN3) for 60 min at 37 C and subsequently exposed to the PNA
derivative dissolved in the same buffer To facilitate comparison, the
values of I were normalized to an exposure at 0.2 l M according to the
linear concentration dependence shown in Fig 7 Each bar represents
the mean of three samples ± SEM.
Fig 4 Cell-associated PNA determined by CE-LIF in the extracts of CHO cells after exposure to 0.5 l M I (r) or 0.2 l M II (j), respectively,
at 37 °C for different periods of time To facilitate comparison, the values of I were normalized to an exposure at 0.2 l M according to the linear concentration dependence shown in Fig 7 Each bar represents the mean of three samples ± SEM.
Fig 5 Cell-associated PNA determined by CE-LIF in the extracts of CHO cells after exposure to various concentrations of I (r) or II (j) for
30 min at 37 °C Each bar represents the mean of three samples
± SEM.
Trang 5II after 60 min could be aggregation of the conjugate and
peptidase cleavage of the MAP-tag However, even after 2 h
exposure to the cells no significant loss of II was detectable
by CE-LIF in the supernatants, ruling out aggregation and
enzymatic breakdown from playing a noticeable role in this
context
Conjugation with MAP significantly augments the
biological activity of a 12-mer antisense PNA directed
against the nociceptin/orphanin FQ receptor
Pretreatment of CM with 0.2 lM of the naked PNA
(I; Table 1) and its MAP-conjugate (II; Table 1) lowered
the chronotropic effect exerted by the neuropeptide
noci-ceptin [24] by 50% and 90%, respectively ( Figs 6 and 7)
Exposure of CM to conjugates of MAP with a scrambled
PNA containing the same base composition
(com-pound III; Table 1) did not negatively influence the
chronotropic effect (Fig 7) These results infer, as anticipa-ted, antisense down-regulation of the nociceptin/orphanin
FQ receptor to be the mechanism of biological activity of compounds I and II The antisense pretreatment remained without any influence on the basal beating rate of the CM, implying that the PNA derivatives are nontoxic at the concentration used Consistent with this notion, no signi-ficant signs of toxicity were found by means of LDH release and trypan blue exclusion throughout all cellular uptake experiments of the present study
Discussion Improved delivery into mammalian cells has been achieved
by covalent coupling of various highly polar bioactive substances with CPPs [10,25–27] The structural require-ments for the delivery activity of peptides, however, remained unclear until now As a prerequisite for an elucidation of such structural requirements in the present work we evaluated the suitability of a conjugate of the synthetic CPP MAP
(KLALKLALKALKAALKLA-NH2) [9,10] with a 12-mer peptide nucleic acid (5¢-GGAGCAGGAAAG-3¢) directed against the mRNA
of the nociceptin/orphanin FQ receptor [12,13] to serve as the parent compound in planned structure–delivery activity relationship investigations
In distinction to earlier studied conjugates of MAP with phosphorothioate oligonucleotides [11] the MAP–PNA conjugates proved nontoxic in the concentration range up
to 1 lM Consistent with previous reports about substan-tially enhanced bioavailability of PNA after conjugation with natural CPPs [3–8], an almost one order of magnitude higher intracellular PNA concentration was achieved after exposing cells to the MAP–PNA conjugate II than after incubation with the naked PNA Correspondingly, pretreat-ment of CM with II impaired the chronotropic effect elicited
by the neuropeptide nociceptin [16–18,24] more than five-fold more than preincubation with the naked PNA
In line with a nonendocytic mode of uptake regarded to
be typical of CPPs [10,25,27], a faint cytosolic and nuclear fluorescence of comparable intensity than measured exter-nally was detected by CLSM in cells treated with II in addition to a clearly visible vesicular fluorescence Surpris-ingly, however, a similar pattern was observed after exposing the cells to the naked PNA, suggesting nonend-ocytic as well as endnonend-ocytic uptake mechanisms to be involved in both cases Quantitation of the intracellular PNA by CE-LIF supported this notion The measured quantities corresponded to more than 20-fold and fourfold enrichments in the cell interior for the MAP-conjugate and the naked PNA, respectively (related to the external concentration and a ratio of about 10 lL cell volumeÆmg protein)1[11]) If the internalization had proceeded primar-ily by an endocytic mechanism, an intracellular concentra-tion of, at maximum, about 10% of the external level should
be expected, considering the total volume of endosomes comprising maximally 10% of the cell volume [28] Thus the commonly accepted interpretation of the different cellular uptake of naked PNA and PNA–CPP conjugates by an endocytic internalization of the former and a nonendocytic one of the latter appears insufficient for explaining our results As one of the possible explanations, our observation
Fig 6 Dose–response curve for the influence of nociception on the
beating rate of CM measured on day 4 of culture without and with
pretreatment with either 0.2 l M of I or II on day 1 and 2 after seeding.
The basal beating rate of the CM on day 4 of culture was 191 ± 12
beatsÆmin)1(SD; n¼ 30) Each point represents the mean of 30–40
cells or cell cluster ± SEM.
Fig 7 Influence of 10)5M nociception on the beating rate of CM
measured on day 4 of culture without and with pretreatment with either
0.2 l M of II or III (Table 1) on day 1 and 2 after seeding Each point
represents the mean of 30–40 cells or cell cluster ± SEM The
differ-ence between the control and the asterisk-marked bar is statistically
significant at P £ 0.05 (Student’s t-test).
Trang 6that energy-dependent and -independent mechanisms
con-tribute differently to the cellular uptake of the naked PNA
and of the conjugate suggests that distinct modes of
nonendocytic uptake should be operative in both cases
Our findings that the MAP–PNA conjugate in contrast to
the naked PNA proved resistant to re-export provide a
further explanation of the different intracellular levels
measured for the conjugate and the naked PNA Analogous
different susceptibilities against re-export were observed for
phosphorothioate oligonucleotides and
MAP–phosphoro-thioate oligonucleotide conjugates [11], suggesting that such
peptide mediated effects are not confined to particular cargo
molecules In addition, the uptake differences between
conjugate and naked PNA were strengthened by serum
influences Similarly Astriab-Fischer et al [29] found that
the presence of serum in the incubation medium did not
affect the uptake of CPP–phosphorothioate oligonucleotide
conjugates in contrast to that of the naked oligonucleotides,
infering that such peptide mediated resistance to serum
influences is a more general reason for the improved
bioavailability of CPP conjugates
In conclusion, our study revealed the ability of the
synthetic CPP MAP to significantly increase the
bioavail-ability and bioactivity of PNA without eliciting enhanced
cell toxicity These results along with the possibility to
measure absolute quantities of internalized PNA by
CE-LIF provide promising preconditions for studies on
struc-ture–delivery activity relationships which are under way
Acknowledgements
We thank H Hans, M Wegener and G Vogelreiter for excellent
technical assistance This work was supported by the European
Commission (QLK3-CT-2002-01989).
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