Cellular delivery of small interfering RNA by a
non-covalently attached cell-penetrating peptide:
quantitative analysis of uptake and biological effect
Sandra Veldhoen, Sandra D. Laufer, Alexander Trampe and Tobias Restle*
Institut fu ¨r Molekulare Medizin, Universita ¨tsklinikum Schleswig-Holstein, Universita ¨t zu Lu ¨beck,
Ratzeburger Allee 160, 23538 Lu ¨beck, Germany
Received October 9, 2006; Accepted October 18, 2006
Cell-penetrating peptides (CPPs) have evolved as
promising new tools to deliver nucleic acids into
cells. So far, the majority of these delivery systems
require a covalent linkage between carrier and cargo.
To exploit the higher flexibility of a non-covalent
strategy, we focused on the characterisation of a
novel carrier peptide termed MPGa, which sponta-
neously forms complexes with nucleic acids. Using
a luciferase-targeted small interfering RNA (siRNA)
as cargo, we optimised the conditions for MPGa-
mediated transfection of mammalian cells. In this
system, reporter gene activity could be inhibited up
to 90% with an IC50 value in the sub-nanomolar
range. As a key issue, we addressed the cellular
ing various approaches. First, transfection of HeLa
cells with MPGa/siRNA complexes in the presence of
several inhibitors of endocytosis showed a signi-
ficant reduction of the RNA interference (RNAi)
effect. Second, confocal laser microscopy revealed
a punctual intracellular pattern rather than a diffuse
distribution of fluorescently labelled RNA-cargo.
These data provide strong evidence of an endo-
cytotic pathway contributing significantly to the
uptake of MPGa/siRNA complexes. Finally, we quan-
tified the intracellular number of siRNA molecules
after MPGa-mediated transfection. The amount of
siRNA required to induce half maximal RNAi was
10000 molecules per cell. Together, the combination
of methods provided allows for a detailed side by
side quantitative analysis of cargo internalisation
and related biological effects. Thus, the overall
efficiency of a given delivery technique as well as
the mechanism of uptake can be assessed.
Today there is a fast growing number of nucleic acid-based
strategies to modulate a vast variety of cellular functions
[for a review see: (1)]. Several classes of oligonucleotides
like aptamers, transcription factor-binding decoy oligo-
nucleotides, ribozymes, triplex-forming oligonucleotides,
immunostimulatory CpG motifs, antisense oligonucleotides
(including peptide nucleic acids), small interfering RNAs
(siRNAs) and microRNAs have attained much interest as a
research tool owing to their highly specific mode of action.
Even more important, these oligomeric nucleic acids do
have a considerable potential to be used as therapeutics.
However, the bottleneck of any nucleic acid-based strategy
remains the cellular delivery of these macromolecules.
Essentially, the nucleic acid delivery techniques available
today comprise various physical and chemical methods,
viral and non-viral vector systems, and uptake of naked
nucleic acids. They all have certain advantages and disad-
vantages and might only be appropriate if particular require-
ments are fulfilled. In general, physical and chemical
methods like microinjection, electroporation or particle
bombardment as well as calcium phosphate co-precipitation
are highly efficient but rather harmful for the target cells
and lack the potential to be applicable in vivo. There is gen-
eral consent that viral vector systems are the most efficient
vehicles to deliver nucleic acids into cells. However, despite
substantial efforts over the last 15 years, up to now research
has failed to develop suitable and especially safe viral sys-
tems [for a review see: (2,3)]. On the contrary, the field has
experienced several setbacks causing important clinical trials
to be put on hold (4–6). As a result of the difficulties
encountered with these viral vectors (e.g. mutagenesis and
immune responses) much attention was paid to the develop-
ment of allegedly safer non-viral delivery systems. This con-
approaches yielding various degrees of enhanced cellular
uptake of nucleic acids. Currently, cationic lipids and poly-
mers are used as a standard tool to transfect cells in vitro.
*To whom correspondence should be addressed. Tel: +49 451 500 2745; Fax: +49 451 500 2729; Email: firstname.lastname@example.org
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
? 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Published online 28 November 2006Nucleic Acids Research, 2006, Vol. 34, No. 226561–6573
However, these approaches are commonly characterised by
a significant lack of efficiency accompanied by a high
level of toxicity and thus rendering them mostly inadequate
for in vivo applications. Nonetheless, there are a few studies
reporting a successful delivery of siRNA in vivo applying
cationic liposomes (7,8), atelocollagen- or PEI-complexed
siRNAs (9–12) as well as cholesterol-conjugated siRNAs
(13,14). Peptides, on the other hand, acting as shuttles for
a controlled cellular delivery of nucleic acids, represent a
new and innovative concept to bypass the problem of poor
bio-availability of these macromolecules. The idea of using
peptides as carriers goes back some 18 years, when two
groups discovered by chance that the HIV-1 transactivating
protein Tat is taken up by mammalian cells (15,16). Just a
fewyears later, theAntennapedia
Drosophila melanogaster was shown to act similarly (17).
Further on, it could be shown that peptides derived from
Tat and Antennapedia as well as other proteins are capable
of transporting macromolecular cargo molecules into cells
(18–20). Based on such promising results, a rapidly expand-
ing field focusing on the so-called cell-penetrating peptides
(CPPs) began to develop. Up to now numerous additional
peptides have been reported to show cell-penetrating proper-
ties and many of them have been used to successfully
deliver a variety of macromolecular cargos into cells [for
a review see: (21,22)].
For all the sequence diversity, CPPs share some common
features beside their ability to cross biological membranes:
(i) a high content of basic amino acids, and (ii) a length of
10–30 residues. Two strategies are utilised for the attachment
of cargo molecules. By far the majority of studies include a
covalent attachment of carrier and cargo [for a review see:
(23)]. This approach might be effective for a specific appli-
cation (e.g. a particular nucleic acid cargo), but it is fairly
limited in terms of flexibility, as a new construct has to be
generated for any given nucleic acid cargo. Alternatively,
the positive charges of certain amphipathic CPPs can be
exploited to bind anionic cargo molecules like nucleic acids
non-covalently via ionic interactions (24–26). Additional
hydrophobic peptide/peptide interactions then drive the mat-
uration of nanoparticles in a sandwich-like assembly reaction.
As a result, such a CPP can in principle be combined with any
For many CPPs, the initial interaction with cells is sup-
posed to be mediated by negatively charged glycosaminogly-
can (GAG) receptors of the extracellular matrix, e.g. heparan
sulphate proteoglycans (27–33). However, the mechanisms
underlying the cellular translocation of CPPs are poorly
understood and subject to controversial discussions. Nonethe-
less, there is considerable evidence that for many CPPs endo-
cytosis is a major route of internalisation (34–38). On the
other hand, there are examples in the literature proposing a
direct penetration of the cell membrane independent of any
endocytotic pathway (39–45), while others suggest both
entry routes are used in parallel or under certain conditions
(33,46–49). Furthermore, as at least four basic routes of
endocytosis can be distinguished to date (50,51), it seems
reasonable to speculate about multiple pathways involved in
cellular entry of CPPs. Then again, currently available data
are based on studies using a variety of different cell lines
and techniques, which renders a direct comparison of
different approaches impossible (37,52–54). It has been
shown that even minor changes of the physical state of a
CPP (e.g. exchange of certain amino acids) can alter translo-
cation properties significantly (41,53). This particularly holds
true for the attachment of large cargo molecules (55,56).
Thus, it might not be possible to generalise results obtained
with a given CPP, and it might be necessary to characterise
each carrier/cargo complex individually. If CPPs are intended
to be used for therapeutic purposes in the future, it is essential
to focus on the attachment of functional cargos and analyse
their biological effects inside the cell. Therefore, a quantita-
tive comparison of the total amount of cargo taken up with
functionally active cargo is an essential requirement in
order to improve delivery. As a prerequisite, there is need
for a sensitive method to quantify intracellular amounts of
cargo in combination with sensitive and easy to handle
In the present study we used the 27 amino acid peptide
MPGa, a derivative of the original MPG peptide (24), as
carrier for siRNA delivery. The primary amphipathic peptide
is composed of a hydrophobic domain, derived from the
N-terminal fusion sequence of the HIV-1 glycoprotein 41,
and a hydrophilic domain equivalent to the nuclear localisa-
tion sequence (NLS) of the SV40 large T antigen linked by
the three amino acid spacer WSQ (57). MPGa differs from
the parent peptide by six amino acids in the hydrophobic
part and is predicted to adopt a partially helical conformation.
The peptide forms stable non-covalent complexes with
nucleic acids in solution, which eventually assemble into
nanoparticles of different sizes (S.V. and A.T., unpublished
data). Utilising this model system, we provide a combination
of methods for detailed quantitative analyses of peptide-
mediated siRNA internalisation along with its biological
effects. To the best of our knowledge, we present for the
first time a detailed analysis on the number of siRNA mole-
cules per cell required to observe half maximal inhibition of
the target for peptide- versus cationic lipid-mediated delivery.
Beyond this, the techniques described here are generally
applicable for a characterisation of delivery systems of
MATERIALS AND METHODS
Oligonucleotides, peptides and cationic lipids
The siRNAs used throughout this study are unmodified as
well as fluorescently labelled oligoribonucleotides with a
30-overhang of two deoxythymidine nucleotides and were
purchased from IBA (Go ¨ttingen, Germany). The sequences
are described in detail elsewhere: si2B and si-sc, (58);
siR206 [Rational fLUC, position 206, (59)]; siGL3 and
siINV [invGL2, (60)]; siLam [lamin A/C siRNA, (61)].
The peptides were each modified with an acetyl group (Ac)
at the N-terminus and a cysteamide group (Cya) at the
C-terminus and were synthesised and purified by Jerini AG
(Berlin, Germany). The sequences are Ac-GALFLAFLAAA-
LSLMGLWSQPKKKRKV-Cya for MPGa and Ac-GALFL-
AFLAAALSLMGLWSQPKSKRKV-Cya for MPGa-mNLS.
LipofectamineTM2000 (LF2000) and LipofectamineTMPlus
were purchased from Invitrogen (Karlsruhe, Germany).
6562Nucleic Acids Research, 2006, Vol. 34, No. 22
Cell lines and cell culture
HeLa-TetOff cells and 293T cells were cultured in Dulbecco’s
Modified Eagle Medium with 4500 mg/l glucose. ECV304
cells, a derivative of human urinary bladder carcinoma cell
line T-24 (ACC 310 DSMZ, Braunschweig, Germany), were
cultured in Medium 199. ECV-GFP-Nuc cells, kindly pro-
vided by Marita Overhoff, stably express green fluorescent
protein tagged with a NLS (pAcGFP1-Nuc, BD Biosciences
Clontech, Heidelberg, Germany) and were cultured in Med-
ium 199 supplemented with 100 mg/ml G418. All media
were supplemented with 10% foetal calf serum (FCS) and
were purchased from Invitrogen. Cells were cultured as expo-
nentially growing subconfluent monolayers in a humidified
atmosphere containing 5% CO2. To establish cell lines stably
expressing firefly luciferase two strategies were followed:
(i) HeLa-TetOff cells were transfected with pTRE2hyg-luc
(cells and plasmid from BD Biosciences, Heidelberg,
Germany) using LipofectamineTMPlus according to the man-
ufacturer’s instructions. Stable transfectants were obtained by
selection with culture medium containing 200 mg/ml
hygromycin B (Invitrogen). (ii) Stably transfected ECV304
cells were generated using a self-inactivating HIV-1 vector
system described by Ja ´rmy et al. (62). Briefly, 293T cells
were transfected with a luciferase-harbouring defective HIV-1
genome and plasmid-encoded packaging functions. Forty
eight hours later, viral particles were harvested from the cul-
ture supernatant by filtration through a 0.45 mm filter and
used to infect 106ECV304cells for 1hat 37?C.The two stable
cell lines generated are referred to as HeLa-TetOff Luc
(HTOL) and ECV GL3, respectively.
Cell viability and cytotoxicity
Cell viability was determined by a fluorescein diacetate
(FDA, Sigma-Aldrich, Deisenhofen, Germany) assay. Non-
fluorescent FDA freely diffuses into cells, where it is cleaved
by cellular esterases to fluorescein. The amount of fluorescein
is thereby proportional to the amount of living cells (63).
Cells were washed twice with phosphate-buffered saline
(PBS) and then overlaid with 20 mM FDA. Fluorescence mea-
surements were performed in a microplate reader (Fluoroskan
Ascent?FL, Thermo Labsystems, Dreieich, Germany).
For specific applications an in vitro toxicology assay
(TOX-2, Sigma-Aldrich) was performed according to the
manufacturer’s instructions. This assay relies on the turnover
mitochondrial dehydrogenases which can be monitored spec-
Delivery of siRNA with LF2000 or MPGa
Twenty four hours prior to transfection, cells were seeded into
12 or 96 well plates (Greiner, Frickenhausen, Germany). Cell
numbers were chosen to finally reach 60% confluency for
MPGa-mediated delivery and 90% confluency for LF2000-
mediated delivery at the time of transfection. LF2000/
siRNA complexes were allowed to form in OptiMEM (Invi-
trogen) according to the manufacturer’s protocol with a final
concentration of 10 mg/ml LF2000. Peptide/siRNA complexes
were formed likewise by mixing the two components at the
individually given concentrations or ratios in OptiMEM and
added to the cell layer after 1 min incubation at room tempera-
ture. In both cases, routinely, the cell culture supernatant
was discarded and replaced by medium supplemented with
10% FCS 4 h after the start of transfection. All information
concerning incubation times given in the manuscript refer to
the time from the beginning of the transfection procedure.
For transfections in the presence of inhibitors/effectors of
endocytosis (all purchased from Sigma-Aldrich), cells were
incubated with the particular compound in OptiMEM at the
concentrations given in Table 1.
Determination of luciferase activity
Luciferase activity was quantified 24 h after transfection,
subsequent to the determination of cell viability as described
above. Luminescence was measured in a microplate reader
(Fluoroskan Ascent?FL, Thermo Labsystems, Dreieich,
Germany) using 50–100 ml of a buffer containing 28 mM
Tricine (pH 7.8), 500 mM ATP, 250 mM coenzyme A,
250 mM D-luciferin, 33 mM DTT, 200 mM EDTA, 15 mM
MgSO4, 1.5% (v/v) Triton X-100 and 5% (v/v) glycerol (all
reagents purchased from Sigma-Aldrich). The luminescence
was normalised to cell viability to account for cell loss due
to cytotoxicity or washing procedures.
Quantification of cellular siRNA uptake via liquid
The detection of siRNA in cellular extracts was performed
essentially as described by Overhoff et al. (64). Briefly, trans-
fections were carried out as described above in a 12 well
format. Four hours after transfection cells were treated
three times with heparin (Fluka/Sigma-Aldrich, 15 U/ml in
OptiMEM) totalling 1 h at 37?C to remove extracellularly
bound peptide/siRNA complexes. Subsequently, cells were
detached by trypsination or by incubation in cold PBS. Ten
percent of each sample was used to measure luciferase acti-
vity while the rest of the sample was subjected to a liquid
hybridisation protocol. Cells were pelleted, incubated in
PBS containing 1% NP-40 for 10 min on ice followed by
total RNA extraction according to standard protocols. The
Table 1. Inhibitors/effectors of endocytosis
Inhibition of energy-dependent processes
Inhibits acidification of endosomes
Disruption of microfilaments, inhibition of
Sterol-binding agent, disrupts caveolar
structure and function
Ionophor, inhibits acidification of
endosomes and therefore prevents
Sterol-binding agent, disrupts caveolar
structure and function
Activation/induction of caveolin-
Inhibition of clathrin-mediated
PI(3)K-inhibitor, inhibits endosome fusion
Filipin complex5 mg/ml
Nystatin 25 mg/ml
Okadaic acid 100 nM
Sucrose 100 mM
The concentrations given in the table are the concentrations used in this study.
Additionally, a brief description of the proposed exerted effect on endocytosis
is given for each substance.
Nucleic Acids Research, 2006, Vol. 34, No. 22 6563
quantification of the siRNA was achieved by hybridisation
with the corresponding32P-labelled sense-strand for 10 min
at 95?C followed by 1 h incubation at 37?C before the
samples were resolved by 20% polyacrylamide gel electro-
phoresis (PAGE) under non-denaturing conditions. After
blotting of the gel onto a nylon membrane (Hybond N+,
Amersham, Freiburg, Germany), the signals were quantified
with a PhosphorImager (TyphoonTM8600 Variable Mode
Imager, GE Healthcare, Mu ¨nchen, Germany). Absolute
amounts of siRNA in the samples were calculated in relation
to the included standards. For this purpose, defined amounts
of siRNA were added to control cell lysates before the
extraction step and treated simultaneously with the other
samples. The amounts of total cellular RNA were determined
spectrophotometrically and used for normalisation.
ECV304, ECV-GFP-Nuc or HeLa cells were seeded in
LabTek?Chamber Slides (8 chambers, Nunc, Wiesbaden,
Germany) at ?18000 cells per chamber and incubated for
24 h. Then cells were washed twice with OptiMEM and fur-
ther incubated for 4 h with MPGa mixed with fluorescently
labelled RNA in OptiMEM (final volume 400 ml). To remove
extracellularly bound complexes, cells were treated with
heparin as described above. Staining of the cell nuclei was
achieved with Hoechst 33342 (12 mg/ml). Finally, microscopy
was performed at room temperature in OptiMEM supple-
mented with 50 mM HEPES, pH 7.0 (Invitrogen). Confocal
images were acquired using a Zeiss LSM510 Meta device.
Semi-confocal images were acquired using a Zeiss Axiovert
200M microscope withApoTome. For processing and analysis
of the images, the LSM510 (Version 3.2SP2) and the AxioVi-
sion (Rel. 4.5) software (both Carl Zeiss) were used for confo-
cal and semi-confocal images, respectively. Fluorescence was
visualised using filters for excitation/emission at 470/525 nm
for Alexa488and GFP-NLS (green fluorescence), 550/605 nm
for Cy3 (red fluorescence) and 365/445 nm for Hoechst 33342
Twenty four hours prior to the experiment, 1 · 106HeLa-
TetOff cells were plated onto glass cover slips (diameter
12 mm) in a 100 mm cell culture dish. The microinjection
setup consisted of a FemtoJet express and a Micromanipula-
tor 5171 (Eppendorf, Hamburg, Germany) mounted on an
Axiovert 100 (Carl Zeiss). The micropipette (Femtotip,
Eppendorf) was loaded with a Microloader (Eppendorf) con-
taining 2–4 ml of either the luciferase harbouring plasmid
pTrehygLuc, siR206 or a mixture of plasmid and siRNA in
varying concentrations. Working pressure for injections into
adherent cells was 90–150 hPa for 0.2–0.3 s. Twenty four
hours after microinjection luciferase activity was evaluated
as described above.
MPGa mediates delivery of siRNA into
The primary amphipathic peptide MPGa can bind virtually
any negatively charged molecule via ionic interactions in a
non-specific manner (S.V. and A.T., unpublished data).
Thus, in principle this peptide is compatible with almost
any given functional oligonucleotide. As RNAi-mediated
inhibition of the reporter gene firefly luciferase is currently
one of the most widely used approaches to study siRNA
delivery, we decided to utilise this approach for our studies
with MPGa. In a first step we established two cell lines stably
expressing the firefly luciferase, namely HTOL and ECV304
GL3 (ECV GL3). These cell lines were used throughout the
study to characterise MPGa-mediated delivery of siRNA. For
comparison, we used the commercially available cationic
Figure 1 shows that MPGa translocates sufficient mole-
cules of the siRNA siR206 (59) into reporter gene expressing
cells to induce a pronounced reduction of the luciferase activ-
ity. Similar results were obtained when using the anti-
luciferase siRNA siGL3 (60) (data not shown). Irrespective
of the cell line used, we observed a maximal extent of
siRNA-mediated inhibition of 80–90% of the target gene
expression, which was equal to or even higher than levels
reached with LF2000. The transfection procedure was rather
straightforward. Briefly, complexes of carrier and cargo were
formed by simple mixing of MPGa and siRNA in OptiMEM.
After a short incubation for 1–3 min, the mixture was added
to the cells for a transfection period of 4 h. Then, the mixture
was removed followed by the addition of fresh medium
supplemented with FCS and the luciferase activity was
measured 20 h later. Hereby, it was irrelevant if peptide
and nucleic acid were mixed at the desired final concentra-
tions or the final concentration was adjusted by sequential
dilution of a concentrated carrier/cargo solution (data not
shown). To ensure that the incubation of cells with the
peptide/siRNA complexes per se did not influence luciferase
expression, additional siRNAs were used as controls. Neither
si2B, targeted to ICAM-1, nor the corresponding scramble
control si-sc (58) or siINV (60), both without a cellular target,
did show any significant change in luciferase activity
when compared to non-transfected control cells (Figure 1
Figure 1. Inhibition of luciferase expression after MPGa- or LF2000-
mediated siRNA delivery. Two different cell lines stably expressing firefly
luciferase, HTOL and ECV GL3, were transfected in duplicates in a 96 well
format with 50 nM siR206 or siINV using 10 mg/ml LF2000 or 4.2 mM
MPGa, respectively. Control cells were incubated with OptiMEM only.
Luciferase expression given as RLU was measured 24 h after transfection and
normalised to cell viability. The graph shows one representative experiment.
Luciferase activity is reduced by 70 and 97% in HTOL cells and 83 and 88%
in ECV GL3 cells after MPGa- or LF2000-mediated delivery of siR206,
6564Nucleic Acids Research, 2006, Vol. 34, No. 22
and Supplementary Figure 1A). The same was true for
LF2000-mediated transfections, evidently excluding any
unspecific effects. As LF2000 and to a certain extent also
MPGa proved to be toxic at higher concentrations, it was
necessary to take this into account. Thus, in each experiment
the values of luciferase activity measured were normalised
for different cell viabilities with the help of a FDA
assay, which was carried out immediately prior to each
An overall positive net charge is supposed to be a prereq-
uisite for the initial interaction of the carrier/cargo complexes
with negatively charged cell surface components. Accord-
ingly, the optimal ratio of peptide to nucleic acid was deter-
mined. When siRNA was mixed at a concentration of 50 nM
with increasing concentrations of MPGa (1.3–12.6 mM), best
results were achieved at an excess ratio of positive charges of
15 (Supplementary Figure 1B). The optimal concentration
of the peptide was determined to be 2–4 mM. In this range,
no significant cellular toxicity occurred as determined via
the XTT assay (data not shown). During optimisation of the
transfection protocol, we observed that cell density at the
time of transfection plays an important role for transfection
efficiency. For transfections with MPGa the maximum
RNAi effect was observed at cell densities of 60–70%,
whereas with LF2000 >90% confluency of the cells at the
time of transfection gave the best results (data not shown).
Recently it was shown by Simeoni et al. (39) that a fluores-
cently labelled siRNA localised rapidly into the nucleus when
transfected with MPG, a related peptide described by Morris
et al. (24). Interestingly, the authors further reported about a
cytoplasmatic localisation when the C-terminal SV40 large
T-antigen NLS sequence was mutated. Since both peptides
MPG and MPGa share the same C-terminal NLS sequence,
we were wondering if comparable results are to be seen
with MPGa. However, we could not observe any differences
between MPGa and a corresponding peptide harbouring a
lysine to serine mutation at position two of the NLS sequence
(MPGa-mNLS) neither by microscopic studies nor analysing
the luciferase activity (data not shown).
In order to compare the transfection efficiency of MPGa
with that of other delivery agents like LF2000, we determined
the apparent value of half maximal inhibition (IC50). In
Figure 2 representative dose-response curves are depicted
for MPGa- and LF2000-mediated siRNA delivery, respecti-
vely. From transfections of both HTOL and ECV GL3 cells
with MPGa/siRNA complexes IC50 values of ?0.8 nM
were calculated (Figure 2A), whereas LF2000-mediated trans-
fections yielded IC50values of 0.02–0.04 nM (Figure 2B).
These results gave a first hint that MPGa-mediated delivery
of siRNA might be less efficient when compared to LF2000.
Consequently, we set out experiments trying to unravel the
underlying mechanisms for this observation.
Quantification of siRNA internalised
The above described functional siRNA-based delivery assay
provided us with the knowledge that MPGa-mediated cellu-
lar transfection of a given extracellular quantity of siRNA
was less efficient in terms of reporter gene inhibition than
LF2000-mediated transfection. One possible explanation
for this observation could simply be a lower rate of siRNA
internalisation. Alternatively, the amount of bio-available
siRNA molecules inside the cell could be lower due to
insufficient release from the carrier/cargo complexes and/or
trapping of complexes in particular cellular compartments.
To investigate this issue in more detail, we analysed the
amount of siRNA taken up side by side with the biological
effect caused by the nucleic acid. For this purpose, we
adapted a highly sensitive method for the quantification of
siRNA described by Overhoff et al. (64) enabling us to detect
the antisense-strand of the siRNA with a sensitivity of
>10 molecules per cell. This so-called liquid hybridisation
assay comprises the extraction of total RNA from the cells
and a hybridisation step of a radioactively labelled probe
Figure 2. IC50of anti-luciferase siRNA delivered by MPGa and LF2000.
ECV GL3 cells were transfected in a 96 well format using a constant
concentration of 4.2 mM MPGa (A) or 10 mg/ml LF2000 (B) and siRNA
concentrations varying from 0.005 nM to 100 nM. Luciferase expression
given as RLU was measured 24 h after transfection and normalised to cell
viability. RNAi-mediated down-regulation of the luciferase activity was
expressed as the percentage of active siR206 versus inactive siINV and the
IC50value was calculated using the GraFit5 software (Erithacus Software,
Surrey, UK). The curves show the best fit of the data of a representative
experiment in duplicates which yielded IC50values of 0.89 nM (±0.15) for
MPGa (A) and 0.018 nM (±0.004) for LF2000 (B).
Nucleic Acids Research, 2006, Vol. 34, No. 226565
with the corresponding antisense-strand of the siRNA in solu-
tion. With the help of PAGE analysis the intact portion of
intracellular siRNA can be quantified.
Recently, it was convincingly demonstrated by Richard
et al. (35) that adequate removal of extracellularly bound
carrier/cargo complexes is crucial for a correct analysis of
intracellular delivery. Taking these findings into account,
we examined several washing procedures. For this matter,
we quantified the amount of siRNA taken up applying the liq-
uid hybridisation assay and in parallel measured the reduction
of the luciferase activity to ensure inhibition reaches the same
level as without such a treatment. Additionally, we analysed
the cells by fluorescence microscopy. Besides a treatment of
the cells with trypsin as suggested by Richard et al. (35), we
tested heparin and a combination of trypsin and heparin under
different conditions (Supplementary Figure 2). By far, the
best results were obtained with a heparin wash (15 U/ml,
three times totalling one hour at 37?C) reducing the amount
of allegedly intracellular siRNA by 60% as compared to a
PBS wash of the cells, while a trypsin treatment increased
the observed signal by 2-fold in our hands and a combination
of both yielded comparable results as heparin alone. The
necessity and efficacy of the heparin wash is impressively
illustrated by the fluorescence microscopy pictures given in
Supplementary Figure 2A. As a result, following the trans-
fection step a heparin wash was included into the protocol,
irrespective of further treatments.
Applying this modified setup we performed quantitative
measurements with ECV GL3 cells. Figure 3 shows the
results of a typical experiment. Here, either 1 or 10 nM
siR206 were transfected for 4 h with either MPGa or
LF2000 in a 12 well format. After 1 h of heparin treatment
and further 19 h of incubation with medium supplemented
with 10% FCS the amount of siRNA taken up by the cells
was quantified utilising the liquid hybridisation assay. In
total, 16.8 fmol siR206 were detectable after transfection of
10 nM siR206 with the peptide as compared to 105.5 fmol
after transfection with LF2000. Reducing the extracellular
concentration of nucleic acid cargo by 10-fold, we observed
a corresponding reduction in uptake by 10-fold. Further
experiments revealed that uptake was linear between
0.1 nM and 100 nM siRNA in the transfection mix for both
transfection reagents (data not shown). Table 2 summarises
a set of data from six independent uptake experiments
(including the results shown in Figure 3). Quantification via
liquid hybridisation was performed 4 or 24 h after trans-
fection with either reagent. With the peptide ?3% of the
siRNA present in the initial transfection mixture was detected
inside the cells after 4 h plus 1 h of heparin treatment. After
24 h this value had dropped to ca. 0.7%. During the first 4 h
of incubation LF2000 delivered twice as much siRNA as
MPGa. After 24 h the amount of intracellular siRNA
exceeded that measured for MPGa-mediated transfection by
a factor of 4–6. However, at half maximal inhibition of
reporter gene activity 10 000 siRNA molecules were detected
inside the cells in case of MPGa-mediated delivery, whereas
in case of LF2000 only 300 molecules per cell were mea-
sured. Thus, the amount of bio-available siRNA molecules
inside the cells was about 30-fold lower for peptide-mediated
delivery as compared to cationic lipid-mediated delivery.
Insights into the mechanism of MPGa-mediated
delivery of siRNAs
As outlined above, quantification of siRNA delivered into
mammalian cells either by LF2000 or MPGa revealed a con-
siderable difference in the absolute amount of molecules
required to trigger an equivalent inhibitory effect. Hence,
we strived for a closer look at the fate of carrier/cargo com-
plexes inside the cells by applying fluorescence microscopy.
All microscopic images shown in this study were obtained
with unfixed living cells. This is particularly important with
Figure 3. Quantification of the cellular uptake of siRNA after MPGa-mediated delivery. ECV GL3 cells were transfected for 4 h in a 12 well format using
2.1 mM MPGa or 10 mg/ml LF2000 and 1 or 10 nM siR206, respectively. Twenty four hours after transfection, intracellular amounts of siRNA were determined
applying the liquid hybridisation protocol as described in the methods section. One representative quantification experiment is shown. (A) 20% non-denaturing
PAGE analysis of the samples. (B) Total amount of siRNA per sample.
6566 Nucleic Acids Research, 2006, Vol. 34, No. 22
respect to earlier publications (35), which had demonstrated
that cell fixation procedures can lead to localisation artefacts
of cargo and carrier inside the cell. Microscopic studies were
performed with unlabelled MPGa peptide and fluorescently
labelled oligonucleotides. To exclude any artefacts, which
might arise from the fluorescence label, we compared differ-
ent fluorophores. Experiments with Cy3 and Alexa488labels
at different positions of the nucleic acid cargo gave inter-
changeable results (data not shown). Figure 4 shows a typical
uptake experiment analysed by confocal laser scanning
microscopy with a punctual non-homogeneous distribution
pattern of the nucleic acid inside the cell indicative of an
endocytotic uptake mechanism. The addition of carboxyfluo-
rescein to the medium facilitates a discrimination of the non-
fluorescent cytoplasm from the green fluorescent extracellular
space. This easily allows for a differentiation of complexes
attached to the cellular surface and complexes internalised
into the cells. Furthermore, as can be seen on the projections
in Figure 4 and when zooming through the image (data not
provided) the vast majority of red dots visible are indeed
inside the observed cell.
Next, we analysed the influence of specific inhibitors/
effectors of different endocytotic pathways on the delivery
of siRNA and in parallel on the inhibition of reporter gene
activity. The compounds used in this study are summarised
in Table 1. Additionally, a brief description of the proposed
mode of action is given for each substance. In general,
these inhibitors/effectors of endocytosis exert a high level
of stress on the cells, so one has to carefully define the con-
centration range, which can be applied with maximal effect
but minimal toxicity. To find out suitable conditions for the
particular cell lines used here, cytotoxicity studies were per-
formed (data not shown). The impact of the substances on
siRNA-mediated down-regulation of luciferase activity after
peptide-mediated delivery was measured at an extracellular
concentration of 1 nM siR206, i.e. in the range of the IC50
value (see above), since under these conditions a maximal
effect would be expected. For transfections with LF2000 a
concentration of 0.1 nM siRNA was applied.
The strongest inhibitory effect on RNAi was observed
when transfections were performed at 4?C. Here, uptake
was drastically reduced and no RNAi could be detected any-
more (Figure 5A and B) clearly indicating an energy-driven
cellular process to be responsible for the uptake of the com-
plexes. The inhibitors/effectors sucrose, cytochalasin B,
nystatin, wortmannin and okadaic acid reduced siRNA-
mediated down-regulation of luciferase activity whereas fil-
ipin complex, monensin and especially chloroquine enhanced
RNAi efficiency. These data provided additional evidences
for endocytotic processes and for the involvement of acidic
compartments in the uptake mechanism of MPGa/siRNA
complexes. Apart from low temperature, only the application
of wortmannin and filipin complex resulted in reduced uptake
of siRNA both after 4 and 24 h. Chloroquine slightly
increased uptake when measured after 4 h. However, no dif-
ference could be observed after 24 h versus transfections
without this compound. This is probably due to the fact,
that chloroquine promotes the release of nucleic acids from
endosomal compartments, which leads to higher amounts of
siRNA detected during the first hours. Upon treatment with
nystatin, the uptake was significantly increased after 4 h of
incubation with even higher values reached after 24 h. Treat-
ment with okadaic acid led to enhanced uptake after 4 h and
slightly reduced uptake after 24 h as compared to untreated
Table 2. Quantification of MPGa- and LF2000-mediated delivery of siRNA
Incubation prior to
siRNA (fmol) detected per mg
of total cellular RNA
siRNA detected inside of
applied outside (%)
siRNA per cell
cell · 105
The table shows averaged data of six independent experiments. For better comparison of different experimental conditions the results were normalised to a
transfection of 1 pmol siRNA with either 2.1 mM MPGa or 10 mg/ml LF2000 in a 12 well format. Quantification via the liquid hybridisation protocol was
performed after 4 or 24 h as described in the methods section. Molecules per cell were calculated on the basis of the cell number seeded for transfection.
Figure 4. Confocal laser scanning microscopy analysis of unfixed HeLa cells
after transfection with MPGa/RNA complexes. Cells were transfected for 3 h
with complexes of 5 mM peptide and 180 nM Cy3-labelled RNA in
OptiMEM. Adding free carboxyfluorescein to the medium leads to a green
staining of the extracellular space. The red and the green lines show the
position within the image of the projections given in the upper and right part,
respectively. Microscopical analysis was performed with a confocal laser
scanning microscope (LSM 510, Carl Zeiss). The white bar equals 10 mm.
Nucleic Acids Research, 2006, Vol. 34, No. 226567
For LF2000, we found a slightly different pattern for
the RNAi effect and siRNA uptake in the presence of these
agents, which nevertheless corroborated the notion that
LF2000/siRNA complexes are taken up by endocytosis
(Supplementary Figure 4).
In an attempt to find out the minimal number of siRNA
molecules needed to trigger RNAi-mediated half maximal
inhibition of luciferase activity we performed nuclear
microinjection studies of a luciferase expression plasmid
together with varying concentrations of siR206 into HeLa,
HTOL and ECV304 cells, respectively. From the IC50value
it could be calculated that ?300 siRNA molecules per cell
were necessary to observe a half maximal inhibition of the
luciferase activity (Figure 6).
The cell membrane is one of the major barriers for an appli-
cation of therapeutically interesting bio-macromolecules like
nucleic acids. In the present study we exploited a CPP-
approach for the delivery of siRNA as a general example
for an oligonucleotide cargo using the peptide MPGa, a
derivative of the original MPG peptide described by Morris
et al. (24). MPGa differs from MPG by six amino acids
in the hydrophobic part (57). These changes result in an
alteration of the overall structure of the peptide towards
a higher tendency of adopting a helical conformation (57).
The peptide forms stable non-covalent complexes with
nucleic acids. Besides the mechanism of cellular uptake of
MPGa/siRNA complexes our main focus was a comparative
parallel analysis of uptake versus functional effects of the
nucleic acid cargo. For this purpose, we used a liquid hybridi-
sation protocol in combination with a simple luciferase
reporter system. For comparison, the commercially available
cationic lipid LF2000 was included into the study.
As shown in Figure 1, MPGa is capable of translocating
siRNA into mammalian cells leading to a down-regulation
of the target protein luciferase. The observed effect was
highly specific for siR206 and maximal inhibition was
achieved with a charge ratio of 15:1, i.e. positive (peptide)
over negative (siRNA) charges (Supplementary Figure 1).
Additionally, we tested MPGa-mediated delivery of siRNA
directed to two other targets, namely ICAM-1 and lamin
A/C applying published protocols (58,61). In both cases we
observed a substantial RNAi effect of the active siRNA as
compared to a control siRNA (data not shown). These data
clearly indicate a specific siRNA-mediated inhibition of
target protein expression rather than an unspecific effect,
which might be caused by the transfection procedure.
Comparing the peptide with LF2000, the maximal achievable
levels of siRNA-mediated target protein down-regulation are
roughly the same. However, an exact determination of the
IC50 values of both carrier/cargo complexes revealed an
?30-fold lower efficiency of MPGa compared to LF2000.
To investigate if this phenomenon was caused by different
Figure 5. RNAi efficiency (A) and intracellular amounts of siRNA (B) after
MPGa-mediated delivery in the presence of inhibitors/effectors of endocy-
tosis. Prior to a 4 h transfection with complexes of 2.1 mM MPGa and 1 nM
siR206, ECV GL3 cells were pre-incubated for 1 h with different modulators
of endocytosis at concentrations given in Table 1. The agents were present
during the entire experiment. Additionally, transfections were performed at
4?C. The data shown are averages of at least three independent experiments.
(A) RNAi efficiency expressed as relative luciferase activity was measured
24 h after transfection in a 96 well format. (B) Uptake of siRNA was analysed
4 or 24 h after transfection in a 12 well format using the liquid hybridisation
protocol. The dashed lines show the situation without treatment.
Figure 6. IC50of anti-luciferase siRNA delivered via nuclear microinjection
into HeLa-TetOff cells. The plasmid pTRE2hyg-luc (130 ng/ml) was co-
injected with varying concentrations (0.0015–15 ng/ml) of siR206 into the
nucleus of ?300 cells per experiment. Twenty four hours after microinjection
the luciferase activity was analysed. Non-injected cells served as control. The
luciferase expression is given as the percentage of active siR206 versus
control cells (mean values of two independent microinjection experiments are
given). The inset shows a fit of the experimental data using the GraFit5
software yielding an IC50value of 15 ng/ml (±0.49). This value translates into
?300 siRNA molecules per cell.
6568Nucleic Acids Research, 2006, Vol. 34, No. 22
levels of uptake or insufficient bio-availability of the siRNA
molecules, we quantified the intracellular amount of cargo.
From a number of diverse procedures described in the litera-
ture (65–68) we adapted a liquid hybridisation protocol
described by Overhoff et al. (64). This assay does not need
any modification of the siRNA and exclusively detects intact
molecules. Thus, artefacts due to detached fluorescence labels
or degraded cargos for example are precluded. Overall, the
liquid hybridisation protocol is a fast, easy to handle and
highly reproducible procedure that can be carried out in
any laboratory without the need of expensive equipment.
Though, before we could perform quantitative uptake
experiments, we had to establish a washing procedure in
order to remove complexes bound to the surface of the
cells. Such extracellularly attached carrier/cargo complexes
are an important source of overestimation of cargo inter-
nalised (35). As opposed to the commonly used trypsin treat-
ment, a heparin wash was much more effective in our hands
(Supplementary Figure 2A and B). Similar results were
reported by Kaplan et al. (69) for the cellular uptake of Tat
peptide in the absence of cargo. The negatively charged
heparin molecules, with a molecular weight of 4–6 kDa, are
much smaller than trypsin and thus might be able to infiltrate
the extracellular matrix more effectively and both detach and,
more importantly, dissociate MPGa/siRNA complexes bound
there. In contrast to this, trypsin considerably increased
the apparent uptake of the complexes (Supplementary
Figure 2B). This might be due to a membrane destabilising
effect of this enzyme which has been shown to enhance the
uptake of small molecules or oligonucleotides (70). For
several CPPs without a cargo it has previously been shown
that the cellular penetration is inhibited by certain GAGs
suggesting that uptake is mediated by GAG receptors on
the cell surface (30,32,56,71). To what extent these receptors
are equally involved in the uptake of MPGa/siRNA com-
plexes cannot be answered with certainty as heparin has a
very strong destabilising effect on the peptide/nucleic acid
complexes (A.T., unpublished data).
With a reliable washing procedure to remove extracellular
complexes in place we were able to accurately quantify the
intracellular amounts of cargo after carrier-mediated delivery
(Figure 3 and Table 2). The amount of siRNA internalised,
increased as expected with the incubation time of transfection
showing a maximum at ?4 h (Supplementary Figure 3B
and C). Similar results were observed with a RNA aptamer
[(72), S.L., unpublished data]. Accordingly, unless otherwise
indicated, all transfections were performed for a period of
4 h before cells were either subjected to quantification
(after an additional 1 h heparin wash) or further incubated
in the absence of carrier/cargo complexes for later analyses.
After 24 h approximately half of the maximal amount of
siRNA internalised was still detectable. All in all <5% of
the siRNA present in the initial transfection mixture was
internalised into the cells regardless of the delivery agent.
After 4 h, twice as much siRNA was detected inside the
cells after transfection with LF2000 when compared to
MPGa-mediated delivery. Surprisingly, 24 h after transfec-
tion this difference rose to a factor of 4–6. In vitro experi-
ments, on the other hand, show that siRNA complexed with
the carrier peptide is strongly protected from degradation
(data not shown). The observed uptake of siRNA was linear
over a tested range of 0.1–100 nM of siRNA. We deliberately
did not apply concentrations >100 nM siRNA to examine if
the process would eventually be saturable, since too high con-
centrations of siRNA lead to off-target effects (73,74). Taken
together, the difference in uptake between the peptide and the
cationic lipid was much smaller than anticipated from the
above described IC50values with a factor of about 2–6 in
favor of LF2000. These findings clearly indicate that uptake
per se was not the limiting factor of the peptide approach.
Combining both techniques, the analysis of uptake and
RNAi effect, it became apparent that for a half maximal
inhibition of reporter gene activity, ?10000 siRNA mole-
cules were necessary in case of MPGa-mediated delivery,
whereas in case of LF2000 only ?300 molecules were
required. Thus, the amount of bio-available siRNA molecules
inside the cells was about 30-fold lower for peptide-mediated
delivery as compared to cationic lipid-mediated delivery. To
the best of our knowledge, this is the first time the number of
siRNA molecules per cell has been determined for half
maximal inhibition of the target for peptide- versus cationic
lipid-mediated delivery. At this point the question arose,
what the cause of this observed discrepancy was. An obvious
reason for these findings would be a difference in the mecha-
nism of uptake. As outlined above, the mechanisms underly-
ing the cellular translocation of CPPs are poorly understood
and remain hotly debated. Nonetheless, there is considerable
evidence that endocytosis is a major route of the internali-
sation of many CPPs. In an attempt to address this question
we performed fluorescence microscopy studies and analysed
the influence of specific inhibitors/effectors of different endo-
cytotic pathways on the delivery of siRNA and the inhibition
of reporter gene activity simultaneously.
A first clear hint for an endocytotic pathway involved in
the uptake of MPGa/siRNA complexes arose from micro-
scopy studies (Figure 4). Consistent with the assumption of
an endocytotic pathway, a typical vesicular distribution
pattern in the cytoplasm could be observed by fluorescence
microscopy. Moreover, the siRNA internalised was observed
to partially co-localise with endosomal/lysosomal compart-
ments (data not shown). The results were very similar for
LF2000-mediated transfection though here a slight diffuse
distribution of fluorescence throughout the cytoplasm was
recognisable but hard to document. To avoid any artefacts
due to cell fixation, all fluorescence microscopy experiments
were performed with living cells. The heparin wash described
above proved to be essential as images of cells not treated
with heparin showed large aggregates on the cell surface
impairing the view on complexes internalised (as clearly
seen in Supplementary Figure 2A). Addition of trypan blue
to quench external fluorescence (32,75) was only suitable
for smaller complexes but larger ones remained visible. Car-
boxyfluorescein in the medium turned out to be very useful to
distinguish between the inside and the outside of the cell and
was used to clearly discriminate between intra- and extracel-
lular complexes. Although MPGa harbors an NLS sequence,
no nuclear localisation could be observed nor was there any
change in the apparent RNAi effect when the NLS sequence
was mutated (data not shown). On the other hand, it cannot be
ruled out that the amount of cargo localised in the nucleus
was below the detection limit. According to a recent study
of Berezhna et al. (76) the intracellular localisation of
Nucleic Acids Research, 2006, Vol. 34, No. 226569
siRNA is suggested to depend on the localisation of the tar-
get. Nonetheless, applying a fluorescently labeled siRNA
against the 7SK RNA, which was shown to strictly localise
in the nucleus (77–80), we did not observe any fluorescence
there (data not shown).
Lowering of the temperature markedly reduces flexibility
and fluidity of the plasma membrane, thereby also slowing
membrane traffic (81) and blocking all endocytotic processes
(82). Consistent with this concept, we could neither detect
MPGa-mediated siRNA delivery at 4?C as shown by liquid
hybridisation analyses as well as by fluorescence microscopy
(Supplementary Figure 3), nor did we observe any RNAi
effect (Figure 5). In accordance with our findings, other
authorsalso suggested an
process for CPP-mediated delivery based on the observation
that uptake was strongly decreased at low temperatures
(31,83,84). In order to address this issue in more detail, we
performed studies on MPGa with different modulators of
endocytosis (Figure 5). First we analysed the effect of
wortmannin, a compound, which is supposed to affect
clathrin-dependent endocytosis (85) and macropinocytosis
(86). Wortmannin led to a strong reduction in the uptake of
MPGa/siRNA complexes. Like with sucrose, which also
blocks clathrin-dependent endocytosis unselectively, the
RNAi effect was reduced significantly. On the other hand,
no clear-cut influence on uptake of siRNA could be seen
applying cytochalasin B, which is supposed to affect the
assembly of actin microfilaments (87,88), whereas the RNAi
effect was strongly diminished in the presence of this com-
pound. Hence, we could not distinguish, whether this was
due to a blockage of macropinocytosis or what appears more
realistic was caused by a block of intracellular vesicle trans-
port. In the presence of nystatin, which is supposed to inhibit
caveolin-dependent lipid-raft-mediated endocytosis (89,90),
the RNAi effect was clearly reduced and could neither be res-
cued by combining nystatin with okadaic acid, chloroquine
nor filipin complex (Figure 5A). Pre-incubation of the cells
with nystatin led to an increase in uptake during the first
4 h, which was even more pronounced when the quantification
was performed after 24 h (Figure 5B). An additional effect of
nystatin is supposed tobe an increase in cell membraneperme-
ability (89). This property of nystatin could account for the
high amount of siRNA taken up. Together with the reduced
RNAi effect observed, this would mean that nucleic acids
internalised this way, are not available for the RNAi machin-
ery. Then again, taking the results of experiments into account
in which different inhibitors were combined to counteract
nystatin, it could be concluded that nystatin itself had a severe
negative effect on the RNAi machinery. Filipin complex like
nystatin shows sterol-binding properties (91–93). However, in
contrast to nystatin almost no change in siRNA uptake was
detected after MPGa-mediated delivery, whereas the RNAi
effect was slightly increased. This may be due to a selective
inhibition of caveolin-mediated endocytosis, which in turn
might shift the balance towards clathrin-mediated endocyto-
sis. This interpretation would be consistent with our idea
that clathrin-mediated endocytosis yields more bio-available
cargo and is further supported by the observation that the
RNAi effect is increased even more by combining filipin
complex with chloroquine (Figure 5A). Okadaic acid, a spe-
cific phosphatase inhibitor, is supposed to stimulate mobility
and internalization of caveolae (94–96). In the presence of
okadaic acid no RNAi effect could be detected. Uptake of
siRNA, on the other hand, was not affected. This could be
interpreted as an increase of caveolin-dependent endocytosis
accompanied by a reduction of clathrin-dependent uptake
leading to an unchanged net amount of intracellular siRNA.
Caveolae direct most of their cargo via caveosomes to the
Golgi apparatus where they are probably beyond reach of
the RNAi machinery. In addition, combining filipin complex
and okadaic acid resulted in levels of luciferase activity
equal to those measured with siRNA alone. Here, induction
of caveolin-mediated endocytosis by okadaic acid was possi-
bly compensated by the inhibitory effect of filipin complex.
Chloroquine, a weak base, is supposed to prevent lysosomal
degradation by inhibiting the acidification of endosomes
(97,98). This eventually leads to the disruption of a large
portion of the endosomes which then release their content
into the cytoplasm (99). In our hands, chloroquine caused a
slightly enhanced RNAi effect leading to the conclusion
that an increased number of siRNA molecules have become
bio-available by endosomal release.
Equivalent experiments with LF2000 as transfection
reagent yielded a slightly different pattern, for example
regarding uptake in the presence of nystatin or chloroquine,
nevertheless strongly indicating uptake via endocytosis
(Supplementary Figure 4). This observation is consistent
with the findings of others concerning the uptake mechanism
of several cationic lipids (100–102).
Overall, ourdata, especially
temperature-dependency of the uptake, strongly indicate
that both MPGa and LF2000/siRNA complexes are taken
up by endocytotic processes. The differences between trans-
fections with MPGa and LF2000 concerning the IC50values
of siRNA could be explained by the property of LF2000 to
promote endosomal escape at least partially, which is consis-
tent with the microscopic observations described above. The
inhibitors of endocytosis described above support the notion
that not one single pathway is responsible for the uptake of
the complexes but rather several pathways are involved.
This may be due to a relatively unspecific interaction of the
complexes with numerous negatively charged molecules on
the cell surface. As a result, various pathways of endocytosis
might be triggered. Accordingly, individual inhibitors used in
this study might show just moderate effects by shifting the
balance in favour of a particular endocytotic pathway. Analo-
gous results were reported by Sa ¨a ¨lik et al. for CPP/avidin
complexes (103). However, it should be kept in mind, that
the interpretation of these particular experimental data is
problematic, as many aspects of endocytosis are still poorly
understood to date. Even more important, the compounds
may have additional unknown effects, which could lead to
unexpected changes in reporter gene activity and/or affect
the RNAi machinery. Besides, the involvement of several
pathways of endocytosis could also be a direct result of an
inhomogeneous size distribution of the MPGa/siRNA com-
plexes (S.V. and A.T., unpublished data), since for each
process only a certain range of particle sizes is believed to
be predominantly taken up (50,104,105). Thus, the size of
the complexes may determine their uptake route and conse-
quently their fate. Then again, we cannot entirely rule out
6570Nucleic Acids Research, 2006, Vol. 34, No. 22
that a certain percentage of complexes are taken up via a direct
penetration of the plasma membrane as reported by others
(39,40,42). In this context it would be very interesting to
know the minimal number of siRNA molecules needed to
trigger RNAi-mediated half maximal inhibition of luciferase
activity. In an attempt to answer this question, we performed
technically challenging nuclear microinjection studies of a
luciferase expression plasmid together with siR206 into
HeLa and ECV304 cells, respectively (Figure 6). From such
experiments it could be calculated that ?300 siRNA mole-
cules per cell were necessary to observe a half maximal inhibi-
tion of luciferase activity. Surprisingly, this number is very
close to what was determined for LF2000-mediated delivery.
Microscopic studies of fluorescently labelled siRNA delivered
with LF2000 on the other hand, showed a vesicular distribu-
tion similar to what has been observed with MPGa, indicative
of the majority of siRNA molecules being not bio-available
either (Supplementary Figure 5). Thus, the microinjection
approach, at least in our hands, turned out not to be practicable
to answer this question. A possible cause could simply be that
the siRNA is retained inside the nucleus. Then again, this
could not be verified by microinjection of fluorescently
labelled siRNA as initially strongly visible fluorescence in
the nucleus completely disappeared within 30 min, probably
either due to quenching effects or transport into cytoplasm.
In spite of this, transiently transfected cells might differ
considerably from stably transfected ones.
In conclusion, our data clearly show that a vesicular
(presumably endosomal) accumulation of the cargo and not
uptake per se is the bottleneck of MPGa-mediated RNA
delivery rendering the vast majority of intracellular siRNA
cargo molecules inactive. Thus, like for other non-viral deliv-
ery systems, solving the problem of vesicular escape remains
the main challenge for this CPP approach. Nonetheless, this
approach yields IC50values for siRNA delivery in the sub-
nanomolar range far superior to other CPP systems together
with the advantage of non-covalent complex formation.
Moreover, this study describes a combination of techniques
which represent easy to handle tools suitable for a detailed
analysis of carrier-mediated delivery of therapeutically inter-
esting nucleic acid molecules, which eventually may lead to
considerably improved delivery.
Supplementary Data are available at NAR online.
We thank Andreas Gebert for his help with the confocal laser
scanning microscope, Laurent Chaloin, Gilles Divita and
Fre ´de ´ric Heitz for their help and advice during initial stages
of the project, Rosel Kretschmer-Kazemi Far and Marita
Overhoff for materials and technical advice and Christian
Tscheik for his contribution to Supplementary Figure 4. T.R.
acknowledges funding by EC-grants QLK2-2001-01451 and
LSHG-CT-2003-503480. Funding to pay the Open Access
publication charges for this article was provided by EC-grant
Conflict of interest statement. None declared.
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