Functional Delivery of siRNA by Disulfide-Constrained Cyclic Amphipathic Peptides

Article abstract of DOI:10.1021/acsmedchemlett.6b00031

The promise of oligonucleotide therapeutic agents to perturb expression of disease-related genes remains unrealized, in part due to challenges with functional cellular delivery of these agents. Herein, we describe disulfide-constrained cyclic amphipathic peptides that complex with short-interfering RNA (siRNA) and affect functional cytosolic delivery and knockdown of target gene products in cell culture and in vivo to mouse lung. Reduction of the constraining disulfide bond and subsequent proteolytic clearance of the peptide are key design features that allow unmasking of the siRNA cargo and presentation to the RNA interference machinery.

Full text of DOI:10.1021/acsmedchemlett.6b00031

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Letter
Functional Delivery of siRNA by Disulfide-
Constrained Cyclic Amphipathic Peptides
Jade J. Welch, Ria J. Swanekamp, Christiaan King, David A. Dean, and Bradley L. Nilsson
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00031 • Publication Date (Web): 30 Mar 2016
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Functional Delivery of siRNA by Disulfide-Constrained Cyclic Am-
phipathic Peptides
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Jade J. Welch,1 Ria J. Swanekamp,1 Christiaan King,2 David A. Dean,2 and Bradley L. Nilsson1,*
1Department of Chemistry, University of Rochester, Rochester, NY 14627
2Department of Pediatrics and Neonatology, University of Rochester Medical Center, School of Medicine and Dentistry,
University of Rochester, Rochester, NY 14642
Supporting Information Placeholder
successful, as the chemical modification to siRNA that is reꢀ
quired can perturb the biological activity of the internalized
oligonucleotide.19ꢀ20 Accordingly, noncovalent CPPꢀsiRNA
ABSTRACT: The promise of oligonucleotide therapeutic
agents to perturb expression of diseaseꢀrelated genes remains
unrealized, in part due to challenges with functional cellular
delivery of these agents. Herein, we describe disulfideꢀ
constrained cyclic amphipathic peptides that complex with
shortꢀinterfering RNA (siRNA) and affect functional cytosolic
delivery and knockdown of target gene products in cell culture
and in vivo to mouse lung. Reduction of the constraining disulꢀ
fide bond and subsequent proteolytic clearance of the peptide
are key design features that allow unmasking of the siRNA
cargo and presentation to the RNA interference machinery.
complexes have been pursued for delivery of siRNA.18,
21ꢀ23
Noncovalent complexation of siRNA with oligoarginine pepꢀ
tides,24ꢀ25 TATꢀdoubleꢀstranded RNA binding protein
fusions,26 or multidomain peptides containing polycationic and
hydrophobic segments25, 27ꢀ30 has provided CPPꢀsiRNA nanoꢀ
particleꢀlike aggregates that facilitate cellular translocation of
the siRNA. These approaches have yielded encouraging reꢀ
sults; yet efficient functional delivery of siRNA remains chalꢀ
lenging, most likely due to localization of CPPꢀsiRNA comꢀ
plexes in the endosome after translocation into the cell.20, 28, 31
Herein we describe the use of redoxꢀsensitive cyclic pepꢀ
tides as an effective method for the functional delivery of
siRNA to cells in culture and to lung cells in an in vivo animal
model. Amphipathic cyclic peptides (cyclized Nꢀterminus to
Cꢀterminus) have recently been shown to carry complexed
small molecule cargo into the nucleus of cells via a nonꢀ
endocytic process.32ꢀ33 We hypothesized that similar disulfideꢀ
constrained cyclic AcꢀC(FKFE)2CGꢀNH2 peptides34 could
form noncovalent complexes with siRNA that would have
advantages over existing CPPꢀsiRNA noncovalent delivery
complexes. First, cyclic peptideꢀsiRNA complexes should be
stabilized to proteolytic and nucleolytic degradation of the
cyclic peptide and its siRNA cargo respectively. Second, Acꢀ
C(FKFE)2CGꢀNH2 and similar sequences are relatively simple
compared to existing CPPs that have been explored for siRNA
delivery. For example, the MPG peptide is 27 amino acids in
(Keywords: siRNA delivery, cellꢀpenetrating peptides, cyꢀ
clic peptides)
The use of oligonucleotides as geneꢀbased therapeutic
agents has been pursued for decades.1 The discovery of RNA
inference (RNAi) mechanisms for gene silencing has inspired
renewed efforts to develop oligonucleotides for therapeutic
applications in the form of exogenous shortꢀinterfering RNA
(siRNA).2ꢀ4 Despite significant effort, the clinical application
of siRNA therapeutics remains extremely limited.5 A major
challenge for the development of siRNA and other oligonucleꢀ
otideꢀbased therapeutics is the transport of these negatively
charged macromolecules into the cell.6 Translocation of siRꢀ
NA into cells requires vectors that facilitate cell binding, inꢀ
ternalization, unpackaging, and presentation of the siRNA
duplex to the RNAi machinery. Numerous siRNA delivery
systems have been explored, including lipid nanoparticles,
cationic polymers, antibody constructs, and RNA aptamers.5ꢀ6
While significant progress in siRNA delivery has been made
with these various transfection agents, none are ideal and sigꢀ
nificant formulation/characterization barriers preclude their
immediate clinical application.
length27 and PepFect14 is 21 amino acids and is side chain
35
conjugated to other agents.31,
In contrast, our disulfideꢀ
constrained cyclic peptides are merely ten amino acids; synꢀ
thesis is straightforward.34 Finally, we predicted that disulfideꢀ
constrained cyclic peptides would undergo reduction by intraꢀ
cellular glutathione, facilitating proteolytic degradation and
presentation of the siRNA cargo to the RNAi machinery.
Cellꢀpenetrating peptides (CPPs) are promising siRNA
transfection agents.7ꢀ9 CPPs have been validated for the transꢀ
location of large macromolecular cargo, including proteins,
into cells without appreciable toxicity.10ꢀ11 CPPs, including
TAT,12ꢀ13 penetratin,14ꢀ15 transportan,16 and oligoarginines,17
have been covalently appended to siRNA to promote cell inꢀ
ternalization.18 These strategies have been only moderately
We chose amphipathic peptides with alternating hydrophoꢀ
bic and hydrophilic residues to evaluate disulfideꢀconstrained
cyclic peptides as siRNA delivery vectors. We initially used
the AcꢀC(FKFE)2CGꢀNH2 peptide;34 we also used the cationic
AcꢀC(WR)4CGꢀNH2 peptide. Trp/Argꢀrich peptides have been
previously exploited as CPPs.32 We further reasoned that
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Trp/Argꢀcontaining peptides would exhibit improved binding
tide did not enter the cells to any appreciable degree (Figure
2A). The cyclic and AcꢀC(FKFE)2CGꢀNH2 (Figure 2B
and C respectively) peptides were found to aid siRNA transloꢀ
cation into the cytosol to a modest degree. In contrast, cyclic
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to siRNA due to attractive charge interactions and based on
the demonstrated ability of Trp to bind to oligonucleotides via
πꢀπ interactions.36 In a linear form, these peptides have a
strong tendency to form βꢀsheet secondary structures that readꢀ
ily selfꢀassemble into fibrils; however, cyclization via a disulꢀ
fide bond between the flanking Cys residues favors the forꢀ
mation of transient, amorphous aggregates that lack the stabiꢀ
lizing hydrogen bond network found in fibrillar assemblies.34
The proposed cyclic peptides present hydrophobic and hydroꢀ
philic side chains on opposite faces of the cyclic peptides
(Figure 1), providing an ideal sequence pattern for binding to
siRNA and facilitating its delivery into the cell. Enantiomeric
Lꢀ
Dꢀ
Lꢀ and DꢀAcꢀC(WR)4CGꢀNH2
(LꢀAcꢀC(WR)4CGꢀNH2 is
shown in Figure 2D) were found to affect significant cytosolic
uptake of RhꢀsiRNA. The chirality of the cyclic peptides did
not significantly influence siRNA uptake. The higher efficienꢀ
cy of cytosolic delivery of siRNA with cyclic AcꢀC(WR)4CGꢀ
NH2 peptides is most likely due to enhanced complexation
affinity of these cationic peptides with siRNA and to enhanced
translocation ability for Arg/Trpꢀcontaining peptides, as has
been previously demonstrated.17, 24 Addition of up to 1 mM
peptide with siRNA to A549, primary rat alveolar epithelial
type II cells, human smooth muscle cells, human fibroblasts,
and mouse lung epithelial MLE15 cells, did not appear to disꢀ
tress the cells based on microscopic analysis.
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L
and D variants of each peptide, as well as a variant with a
redoxꢀinsensitive thioether constraint (which cannot be reꢀ
duced), were prepared to determine if the proteolytic degradaꢀ
tion of the reduced peptide in the cell is necessary for funcꢀ
tional knockdown by internalized siRNA.
Peptides were prepared by solid phase peptide synthesis and
cyclization was performed as previously described (see Supꢀ
porting Information for experimental details and structures and
characterization data for all peptides (Figures S1–S11).34 The
cyclic AcꢀC(FKFE)2CGꢀNH2 and AcꢀC(WR)4CGꢀNH2 pepꢀ
tides were tested for stability in water, cell culture media, and
fetal bovine serum (FBS) supplemented media (Figure S12).
Neither peptide showed evidence of degradation in water or in
cell culture media, but both were slowly degraded over 24 h in
FBSꢀsupplemented media, most likely due to the presence of
glutathione in this media.37 The rate of degradation was slow,
however, related to the length of time peptide/siRNA comꢀ
plexes were incubated with cells in subsequent siRNA delivꢀ
ery analyses.
Figure 1. Structural models of the cyclic, disulfideꢀconstrained
AcꢀC(FKFE)2CGꢀNH2 peptide shown the hydrophobic face (A)
and the hydrophilic face (B). Hydrophobic Phe side chains are
shown in green, hydrophilic Lys and Glu side chains are shown in
blue, the cystine disulfide sulfurs are shown in yellow, and backꢀ
bone and other functionality is shown in grey. This class of cyclic
amphipathic peptide presents a hydrophobic and a hydrophilic
face, as is apparent in these structural representations.
Figure 2. Transport of rhodamineꢀlabeled (Rh) siRNA into A549
lung cancer cells by disulfideꢀconstrained cyclic amphipathic
peptides. Nuclei are stained with DAPI. Scale bar is 20 µm and
corresponds to all images. A. RhꢀsiRNA alone, B. cyclic LꢀAcꢀ
C(FKFE)2CGꢀNH2 + RhꢀsiRNA, C. cyclic DꢀAcꢀC(FKFE)2CGꢀ
NH2 + RhꢀsiRNA, D. cyclic LꢀAcꢀC(WR)4CGꢀNH2 + RhꢀsiRNA.
Peptide concentrations were 100 µM, RhꢀsiRNA concentrations
were 100 nM.
Transport of siRNA into cells using the proposed cyclic
amphipathic peptides was assessed in vitro by fluorescence
imaging of A549 lung cancer cells incubated with rhodamine
(Rh)ꢀlabeled siRNA in the presence or absence of the cyclic
peptides (Figure 2). The Rhꢀlabeled siRNA was incubated
with cyclic peptide (100 µM peptide, 100 nM siRNA in phosꢀ
phateꢀbuffered saline) at room temperature for 30 minutes
after which these mixtures were applied to A549 lung cancer
cells in culture for 2 hours. Upon rinsing, the cells were alꢀ
lowed to stand for 48 h, after which the cells were washed and
analyzed by fluorescence microscopy to determine siRNA
transport efficiency. It was found that RhꢀsiRNA without pepꢀ
Functional gene knockdown analyses were conducted in orꢀ
der to determine whether this siRNA has been delivered effecꢀ
tively to RNAi machinery (Figure 3). Cyclic LꢀAcꢀ
C(FKFE)2CGꢀNH2, DꢀAcꢀC(FKFE)2CGꢀNH2, and LꢀAcꢀ
C(WR)4CGꢀNH2 (100 µM) were premixed for 30 min with
600 nM siRNA targeted to the TTFꢀ1 transcription factor,
which is a master regulator of cells in the thymus and lung.
Human pulmonary epithelial A549 and H441 cells were treatꢀ
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ACS Medicinal Chemistry Letters
ed with the resulting cyclic peptideꢀsiRNA complexes for 48 ing Information), was synthesized in order to test the hypotheꢀ
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h, at which time TTFꢀ1 expression was quantified by Western
blot and densitometry analysis normalized to actin expression
(Figure 3). This time (48 h) was determined previously to
show optimal TTFꢀ1 knockdown using standard liposomal
reagents to delivery TTFꢀ1 siRNA.38 While naked siRNA
failed to knockdown TTFꢀ1 expression, each of the peptideꢀ
siRNA complexes displayed knockdown efficiencies of varyꢀ
ing levels in both cell lines. In A549 cells, cyclic DꢀAcꢀ
C(FKFE)2CGꢀNH2 and LꢀAcꢀC(FKFE)2CGꢀNH2 provide TTFꢀ
1 knockdown of ~20% and ~85% respectively. These results
are interesting, since both enantiomers appeared to transport
siRNA into the cytosol at similar levels. We hypothesize that
the higher proteolytic stability of the Dꢀpeptide results in less
efficient unpackaging of the siRNA cargo, and thus less effiꢀ
cient knockdown due to lack of siRNA access to the RNAi
machinery. We were gratified to find that the cyclic LꢀAcꢀ
C(WR)4CGꢀNH2 peptide promoted >90% siRNA knockdown
of TTFꢀ1, consistent with the high transport capability of this
peptide. Interestingly, knockdown of TTFꢀ1 with cyclic LꢀAcꢀ
C(FKFE)2CGꢀNH2 delivery is still surprisingly effective conꢀ
sidering the difference in siRNA delivery efficiency of this
peptide relative to cyclic AcꢀC(WR)4CGꢀNH2.
sis that reductive ringꢀopening and proteolytic degradation of
the cyclic CPPs is necessary for efficient gene knockdown.
Gene knockdown with thioetherꢀ(WR)4CGꢀNH2 was tested in
human H441 cells. It was found that thioetherꢀ(WR)4CGꢀNH2
displayed negligible knockdown in comparison to cyclic Acꢀ
C(WR)4CGꢀNH2, with ~0% and ~45% TTFꢀ1 knockdown
with these peptides respectively (Figure S13). These findings,
coupled with the poor knockdown observed with cyclic DꢀAcꢀ
C(FKFE)2CGꢀNH2, further indicate that reductive cleavage of
the constraining disulfide bond and proteolytic degradation of
the delivery peptide are essential for unpackaging of siRNA
leading to functional gene knockdown in the cell.
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Knockdown analyses utilizing human H441 cells showed a
similar trend (Figure S13, Supporting Information). It was
found that cyclic DꢀAcꢀC(FKFE)2CGꢀNH2 and LꢀAcꢀ
C(FKFE)2CGꢀNH2 provide TTFꢀ1 knockdown of ~30% and
40% respectively. Commercial transfection agents, Pepfect 14
and Lipfectamine, were assessed as comparative controls.
Pepfect 14 (~50% TTFꢀ1 knockdown) and Lipofectamine
2000 (~40% TTFꢀ1 knockdown) provided knockdown levels
that were comparable to the AcꢀC(WR)4CGꢀNH2 (Figure S13).
Significantly, these preliminary studies indicate favorable
RNAi knockdown of a gene target in cells compared to existꢀ
ing CPPꢀsiRNA transport vectors that range in effectiveness
from 20–90% knockdown.22, 28, 31, 39 The simplicity of the cyꢀ
clic peptides described herein are a distinct advantage of existꢀ
ing CPPs for siRNA delivery.
Figure 4. In vivo knockdown of TTFꢀ1 expression in mouse lung
by TTFꢀ1ꢀtargeted siRNA. TTFꢀ1 expression was quantified by
Western blot analysis relative to actin expression in mouse lung
(see Supporting Information and Figure S14 for details). T indiꢀ
cates TTFꢀ1 specific siRNA while Sc indicates a scrambled conꢀ
trol (n=4, * p<0.05).
In order to further validate the functional ability of the disulꢀ
fideꢀconstrained cyclic peptides AcꢀC(FKFE)2CGꢀNH2 and
AcꢀC(WR)4CGꢀNH2 as effective siRNA transfection agents,
we delivered TTFꢀ1 siRNAꢀpeptide complexes in vivo to the
lungs of C57B6 mice and assessed whole lung expression of
TTFꢀ1 via western blot (Figure 4, and Figure S14). As identiꢀ
fied earlier either the Dꢀ or Lꢀisoform of both C(FKFE)2CGꢀ
NH2 and AcꢀC(WR)4CGꢀNH2 (100 µM) were complexed with
TTFꢀ1 siRNA (100 nM), and then suspended in a total volume
of 50 µL of saline solution. Mice were anesthetized by IP inꢀ
jection with isofluorine and placed in the supine position. The
tongue of each animal was pulled out, gently, with bluntꢀ
ended forceps, and the 50 µL siRNA solution was delivered
via pipette to the back of the tongue. Using a second pair of
forceps the animals’ noses were gently pinched shut, and the
tongues remained held until the animal aspirated the solution
into their lungs. After aspiration animals were allowed to reꢀ
cover for 48 hours, at which time they were euthanized and
lungs were harvested and lysed for western blot detection of
TTFꢀ1. Similar to the in vitro studies, animal lungs treated
with either the Dꢀ or Lꢀenantiomer of both AcꢀC(FKFE)2CGꢀ
NH2 and AcꢀC(WR)4CGꢀNH2 showed reduced TTFꢀ1 protein
expression compared to control groups, with the Lꢀenantiomer
of both groups showing the least expression of TTFꢀ1, sugꢀ
gesting functional delivery of siRNA into cells and targeted
Figure 3. Knockdown efficiency of cyclic peptideꢀsiRNA comꢀ
plexes against TTFꢀ1 expression in A549 lung cancer cells. Lꢀ
peptide is cyclic LꢀAcꢀC(FKFE)2CGꢀNH2, Dꢀpeptide is cyclic Dꢀ
AcꢀC(FKFE)2CGꢀNH2, and WR4 is cyclic LꢀAcꢀC(WR)4CGꢀNH2.
A. Western blot densitometry of TTFꢀ1 expression relative to
actin expression. B. Relative TTFꢀ1 levels expressed as percent
knockdown normalized against actin expression (n=3).
A thioether cyclized peptide that is insensitive to reductive
ringꢀopening, thioetherꢀ(WR)4CGꢀNH2 (Figure S1.F, Supportꢀ
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degradation of TTFꢀ1 mRNA. These results also identify the
that disulfide bond reduction and proteolytic degradation of
lung as an amenable target for RNAi based therapies.40ꢀ41
the delivery peptides contributes to unpackaging and presentaꢀ
tion of siRNA to the RNAi apparatus. The mechanism by
which disulfideꢀconstrained cyclic peptides assist siRNA deꢀ
livery to the cytosol is as yet unknown. Previous work by Paꢀ
rang and coworkers with delivery of small molecule drugs
using NꢀtoꢀC terminal cyclic Trp/Arg peptides suggests that
this class of material does not utilize endocytic pathways, thus
avoiding problems with endosomal escape.32 However, the
possibility that our disulfideꢀconstrained cyclic peptides exꢀ
ploit endocytic pathways for cargo translocation cannot, at
present, be ruled out. Studies to elucidate the mechanism(s) by
which our cyclic peptides enable siRNA transport into the
cytosol are currently underway.
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Studies were conducted to characterize the formation of cyꢀ
clic peptideꢀsiRNA noncovalent complexes. A gel shift assay
was utilized in which varying concentrations of cyclic LꢀAcꢀ
C(FKFE)2CGꢀNH2 and LꢀAcꢀC(WR)4CGꢀNH2 were incubated
with siRNA (Figure S15). It was found that cyclic LꢀAcꢀ
C(FKFE)2CGꢀNH2 complexed with siRNA only weakly, reꢀ
quiring much greater than 100ꢀfold excess of peptide to siRꢀ
NA before shifting of the siRNA band was observed on the
agarose gel; even at large excesses of peptide, the shifted siRꢀ
NA band was observed to streak on the gel, suggesting that the
complex is weak. In contrast, at 100ꢀfold excess of cyclic Lꢀ
AcꢀC(WR)4CGꢀNH2, the siRNA band was cleanly shifted
indicating higher binding affinity for the Trp/Argꢀpeptide to
siRNA; this may account, in part, for its higher translocation
efficiency for siRNA into the cytosol. These results support
noncovalent complexation between the peptide and siRNA as
the mode of siRNA transport, as opposed to independent poraꢀ
tion of the membrane by the peptides independent of siRNA
complex formation.
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In conclusion, we have demonstrated that disulfideꢀ
constrained cyclic amphipathic peptides affect functional deꢀ
livery of siRNA to cells both in vitro and in vivo. Our data
supports the formation of noncovalent peptideꢀsiRNA comꢀ
plexes that are able to penetrate into the cytosol. Our data also
suggests that disulfide bond reduction in by chemical reductꢀ
ants in the cytosol likely plays a role in peptide linearization
and proteolytic clearance, enabling unpackaging of the siRNA
cargo. Functional knockdown of gene targets is highly effiꢀ
cient and is comparable to the best available siRNA transfecꢀ
tion agents. Our simple peptides appear to be ideal for in vitro
siRNA delivery in cell culture and for localized in vivo delivꢀ
ery to lung tissues. However, the peptideꢀsiRNA complexes
appear to be weak, suggesting that these peptides may not be
ideal for systemic in vivo delivery siRNA. Evidence shown
herein that the cyclic peptides are slowly degraded in fetal
bovin serum (most likely due to the presence of glutathione)
suggests that applications that require prolonged exposure of
siRNA/cyclic peptide complexes to blood prior to reaching
target tissues may be problematic. Additional studies are curꢀ
rently underway to characterize the mechanisms of siRNA
delivery by these peptides as well as to correlate the strength
of the noncovalent complexes to functional gene knockdown
efficiency. These studies will enable the design of nextꢀ
generation siRNA delivery agents based on the disulfideꢀ
constrained cyclic peptides described herein.
Dynamic light scattering (DLS) and transmission electron
microscopy (TEM) were employed to determine the physical
characteristics of the siRNA complexes formed with 10ꢀfold
and 1000ꢀfold cyclic
LꢀAcꢀC(WR)4CGꢀNH2. DLS indicated
the presence of polydisperse nanoparticles with an average
hydrodynamic radius of 84 ± 12 nm (Figure S16). TEM imꢀ
ages showed spherical nanoparticles with an average diameter
of 18 ± 2 nm (Figure S15). These nanoparticles formed higher
order aggregates consistent with the particle sizes observed by
DLS experiments. These cyclic amphipathic peptides presumꢀ
ably encapsulate the siRNA duplex via hydrophobic/aromatic
and/or charge interactions between the peptide and the siRNA.
The opposite facial orientation of hydrophobic and hydrophilic
side chain functionality in these peptides also enables presenꢀ
tation of functionality (guanidinium or ammonium) to the cell
surface that promotes siRNA translocation into the cell. In
contrast, TEM showed no evidence of nanoparticle formation
with between cyclic LꢀAcꢀC(FKFE)2CGꢀNH2 and siRNA (in
1000ꢀfold peptide excess); DLS shows polydisperse aggreꢀ
gates with no consistent radius. These results are consistent
with a weaker binding affinity of cyclic AcꢀC(FKFE)2CGꢀNH2
compared to cyclic AcꢀC(WR)4CGꢀNH2 to siRNA. The correꢀ
lation of cyclic peptide/siRNA binding affinity to translocation
efficiency and a more detailed characterization of the resulting
complexes are topics of ongoing research in our group.
ASSOCIATED CONTENT
Supporting Information
Additional figures, experimental procedures, and characterization
data. This material is available free of charge via the internet at
http://pubs.acs.org.
Our data provides early insight into the basis for functional
of delivery of siRNA by these cyclic amphipathic peptides.
While fluorescence microscopy indicates that cyclic Acꢀ
C(WR)4CGꢀNH2 enables siRNA translocation much more
efficiently than cyclic AcꢀC(FKFE)2CGꢀNH2, TTFꢀ1 knockꢀ
down in vitro and in vivo indicates that gene knockdown with
cyclic LꢀAcꢀC(WR)4CGꢀNH2 peptide was only slightly more
efficient than was observed with cyclic LꢀAcꢀC(FKFE)2CGꢀ
NH2. This suggests that highꢀdensity siRNA delivery is not
absolutely essential for functional downregulation of gene
AUTHOR INFORMATION
Corresponding Author
nilsson@chem.rochester.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
expression. Additionally,
D and L enantiomers of these cyclic
peptides affected similar levels of siRNA translocation into the
cytosol, but functional gene knockdown by siRNA was signifꢀ
icantly more effective with the L enantiomers. This data, comꢀ
bined with the inability of the permanently cyclized peptide to
knockdown gene expression, is consistent with our hypothesis
This work was supported by grants EB9903 and HL120521 from
the National Institutes of Health and by grants from the National
Science Foundation (DMRꢀ1148836, CHEꢀ0840410, and CHEꢀ
0946653).
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(24) Wang, Y. H.; Hou, Y. W.; Lee, H. J., An intracellular delivery
method for siRNA by an arginineꢀrich peptide. J. Biochem. Biophys.
Methods 2007, 70 (4), 579ꢀ586.
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Figure 1. Structural models of the cyclic, disulfide-constrained Ac-C(FKFE)2CG-NH2 peptide shown the
hydrophobic face (A) and the hydrophilic face (B). Hydrophobic Phe side chains are shown in green,
hydrophilic Lys and Glu side chains are shown in blue, the cystine disulfide sulfurs are shown in yellow, and
backbone and other functionality is shown in grey. This class of cyclic amphipathic peptide presents a
hydrophobic and a hydrophilic face, as is apparent in these structural representations
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Figure 2. Transport of rhodamineꢀlabeled (Rh) siRNA into A549 lung cancer cells by disulfideꢀconstrained
cyclic amphipathic peptides. Nuclei are stained with DAPI. Scale bar is 20 µm and corresponds to all images.
A. RhꢀsiRNA alone, B. cyclic LꢀAcꢀC(FKFE)2CGꢀNH2 + RhꢀsiRNA, C. cyclic DꢀAcꢀC(FKFE)2CGꢀNH2 + RhꢀsiRNA,
D. cyclic LꢀAcꢀC(WR)4CGꢀNH2 + RhꢀsiRNA. Peptide concentrations were 100 µM, RhꢀsiRNA concentrations
were 100 nM.
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Figure 3. Knockdown efficiency of cyclic peptide-siRNA com-plexes against TTF-1 expression in A549 lung
cancer cells. L-peptide is cyclic L-Ac-C(FKFE)2CG-NH2, D-peptide is cyclic D-Ac-C(FKFE)2CG-NH2, and WR4 is
cyclic L-Ac-C(WR)4CG-NH2. A. Western blot densitometry of TTF-1 expression relative to actin expression. B.
Relative TTF-1 levels expressed as percent knockdown normalized against actin expression (n=3).
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Figure 4. In vivo knockdown of TTF-1 expression in mouse lung by TTF-1-targeted siRNA. TTF-1 expression
was quantified by Western blot analysis relative to actin expression in mouse lung (see Supporting
Information and Figure S12 for details). T indicates TTF-1 specific siRNA while Sc indicates a scrambled
control (n=4, * p<0.05).
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