Light-responsive helical polypeptides capable of reducing toxicity and unpacking DNA: Toward nonviral gene delivery

Article abstract of DOI:10.1002/anie.201302820

Gene delivery vehicles: Helical, cationic polypeptides with light-responsive domains showed potent membrane activity and promoted efficient cellular internalization of DNA (see picture). After transfection, a light trigger induces protecting-group removal to expose anionic carboxylate groups. The reduced cationic charge and helix distortion of the polypeptides result in enhanced DNA unpacking and reduced material toxicity because of the eliminated membrane activity. Copyright

Full text of DOI:10.1002/anie.201302820

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DOI: 10.1002/anie.201302820
Gene Delivery
Light-Responsive Helical Polypeptides Capable of Reducing Toxicity
and Unpacking DNA: Toward Nonviral Gene Delivery**
Lichen Yin, Haoyu Tang, Kyung Hoon Kim, Nan Zheng, Ziyuan Song, Nathan P. Gabrielson,
Hua Lu, and Jianjun Cheng*
Nonviral gene delivery with synthetic cationic polymeric
vectors is widely recognized as an attractive alternative to
viral gene delivery, which suffers from inherent immunoge-
nicity and various side effects.[1] The transfection efficiency
and chemotoxicity of these polymeric vectors are often
closely related to the density of their cationic charge.[2]
Materials with low charge density usually show low toxicity
but are often poor transfection agents. Polycations with high
charge density could mediate effective gene transfer, which is
however often associated with significant, charge-induced
toxicity.[3] When modified with various charge-reducing
moieties, including saccharides,[4] hydrocarbons,[5] and poly-
(ethylene glycol) (PEG),[6] polycations often benefit from
improved safety profiles while in the meantime suffer from
significantly reduced gene delivery capabilities. In addition to
the charge-induced toxicity, excessive positive charges on
polycations would also enhance the electrostatic attraction
with the nucleic acids to restrict intracellular gene release.[3g,7]
Therefore, it would be of great interest to develop a highly
charged polycation that possesses full transfection capacity
and membrane activity during gene transfer, but can be
triggered to transform to a less charged or uncharged material
with low membrane activity after transfection, such that
intracellular DNA unpacking can be facilitated and toxicity
can be reduced.[8]
CPPs are often too short (10–25 peptide residues) and lack
adequate cationic charge to efficiently condense and deliver
genes by themselves. As such, CPPs often serve as membrane-
active ligands incorporated or conjugated to delivery vehicles
to improve delivery efficiencies.[11]
We recently developed high-molecular-weight (MW),
cationic, cell-penetrating, a-helical polypeptides, termed
PVBLG-8 (Scheme 1A).[12] By maintaining a minimum sep-
Cell-penetrating peptides (CPPs), notable for their excel-
lent membrane activities, have been developed and used in
drug and gene delivery.[9] Helical structures are often
observed in CPPs or formed in CPPs during membrane
transduction, and have been tied to their membrane activ-
ity.[10] Mechanistically, the helical CPP presents a rigid
amphiphilic structure that interacts with and destabilizes
lipid bilayers, creating transient pathways to facilitate the
passive diffusion of exogenous materials.[9a] Well-known
examples of CPPs include oligoarginine, HIV-TAT, and
penetratin. Despite their excellent membrane permeability,
Scheme 1. A) Schematic illustration of cationic helical PVBLG-8.
B) Synthetic route of PDMNBLG-r-PVBLG-8.
[*] Dr. L. Yin,[+] Dr. H. Tang,[+] K. H. Kim, N. Zheng, Z. Song,
Dr. N. P. Gabrielson, Dr. H. Lu, Prof. Dr. J. Cheng
Department of Materials Science and Engineering
University of Illinois at Urbana-Champaign
1304 W. Green Street, Urbana, IL 61801 (USA)
E-mail: jianjunc@illinois.edu
aration distance of 11 s-bonds between the backbone and the
side chain charge, the helical structure of PVBLG-8 is
stabilized by increased hydrophobic interaction of the side
chains and reduced side-chain charge repulsion. Because of
the high charge density and higher MW compared to tradi-
tional CPPs, PVBLG-8 can condense and deliver DNA to
mammalian cells much more effectively, making it a better
vector for gene delivery.[12b] However, PVBLG-8 shows
notable cytotoxicity at high concentrations and therefore
shares the same concerns as many other polycations. In
Homepage: http://cheng.matse.illinois.edu/
[+] These authors contributed equally to this work.
[**] J.C. acknowledges support from the NSF (CHE-1153122) and the
NIH (NIH Director’s New Innovator Award 1DP2OD007246,
1R21EB013379).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302820.
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consistence with previous reports on the correlation between
cytotoxicity and helicity of CPPs, the helical structure of
PVBLG-8 also contributes to its observed toxicity. Moreover,
because of the high density of its cationic charge, PVBLG-8
also suffers from inefficient DNA release. Thus, in attempts to
reduce material toxicity as well as facilitate intracellular DNA
release, we sought a strategy to reduce both the charge
density and the helical content of PVBLG-8 at the post-
transfection state. Here, we report the design of cationic a-
helical poly(g-(4,5-dimethoxy-2-nitrobenzyl)-l-glutamate)-r-
PVBLG-8 (PDMNBLG-r-PVBLG-8, Scheme 1B), which
maintains high membrane activity during transfection
because of high charge density and helical contents, and
transforms to a toxicity-reduced and DNA-repelling state
with a distorted helix and diminished density of the cationic
charge after transfection in response to external triggers.
PVBLG-8 contains stable pendant benzyl ester bonds that
are difficult to cleave under physiological conditions, thus
prohibiting the conversion of the material into the desired
nontoxic, negatively charged, random-coiled poly(glutamic
acid). By incorporating various amounts of light-sensitive 4,5-
dimethoxy-2-nitrobenzyl-glutamate
(DMNBLG)
into
PVBLG-8 to prepare random PDMNBLG-r-PVBLG-8
copolymers, we could enable the photonic manipulation of
the toxicity and gene-release profiles of the material after
transfection. Because the DMNBLG residues are uncharged
and hydrophobic, the polycationic nature and helical struc-
ture would be well maintained in PDMNBLG-r-PVBLG-8 to
exert membrane activity. When a photonic stimulus is applied
after transfection, the PDMNBLG domain would yield
pendant carboxylate groups (blue segment of the illustration
and chemical structure in Figure 1) through light-triggered
de-esterification, and the polypeptide would thus have a much
reduced density of cationic charge. The intramolecular
electrostatic attraction between the negatively charged car-
boxylate groups and the positively charged amine side groups
of the original PVBLG-8 would transform the helical
conformation of the parental polypeptide to the helix-
disrupted conformation of the light-treated polypeptide.
Collectively, the irradiation of PDMNBLG-r-PVBLG-8
would lead to a net effect of reducing the cytotoxicity of the
material and promoting intracellular gene release (Figure 1).
While light-enhanced gene transfection either by charge-
switching multivalency[8a,b] or by supramolecular recogni-
tion[8d] has been reported, the current study provides a novel
strategy to modulate the gene transfection and cytotoxicity by
regulating the polypeptide helicity.
Figure 1. Intracellular kinetics of PDMNBLG-r-PVBLG-8/DNA com-
plexes, including uptake by endocytosis and passive diffusion, endo-
somal escape by membrane destabilization, light-triggered conversion
of the charge and secondary structure of the polypeptide, subsequently
facilitating intracellular DNA unpacking, and nuclear transport.
curves (Figure S2 in the Supporting Information), well-
defined MWs, and the low polydispersity index (PDI < 1.1,
Table 1).[13] All synthesized polypeptides exhibited excellent
solubility in aqueous solution at pH < 9 and adopted a-helical
conformations (Figure 2A). The helicities were as high as
90% (Figure S6 in the Supporting Information) and the
helical structures were remarkably stable against pH changes
between 1 and 9 (Figure 2B), demonstrating that the addition
of up to 40 mol% DMNB-l-Glu residues to the random
copolymer did not affect the helical conformation of PVBLG-
8.
The helicities of PDMNBLG-r-PVBLG-8 were demon-
strated to be photo-responsive. When an aqueous solution of
the polypeptide was irradiated with UV light (l = 365 nm,
20 mWcmÀ2), an efficient model light trigger, the absorption
at 346 nm in the UV/Vis spectroscopy decreased whereas the
absorption at 400 nm increased (Figure S5 in the Supporting
Information), indicating cleavage of the photo-labile ester
bond and generation of nitrobenzaldehyde.[14] By plotting the
OD346 value against the irradiation time, we determined that
the photochemical reaction approached maximum conversion
after 10 min of irradiation with UV light. UV irradiation also
To test the above-mentioned design, photo-responsive
PDMNBLG-r-PVBLG-8 with a fixed degree of polymeri-
zation of 200 and various molar contents of DMNBLG (10%,
20%, 30%, and 40%, designated as P10, P20, P30, and P40,
respectively) were synthesized by ring-opening copolymer-
ization of DMNB-l-Glu-NCA and VB-l-Glu-NCA. Side
chains of the resulting PDMNBLG-r-PABLG were aminated
to yield PDMNBLG-r-PVBLG-8 (Scheme 1B). In addition,
P0,
a nonresponsive control polymer containing no
DMNBLG (P0 = PVBLG-8) was also prepared.[12b] Hexam-
ethyldisilazane (HMDS) as the initiator ensured well-con-
trolled polymerization, evidenced by the monomodal GPC
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Table 1: Properties of PDMNBLG-r-PVBLG-8.
thiourea (FITC-Tris, a membrane-impermeable fluorescent
dye in the nonreactive form of FITC after reaction of FITC
with Tris) was used as a biomarker for membrane pore
formation.[9b] The polypeptides facilitated the uptake of
FITC-Tris in HeLa and COS-7 cells by 8–10 fold (Figure 3A),
M/I[a]
Mn (Mn*)
PDI[c] Composition[d]
ꢀ103[b,c]
P0
(200+0)/1
47.7 (49.0) 1.08
PVBLG-8194
P10 (180+20)/1 47.9 (50.6) 1.02
P20 (160+40)/1 57.2 (52.2) 1.02
P30 (140+60)/1 55.6 (53.8) 1.03
P40 (120+80)/1 52.7 (55.4) 1.06
PDMNBLG19-r-PVBLG-8170
PDMNBLG44-r-PVBLG-8175
PDMNBLG62-r-PVBLG-8145
PDMNBLG76-r-PVBLG-8114
[a] Feed ratio of (DMNB-l-Glu-NCA+VB-l-Glu-NCA)/HMDS.
[b] Obtained MW for PDMNBLG-r-PVBLG (expected MW*). [c] Obtained
MW and PDI were determined by GPC. [d] The composition was
1
determined by H NMR spectroscopy.
Figure 3. Polypeptides display diminished membrane activity and cyto-
toxicity upon UV irradiation. A) Uptake of FITC-Tris in HeLa and COS-7
cells in the presence of P20 and UV-treated P20 (n=3). B) Cytotoxicity
of polypeptide/DNA complexes in HeLa cells (N/P ratio of 20, 5 mg
DNA/mL) with/without in situ UV irradiation (20 mWcmÀ2, 5 min) as
assessed by the MTT assay (n=3); ns=no significant difference
(p>0.05).
presumably because P20 induced pore formation on cell
membranes.[9b] To validate this hypothesis, FITC-Tris inter-
nalization in the presence of UV-pretreated P20
(20 mWcmÀ2, 10 min) was studied, showing that light-trig-
gered reduction of the density of cationic charges and loss of
a-helicity indeed reduced the membrane activity and pore-
formation capability of P20 (P20(UV) + FITC-Tris group vs.
P20(non-UV) + FITC-Tris group, p < 0.05; p = estimate of
the probability that the result has occurred by statistical
accident; the lower the p value, the higher the statistical
significance). It was therefore not surprising to observe that
the cytotoxicities of all photo-responsive polypeptides (P10–
P40) were notably reduced upon UV irradiation (Figure S9 in
the Supporting Information) as determined by the MTTassay.
In a typical experiment in accordance with the transfection
process, polypeptide/DNA complexes (N/P ratio of 20) were
first incubated with cells for 4 h, followed by UV irradiation
for 5 min and further culturing for 20 h before viability
assessment (Figure 3B, and Figure S10 in the Supporting
Information). Consistently, complexes formed from P10–P40,
but not the nonresponsive P0, showed diminished cytotoxicity
in response to UV irradiation. These results collectively
validated the proposed strategy of eliminating the membrane
activity and improving the cell tolerability of polypeptides by
post-transfection light exposure. UV light is not biocompat-
ible; but cells that receive low-intensity UV irradiation for
a short period of time (20 mWcmÀ2, 5 min) showed uncom-
promised viability ((96.8 Æ 4.2)%, n = 3, n = number of
experiments), thus indicating that UV irradiation in this
proof-of-concept model system did not induce appreciable
cytotoxicity that would otherwise jeopardize the analysis of
trigger-induced toxicity reduction. The cleaved DMNB also
showed minimal toxicity to HeLa cells following 24 h
incubation at 0.1 mmolmLÀ1, corresponding to the concen-
tration of DMNB generated from the UV-irradiated P40/
DNA complexes (N/P ratio of 20) at 5 mgDNAmLÀ1 during
Figure 2. PDMNBLG-r-PVBLG-8 with reduced helicity and cationic
charge upon UV irradiation (20 mWcmÀ2, 10 min). A) CD spectra of
PDMNBLG-r-PVBLG-8 in water before UV irradiation. B) Helices of
PDMNBLG-r-PVBLG-8 at different pH values. C) CD spectra of
PDMNBLG-r-PVBLG-8 in water after irradiation. D) Change of the zeta
potential of polypeptide/DNA complexes (N/P ratio=20) upon UV
irradiation.
significantly attenuated the a-helicities of P20, P30, and P40,
which contain significant portions of photo-responsive
DMNBLG residues, while minimal changes were seen in P0
and P10 (Figure 2C, and Figure S6 in the Supporting Infor-
mation). The observed helix disruption was attributed to the
intramolecular charge attraction between amine groups of
PVBLG-8 and carboxylate groups generated through the
removal of the DMNB group. The formation of carboxylate
groups and the reduction of positive charge of the materials
were also supported by the observation that the zeta
potentials of all polypeptide/pCMV-Luc/DNA complexes at
the optimal N/P (positively charged amine group (N) of each
polycation residue/negatively charged phosphate group (P) of
each DNA residue) ratio of 20 (diameter of 130–170 nm) were
decreased upon UV irradiation (Figure 2D).
P20, which has the highest density of cationic charges
among the three PDMNBLG-r-PVBLG-8 analogues (P20,
P30, and P40) that are susceptible to light-induced helicity
reductions (Figure 2C), was selected to study the light-
triggered changes of membrane activity and cytotoxicity.
Fluorescein
isothiocyanate/tris(hydroxymethyl)methane-
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the transfection process (Figure S11 in the Supporting Infor-
mation).
and random-coil for PVBDLG-8 prepared with racemic d,l-
Glu[12b]), were allowed to form complexes with DNA (N/P
ratio of 20). PVBLG-8 showed higher DNA condensation
than PVBDLG-8 (Figure S14 in the Supporting Information),
thus suggesting that the reduction in both charge and helicity
in UV-treated PDMNBLG-r-PVBLG-8 could synergistically
promote the DNA release. By labeling P20 with rhodamine
(RhB) and DNA with YOYO-1, we further studied the
intracellular DNA unpacking using confocal laser scanning
microscopy (CLSM). Compared to nontreated cells, inside
which red and green fluorescence were largely overlapped,
UV-treated cells exhibited notably enhanced separation of
green fluorescence from red fluorescence (Figure 4B), thus
suggesting trigger-induced intracellular DNA release. The
unpacked DNA spread to the entire cytoplasm and some was
localized inside the nuclei (Figure 4B). Since DNA needs to
enter the nuclei before it can be transcribed, we further
quantified the nuclear distribution of YOYO-1-DNA.[7]
Following the treatment of the cells with the complex for
4 h and subsequent irradiation with UV light, 30% of the
internalized DNA was distributed to the nuclei, representing
a 2.3-fold increase over nontreated cells (Figure 4C). As
a result, UV irradiation led to up to 8.5- and 5.6-fold increase
in luciferase expression in HeLa (Figure 4D) and COS-7 cells
(Figure S16 in the Supporting Information), respectively.
Maximal transfection efficiency was noted for UV-treated
P20, outperforming commercial reagent LipofectamineTM
2000 (LPF2000) by 10–18 fold. The promoted gene trans-
fection of P20/DNA complexes was also noted by flow
cytometry when plasmid encoding enhanced green fluores-
cent protein (pEGFP) was used (Figure S17 in the Supporting
Information). UV irradiation did not alter the transfection
efficiency of the nonresponsive P0, thus indicating that
irradiation itself did not improve gene expression.
We next investigated whether light-triggered alteration of
the charge and conformation of the polypeptides would
promote intracellular DNA unpacking and gene transfection.
As expected, the incorporation of DMNBLG moieties did not
compromise the capacity of the polypeptides for DNA
delivery, leading to a notable uptake level of YOYO-1-
labeled DNA in both HeLa and COS-7 cells as a result of
caveolae-mediated endocytosis and energy-independent non-
endocytosis (Figure S12 in the Supporting Information). UV-
induced DNA release was monitored by the heparin replace-
ment assay.[15] UV irradiation for 5 min notably facilitated the
DNA release from the P20/DNA complexes, leading to
almost complete DNA dissociation within 12 h (Figure 4A,
Figure 4. UV (365 nm, 20 mWcmÀ2, 5 min)/NIR (750 nm, 3.2 mJcmÀ2
/
Because UV irradiation often suffers from low penetra-
tion and potential genotoxic effects when clinically applied,
we went on to evaluate the applicability of near-infrared
(NIR) modulation in this system. NIR irradiation (750 nm,
3.2 mJcmÀ2/pulse) eliminated the helicity of P20 (Figure S19
in the Supporting Information), which reached the lowest
level following protracted irradiation (1.5 h). In accordance,
NIR irradiation for 1.5 h triggered a 6.9-fold increment in the
transfection efficiency of P20/DNA complexes in HeLa cells,
compared to the unappreciable enhancement of the non-
responsive P0/DNA complexes (Figure 4E). As expected,
NIR irradiation did not induce cell death (viability of (95.6 Æ
6.2)%, n = 3). These results validated the potential of
regulating the transfection performance of photo-responsive
polypeptides using highly penetrating and more biocompat-
ible light. DMNB has negligible absorption in the NIR region,
and the slower responsiveness was mainly due to the low two-
photon uncaging cross section of the DMNB group.[14] A more
NIR-sensitive Glu-protecting ligand (under development in
our group) would substantially enhance the applicability of
this class of smart, trigger-responsive, nonviral delivery
vectors.
pulse, 1.5 h) irradiation improves transfection efficiency by facilitating
intracellular DNA unpacking and nuclear transport. A) DNA release
from nontreated and UV-treated complexes (n=3). B) CLSM images
of HeLa cells incubated with RhB-P20 (red)/YOYO-1-DNA (green)
complexes with/without UV irradiation (bar=20 mm). C) Subcellular
distribution of YOYO-1-DNA in HeLa cells following treatment with
P20/DNA complex and UV irradiation. D) Transfection efficiency in
HeLa cells (N/P=20, 5 mg DNA/mL) with/without UV irradiation
(n=3). E) Transfection efficiency of P20/DNA complexes in HeLa cells
(N/P=20, 5 mg DNA/mL) with/without NIR irradiation (n=3).
ns=no significant difference (p>0.05).
and Figure S13 in the Supporting Information). Compara-
tively, UV irradiation exerted no effect on the P0/DNA
complexes, confirming that the helix-distorted and cationic-
charge-reduced polypeptides promoted DNA unpacking. The
size of the complex was markedly augmented upon UV
irradiation, consistently signifying reduced DNA condensa-
tion by the polypeptides (Figure S8 in the Supporting
Information).
Apart from the charge conversion that would facilitate
DNA unpacking, we also examined the impact of secondary
structure. PVBLG-8 and PVBDLG-8, two homo-polypep-
tides that possess the same charge density but different
conformation (a-helix for PVBLG-8 prepared with l-Glu,
In summary, we developed a class of cationic helical
polypeptides with built-in trigger-responsive domains that
control the charge and conformational change of the poly-
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Piepmeier, Z. Z. Jiang, W. M. Saltzman, Nat. Mater. 2012, 11,
82 – 90.
[4] P. M. McLendon, K. M. Fichter, T. M. Reineke, Mol. Pharm.
2010, 7, 738 – 750.
[5] D. F. Zhi, S. B. Zhang, B. Wang, Y. N. Zhao, B. L. Yang, S. J. Yu,
Bioconjugate Chem. 2010, 21, 563 – 577.
[6] S. Takae, K. Miyata, M. Oba, T. Ishii, N. Nishiyama, K. Itaka, Y.
Yamasaki, H. Koyama, K. Kataoka, J. Am. Chem. Soc. 2008, 130,
6001 – 6009.
peptides upon external stimuli. The reduction of cationic
charge and helix disruption subsequently resulted in the
reduction of membrane activities of the polypeptides and
enhancement of the DNA unpacking capacities, thus sub-
stantially improving the efficiency of gene delivery with
diminished cytotoxicity. As such, one trigger stimulates
multiple gene transfection/delivery-relevant changes of mate-
rial properties, thus provoking desired intracellular responses
to overcome various barriers against nonviral gene delivery.
[7] X. Zhao, L. Yin, J. Ding, C. Tang, S. Gu, C. Yin, Y. Mao, J.
Controlled Release 2010, 144, 46 – 54.
[8] a) M. A. Kostiainen, D. K. Smith, O. Ikkala, Angew. Chem. 2007,
119, 7744 – 7748; Angew. Chem. Int. Ed. 2007, 46, 7600 – 7604;
b) G. Han, C. C. You, B. J. Kim, R. S. Turingan, N. S. Forbes,
C. T. Martin, V. M. Rotello, Angew. Chem. 2006, 118, 3237 –
3241; Angew. Chem. Int. Ed. 2006, 45, 3165 – 3169; c) X. A.
Jiang, Y. R. Zheng, H. H. Chen, K. W. Leong, T. H. Wang, H. Q.
Mao, Adv. Mater. 2010, 22, 2556 – 2560; d) S. K. M. Nalluri, J.
Voskuhl, J. B. Bultema, E. J. Boekema, B. J. Ravoo, Angew.
Chem. 2011, 123, 9921 – 9925; Angew. Chem. Int. Ed. 2011, 50,
9747 – 9751; e) X. H. Liu, J. W. Yang, D. M. Lynn, Biomacro-
molecules 2008, 9, 2063 – 2071.
[9] a) K. M. Stewart, K. L. Horton, S. O. Kelley, Org. Biomol.
Chem. 2008, 6, 2242 – 2255; b) G. Ter-Avetisyan, G. Tuenne-
mann, D. Nowak, M. Nitschke, A. Herrmann, M. Drab, M. C.
Cardoso, J. Biol. Chem. 2008, 284, 3370 – 3378.
[10] a) P. Ruzza, A. Calderan, A. Guiotto, A. Osler, G. Borin, J. Pept.
Sci. 2004, 10, 423 – 426; b) D. S. Daniels, A. Schepartz, J. Am.
Chem. Soc. 2007, 129, 14578 – 14579.
[11] a) R. N. Johnson, D. S. H. Chu, J. Shi, J. G. Schellinger, P. M.
Carlson, S. H. Pun, J. Controlled Release 2011, 155, 303 – 311;
b) M. Meyer, A. Philipp, R. Oskuee, C. Schmidt, E. Wagner, J.
Am. Chem. Soc. 2008, 130, 3272 – 3274.
[12] a) H. Lu, J. Wang, Y. G. Bai, J. W. Lang, S. Y. Liu, Y. Lin, J. J.
Cheng, Nat. Commun. 2011, 2, 206; b) N. P. Gabrielson, H. Lu,
L. C. Yin, D. Li, F. Wang, J. J. Cheng, Angew. Chem. 2012, 124,
1169 – 1173; Angew. Chem. Int. Ed. 2012, 51, 1143 – 1147.
[13] H. Lu, J. J. Cheng, J. Am. Chem. Soc. 2007, 129, 14114 – 14115.
[14] N. Fomina, C. McFearin, M. Sermsakdi, O. Edigin, A. Almutairi,
J. Am. Chem. Soc. 2010, 132, 9540 – 9542.
Received: April 5, 2013
Revised: May 25, 2013
Published online: July 5, 2013
Keywords: cell-penetrating peptides · charge conversion ·
.
helical structures · nonviral gene delivery
[1] a) A. K. Salem, P. C. Searson, K. W. Leong, Nat. Mater. 2003, 2,
668 – 671; b) K. Roy, H. Q. Mao, S. K. Huang, K. W. Leong, Nat.
Med. 1999, 5, 387 – 391; c) M. A. Mintzer, E. E. Simanek, Chem.
Rev. 2009, 109, 259 – 302.
[2] a) Y. Lee, K. Miyata, M. Oba, T. Ishii, S. Fukushima, M. Han, H.
Koyama, N. Nishiyama, K. Kataoka, Angew. Chem. 2008, 120,
5241 – 5244; Angew. Chem. Int. Ed. 2008, 47, 5163 – 5166; b) K.
Miyata, M. Oba, M. Nakanishi, S. Fukushima, Y. Yamasaki, H.
Koyama, N. Nishiyama, K. Kataoka, J. Am. Chem. Soc. 2008,
130, 16287 – 16294; c) H. M. Liu, H. Wang, W. J. Yang, Y. Y.
Cheng, J. Am. Chem. Soc. 2012, 134, 17680 – 17687.
[3] a) B. R. McNaughton, J. J. Cronican, D. B. Thompson, D. R. Liu,
Proc. Natl. Acad. Sci. USA 2009, 106, 6111 – 6116; b) H. Uchida,
K. Miyata, M. Oba, T. Ishii, T. Suma, K. Itaka, N. Nishiyama, K.
Kataoka, J. Am. Chem. Soc. 2011, 133, 15524 – 15532; c) S.
Srinivasachari, K. M. Fichter, T. M. Reineke, J. Am. Chem. Soc.
2008, 130, 4618 – 4627; d) H. Wei, J. G. Schellinger, D. S. H. Chu,
S. H. Pun, J. Am. Chem. Soc. 2012, 134, 16554 – 16557; e) J. Li, C.
Yang, H. Z. Li, X. Wang, S. H. Goh, J. L. Ding, D. Y. Wang,
K. W. Leong, Adv. Mater. 2006, 18, 2969 – 2974; f) J. C. Sunshine,
D. Y. Peng, J. J. Green, Mol. Pharm. 2012, 9, 3375 – 3383; g) M. S.
Shim, Y. J. Kwon, J. Controlled Release 2009, 133, 206 – 213;
h) J. B. Zhou, J. Liu, C. J. Cheng, T. R. Patel, C. E. Weller, J. M.
[15] W. Y. Seow, Y. Y. Yang, A. J. T. George, Nucleic Acids Res. 2009,
37, 6276 – 6289.
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