Efficient γ-amino-proline-derived cell penetrating peptide-superparamagnetic iron oxide nanoparticle conjugates via aniline-catalyzed oxime chemistry as bimodal imaging nanoagents

Article abstract of DOI:10.1039/c2cc17937g

Aniline-catalyzed oxime chemistry was employed to conjugate a γ-amino-proline-derived cell penetrating peptide to superparamagnetic iron oxide nanoparticles (SPIONs). Internalization of the novel nanoconjugate into HeLa cells was found to be remarkably higher compared to the analogous TAT-SPION conjugate.

Full text of DOI:10.1039/c2cc17937g

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COMMUNICATION
Efficient c-amino-proline-derived cell penetrating
peptide–superparamagnetic iron oxide nanoparticle conjugates
via aniline-catalyzed oxime chemistry as bimodal imaging nanoagentsw
Silvia Cavalli,z* Daniel Carbajo,z Milena Acosta, Silvia Lope-Piedrafita,ea
ab
c
ad
adf
daf
ac
abg
Ana Paula Candiota, Carles Aru
´
s, Miriam Royo and Fernando Albericio*
Received 19th December 2011, Accepted 3rd April 2012
DOI: 10.1039/c2cc17937g
Aniline-catalyzed oxime chemistry was employed to conjugate a
c-amino-proline-derived cell penetrating peptide to superpara-
magnetic iron oxide nanoparticles (SPIONs). Internalization
of the novel nanoconjugate into HeLa cells was found to be
remarkably higher compared to the analogous TAT–SPION
conjugate.
TAT protein-derived peptide sequences, in particular
GRKKRRQR48–57, have been extensively used as an efficient
1
0,11
way of internalizing SPIONs into cells.
Few years ago, a
new class of non-natural cis-g-amino-L-proline-derived peptides
1
2
has been developed in our laboratories. These types of
membrane translocating peptides displayed relatively good
ability to internalize into HeLa and COS-1 cells, however
their cellular uptake was still lower than TAT. Interestingly,
Recent advances in nanotechnology have offered new oppor-
tunities for detection, prevention, and treatment of disease, in
1
particular cancer. In this respect engineered superparamagnetic
1
2
those peptides were less toxic compared to TAT. Another
important advantage of this family of CPPs is related to the
fact that they showed stability towards protease degradation,
imparted by the g-peptide skeleton, which permitted us to
circumvent one of the major limitations of CPPs associated
iron oxide nanoparticles (SPIONs) represent an advanced tool
in nanomedicine because they can be loaded in a multiple and
orthogonal way with drugs and probes in order to simulta-
13
2
,3
with enzymatic liability. Thus, in this work we selected the best
cell penetrating g-amino-proline-derived peptide, previously
discovered in our laboratory, to synthesize a suitable derivative
neously act as molecular imaging agents and drug carriers.
In fact, thanks to their superparamagnetic character, SPIONs
can be used as negative contrast agents in magnetic resonance
a
a
2
–7
of {gAmp[N -CH CH CH(CH ) ]–gAmp(N -CH CH Ph)} –
imaging (MRI).
Among other relevant applications, they
2
2
3 2
2
2
3
2
can be exploited for hyperthermia therapy (HT), or as
8
magnetic vectors for targeted drug delivery. When SPIONs
PEG–NH (peptide a in Chart 1) with the aim of engineering
2
a novel type of CPP–SPION conjugates useful as bimodal
imaging nanoagents.
are loaded with a fluorescent dye they can act as bimodal
nanodevices for both magnetic resonance and fluorescence
Developing new straightforward and chemoselective
routes, which occur under conditions close to physiological
medium, for functionalizing SPIONs could result in obtaining
9
microscopy imaging.
Cell penetrating peptides (CPPs) have been used to increase
1
4
1
cellular uptake of nanoparticles. As an example HIV-1
0
medical breakthroughs that target in vivo nanomedicine.
A growing area of chemical biology focuses on developing
reactions that do not interfere with biological processes,
a
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials,
and Nanomedicine, 08028 Barcelona, Spain.
E-mail: silviacavalliIRB@gmail.com, albericio@irbbarcelona.org;
Fax: +34 93403 7126; Tel: +34 93403 7088
Institute for Research in Biomedicine, 08028 Barcelona, Spain
Combinatorial Chemistry Unit, Barcelona Science Park,
08028 Barcelona, Spain
Department of Biochemistry and Molecular Biology, Universitat
Auto`noma de Barcelona (UAB), 08193 Cerdanyola del Valle`s, Spain
Nuclear Magnetic Resonance Service, Universitat Auto`noma de
Barcelona (UAB), 08193 Cerdanyola del Valle`s, Spain
Institute of Biotechnology and Biomedicine, Universitat Auto`noma de
Barcelona, 08193 Cerdanyola del Valle`s, Spain
Department of Organic Chemistry, University of Barcelona,
b
c
d
e
f
g
08028 Barcelona, Spain
w Electronic supplementary information (ESI) available: All synthetic
and biological procedures and additional relevant data. See DOI:
10.1039/c2cc17937g
zw oT r hk e. se authors contributed equally to the experimental part of the
Chart 1 Molecular structure of aminooxyacetyl-derived peptides a–c.
This journal is c The Royal Society of Chemistry 2012
5
322 Chem. Commun., 2012, 48, 5322–5324

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1
5,16
so called bioorthogonal chemical reactions.
In this respect
Dirksen and Dawson have shown that the use of aniline as a
1
nucleophilic catalyst activates aldehydes towards aminooxy
7
groups to form bioconjugates via oxime ligation that occurs at
1
8,19
neutral pH.
In light of these results, several groups have
recently explored the scope of oxime ligation for in vivo
0–22
2
labeling.
Having those in mind as well as other applica-
tions of oximation as an efficient chemoselective type of
2
3,24
ligation,
we used the aniline-catalyzed oxime chemistry
to functionalize SPIONs with the most efficient aforementioned
g-amino-proline-derived peptide. This approach allowed for an
easy and chemoselective conjugation of the peptide under
conditions that are close to physiological medium. Another
advantage of this strategy is that crude peptide could be used
during the ligation, thanks to the chemoselectivity of this type
of reaction. Thus, purification occurred only during dialysis of
the final conjugate.
In brief, commercially available crosslinked dextran coated
2
iron oxide nanoparticles (SPION-NH ) were first modified
Fig. 1 (A) Cellular uptake quantification with flow cytometry. HeLa cells
with benzaldehyde groups (SPION-CHO) by reaction with
the N-hydroxysuccinimidyl ester of 4-formylbenzoic acid in
citrate buffer at pH 8 with a small amount of N,N-diisopropyl-
were incubated with samples 1a–c at a final concentration of 1 mM of
CF for 6 h. Results were normalized by subtracting the autofluorescence
control value and dividing by the CF-fluorescence value for the same
experiment. Error bars represent standard deviation (SD) from the
mean value of three independent experiments. Sample 1a was kept as
reference. (B) Confocal microscopy image of HeLa cells incubated with
nanoparticle solution 1a at 1 mM of CF for 6 h. (C) MRI signal intensity
at increasing echo times (TE) in 1% agarose phantoms of (a) PBS,
(b) unlabeled and (c) labeled cells with nanoparticle 1a at 250 mM Fe.
Cells were incubated for 4 h. Samples were measured at 7 T using a fast
spin echo sequence. Images were acquired at different effective echo
times (TE eff.): 10, 50, 90 and 130 ms. (D) Viability of HeLa cells in the
2
5
ethylamine (DIPEA) as shown in Scheme 1. Furthermore,
the aminooxyacetyl-derived peptide a (Chart 1), labeled with
e
the 5(6)-carboxyfluorescein (CF) dye at the N of a terminal
lysine (K) residue, was reacted with the nanoparticle solution
SPION-CHO in phosphate buffer at pH 7.5 in the presence
1
of 100 mM of aniline to form CPP–SPION conjugate 1a
8
(
Scheme 1).
To prove the occurrence of the oxime ligation a control was
also prepared exploiting the non-reactive nature of an acetylated
0
2
presence of solution of SPION-NH and conjugates 1a–c at 250 mM of
version of the peptide a (ESIw). As further control constructs,
Fe measured by MTT assay. Data are normalized considering BL
(untreated cells) as 100. Error bars represent standard deviation (SD)
from the mean value of three independent experiments.
an aminooxyacetyl-(CF)-K-modified version of the well-known
cell-penetrating TAT peptide (b) was conjugated to SPION via
oxime bond formation (1b) to be used as a positive CPP
control. A similar conjugate of (CF)-K only (1c) was prepared
as well that functioned as a negative control. In both cases the
High-Resolution Magic Angle Spinning NMR (HRMAS-
NMR) was recently proved to be a successful technique for the
26
0
0
corresponding acetylated peptides b and c were also used to
verify the occurrence of the oximation. All samples were
extensively dialysed in ultrapure water or PBS buffer. Dialysed
nanoparticle solutions were further analyzed by fluorescence
microscopy to check the incidence of the ligation. Conjugated
sample 1a displayed fluorescence upon irradiation at 492 nm,
characterization of molecules bound to the SPION surface.
In the case of benzaldehyde-functionalized SPION (SPION-
CHO) aromatic and aldehyde protons were detected at
7.83–7.94 and 10.00 ppm, respectively (ESIw). Moreover, using
this technique, oxime bond formation was thus further demon-
strated by the presence of a singlet at 8.38 ppm as shown in the
HRMAS-NMR spectrum of the lyophilized nanoparticle
sample 1a dispersed in DMSO (ESIw).
0
while no fluorescent signal was observed in the control 1a
applying the same settings (ESIw).
First, cellular uptake in HeLa cells was estimated using flow
cytometry quantification techniques. Cells were incubated
with nanoparticles in PBS dispersed in cellular medium for
6
hours at 37 1C at a final concentration of 1 mM of CF.
Cellular uptake of 1a was compared to conjugates 1b and 1c,
used as a positive and a non-cell penetrating construct,
respectively. As summarized in Fig. 1A, flow cytometry analysis
revealed that conjugate 1a was the most efficient at crossing
the HeLa cell membranes. Cells incubated with 1a displayed
Scheme 1 Schematic representation of the synthesis of CCP–SPION
conjugates 1a–c. Reagents and conditions: (1) 4 mg N-hydroxysuccinimidyl
ester of 4-formylbenzoic acid, 10 mM DIPEA in 0.1 M citrate buffer at
pH 8. (2) 2 mg of aminooxyacetyl-derived peptides a–c, 100 mM aniline
in 0.1 M phosphate buffer at pH 7.5. 1a–c stand for CPP–SPION
conjugated with aminooxyacetyl-derived peptides a–c (Chart 1),
respectively.
2
.4 times more fluorescence than those incubated with TAT-
derivative 1b. To exclude any contribution of surface-bound
conjugates in the uptake measurements, flow cytometry experi-
ments were performed by lowering the pH of the suspension to
pH 6 by adding 1 N HCl, because g-peptides are not sensitive
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 5322–5324 5323

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1
2
to trypsin treatment. In this case only a small decrease of
fluorescence was observed by varying pH from 7.4 to 6,
suggesting that the fluorescence detected during the measure-
ment mainly came from internalized nanoconstructs. Flow
cytometry experiments were also conducted in the presence of
propidium iodide (PI) in order to exclude any contribution
of dead cells to fluorescence. Also in this case the addition of
PI did not dramatically alter the initial flow cytometry results
working as a bimodal probe for both confocal laser micro-
scopy and magnetic resonance imaging (MRI) techniques.
Importantly, we expect that the generic nature and versatility
of this approach based on aniline-catalyzed oximation described
in this work will allow any aldehyde-functionalized SPION to
be conjugated to any aminooxyacetyl-derived reagent in a
straightforward manner, using a similar synthetic protocol.
Furthermore, we are planning to investigate the relevance of
this type of bioorthogonal conjugation for in vivo imaging
purposes.
(
ESIw).
Next, confocal microscopy experiments were performed to
confirm the good cellular uptake of nanoconjugate 1a in HeLa
cells and prove the use of such nanodevices for optical micro-
scopy measurements. Cells were incubated for 6 h with
nanoparticles at a final concentration of 1 mM of CF. As
shown in Fig. 1B cells exposed to nanoparticle 1a were
strongly fluorescent (green dots).
We thank M. A. Molins for the help in performing the
HRMAS-NMR measurements and Roger Prades for the
fruitful discussions about the preparation of the MRI samples.
This work was supported by CICYT (CTQ2009-07758 and
2008-00177), ISC III (CB06_01_0074), the Generalitat Catalunya
(2009SGR 1024).
Iron-labeled HeLa cells were studied by MRI in 1% agarose
2
7
phantoms in order to verify if the good cellular uptake
observed by flow cytometry and confocal microscopy could
also give an MRI response. HeLa cells were incubated with
nanoparticle solution 1a at a final concentration of 250 mM of
iron for 4 h, washed, and immobilized in 1% agarose gel. T2
measurements of unlabeled and labeled cells were made using
a multi-slice multi-echo (MSME) sequence on a 7 T MRI
scanner. T2 measured values were 239.1 ms for PBS only,
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microscopy. Furthermore, the high level of cellular uptake was
demonstrated to provide a decrease in the T2 of labeled cells
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