Peptide Nanoconjugation
DOI: 10.1002/anie.201209662
Orientation-Controlled Conjugation of Haloalkane Dehalogenase
Fused Homing Peptides to Multifunctional Nanoparticles for the
Specific Recognition of Cancer Cells**
Serena Mazzucchelli,* Miriam Colombo, Paolo Verderio, Ewa Rozek, Francesco Andreata,
Elisabetta Galbiati, Paolo Tortora, Fabio Corsi, and Davide Prosperi*
Multifunctional nanoparticles (MNPs) that combine unique
superparamagnetic properties and fluorescence emission are
promising bimodal tracers for the noninvasive diagnosis of
malignant cells both in vitro and in vivo.[1–4]
The selective
recognition of specific cancer cells impacts diagnostic sensitivity,
and it can be accomplished by the functionalization of
MNPs with molecules that have a high affinity for specific
membrane receptors.[5–7]
However, the mode in which individual
homing ligands are immobilized at the interface
between the inorganic core and the biological environment,
may strongly affect the actual targeting efficiency of the
nanoconjugate.[8–10]
A generally underestimated concern is
the molecular organization at the nanoscale, which is
a relevant consideration for protein ligands and is even
crucial when short peptides are used. To optimize the
recognition by a specific biological receptor, the immobilized
peptide needs to be stably ligated to the nanoparticle but
sufficiently mobile to interact with the receptor. Indeed,
peptides tend to bind to the surface of an MNP through
hydrophobic residues, which is promoted by entropic stabilization
and electrostatic interactions and exploits polar and
dissociated groups in the peptide sequence .[11]
This often
results in a loss or reduction of delivery efficiency and, more
importantly, of target selectivity. For this reason, the development
of effective and reliable strategies to afford ordered
ligand orientation on the nanoparticle surface has attracted
a lot of interest in nanomedicine.[8,12,13]
Several approaches
have been explored to control ligand positioning, including
conjugation mediated by affinity tags inserted into the protein
primary sequence,[14,15]
oriented immobilization on MNPs
driven by recombinant protein linkers,[16,17]
and site-specific
chemo-selective ligation.[18,19]
Recently, we have proposed
a novel approach that relies on engineered proteins consisting
of a receptor-targeting domain genetically fused with a nanoparticle-capture
domain, in which the capture module should
be an enzyme capable of irreversibly reacting with a suicide
inhibitor covalently anchored on MNP. As a proof of concept,
we have immobilized an scFv antibody fused to a SNAP tag
onto MNPs.[20]
The same approach was exploited for ligand
functionalization of pegylated capsules.[21]
In principle, this
bimodular orthogonal bioreaction could present two important
advantages when the homing ligand is a short peptide
(i.e., 5–30 aa): 1) the peptide is separated from the nanoparticle
surface by a protein spacer, which prevents undesired
interactions and thus optimizes the ligand availability for
molecular recognition; 2) the introduction of globular proteins
(i.e., the reacting enzyme) enhances the solubility of the
nanoconstruct.
Haloalkane dehalogenase (HALO) from Rhodococcus
rhodochrous forms an ester bond between aspartate 106 in the
enzyme and the substrate, concomitantly removing halides
from aliphatic hydrocarbons. Substitution of His272 with
a phenylalanine prevents the substrate release, which usually
occurs in native HALO, and thus an stable bond can be
formed between HALO and an alkyl conjugate (Figure 1).[22]
Hence, we reasoned that a chloroalkane linker could be
a good candidate to mediate the covalent, oriented immobilization
on MNP of homing peptides genetically fused with
HALO.
We designed a bimodular genetic fusion (HALO–U11)
comprising a small peptide of 11 amino acids (U11) that has
a high affinity for urokinase plasminogen activator receptor
(uPAR), which is overexpressed in several metastasizing
tumors, as a targeting module, and HALO, as an MNP
capture module (Figure 1). We demonstrate the utility of this
method for peptide nanoconjugation and cellular imaging by
evaluating the capability of MNP covalently bound with
HALO–U11 to specifically recognize uPAR-positive cancer
cells. UPAR is up-regulated in a broad range of cancer cell
types and tumor-associated stromal cells, and mediates
various biological processes at the cell surface, including
plasminogen activation, extracellular matrix invasion, cell
adhesion, and metastasis.[23]
U11 is believed to be the primary
[*] Dr. M. Colombo, P. Verderio, E. Rozek, F. Andreata, E. Galbiati,
Prof. P. Tortora, Dr. D. Prosperi
NanoBioLab, Dipartimento di Biotecnologie e Bioscienze
Università di Milano Bicocca
Piazza della Scienza 2, 20126 Milano (Italy)
E-mail: davide.prosperi@unimib.it
Homepage: http://www.nanobiolab.btbs.unimib.it
Dr. D. Prosperi
Labion, Polo Tecnologico, Fondazione Don Gnocchi IRCCS-ONLUS
Via Capecelatro 66, 20148 Milano (Italy)
Dr. S. Mazzucchelli, Dr. M. Colombo, Prof. F. Corsi
Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”
Università di Milano, Ospedale L. Sacco
Via G.B. Grassi 74, 20157 Milano (Italy)
E-mail: serena.mazzucchelli@gmail.com
[**] M.C. acknowledges the research fellowships of CMENA. This work
was partly supported by Fondazione Regionale per la Ricerca
Biomedica (FRRB), NanoMeDia Project, and Fondazione “Romeo
ed Enrica Invernizzi”.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201209662.
Angewandte
Chemie
3121Angew. Chem. Int. Ed. 2013, 52, 3121 –3125  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
uPAR binding motif with a dissociation constant of 1.3–
1.4 mm.[24,25]
The HALO sequence was modified by selective mutagenesis
of the native protein (see the Supporting Informa-
tion).[22]
Moreover, SalI and XhoI restriction sites were
inserted at the 5’- and 3’-positions, respectively, and the
modified gene was cloned in a pGEX-6-P-1 vector to express
HALO fused with glutathione S-transferase (GST). HALO
was expressed in BL21(DE3) codon plus E. coli strain. After
induction with isopropyl b-d-1-tiogalactopiranoside (IPTG),
cells were collected and disrupted, HALO was isolated from
the crude extract by using a glutathione–sepharose column
and eluted by PreScission protease cleavage (1 mgLÀ1
yield).
Purified fractions showed an excellent degree of purity,
despite the presence of a residual GST contaminant (see the
Supporting Information, Figure S3).
The anchor ligand L1 containing a chlorohexane moiety,
which is reactive toward the HALO binding site, was
synthesized in three steps from simple precursors (see the
Supporting Information, Scheme S1). Magnetite nanoparticles
with narrow size distribution (10.1 Æ 1.3 nm, MNP0)
capped by oleate surfactant were obtained by solvothermal
decomposition in organic solvents,[26]
and they were transferred
to the water phase by coating them with an amphiphilic
polymer (PMA) in sodium borate buffer (pH 12).[20,27]
The
resulting PMA-coated nanoparticles (MNP1) were superparamagnetic
and exhibited excellent solubility in aqueous
media. Amino groups were introduced on MNP1 by using
a homobifunctional linker (2,2-(ethylenedioxy)bisethylamine;
EDBE) to give MNP2. L1 was linked to the amines
on the polymer envelope through nucleophilic addition to the
p-nitrophenyl carbonate
group by incubation overnight
at 48C (MNP3,
Scheme 1). MNP3 were
characterized by dynamic
light scattering (DLS),
and exhibited a mean
hydrodynamic size of
40.1 Æ 2.7 nm in PBS
(5 mgmLÀ1
, pH 7.4) with
a zeta potential of
À28.5 Æ 3.0 mV. MNP3
was very stable in PBS
buffer and formed a dark
transparent solution.
The optimal conditions
for the conjugation
of the fusion protein with
L1 on MNP was determined
by varying several
experimental parameters,
including the protein/
nanoparticle ratio, time,
temperature, and incubation
buffer (see the Supporting
Information,
Figure 1. A) Schematic representation of a HALO-conjugated multifunctional nanoparticle (MNP-H). a) Interaction
of haloalkane anchor ligand with the genetically modified HALO binding site. b) TEM image of MNP-H11
(inset: size distribution of the nanoparticles).
Scheme 1. Synthesis of HALO-functionalized multifunctional nanoparticles
(MNP-H and MNP-H11). a) PMA, SBB, pH 12; b) EDBE, EDC,
water. EDBE=2,2-(ethylenedioxy)bisethylamine, EDC=N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride, PMA=amphiphilic
polymer, SBB=sodium borate buffer.
.AngewandteCommunications
3122 www.angewandte.org  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2013, 52, 3121 –3125
Table S1). HALO-functionalized MNP (MNP-H) were
obtained by treating purified fluoresceine isothiocyanate
(FITC) labeled HALO with MNP3 in a 1:1 ratio (w/w) in
PBS, pH 7.4 (Scheme 1). After 1 h incubation at 258C,
unconjugated HALO was removed by centrifuging the
mixture in amicon YM-100 tubes and the concentrated
nanoparticles were further reacted with a-methoxy-w-aminoPEG
(2 kDa, mPEG2k-NH2), after activation of the carboxylate
groups of the polymer by EDC, to minimize possible
nonspecific adsorption. The nanoparticles were then washed
three times with PBS. The amount of unreacted dye-labeled
HALO was fluorometrically measured after first establishing
a standard calibration curve, which provided the number of
HALO molecules attached to each nanoparticle. We determined
the presence of an average of about 5 HALO
molecules per MNP-H. DLS analysis showed an increment
in the hydrodynamic size upon conjugation (62.9 Æ 7.2 nm),
consistent with the attachment of protein molecules, and the
nanoparticles were stable owing to a negative zeta potential
of À32.3 Æ 0.4 mV. To assess whether the conjugation occurred
specifically to L1, HALO was incubated with MNP2,
as a control. No binding, within the fluorescence assay
sensitivity, occurred to nanoparticles in the absence of L1,
thus demonstrating that HALO immobilization on the nanoparticles
was indeed mediated by ligand interaction with the
active site of the enzyme. Moreover, sodium dodecyl sulphate–polyacrylamide
gel electrophoresis (SDS-PAGE) (Figure
S4) showed that, whereas protein incubated with MNP2
was able to migrate upon the application of a current, no
HALO molecules were released from MNP-H.
After preliminary assessment of the efficiency of the
HALO conjugation, a HALO capture module was engineered
by the introduction of a targeting element that consists
of the 11 amino acid sequence VSNKYFSNIHW (U11)
involved in uPAR recognition, through a C-terminal insertion
of a GGGGSGGGG loop, which provides sufficient freedom
to U11 (Figure S5). HALO–U11 fusion protein was produced
in BL21(DE3) E. coli and purified by using the same
procedure described above for HALO, and then HALO–
U11 was reacted with FITC-labeled MNP3 by using the
conjugation protocol illustrated in Scheme 1, to give MNPH11
(size = 67.6 Æ 3.1 nm, zeta potential = À27.8 Æ 2.6 mV).
In this case, the fluorescent label was covalently incorporated
inside the polymer layer to avoid contact of the dye with the
external environment, which could affect the nanoparticle
affinity for cellular receptors.
U937 cell lines were selected as the cellular model to
assess the targeting efficiency of MNP-H11, because these
cancer cells are available both as uPAR-positive (U937_13)
and as uPAR-negative (U937_10). The only difference
between them was the membrane expression of a U11specific
receptor. U937_13 cell lines were first treated in
parallel with dye-labeled MNP2 and HALO to evaluate
nonspecific interactions of the pegylated nanoparticles and of
the capture protein, respectively, with uPAR+
cells. In both
cases, no evidence of cell labeling was detected by flow
cytometry (see the Supporting Information, Figure S8). To
assess the influence of the controlled orientation of ligand
presented HALO–U11, MNP were also directly conjugated
with U11 peptide (4–6 molecules per MNP) by introducing
a Cys residue at the C-terminal (MNP-U11). MNP-H11 and
MNP-U11 were each incubated for 1 h with U937_13 and
with U937_10 (control) cancer cells at two different concentrations
(20 mgmLÀ1
and 100 mgmLÀ1
). Flow cytometry performed
on the U937_13 cells treated with MNP-H11 evidenced
a twentyfold increase in the percentage of cells in the
positive region compared to MNP-U11-treated cells
(Figure 2). Quite surprisingly, MNP-U11 were not able to
bind uPAR+
cells any more than to uPARÀ
, probably owing
to a low availability of the short peptides for recognition.
U937_10 cells remained mostly unlabeled after MNP-H11
treatment, even at 100 mgmLÀ1
. These results demonstrated
that the controlled peptide orientation is crucial for optimal
target specific recognition, as MNP-H11 were captured
selectively by uPAR-expressing U937_13 cells.
The specificity of the binding between MNP-H11 and
uPAR was confirmed by confocal laser scanning microscopy.
U937_13 and U937_10 cells (CTRL-) were treated in parallel
with MNP-H11 (100 mgmLÀ1
) for 1 h at 378C. As a uPAR
expression control, U937_13 cells were immunodecorated
with anti-uPAR antibody (CTRL +). MNP-H11 were localized
in the proximity of the cell membrane and inside the
cytoplasm of uPAR+
cells only, showing a uPAR recognition
pattern similar to the positive control; this finding confirmed
that MNP-H11 adhesion to the cell membrane and internalization
were actually mediated by specific interactions with
the U11 peptide (Figure 3). Finally, cell-death experiments
performed on U937_13 cells after 24 h incubation with MNPH11
at 20 mgmLÀ1
and 100 mgmLÀ1
, suggested that MNP-H11
were nontoxic within this range of concentrations; this finding
is significant for in vitro and in vivo applications (Figure 4).
In summary, we have established a new bimodular
strategy for controlled peptide positioning on multifunctional
nanoparticles. The advantages of our approach are: 1) the
peptide was produced fused to a capture domain (HALO) by
recombinant expression, which afforded the active targeting
ligand in high purity and avoided chemical synthesis and
Figure 2. MNP-H11 and MNP-U11 binding specificity to uPAR. U937
uPAR+
(U937_13) and uPARÀ
(U937_10) cells were incubated at 378C
with MNP-H11 and MNP-U11 at two different concentrations
(0.02 mgmLÀ1
and 0.1 mgmLÀ1
) for 1 h and then processed for flow
cytometry. Untreated cells were used to set the positive region. Data
are expressed as means Æ standard error (SE) of three individual
experiments.
Angewandte
Chemie
3123Angew. Chem. Int. Ed. 2013, 52, 3121 –3125  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org
purification; 2) the recombinant peptide was designed to
achieve an efficient covalent conjugation to MNP functionalized
with simple linkers in an orientation-controlled
manner; 3) selective immobilization was accomplished by an
enzymatic biorecognition event, which prevented nonspecific
adsorption provided that MNP were properly pegylated. In
this way, highly active MNP-H11 were engineered, and
proved to be very effective in selectively targeting uPARpositive
cancer cells. This method is simple and versatile and
offers a new solution for the covalent immobilization on MNP
of active homing ligands directed to a broad spectrum of
specific biomarkers; these nanoparticles can be exploited for
the development of nanoparticle-based diagnosis and treatments
of human malignancies.
Received: December 3, 2012
Revised: January 1, 2012
Published online: February 5, 2013
.Keywords: bioconjugation · cell targeting · peptides ·
nanoparticles · receptors
[1] K. Park, S. Lee, E. Kang, K. Kim, K. Choi, I. C. Kwon, Adv.
Funct. Mater. 2009, 19, 1553 – 1566.
[2] J.-H. Lee, Y.-M. Huh, Y.-w. Jun, J.-w. Seo, J.-t. Jang, H.-T. Song, S.
Kim, E.-J. Cho, H.-G. Yoon, J.-S. Suh, J. Cheon, Nat. Med. 2007,
13, 95 – 99.
[3] J. Kim, S. Park, J. E. Lee, S. M. Jin, J. H. Lee, I. S. Lee, I. Yang, J.S.
Kim, S. K. Kim, M.-H. Cho, T. Hyeon, Angew. Chem. 2006,
118, 7918 – 7922; Angew. Chem. Int. Ed. 2006, 45, 7754 – 7758.
[4] F. Corsi, L. Fiandra, C. De Palma, M. Colombo, S. Mazzucchelli,
P. Verderio, R. Allevi, A. Tosoni, M. Nebuloni, E. Clementi, D.
Prosperi, ACS Nano 2011, 5, 6383 – 6393.
[5] M. Lewin, N. Carlesso, C.-H. Tung, X.-W. Tang, D. Cory, D. T.
Scadden, R. Weissleder, Nat. Biotechnol. 2000, 18, 410 – 414.
[6] N. T. K. Thanh, L. A. W. Green, Nano Today 2010, 5, 213 – 230.
[7] D. Simberg, T. Duza, J. H. Park, M. Essler, J. Pilch, L. Zhang,
A. M. Derfus, M. Yang, R. M. Hoffman, S. Bhatia, M. J. Sailor,
E. Ruoslahti, Proc. Natl. Acad. Sci. USA 2010, 104, 932 – 936.
[8] E. Occhipinti, P. Verderio, A. Natalello, E. Galbiati, M.
Colombo, S. Mazzucchelli, A. Salvad›, P. Tortora, S. Maria Doglia,
D. Prosperi, Nanoscale 2011, 3, 387 – 390.
[9] M. E. Aubin-Tam, K. Hamad-Schifferli, Biomed. Mater. 2008, 3,
034001.
[10] K. Boeneman, J. R. Deschamps, S. Buckhout-White, D. E.
Prasuhn, J. B. Blanco-Canosa, P. E. Dawson, M. H. Stewart, K.
Susumu, E. R. Goldman, M. Ancona, I. L. Medintz, ACS Nano
2010, 4, 7253 – 7266.
[11] K. H. Lee, F. M. Ytreberg, Entropy 2012, 14, 630 – 641.
[12] I. Medintz, Nat. Mater. 2006, 5, 842.
[13] W. R. Algar, D. E. Prasuhn, M. H. Stewart, T. L. Jennings, J. B.
Blanco-Canosa, P. E. Dawson, I. L. Medintz, Bioconjugate
Chem. 2011, 22, 825 – 858.
[14] C. Xu, K. Xu, H. Gu, X. Zhong, Z. Guo, R. Zheng, X. Zhang, B.
Xu, J. Am. Chem. Soc. 2004, 126, 3392 – 3393.
[15] M. J. C. Long, Y. Pan, H.-C. Lin, L. Hedstrom, B. Xu, J. Am.
Chem. Soc. 2011, 133, 10006 – 10009.
[16] S. Mazzucchelli, M. Colombo, C. De Palma, A. Salvad›, P.
Verderio, M. D. Coghi, E. Clementi, P. Tortora, F. Corsi, D.
Prosperi, ACS Nano 2010, 10, 5693 – 5702.
[17] I. García, J. Gallo, N. Genicio, D. Padro, S. PenadØs, Bioconjugate
Chem. 2011, 22, 264 – 273.
[18] C.-C. Yu, P.-C. Lin, C.-C. Lin, Chem. Commun. 2008, 1308 –
1310.
[19] M. Colombo, S. Sommaruga, S. Mazzucchelli, l. Polito, P.
Verderio, P. Galeffi, F. Corsi, P. Tortora, D. Prosperi, Angew.
Chem. 2012, 124, 511 – 514; Angew. Chem. Int. Ed. 2012, 51, 496 –
499.
[20] M. Colombo, S. Mazzucchelli, J. M. Montenegro, E. Galbiati, F.
Corsi, W. J. Parak, D. Prosperi, Small 2012, 8, 1492 – 1497.
[21] M. K. M. Leung, C. E. Hagemeyer, A. P. R. Johnston, C. Gonzales,
M. M. J. Kamphuis, K. Ardipradja, G. K. Such, K. Peter, F.
Caruso, Angew. Chem. 2012, 124, 7244 – 7248; Angew. Chem. Int.
Ed. 2012, 51, 7132 – 7136.
Figure 3. Confocal microscopy images of U937 uPAR+
and uPARÀ
(CTRLÀ) cells, incubated for 1 h at 378C with MNP-H11 (0.1 mgmLÀ1
,
green). As a positive uPAR-expression control (CTRL+), U937 uPAR+
cells were immunodecorated with anti-uPAR primary antibody, revealed
with an anti-mouse secondary antibody conjugated with Alexa fluor
488 (Invitrogen). Nuclei were stained with 4’,6-diamidino-2-phenylindole
(DAPI). Scale bar: 10 mm.
Figure 4. Cell-death assay. U937 uPAR+
cells were treated with MNPH11
(0.02 mgmLÀ1
and 0.1 mgmLÀ1
) for 24 h (light and dark grey,
respectively). Cell death was assessed by measuring the exposure of
Annexin V and the incorporation of 7-aminoactinomycin D and evaluated
by flow cytometry. An untreated sample is shown as negative
control. All results are expressed as means Æ SE of five individual
experiments.
.AngewandteCommunications
3124 www.angewandte.org  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2013, 52, 3121 –3125
[22] G. V. Los, L. P. Encell, M. G. McDougall, D. D. Hartzell, N.
Karassina, C. Zimprich, M. G. Wood, R. Learish, R. Friedman
Ohana, M. Urh, et al., ACS Chem. Biol. 2008, 3, 373 – 382.
[23] F. Blasi, N. Sidenius, FEBS Lett. 2010, 584, 1923 – 1930.
[24] Q. Huai, A. P. Mazar, A. Kuo, G. C. Parry, D. E. Shaw, J.
Callahan, Y. Li, C. Yuan, C. Bian, L. Chen, et al., Science 2006,
311, 656 – 659.
[25] M. Wang, D. W. P. M. Lçwik, A. D. Miller, M. Thanou, Bioconjugate
Chem. 2009, 20, 32 – 40.
[26] J. Park, K. An, Y. Hwang, J.-G. Park, H.-J. Noh, J.-Y. Kim, J.-H.
Park, N.-M. Hwang, T. Hyeon, Nat. Mater. 2004, 3, 891 – 895.
[27] C.-A. J. Lin, R. A. Sperling, J. K. Li, T.-Y. Yang, P.-Y. Li, M.
Zanella, W. H. Chang, W. J. Parak, Small 2008, 4, 334 – 341.
Angewandte
Chemie
3125Angew. Chem. Int. Ed. 2013, 52, 3121 –3125  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org