Angewandte
Chemie
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-amino-
PEG (2 kDa, mPEG2k-NH2), after activation of the carbox-
ylate 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 deter-
mined 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 oc-
curred 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 nano-
particles was indeed mediated by ligand interaction with the
active site of the enzyme. Moreover, sodium dodecyl sul-
phate–polyacrylamide gel electrophoresis (SDS-PAGE) (Fig-
ure 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 engi-
neered 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 MNP-
H11 (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.
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 concen-
trations (20 mgmLÀ1 and 100 mgmLÀ1). Flow cytometry per-
formed on the U937_13 cells treated with MNP-H11 evi-
denced 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
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.
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 local-
ized 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 internal-
ization 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 MNP-
H11 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
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 U11-
specific 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
Angew. Chem. Int. Ed. 2013, 52, 3121 –3125
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Mazzucchelli, Serena
