Gold Nanoparticles Functionalized with RGD-Semipeptides: A Simple yet Highly Effective Targeting System for αVβ3 Integrins

Article abstract of DOI:10.1002/chem.201801823

Effective and selective targeting of the α_Vβ₃ integrin subtype is of high relevance in cancer research for the development of therapeutic systems with improved efficacy and of diagnostic imaging probes. We report here a new class of highly selective, α_Vβ₃-targeted gold nanoparticles (AuNPs), which carry cyclic 4-aminoproline-RGD semipeptides (cAmpRGD) as the targeting moiety immobilized at low surface density on the poly(ethylene glycol) (PEG)-based nanoparticle coating. We show that these nanoparticles are potent inhibitors of the integrin-mediated melanoma tumor cell adhesion to vitronectin and are selectively internalized via receptor-mediated endocytosis. Furthermore, we have developed bifunctional cAmpRGD-functionalized AuNPs by conjugation of a fluorophore (FAM or TAMRA) to a separate set of reactive groups on the PEG-based coating. These bifunctional AuNPs not only recapitulate the binding properties of cAmpRGD-AuNPs but also can be visualized via confocal laser microscopy, allowing direct observation of nanoparticle internalization. The peculiar molecular design of these nanoparticles and their precisely defined architecture at the molecular level accounts for their selective integrin binding with very low nonspecific background.

Full text of DOI:10.1002/chem.201801823

A Journal of
Accepted Article
Title: Gold nanoparticles functionalized with RGD-semipeptides: a
simple yet highly effective targeting system for αVβ3 integrins
Authors: Vito Maggi, Francesca Bianchini, Elisabetta Portioli, Silvia
Peppicelli, Matteo Lulli, Daniele Bani, Roberta Del Sole,
Franca Zanardi, Andrea Sartori, and Roberto Fiammengo
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To be cited as: Chem. Eur. J. 10.1002/chem.201801823
Link to VoR: http://dx.doi.org/10.1002/chem.201801823
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Chemistry - A European Journal
Gold nanoparticles functionalized with RGD-semipeptides: a
simple yet highly effective targeting system for V3 integrins
Vito Maggi,‡[a, b] Dr. Francesca Bianchini,‡ [c] Dr. Elisabetta Portioli,[d] Dr. Silvia Peppicelli,[c]

Dr. Matteo Lulli,[c] Prof. Dr. Daniele Bani,[e] Dr. Roberta Del Sole,[b] Prof. Dr. Franca Zanardi,[d]
[d]
[a]
Prof. Dr. Andrea Sartori, and Dr. Roberto Fiammengo
[a]
Center for Biomolecular Nanotechnologies@UniLe, Istituto Italiano di Tecnologia (IIT), Via
Barsanti, 73010 Arnesano, Lecce (Italy); [b] Department of Engineering for Innovation, University
[c]
of Salento, Via per Monteroni Km 1, 73100, Lecce (Italy); Department of Experimental and
Clinical Biomedical Sciences "Mario Serio", University of Florence, Viale Morgagni 50, 50134,
[d]
Florence (Italy); Food and Drug Department, University of Parma, Parco Area delle Scienze
[e]
27a, 43124 Parma (Italy); Department of Experimental and Clinical Medicine, University of
Florence, Viale Pieraccini 6, 50139 Florence (Italy).
Corresponding Author Emails: *francesca.bianchini@unifi.it, *andrea.sartori@unipr.it, and
*roberto.fiammengo@iit.it.
Author Contributions: ‡These authors contributed equally.
Supporting Information Contents: Description of materials, methods, and experimental
procedures. NMR spectra of compounds 1, 3, 3S, 6, 7, 20, 21, and THPTA and MALDI-TOF/TOF
of 3.
1
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Abstract: Effective and selective targeting of the V3 integrin subtype is of high relevance in
cancer research for the development of therapeutic systems with improved efficacy and of
diagnostic imaging probes. We report here a new class of highly selective, V3-targeted gold
nanoparticles (AuNPs), which carry cyclic 4-aminoproline-RGD semipeptides (cAmpRGD) as the
targeting moiety immobilized at low surface density on the poly(ethylene glycol) (PEG)-based
nanoparticle coating. We show that these nanoparticles are potent inhibitors of the integrin-
mediated melanoma tumor cell adhesion to vitronectin and are selectively internalized via
receptor-mediated endocytosis. Furthermore, we have developed bifunctional cAmpRGD-
functionalized AuNPs by conjugation of a fluorophore (FAM or TAMRA) to a separate set of
reactive groups on the PEG-based coating. These bifunctional AuNPs not only recapitulate the
binding properties of cAmpRGD-AuNPs but also can be visualized via confocal laser microscopy,
allowing direct observation of nanoparticle internalization. The peculiar molecular design of these
nanoparticles and their precisely defined architecture at the molecular level accounts for their
selective integrin binding with very low nonspecific background.
Introduction
Integrins constitute a large family (24 members) of cell surface receptors, which allow cells to
sense and respond to physicochemical changes of the extracellular matrix triggering cell adhesion,
migration, proliferation, and differentiation.[1] Integrins are heterodimers composed of one  and
one  subunit. Eight of these heterodimers bind to the tripeptide sequence RGD presented by
extracellular matrix proteins such as fibronectin, vitronectin, fibrinogen, etc.[2] RGD-binding
integrins are involved in cancer progression and subtypes V3, V5, 51, in particular, play also
a key role in tumor angiogenesis.[3] Integrin V3 is highly expressed on many different types of
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tumor cells (including glioblastomas, melanomas, ovarian, breast and prostate cancers), and on the
endothelial cells of tumor neovasculature and V3 expression generally correlates with tumor
progression and metastatic potential.[4] This distinctive expression profile explains the high
attention dedicated to V3 targeting for the development of therapeutic systems and diagnostic
imaging probes with improved efficacy. A large variety of synthetic ligands have been developed
as selective binders for the V3 subtype, including cyclopentapeptides such as c(RGDfV) and
related analogs,[2] rigid semipeptides and peptidomimetic scaffolds.[5] Yet, only few of these
ligands have been exploited to confer targeting ability to nanoparticulate materials for diagnostic
imaging, mainly c(RGDfV) and related analogs.[6] Inorganic nanoparticles, are well-suited to be
developed into contrast agents,[7] but a widespread use in clinical diagnostic imaging has yet to
come.[8] A number of issues remain unresolved in this field, including the reproducibility of
nanoparticle-based formulations, concerns about nanoparticle biocompatibility, and the
implementation of a sensible molecular design to confer the desired targeting ability with low
background. In this respect, the chemical identity of the organic coating on nanoparticles is
primarily responsible for their interaction with the biological milieu and for their targeting
performances.[9] With these arguments in mind, we have focused on developing gold nanoparticles
(AuNPs) targeted to the V3 integrin subtype with a novel chemical design. More in detail, in this
article we report the immobilization of cyclic 4-aminoproline based RGD semipeptides
(cAmpRGD)[10] at low surface density on top of a dense, poly(ethylene glycol) (PEG)-based AuNP
coating.[11] The use of cAmpRGD semipeptides as targeting moiety on inorganic nanoparticles is
unprecedented despite their excellent binding affinity and selectivity. Compared to previously
reported RGD-functionalized AuNPs,[12] these AuNPs present a low number of targeting ligands
(approx. 10 cAmpRGD/NP vs. 250-350 RGD/NP for previously reported AuNPs of comparable
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size[12h, 12j]), which maximize targeting while allowing to preserve the stealth properties of their
peculiar PEG coating. In this work, we show that they effectively compete for vitronectin binding
to V3 integrins inhibiting the adhesion of human melanoma cells M21 with an IC50 of around 1
nM nanoparticle (or 10 nM cAmpRGD), approximately 200 times more potently than free
cAmpRGD semipeptides in solution. This is the highest ever-measured potency increase for RGD-
targeted nanoparticles and demonstrates that outstanding targeting performances can be achieved
via a sensible molecular design. Notably, although appealing from the experimental point of view,
the preparation of targeted AuNPs by simply mixing thiolated targeting moieties with
commercially available PEG-thiols, generally results in lowering targeting performances. For
instance, relatively low multivalent enhancement factor (MVE), defined as IC50(soluble ligand)/
IC50(ligand when displayed on the nanoparticle)[13] was observed by Arosio et al.[12i] (MEV~7),
while MVE<1 (~0.5) can be calculated from the data reported by Kim and co-workers.[12h] High
background uptake was instead reported by Morales-Avila et al.[12j] (~ 15% cell uptake for
non-targeted AuNPs vs. ~35% for AuNPs carrying c[RGDfK(C)] as the targeting peptide).
In other cases, it is difficult to comparatively assess the targeting performances because
data on the RGD loading per AuNP are not available.[12a, 12b, 12d]
In this article, we also show V3-directed nanoparticle internalization into melanoma tumor
cells and the absence of toxicity at all investigated concentrations. Finally, we describe the
preparation of bifunctional cAmpRGD AuNPs by conjugation of fluorophores to a separate set of
reactive groups on the PEG-based coating. These fluorescently labelled bifunctional AuNPs
recapitulate the excellent binding properties of cAmpRGD-AuNPs but can be visualized via
confocal laser microscopy affording direct and valuable information about nanoparticle
localization.
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Results and discussion
Clickable cyclic aminoproline-based RGD semipeptides. Cyclic 4-aminoproline based RGD
semipeptides (cAmpRGD) are selective, high affinity ligands for V3 integrins.[10, 14] Previous
work has shown that functionalization of the Nα-proline site with various linkers does not
negatively impact integrin binding affinity. Therefore, this position is ideally suited for tethering
the RGD motif to biologically active molecules[5f, 15] or contrast agents for diagnostic imaging.[16]
Along these lines, fully deprotected 4-azidobutyl functionalized cAmpRGD derivative 1 (Chart 1)
was prepared from the known protected precursor 1S[5f] (Scheme S1) for immobilization onto
PEGylated AuNPs via the CuI-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.
Interestingly, cAmpRGD semipeptides possess good stability against proteolytic enzymes.[17]
Chart 1. cAmpRGD azide 1 and -functionalized alkyl-PEG600 thiols 2, 3, and 4 (n ~ 13)
employed for AuNP coating and functionalization.
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Preparation of mono- and bi-functionalizable AuNPs. PEG-based coatings greatly reduce or
even suppress the formation of a protein corona and impart stealth properties to AuNPs thus
preventing their sequestration by cells of the mononuclear phagocyte system (MPS).[11b, 18]
Some years ago, we developed PEGylated AuNPs coated by mixed self-assembled monolayers
(SAMs) of alkylPEG600 thiols.[19] The structural features of these AuNPs allow for
immobilization of a controlled number of receptor ligands on top of negatively charged (in neutral
or mildly basic aqueous solutions) hydrophilic coatings.[11c] SAMs of PEG-thiols with a long alkyl
chain (here C11) between the thiol group and the PEG segment are more compact that those
obtainable from PEG-thiols lacking this chain, resulting in the effective shielding of the Au surface
and strong suppression of nonspecific adsorption of proteins from the biological milieu.[11, 20] In
this study, spherical, citrate-capped AuNPs with a diameter of 13 nm[21] were coated (passivated)
with mixed SAMs of -functionalized alkyl-PEG600 thiols 2 and 3 (Chart 1). The typical
composition of the passivation reaction was 90% (molar fraction) of carboxy-terminated thiol 2
and 10% alkyne-terminated thiol 3, resulting in PEGylated AuNPs (5). Alternatively, the surface
passivation procedure was performed with a ternary mixture of alkyl-PEG600 thiols 2, 3, and 4.
In this latter case, the alkyne and amine functional groups displayed on the AuNP coating have
orthogonal reactivity and can be exploited for the preparation of bifunctionalized AuNPs (vide
infra).
Functionalization of PEGylated AuNPs with cAmpRGD motifs via click-chemistry. The
CuAAC reaction (“click chemistry”) is a very versatile approach for the preparation of
bioconjugates and has been exploited for the immobilization of biomolecules on the surface of
nanoparticles.[22] This reaction is compatible with all functional groups displayed on biomolecules
and has been applied even for the direct functionalization of living cells.[23] Despite its great
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potential, the exploitation of this reaction for nanoparticle functionalization is not always
straightforward, especially when the reaction is carried out in buffered aqueous medium employing
CuSO4 as the catalyst precursor. The use of nitrogen donor ligands stabilizes the CuI oxidation
state, accelerates the reaction and has been reported to scavenge the formation of reactive oxygen
species, which forms upon the re-oxidation of CuI to CuII, typically occurring in aqueous media.[24]
Still, these conditions may be problematic in case of colloidal solutions of nanoparticles resulting
in aggregation.
We initially investigated the CuAAC reaction between cAmpRGD 1 and the -alkyne-
terminated carboxylic acid 6 in solution (Schemes S8). These experiments showed that the
semipeptide part of the adduct remains untouched under the explored CuAAC conditions, an
important pre-requisite for maintaining its biological activity. We also found out that
hydroxylamine hydrochloride can be used as an alternative to ascorbic acid for reducing the CuII
precursor to CuI without major differences in adduct yield. Thus, the use of aq. hydroxylamine at
slightly basic pH (50 mM NaHCO3, pH 8.4) is expected to be of particular relevance for CuAAC-
based bioconjugations because it circumvents the generation of dehydroascorbate, which has been
reported to modify the side chains of several amino acids, in particular of arginine.[25] According
to the results obtained in solution, 20 M aq. CuSO4, 100 M THPTA ligand, 5 mM reducing agent,
in 50 mM aq. NaHCO3 for 2-4 h at room temperature were the reaction conditions chosen for the
conjugation of cAmpRGD 1 to AuNPs 5 displaying terminal alkynes on the PEG-based coating
(Figure 1). Under these conditions, we obtained AuNPs 9 carrying 10±2 cAmpRGD/AuNP as
determined by amino acid analysis (Table 1). As a complementary approach to estimate
cAmpRGD loading on AuNPs, we employed semipeptide 7, a fluorescently labeled variant of 1
bearing 5(6)-carboxyfluorescein on the  carboxyl group of the Asp residue (Scheme S4). From
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the corresponding AuNPs 10, quantification of the fluorescence signal, after displacement of the
PEG-based SAM from the gold surface,[19] resulted in 11±3 labeled cAmpRGD/AuNP in excellent
agreement with the amino acid analysis data. Successful conjugation of 1 and 7 to AuNPs 5 was
also evident from the reduced electrophoretic mobility of cAmpRGD-conjugated AuNPs 9 and 10
compared to 8, the negative control AuNPs obtained by omitting the Cu catalyst in the reaction
mixture (Figure 1).
Figure 1. A) Functionalization of PEGylated AuNPs 5 with cAmpRGD motifs via CuAAC.
THPTA= tris(3-hydroxypropyltriazolylmethyl)amine; FAM= 5(6)-carboxyfluorescein. B)
Agarose gel electrophoresis of AuNPs 8–10 (0.6%, sodium borate buffer). Successful conjugation
of semipeptides 1 or 7 is evident from the reduced electrophoretic mobility of AuNPs 9 and 10
compared to control AuNPs 8.
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Table 1. Physicochemical characterization of the AuNPs used in this work (Au-core diameter 13
± 1 nm, transmission electron microscopy - TEM).[a]
DLS[b]
Ø (nm)[c]
NcAmpRGD/AuNP
-Potential[b]
AuNPs
PDI[d]
(mV)
AA[e]
31 ± 2
31 ± 2
32 ± 3
36 ± 3
0.29 ± 0.05
0.35 ± 0.07
0.33 ± 0.07
0.31 ± 0.01
- 35 ± 5
- 35 ± 6
- 34 ± 5
- 42 ± 5[f]
--
5
8 (Neg. ctrl.)
< 2[f]
10 ± 2[f]
11 ± 2[g]
9
10
[a]
Reported values are averages and standard deviations of at least 4 independent batches of
[b]
[c]
AuNPs, unless otherwise stated;
Measurements in 10 – 50 mM NaHCO3;
Derived from
intensity distributions. [d] PDI= polydispersity index; [e] AA= amino acid analysis. [f] Averages of
two values ± 0.5 × (absolute difference). [g] 11 ± 3 from fluorescence analysis.
It is worth noting that these nanoparticles have a relatively low surface grafting density of
cAmpRGD motifs. In fact, considering a nanoparticle diameter of approximately 30 nm (Au core
+ PEG shell, as measured by dynamic light scattering, DLS), a loading of approx. 10
cAmpRGD/NP corresponds to a surface grafting density of 1 cAmpRGD over a surface area of
280 nm2 or to an approximate average distance of 18 nm between two neighbouring cAmpRGD
units. It has been reported that ligand density on nanoparticles may affect their receptor targeting
ability and in particular, a high surface density is detrimental for targeting.[13, 26] The surface
grafting density of cAmpRGD moieties on our AuNPs compares remarkably well with the density
of cyclicRGD derivatives on aminated cross-linked iron oxide nanoparticles for which the best
targeting performances were described in the seminal work of Josephson[13] The agreement
between these results imply that a higher RGD grafting density is most probably unnecessary or
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may even be detrimental. Furthermore, a low RGD grafting density produces a minimal impact on
nanoparticle surface properties, which remain dictated by the peculiar PEG coating (see Table 1).
Double functionalization of PEGylated AuNPs via click-chemistry and amide bond
formation. AuNPs 11, passivated with a ternary mixture of alkyl-PEG600 thiols composed of
89% (molar fraction) carboxy-terminated thiol 2, 10% alkyne-terminated thiol 3, and 1% amino-
terminated thiol 4, were reacted with the N-hydroxysuccinimidyl ester of 6-carboxyfluorescein
(FAM) (or with 5(6)-carboxytetramethyl rhodamine, TAMRA) to install the desired fluorophore
(Figure 2). The corresponding, fluorescently labeled AuNP intermediates 12 and 13 underwent a
coarse purification to remove most of the excess fluorophore before carrying out the CuAAC
reaction with cAmpRGD-azide 1. A final extensive purification procedure involving ultrafiltration
and gel filtration afforded bifunctional AuNPs 14 and 15, respectively.
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Figure 2. A) Synthetic scheme for the preparation of bifunctional AuNPs 14 (FAM, cAmpRGD)
and 15 (TAMRA, cAmpRGD). PEGylated AuNPs 11 displaying primary amino groups and
terminal alkynes on their passivation layer are first reacted with the NHS-ester of the desired
fluorophore and then decorated with cAmpRGD motifs via CuAAC. FAM= 6-carboxy fluorescein,
TAMRA= 5(6)-carboxy tetramethylrhodamine. B) Agarose gel electrophoresis of AuNPs 11-15
(0.6%, sodium borate buffer). Successful conjugation of semipeptide 1 is evident from the reduced
electrophoretic mobility of AuNPs 14 and 15 compared to precursor AuNPs 12 and 13.
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We deliberately kept low the molar fraction of the amino terminated thiol 4 during the
passivation reaction to limit the number of fluorophores that could be coupled and hence their
potential impact on nanoparticle surface properties. Indeed, quantification of FAM bound to
AuNPs 14 and of TAMRA bound to AuNPs 15 resulted in 13 ± 8 fluorophores/NP.[19] Further
physicochemical characterization revealed that these fluorescently labeled AuNPs do not
significantly differ from cAmpRGD-AuNPs 9 in terms of size, -potential and loading of RGD
motifs (see Table S5). Thus, it is possible to address independently the two reactive groups on the
PEG-based coating of these AuNPs. In the following paragraphs, we show that fluorescently
labeled cAmpRGD-AuNPs 14 and 15 are valuable materials for investigation of the AuNP
interactions with cells via fluorescence microscopy.
In-vitro testing of AuNP biocompatibility. Biocompatibility of cAmpRGD-functionalized
AuNPs 9 and negative control AuNPs 8 was assessed via flow cytometry through the Annexin
V/propidium iodide (PI) assay[27] on the human melanoma M21 cell line. These cells are chosen
as a model cancer cell line for our study because they are known to express high levels of V3
heterodimers,[28] which we experimentally confirmed by flow cytometry analysis. In addition,
these cells do not express V5 integrins (Figure S6). Incubation for 24 h with up to 10 nM
cAmpRGD-functionalized AuNPs 9 or negative control AuNPs 8 did not induce M21 cells to
undergo early apoptosis (PI-/FITC+) or late apoptosis/necrosis (PI+/FITC+) (Figure S7).
Additionally, under these conditions there were no significant effects on cell cycle (Figure S8).
Taken together these results demonstrate the excellent biocompatibility of these PEGylated
AuNPs.
Inhibition of cell adhesion by cAmpRGD-AuNPs. Synthetic, soluble RGD ligands can inhibit
integrin-mediated cell adhesion. Since cAmpRGD motifs are selective, high affinity ligands for
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V3 integrins (see Table S6),[10, 14] we investigated whether this property is retained also after
their immobilization on PEGylated AuNPs. We assessed the adhesion inhibition potency of
cAmpRGD-AuNPs 9 through an in vitro assay on M21 cells expressing V3 integrins but not
V5 as described in the previous section. Suspended, nanoparticle-treated M21 cells were allowed
to adhere for 1 h on vitronectin (the natural V3 integrin ligand) coated wells, before
quantification. We observed that AuNPs 9 are effective in inhibiting M21 cell adhesion with
approx. 50% inhibition (compared to untreated M21 cells) at 1 nM of AuNPs 9 (Figure 3A). In
other words, cAmpRGD-AuNPs 9 in solution effectively compete with surface immobilized
vitronectin for V3 integrin binding. As a comparison, the IC50 for the unconjugated cAmpRGD
semipeptide 24 is around 2 M (Figure S9). Thus, the activity of the immobilized cAmpRGD on
AuNPs 9 in suppressing cell adhesion is 200 times higher than that of the soluble cAmpRGD
ligand implying a strong multivalent enhancement factor.[13]
Conversely, negative control AuNPs 8 did not significantly inhibit cell adhesion at all
investigated concentrations. Finally, both fluorescently labeled cAmpRGD-AuNPs 14 and 15
inhibit cell adhesion with an IC50 of around 1 nM nanoparticle (or 10 nM cAmpRGD), with no
measurable differences compared to AuNPs 9 (Figure 3B and C). It is also interesting to note that
AuNPs 10, which differ from AuNPs 14 only by the position of the FAM label, do not inhibit cell
adhesion (see Figure S9). As known from literature, derivatization of the  carboxyl group of the
Asp residue (here with the FAM fluorophore, see structure 7 in Scheme S4 vs. 1) results in
complete loss of αVβ3 binding capability.[10, 29] This result further support the specificity of
interaction between cAmpRGD-AuNPs and the V3 integrin receptor.
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Figure 3. Inhibition of cell adhesion: suspensions of V3-expressing, human melanoma M21 cells
were treated with increasing concentrations of cAmpRGD-functionalized AuNPs (or control
AuNPs) before plating them on a vitronectin-coated surface. Adhesion was colorimetrically
evaluated after 1 h and normalized to that of untreated control cells. Cells were treated with: A)
cAmpRGD-AuNPs 9, control AuNPs 8, or soluble cAmpRGD-NH2 24; B) cAmpRGD-FAM-
AuNPs 14 or control FAM-AuNPs 16; C) cAmpRGD-TAMRA-AuNPs 15 or control TAMRA-
AuNPs 17.
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V3-Directed cellular internalization of AuNPs. Functionalization of nanoparticles with
RGD motifs may enhance cellular uptake via receptor-mediated endocytosis.[6c] Negatively
charged, PEGylated AuNPs are generally poorly internalized by mammalian cells.[30] Differently
from other commonly employed PEGylated nanoparticles, our AuNPs are coated by a very
compact self-assembled monolayer of PEG thiols. In fact, our alkylPEG600 thiols possess a C11
alkyl chain between the PEG600 segment and the thiol group and we showed that this design
completely suppress the place-exchange reaction with either thiolated peptides[11c] or
oligonucleotides.[31] We suggested that a hydrophobic region, created by the C11-alkyl chains,
shields the Au surface. These results were confirmed and extended by the group of Sokolov, who
showed that alkylPEG coatings on AuNPs are effective in preventing protein adsorption and
macrophage uptake, and perform much better than standard PEG coatings (with no alkyl
chains).[11b] Accordingly, the design of cAmpRGD-AuNPs 9 should be ideal for the targeting of
V3 integrins and cellular uptake should reflect selective, receptor-mediated endocytosis. In fact,
the low surface grafting density of cAmpRGD motifs only marginally influence nanoparticle
surface properties, which remain dictated by the negatively charged PEG-based coating. The
uptake experiments (2 h or 24 h incubation time) carried out on M21 cells in complete cell culture
medium confirm this hypothesis. At both incubation times, cAmpRGD-AuNPs 9 are taken up in
significantly higher amounts than AuNPs 8 and in a dose-dependent manner (Figure 4). In
particular, large differences are observed after 24 h with the amount of internalized 9 being
approximately 6-7 times the amount of 8. The results of these experiments also confirm the very
low uptake of control AuNPs 8, which is only ~1% the amount of Au incubated with the cells for
24 h and at the highest nanoparticle concentration.
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Figure 4. Uptake of cAmpRGD-AuNPs 9 compared to control AuNPs 8 by M21 cells after 2 h
and 24 h incubation in complete cell culture medium evaluated via inductively coupled plasma-
optical emission spectroscopy (ICP-OES) analysis. Values are averages of 3 replicates ± standard
deviation.
To confirm that the uptake of cAmpRGD-AuNP 9 is indeed directed by V3 integrin binding,
the same experiment was repeated using the human prostate carcinoma PC-3 cell line. In our
experimental conditions, PC-3 cells do not express integrins V3 and have only very low levels
of integrins V5, as verified by flow cytometry analysis (Figure S6).[32] As expected, for these
cells no significant differences in uptake between AuNPs 9 and 8 were observed both at 2 h and
24 h for all tested concentrations (Figure S10). Furthermore, AuNP uptake (both for 9 and 8) by
PC-3 cells, is low and only slightly higher (≤1.5-fold) than for negative control AuNPs 8 by M21
cells. This result demonstrates that the expression of V3 integrins is necessary for substantial
AuNP uptake, and supports the active role of the V3 receptor in the internalization of targeted
cAmpRGD-AuNPs 9.
We also evaluated nanoparticle internalization by human melanoma cells via fluorescence
confocal microscopy and transmission electron microscopy (TEM). Briefly, M21 cells were
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exposed for 24 h to 5 nM cAmpRGD-FAM-AuNPs 14 or cAmpRGD-TAMRA-AuNPs 15 and
compared with cells exposed to control TAMRA-AuNPs 16 or TAMRA-AuNPs 17.
cAmpRGD-FAM-AuNPs 14
cAmpRGD-TAMRA-AuNPs 15
FAM-AuNPs 16
TAMRA-AuNPs 17
Figure 5. Confocal laser microscopy images of M21 cells treated for 24 h at 37 °C with 5 nM of
cAmpRGD-FAM-AuNPs 14 or cAmpRGD-TAMRA-AuNPs 15 (top) and with FAM-AuNPs 16
or TAMRA-AuNPs 17 (bottom). After treatment, cells were rinsed, fixed and stained with DAPI
to make cell nuclei visible. Scale bar = 20 m.
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Fluorescence images show that cells treated with AuNPs 14 and 15 present significant
fluorescent labelling compared to cells exposed to control AuNPs 16 and 17 (Figure 5). The green
staining pattern observed for cAmpRGD-conjugated AuNPs 14 strongly suggests the involvement
of vesicles, such as endosomes, typical for receptor-mediated internalization. Identical conclusions
can be made observing the red staining pattern caused by cAmpRGD-conjugated AuNPs 15.
Further information about the intracellular localization of the nanoparticles was obtained via TEM.
In this case, M21 cells were exposed for 2 h or 24 h to 5 nM of cAmpRGD-AuNPs 9 and control
AuNPs 8. After incubation, the cells were washed, pelleted and further processed for TEM
analysis. We found that cells exposed to cAmpRGD-AuNPs 9 for 2 h clearly show nanoparticles
in contact with the cell membrane and embedded in endosomal vesicles (Figure 6A). Interestingly,
after 24 hours, endosomal vesicles containing nanoparticles are also found close to the
extracellular membrane, probably involved in short recycling of receptor-mediated internalization
vesicles (Figure 6B). This deduction is also supported by the observation of elongated, pear-shaped
vescicles, typical of receptor recycling (Figure 6B inset). In addition, late endosomes loaded with
nanoparticles are found close to the nucleus and the Golgi apparatus (Figure 6). Conversely,
images of cells exposed to control AuNPs 8 show only negligible amounts of internalized
nanoparticles at 24 h incubation (Figure S12), which correlates well with the much lower degree
of internalization measured via ICP-OES.
18
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Figure 6. Transmission electron micrographs of M21 treated for 2 h (A) or 24 h (B) with 6.7 nM
cAmpRGD-AuNPs 9. Black arrows show representative AuNPs at the cell membrane or in early
endocytic vesicles after 2 h (A) or AuNPs in late endosomes, close to the nucleus, and the Golgi
apparatus after 24 h (B). The white arrow points at AuNPs in an endosomal vesicle close to the
19
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10.1002/chem.201801823
Chemistry - A European Journal
extracellular membrane after 24 h, suggesting involvement in short recycling of receptor-mediated
internalization vesicles. The elongated, pear-shaped vescicle observable in the inset is also
characteristic of receptor recycling.
Finally, we investigated how cAmpRGD-AuNPs 9 affect the cytoskeleton of vitronectin-
adherent M21 cells. Well-spreaded M21 cells were treated with 6.7 nM of AuNPs 9 or control
AuNPs 8. After 1 h treatment, cAmpRGD-AuNPs 9 caused cytoskeletal disorganization, as
revealed by staining with rhodamine-conjugated phalloidin (Figure 7). In particular, we observed
a clear reduction in the number of actin filaments and a diffuse microgranular pattern of the
rhodamine stain suggesting transition from F-actin to G-actin structures.[33] Cells treated with
control AuNP 8 did not show cytoskeleton changes and behaved as untreated cells.
untreated
cAmpRGD-AuNPs 9
control-AuNPs 8
Figure 7. cAmpRGD-AuNPs 9 interact with the integrin receptors of M21 cells that adhere to
vitronectin-coated surfaces. Treatment with 6.7 nM of AuNPs 9 for 1 h causes significant
cytoskeletal disorganization as evident by the reduction of actin filaments and by the diffuse
microgranular pattern of the staining. Treatment with control AuNPs 8 does not induce any
significant change compared to untreated cells. Actin fibers were stained with rhodamine-
conjugated phalloidin and nuclei with DAPI. Scale bar: 20 m.
20
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10.1002/chem.201801823
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This experiment suggests that cAmpRGD-AuNPs 9 are able to engage the integrin receptors of
cells that are already interacting with the extracellular matrix (here vitronectin). Notably, this test
is performed under more physiologically relevant conditions compared to the adhesion inhibition
assay, and confirms the antagonist behavior of AuNPs 9 towards the V3 receptor.
Conclusions
In summary, we have developed a new class of AuNPs for the selective targeting of the V3
integrin subtype, which exploits the excellent binding properties of cyclic cAmpRGD
semipeptides grafted at low surface density on the PEG-based coating of the nanoparticles. This
nanoparticle design allows for excellent selectivity with low nonspecific background. We have
shown that cAmpRGD-AuNPs inhibit the adhesion of human melanoma cells M21 (expressing
high levels of the V3 integrin subtype) to vitronectin-coated surfaces with a multivalent
enhancement factor of approximately 200. Furthermore, cAmpRGD-AuNPs are selectively
internalized by M21 cells but not by the human prostate carcinoma PC-3 cell line for which this
integrin subtype is practically absent. We have also shown via TEM that at early times (2 h)
cAmpRGD-AuNPs 9 are located both at the cell membrane and in early endosomes, while at longer
times (24 h) they are found in late endosomes close to the nucleus as well as close to the
extracellular membrane, probably involved in short recycling of receptor-mediated internalization
vesicles as expected for specific binding to integrin receptors. Finally, we have demonstrated that
cAmpRGD-AuNPs 9 can target the integrin receptors of vitronectin-adherent cells inducing
evident cytoskeletal disorganization, thus supporting the notion of an antagonist effect toward the
V3 receptor. Last but not least, we have developed bifunctional cAmpRGD-functionalized
AuNPs by conjugation of a fluorophore (FAM or TAMRA) to a separate set of reactive groups on
the PEG-based coating. These bifunctional AuNPs recapitulate the binding properties of
21
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10.1002/chem.201801823
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cAmpRGD-AuNPs 9 and can be visualized via confocal laser microscopy. These results together
with the absence of cytotoxicity at all concentrations and times used in this study suggest that our
cAmpRGD-AuNPs could be developed into useful contrast agents with low background for
visualization of solid tumors overexpressing V3 receptors and of tumor angiogenesis (e.g. via
computed tomography, CT).[34]
Acknowledgments
The authors thank Mr. Arthur Lint for technical assistance in organic synthesis, Dr. Maria
Rosaria Lazzoi and Prof. Giuseppe Mele (University of Salento) for the NMR of compound 21,
Dr. Andrea Faccini (University of Parma) for MS/MS analysis, the Centro Interdipartimentale
Misure “G. Casnati” (University of Parma) for instrumental facilities, and Prof Lido Calorini
(University of Florence) for revising the manuscript. This work was financially supported by the
Italian Ministry of Education, University and Research (MIUR) (PRIN 2015 contract nr.
2015RNWJAM and 20157WW5EH). This work was also supported by Ente Cassa di Risparmio
di Firenze and the Istituto Toscano Tumori (Calorini 2013).
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Table of Contents Graphic
Gold nanoparticle (AuNP) design is essential for superior V3 integrin targeting. Cyclic
cAmpRGD semipeptides are used as selective targeting units at low surface density on top of a
compact alkylPEG coating. cAmpRGD-AuNPs inhibit integrin-mediated cell adhesion to
vitronectin with an IC50~1 nM indicating a high multivalent enhancement factor (~200). At 24 h ,
these AuNPs are internalized 6-7 times more efficiently than non targeted nanoparticles, only by
cells expressing the V3 integrin subtype.
Keywords: Gold nanoparticles, integrins, melanoma, RGD, click-chemistry.
26
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