Editorial
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Targeted nanoscale magnetic
hyperthermia: challenges and potentials of
peptide-based targeting
“
Recent reports suggest that diagnosis and therapy of other malignancies using
superparamagnetic iron oxide nanoparticles is feasible through active targeting and
accumulation of the magnetic nanoparticles to tumor sites.
Keywords: cancer • cell death • hyperthermia • magnetic field • peptide • receptor
• targeting
Magnetic nanoparticles (MNPs) are the sub-
ject of an increasing attention in oncology.
Indeed, MNPs enable MRI of malignancies
and, when exposed to a high frequency alter-
nating magnetic field, generate hyperther-
mia. Superparamagnetic iron oxide nanopar-
ticles (SPION) have been used as contrast
agent for liver, spleen and lymph node MRI
imaging. In such clinical applications, intra-
venously administrated SPIONs accumulate
preferentially to malignant tissues because of
capture by reticuloendothelial system.
Recent reports [1–3] suggest that diagnosis
and therapy of other malignancies using SPI-
ONs is feasible through active targeting and
accumulation of the MNPs to tumor sites.
Active targeting exploits the fact that cells
composing tumors (tumor and microenvi-
ronment cells) express and even overexpress
biological markers that are not expressed, or
less expressed in normal cells. Ligand mol-
ecules recognizing biological markers of the
tumor are thus grafted at the MNP surface so
that intravenously injected MNPs can reach
tumor cells and accumulate in these cells.
The strategy reported with EGF- and gas-
trin-grafted SPIONs [1–3] presents the origi-
nality of using the physiological process of
ligand-induced internalization of receptors
and their sorting to lysosomes. It is assumed
that exposure, to a high frequency alternat-
ing magnetic field, of cells containing minute
amounts of MNPs in their lysosomes, gener-
ates nanoscale hyperthermia at the surface of
MNPs, leading to activation of death signal-
10.2217/NNM.14.236 © 2015 Future Medicine Ltd
”
ing pathways originating from the interior of
the lysosomes [4] . At the difference of mag-
netic fluid hyperthermia experienced with
nontargeted SPIONs directly injected into
glioma  [5] and prostate cancers [6] , targeted
nanoscale hyperthermia requires minute
amounts of MNPs, making this approach
an attractive therapeutic option. However,
it remains to establish if targeted nanoscale
hyperthermia generated inside lysosomes of
tumor cells will be sufficient to effectively
Daniel Fourmy
eradicate tumors in vivo. So far, only SPI-
Author for correspondence:
ONs with a low thermal power were used to University of Toulouse 3, EA4552
generate nanoscale hyperthermia. We assume Receptor & Therapeutic Targeting of
that substitution of SPIONs with MNPs dis- Cancers, Toulouse, France
playing an enhanced thermal power [7] will Tel.: +33 561 323 057
Daniel.Fourmy@ inserm.fr
improve the outcome of treatment.
Beyond the nature of the MNP magnetic
Julian Carrey
core, one crucial aspect in the strategy of tar- Université de Toulouse 3, INSA, CNRS
geted nanoscale hyperthermia is the design UMR5215, Laboratoire de Physique
of the targeting moiety of the MNPs. In this et Chimie des Nano-Objets (LPCNO),
respect, receptors for peptidic hormones and Toulouse, France
growth factors being frequent cancer mark-
ers, the grafting of peptides to MNPs repre- Véronique Gigoux
University of Toulouse 3, EA4552
sents a usual way to target MNPs to tumors. Receptor & Therapeutic Targeting of
Peptide grafting of MNPs offers a large spec- Cancers, Toulouse, France
trum of opportunities and also presents some
potential problems.
Efficient covalent attachment of peptidic
ligands to MNPs is made possible, thanks to
several types of functionalization and cou-
pling chemistry [8] . The choice of coupling
strategy must however take into account the
requirement of maintaining the integrity of
part of
receptor-binding and -activation domain(s)
Nanomedicine (Lond.) (2015) 10(6), 893–896 ISSN 1743-5889 893
Editorial  Fourmy, Carrey & Gigoux
of the peptidic ligand. For most targeted surface recep-
tors, this information is available from structure–
activity relationship studies and/or from 3D structure
determination of liganded receptors. In fact, depend-
ing on the targeted receptor, binding and activation
domains can be contained in the same peptidic frag-
ment, which is sometimes composed of a relatively
small number of amino acids, or they can be partly
distinct. Having in mind these data, next challenge
will be to orientate the coupling reaction so that no or
minimal undesired chemical reaction could occur with
amino acid side chains of the binding and activation
domain(s). By using adequate coupling chemistry [8]
and eventually, by modifying peptide sequence of
the ligand in a way that preserves its pharmacophore,
efficient coupling of peptidic ligands to MNPs can be
expected. An additional major point to consider is the
steric hindrance. Indeed, the ligand’s pharmacophore
must remain sufficiently away from the MNP and
freely exposed to its environment to enable interac-
tions in the binding cavity of the cell surface receptors.
This is generally achieved by coupling the ligands to
the MNPs via functional groups located at the extrem-
ity of poly(ethyleneglycol) coating (PEG 2000) used to
enhance circulation time of the MNPs. However, such
traditional approach may not be optimal for all target-
ing purposes, especially in the case of ligands having
moderate to low affinities for their targeted receptor.
For example, a study aimed at targeting HER-2-over-
expressing cancer cells revealed that coating of MNPs
with PEG350 and ligand grafting through a linker
equivalent to PEG500 significantly enhance specific
MNP uptake by cells [9] .
“...such traditional approach may not be optimal
for all targeting purposes, especially in the case
of ligands having moderate to low affinities for
their targeted receptor. ”
The ligand density at MNP surface is an additional
critical point conditioning both effectiveness and
specificity of the targeting. An optimal ligand density
is that leading to the highest amount of MNPs accu-
mulation in cells expressing variable levels of receptors
while keeping the amount of added MNPs as low as
possible for minimal nonspecific uptake and toxicity.
Successful targeting of cell expressing EGF receptor
was documented with SPIONs bearing an average of
20–50 EGF molecules per MNP. For CCK2R target-
ing, a maximum uptake and accumulation in lyso-
somes was achieved with MNPs bearing 100 ligand
molecules per MNP. In the later example, it was also
noticed that association kinetic of the targeted MNPs
to the cells was dramatically slowed relative to the free
894 Nanomedicine (Lond.) (2015) 10(6)
ligand. Such slow binding, which is probably due to
the size and solid nature of the MNPs, could negatively
influence MNP uptake by tumors in vivo. Interest-
ingly, multivalency resulting from grafting of a large
number of ligands can counterbalance ligand affinity
loss caused by ligand coupling, especially in the case of
very small ligand. This was illustrated for angiotensin
II receptor type 1 and αvβ integrins targeting [10,11] .
Ligand multivalency at the MNP surface can also
favor receptor clustering or oligomerization, which
are molecular events required for, or contributing to
receptor activation and internalization. Furthermore,
magnetic field can cause clustering of MNP-bound
receptors, as reported with EGF receptors [12] , T-cell
receptors  [13] and death receptor 4 [14] . In the two last
examples, receptor clustering was used to cause tumor
cell death.
The pharmacological profile of the peptidic ligand
used to target MNPs to tumor cell surface receptors
is an additional critical point which must be consid-
ered with regard to the signaling pathways that provide
advantage in the uptake of the MNPs. Peptidic antago-
nists of G-protein-coupled receptors recognize equally
both coupled and uncoupled states of the receptors.
Consequently, they generally show enhanced binding
activity relative to agonists. However, since they do not
trigger cell signaling involved in receptor internaliza-
tion, they remain at the cell surface. Peptidic antag-
onists are therefore adequate to deliver MNPs at the
surface of tumor cells but not in their lysosomes. The
concept of biased ligands of G-protein-coupled recep-
tors which are ligands having distinct efficacy and
potency on the different signaling pathways of the tar-
geted receptors makes theoretically possible the design
of peptide-targeted MNPs with more predictable
activity.
Peptide-targeted MNPs present also the particu-
larity of being potentially sensitive to proteases, even
though MNP-grafted peptides are presumably less
sensitive than free peptides. The stability of the grafted
peptide is an important parameter which can be either
detrimental or beneficial. In the case of ligand cleavage
in the blood, the targeting advantage is lost, even if the
MNPs are designed to have a long circulating time.
On the contrary, if peptide cleavage occurs in endocy-
tosic vesicles following receptor-mediated internaliza-
tion of the ligand-targeted MNPs, ligand (and MNP)
dissociation from the receptor can be expected, even-
tually leading to receptor resensitization and recycling
to the cell surface [15] . Therefore, cancer cell target-
ing with MNPs grafted with peptidic ligands sensi-
tive to endosomal proteases can be a way to enhance
MNP accumulation in tumors since this formulation
permits at the same time internalization of the MNPs
future science group

and reappearance of the targeted receptors at the cell
surface.
In vivo studies on animal models of cancers have
consistently shown that uptake of targeted MNPs by
solid tumors is not only dependent on effectiveness of
targeting measured in vitro, but is also conditioned
by the ability of the MNPs to escape capture by the
reticuloendothelial system, remain in the blood for
enough time and cross biological barriers existing
between the blood stream and tumor cells [16] . It has
been appreciated that MNP coating by PEG or dex-
tran significantly increases their stealthiness and cir-
culating time. Whether the presence of peptides on
MNP surface affects the nature of the protein corona
surrounding MNPs in biological fluids and circulating
time is case dependent and therefore requires specific
investigations. Concerning the ability of the targeted
MNPs to cross tumor microenvironment and to pene­
trate deeply in the tumor, recent findings suggest that
their decoration with peptides carrying motifs target-
ing αvβ3/5 integrins and neuropilin-1, which have
reported to enhance tumor homing and penetration of
pharmaceutics, represents an attractive option [17] .
Anticancer therapy with peptide-targeted MNPs
also offers the possibility of minimizing undesired
accumulation of the MNPs in healthy tissues express-
ing the targeted receptor, albeit at low levels relative
to tumor cells. This can be achieved by coupling
ligands aimed at increasing homing of the MNPs near
the tumor (for instance, RGD motifs targeting αvβ
integrins) or by taking advantage of acidosis of tumor
microenvironment. The introduction of a chemical
References
Papers of special note have been highlighted as:
• of interest; •• of considerable interest
1 Sanchez C, El Hajj Diab D, Connord V et al. Targeting
a G-protein-coupled receptor overexpressed in endocrine
tumors by magnetic nanoparticles to induce cell death. ACS
Nano 8(2), 1350–1363 (2014).
2 Creixell M, Bohorquez Ac, Torres-Lugo M, Rinaldi C.
EGFR-targeted magnetic nanoparticle heaters kill cancer
cells without a perceptible temperature rise. ACS Nano 5(9),
7124–7129 (2011).
3 Domenech M, Marrero-Berrios I, Torres-Lugo M, Rinaldi C.
Lysosomal membrane permeabilization by targeted magnetic
nanoparticles in alternating magnetic fields. ACS Nano 7(6),
5091–5101 (2013).
4 Iovino N, Bohorquez Ac, Rinaldi C. Magnetic nanoparticle
targeting of lysosomes: a viable method of overcoming tumor
resistance? Nanomedicine (Lond.) 9(7), 937–939 (2014).
5 Silva Ac, Oliveira Tr, Mamani Jb et al. Application of
hyperthermia induced by superparamagnetic iron oxide
nanoparticles in glioma treatment. Int. J. Nanomed. 6,
591–603 (2011).
future science group
Targeted nanoscale magnetic hyperthermia  Editorial
moiety protecting the ligand from receptor recognition
in neutral environment but enabling ligand deprotec-
tion in acidic pH represents an original approach [18] .
“Peptide-targeted magnetic nanoparticles have
the potential to enable personalized anticancer
therapy since ligands recognizing several targets
can be simultaneously coupled to magnetic
nanoparticles. ”
To conclude, we believe that the different opportuni-
ties and constraints briefly summarized here highlight
how peptides are ideal ligands for targeting MNPs
to tumors. Peptides enable engineering of MNPs to
perform cooperative functions such as self-amplified
homing and penetration of the MNPs, amplification
of the targeting by ligand combination and enhanced
tumor uptake while minimizing undesired targeting
and uptake. Peptide-targeted MNPs have the potential
to enable personalized anticancer therapy since ligands
recognizing several targets can be simultaneously
coupled to MNPs.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involve-
ment with any organization or entity with a financial inter-
est in or financial conflict with the subject matter or mate-
rials discussed in the manuscript. This includes employment,
consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this
manuscript.
6 Johannsen M, Thiesen B, Wust P, Jordan A. Magnetic
nanoparticle hyperthermia for prostate cancer.
Int. J. Hyperthermia 26(8), 790–795 (2010).
7 Meffre A, Mehdaoui B, Kelsen V et al. A simple chemical
route toward monodisperse iron carbide nanoparticles
displaying tunable magnetic and unprecedented
hyperthermia properties. Nano Lett. 12(9), 4722–4728
(2012).
8 Conde J, Dias JT, Grazu V, Moros M, Baptista PV, De
La Fuente JM. Revisiting 30 years of biofunctionalization
and surface chemistry of inorganic nanoparticles for
nanomedicine. Front. Chem. 2, 48 (2014).
9 Stefanick Jf, Ashley Jd, Kiziltepe T, Bilgicer B. A systematic
analysis of peptide linker length and liposomal polyethylene
glycol coating on cellular uptake of peptide-targeted
liposomes. ACS Nano 7(4), 2935–2947 (2013).
10 Hennig R, Pollinger K, Veser A, Breunig M, Goepferich
A. Nanoparticle multivalency counterbalances the ligand
affinity loss upon PEGylation. J. Control. Release 194C,
20–27 (2014).
11 Bolley J, Guenin E, Lievre N et al. Carbodiimide versus
click chemistry for nanoparticle surface functionalization:
www.futuremedicine.com 895
Editorial  Fourmy, Carrey & Gigoux
a comparative study for the elaboration of multimodal
superparamagnetic nanoparticles targeting alphavbeta3
integrins. Langmuir 29(47), 14639–14647 (2013).
12 Bharde AA, Palankar R, Fritsch C et al. Magnetic
nanoparticles as mediators of ligand-free activation of EGFR
signaling. PLoS ONE 8(7), e68879 (2013).
13 Perica K, Tu A, Richter A, Bieler JG, Edidin M, Schneck
Jp. Magnetic field-induced T cell receptor clustering by
nanoparticles enhances T cell activation and stimulates
antitumor activity. ACS Nano 8(3), 2252–2260 (2014).
14 Cho MH, Lee EJ, Son M et al. A magnetic switch for the
control of cell death signalling in in vitro and in vivo systems.
Nat. Mater. 11(12), 1038–1043 (2012).
896 Nanomedicine (Lond.) (2015) 10(6)
15 Murphy JE, Padilla BE, Hasdemir B, Cottrell GS, Bunnett
Nw. Endosomes: a legitimate platform for the signaling train.
Proc. Natl Acad. Sci. USA 106(42), 17615–17622 (2009).
16 Lammers T, Kiessling F, Hennink WE, Storm G. Drug
targeting to tumors: principles, pitfalls and (pre-) clinical
progress. J. Control. Release 161(2), 175–187 (2012).
17 Ruoslahti E. Peptides as targeting elements and tissue
penetration devices for nanoparticles. Adv. Mater. 24(28),
3747–3756 (2012).
18 Han L, Guo Y, Ma H et al. Acid active receptor-specific
peptide ligand for in vivo tumor-targeted delivery. Small
9(21), 3647–3658 (2013).
future science group