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Research Highlights
Highlights from the latest articles in nanomedicine

Herceptin-conjugated nanocarriers
for targeted imaging and treatment of
HER2-positive cancer
One of the essential distinctions between
human beings and animals is that we
possess the ability to make drugs to cure
diseases. Over the thousands of years of
human history, a great variety of drugs,
from natural herbs to semi- and totally
synthesized chemicals, and then to purified and synthesized biological macromolecules, have been successfully developed
as effective drugs to treat various diseases.
These drugs, however, are so far blind
therapeutic agents, which not only work on
the diseased cells, but also attack healthy
cells and thus cause side effects, which are
serious and sometimes fatal. In most cases,
it is difficult to distinguish between drugs
and poisons, as asserted by an old Chinese
idiom: Every drug has at least 30% poisonous effects (, pronounced as
shi-yao-san-fen-du). It has been a dream
that any drug can be made of targeting
effects, which cure the diseased cells only
and leave the healthy cells untouched.
The side effects can thus be avoided and,
moreover, the maximum tolerable dose
(MTD; i.e., the highest dose of a drug or
treatment that does not cause unacceptable side effects [1]) can be greatly raised,
thus enhancing the therapeutic effects of
the drug. It can be predicted that drug
targeting will become one of the major
achievements in 21st Century medicine.
Drug targeting has thus become an
area of high interest, especially in the past
decades when strategic progress has been
made in pharmacodynamics that is, to
study what drugs do to our body or how
drugs act at their targets. In the late 1990s,
it was made clear that drugs interact with
diseased cells through receptors, where
they act either as agonists to mimic the
action of neurotransmitters or to facilitate
10.2217/NNM.11.1 2011 Future Medicine Ltd

binding of endogenous neurotransmitters,


or as antagonists to block access of transmitters to the binding site. Although
hundreds of drugs have been of use, surprisingly, they act on only a few different
types of receptors and only 480 targets
have been identified as of 1996. This is
an historic progress in medicine that triggered an intensive investigation on drug
targeting. Various ligands, such as folic
acids, peptides, sugar residues, aptamers,
proteins and antibodies, have been found
and applied to conjugates with the drug
itself or the drug carrier for drug targeting.
Among the various ligands, in current intensive investigation, trastuzumab
(Herceptin), a humanized recombinant
anti-HER2 monoclonal antibody, is unique
and the most successful one, and has been
widely used in the various nanocarriers of
anticancer drugs. HER2/neu is a member of the EGF receptor (EGFR) family,
which is a receptor tyrosine-specific protein
family consisting of four semihomologous
receptors: EGFR, HER2/neu, HER3 and
HER4. It is well known that HER2 is
overexpressed in 2530% of invasive breast
cancers. Herceptin can specifically bind to
the membrane region of HER2/neu with
a high affinity and inhibit signal transduction as well as cell proliferation. As a US
FDA-approved first-line therapeutic agent
for HER2-overexpressing breast cancer, it
not only shows significant biostatic activity as a single agent for immunotherapy,
but also demonstrates synergistic antitumor effects with chemotherapeutic agents
such as docetaxel. Moreover, Herceptin has
been widely applied to the various nanocarriers to realize sustained, controlled and
targeted drug delivery for HER2-positive
cancer therapy.
Nanomedicine (2011) 6(2), 311315

Yu Mi1, Yutao Liu1, Yajun Guo2,3


&Si-Shen Feng1,4,5
Department of Chemical & Biomolecular Engineering,
National University of Singapore, Singapore
2
International Joint Cancer Institute, The Second
Military Medical University, Shanghai, PR China
3
National Engineering Research Center for Antibody
Medicine & Shanghai Key Laboratory of Cell Engineering
& Antibody, Shanghai, PR China
4
Division of Bioengineering, Faculty of Engineering,
National University of Singapore, Singapore
5
Nanoscience & Nanoengineering Initiative (NUSNNI),
National University of Singapore, Singapore

Author for correspondence:


chefss@nus.edu.sg
1

Financial & competing


interestsdisclosure
The authors have no relevant affiliations or financial involvement with any organization or entity
with a financial interest in or financial conflict with
the subject matter or materials discussed in the
manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending,
orroyalties.
No writing assistance was utilized in the
production of this manuscript

part of

ISSN 1743-5889

311

News & Views Research Highlights


Herein, we highlight four articles that
demonstrate excellent proof-of-concept
experimental results for feasibility and
efficiency of application of Herceptinconjugated nanocarriers for targeted imaging and treatment of HER2-positive cancers, which include three articles involving
micelles, liposomes and nanoparticles of
biodegradable polymers, respectively, and
one article discussing the mechanism of

nanoconjugated antibody-induced receptor endocytosis. These articles represent


the latest progress at the cutting edge of
nanomedicine.

Website
1

National Cancer Institute: Dictionary of


Cancer Terms Maximum tolerated dose
www.cancer.gov/dictionary/?CdrID
=5 46597

Benefit of anti-HER2-coated paclitaxel-loaded


immuno-nanoparticles in the treatment of
disseminated ovarian cancer: therapeutic efficacy and
biodistribution in mice
Evaluation of: CirstoiuHapcaA,
Buchegger F, Lange N, Bossy L,
Gurny R, DelieF: Benefit of
anti-HER2-coated paclitaxelloaded immuno-nanoparticles in
the treatment of disseminated
ovarian cancer: therapeutic
efficacy and biodistribution in
mice. J.Control. Release 144(3),
324331 (2010).
In this article, the authors aimed to
explore the therapeutic efficacy and biodistribution of Herceptin-conjugated
poly(dl-lactic acid) nanoparticles (NPs)
in a mouse xenograft ovarian cancer
model induced by intraperitoneal inoculation of SKOV-3 cells overexpressing
HER2 antigens. Paclitaxel (Tx) was
used as a model drug encapsulated in the
poly(dl-lactic acid) NPs (NPTx). NP
Tx was then thiolated and conjugated to
sulfo-MBS-activated anti-HER2 mono
clonal antibodies (mAbs) to obtain
polymeric immunonanoparticles (NP
TxHER). The drug encapsulation
efficacy was found to be 7810%, with a
drug loading of 7.80.8% (w/w). After
covalent coupling of anti-HER2 mAbs
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on the NPTx surface, the size of NPTx


HER was 237 43 nm. Approximately
250molecules of mAbs were bound per
NP. The treatment in mice with NP
TxHER followed the rule of an initial
dose of 20 mg/kg Tx administered as
10 mg/kg intravenously and 10 mg/kg
intraperitoneally, followed by five alternative intraperitoneal and intravenous
injections of 10mg/kg Tx every 3days.
The therapeutic eff icacy was determined by bioluminescence imaging and
survivalrate.
The therapeutic efficacy of NPTxHER
was first demonstrated in the bioluminescence imaging results. The bioluminescent
signal decreased in one mouse and remains
stable in two others in the group receiving free Tx after 1week treatment, whereas
it disappeared in two mice and decreased
significantly in the third one in the group
receiving NPTxHER. The quantitative analysis of signal demonstrated that
while free Tx treatment caused a relatively
constant bioluminescence over 70 days
without any significant change, NPTx
HER treatment gave a significantly lower
bioluminescence, which indicated a notable decrease of tumor size. The targeting
effect of Herceptin was further confirmed
Nanomedicine (2011) 6(2)

by comparing the survival rate among different Tx formulations of NPTxHER,


NPTxRIT (RIT stands for Mabthera,
rituximab, an irrelevant mAb), free Tx and
Herceptin, in which NPTx-HER showed
the highest survival rate among all the
formulations. It also showed that a higher
survival rate of animals was observed when
treatments were initiated on day 3 compared with day5, which highlighted that
the efficacy of the treatments depends on
its onset.
As for the biodistribution study, free Tx,
NPTx and NPTxHER were analyzed
in both of the healthy and tumor-bearing
mice. In the healthy mice, NPTx showed
higher tissue concentration compared with
free Tx with a preferential distribution in
the reticuloendothelial system organs, such
as the liver and spleen, while biodistributions of NPTxHER and NPTx were not
significantly different. In tumor-bearing
mice, for intravenous injection, the NPTx
and NPTxHER was inclined to selectively distribute in the spleen and tumor
compared with the nonspecific tissue distribution of free Tx. Moreover, the NP
TxHER showed a higher tumor accumulation than NPTx. For interperitoneal
injection, free Tx appeared to accumulate
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Research Highlights
in the spleen, while the concentration of
encapsulated Tx in tumor was tenfold
higher compared with the free Tx.
This study applied a commonly used
US FDA-approved polymer PLA to form
NPs as nanocarriers, which were successfully conjugated with Herceptin on their
surface. The work compared the effect of
different injection administration (intravenous vs intraperitoneal) for biodistribution and demonstrated a selective distribution of Herceptin-conjugated NPs in
tumor. The improvement of the therapeutic efficacy was clearly demonstrated by

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the bioluminescence imaging and survival


rate. However, no treated mice were fully
recovered by using any of the fomulations. Both bioluminescence imaging and
necropsy of treated mice showed that the
presence of tumors, especially under the
liver, was difficult to reach. Therefore, the
therapeutic efficacy of such Herceptinconjugated PLA NPs should be further
improved by either increasing the drug
loading, encapsulation efficiency and
controlled release kinetics, or precise control of the targeting ligand for a better NP
accumulation in tumors.

Targeted near-infrared quantum dot-loaded micelles for


cancer therapy and imaging
Evaluation of: Nurunnabi M,
Cho KJ, Choi JS, Huh KM,
LeeYK: Targeted near-IR
QDs-loaded micelles for cancer
therapy and imaging.
Biomaterials 31(20), 54365444
(2010).
In this article, Herceptin-conjugated
micelles loaded with near-infrared (IR)
quantum dots (QDs) were developed for
cancer detection and treatment, which
were formulated by a mixture of poly
ethylene glycol10,12-pentacosadiynoic
acid (PEGPCDA) and PCDAHerceptin
conjugates that were further stabilized
under UV irradiation for intram icellar
cross-linking between PCDAs. The cytotoxicity and cellular selectivity of these
near-IR QD-loaded micelles were detected
with human nasopharyngeal epidermoid
cancer (KB) cells, which are HER2 negative, and SK-BR3 breast cancer cells,
which are HER2 positive. Confocal
microscopy results showed a significant
increase of fluorescent intensity in the
SK-BR3 cells compared with that in the
KB cells, which demonstrated the HER2mediated endocytosis of the micelles by

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the SK-BR3 cells. The targeting effect was


further confirmed by cell viability evaluation, which indicated less cytotoxicity of
the micelles to the KB cells than to the
SK-BR3 cells.
The therapeutic effect of the micelles
was determined in athymic BALB/c-nu/nu
female nude mice bearing HER2-positive
MDA-MB-231 tumors. The tumor volume of mice treated with such near-IR
QD-loaded micelles showed shrinkage
in initial tumor volume and inhibition of
tumor growth by 77.3% compared with
that of the control group, which showed a
significant increase of the tumor volume.
The biodistribution of these near-IR
QD-loaded micelles was also assessed
in this article. The micelles were intra
venously injected into tumor-bearing
mice. The results showed that the fluorescence signal was first observed throughout the body at 10 min postinjection, and
the signal disappeared after 1day, except
at the tumor site. A strong signal was
found at the tumor site and this signal
remained up to 5days. The quick clearance of the micelles ensured that they
would not be as harmful to the animal,
while the accumulation of the targeted
micelles in tumors ensured that such

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QD-loaded micelles can be of dual function as imaging and therapeutic agent for
HER2-overexpressed cancer cells. Such
a system could have much better therapeutic effects if anticancer drugs are also
coencapsulated in the micelles.
This study can be considered as a
model of Herceptin-conjugated micelles
for cancer imaging. The micelles were
cross-linked for stability and reduction
of toxicity of QDs. With the help of
Herceptin as the targeting ligand, it was
demonstrated in vivo that the micelles
were accumulated into the HER2 receptor-overexpressing cancer cells. However,
the therapeutic effect of this agent needs
to be further discussed. Whether the
inhibition of tumor growth was caused
by the toxicity of QDs or the therapy of
Herceptin, and how the cytotoxicity of
near-IR QD-loaded micelles for normal
cells is affected, should be further investigated. Besides, the design of the micelles
of the mixed PEGPCDA and PCDA
Herceptin may also lead to Herceptin
encapsulation by long PEG chains on the
surface. A mixture of PEGPCDA and
HerceptinPEGPCDA for the micelles
formulation may be recommended for
this targeted delivery.

313

News & Views Research Highlights


Listeriolysin O enhances cytoplasmic
delivery by HER2-targeting liposomes
Evaluation of: Kullberg M,
Owens JL, Mann K: Listeriolysin
O enhances cytoplasmic delivery
by Her-2 targeting liposomes.
J.Drug Target 18(4) 313320
(2010).
This work represents an example of
Herceptin-conjugated liposomes for targeted delivery of bioactive molecules into
the cytoplasm for cellular and molecular
imaging and treatment, in which liposomes were made with lipids DPPC/
MPPC/DPPG/DSPE-PEG(3400)-NHS
at a molar ratio of 82:10:3.5:4. Herceptin
was postconjugated to the surface of the
liposomes by carbodiimide chemistry.
A pore-forming protein, listeriolysin O
(LLO), was also added in the formulation
to enhance cytoplasmic delivery of liposomal contents to breast cancer cells. LLO is
a protein that can form pores with the help
of cholesterol in the endosomal membrane
to increase the delivery of liposomal contents from the endosomal compartments
to the cytoplasm of the targeted cells.
Addition of this LLO product to the liposome surface increased the diameter of the
liposomes from 2138 to 2355nm. The
article discussed heat-triggered cytoplasmic

delivery and nonthermosensitive delivery


from the targeting LLO liposomes in two
mammary epithelial cell lines, ce2 and
MTSV17, which are identical except that
ce2 has been transfected with HER2 DNA
and expresses tenfold more HER2 receptors than MTSV17. Calcein was used as
a model cargo, and fluorescence micro
scopy analysis was performed to determine
the relative c ytoplasmic c oncentrations
ofcalcein.
The result showed that the relative
fluorescence signal in ce2 was stronger
than that in MTSV17 for both of the
control liposomes and the LLO liposomes
at 37 and 42C, which demonstrated the
effect of targeting delivery for HER2overexpressed cells. Meanwhile, both of
the control liposomes and the LLO liposomes showed higher fluorescence intensity
in cytoplasm at 42C than at 37C, and
the addition of LLO to the liposomes had
a more significant effect at 42C, which
delivered over 22-fold more calcein into
the cytoplasm of ce2 cells than into the
cytoplasm of MTSV17 cells. The above
consequence suggested the heat-triggered
delivery of the liposomes and the enhanced
cytoplasmic delivery effect of LLO. For
nonthermosensitive delivery of these liposomes, at 1.5h the amount of calcein in

the cytoplasm of both the ce2 cells and


the MTSV17 cells was minor. At 4 and
10h there were increasing amounts of fluorescent calcein. At the 10h time point,
the ce2 cells treated with LLO liposomes
showed a 12-fold greater concentration
of cytoplasmic calcein than that of the
control liposomes. Moreover, the ce2 cells
exhibited a sevenfold greater cytoplasmic
calcein concentration than the MTSV17
cells did.
The strategy of this article provided
us with the possibility of targeted drug
delivery to HER2-overexpressed cancer
cells by thermoactive liposomes, while
restricting the drug release within the
tumor site by localized hyperthermia.
However, after an extended incubation at
37C, the liposomes also delivered calcein
to the cytoplasm of HER2-overexpressed
cells even without heating. The authors
suggested that separating the LLO into
one set of liposomes, and the drug being
delivered into another set would be a possible solution to reduce drug delivery to
cells expressing normal levels of HER2.
This article would have been more convincing if a model anticancer drug could
be encapsulated in such targeting liposomes to test their possible advantages for
ta rgeted delivery of therapeutic effects.

Nanoconjugation modulates the trafficking and


mechanism of antibody-induced receptor endocytosis
Evaluation of: Bhattacharyya S,
Bhattacharya R, Curley S,
McNivenMA, Mukherjee P:
Nanoconjugation modulates the
trafficking and mechanism of
antibody induced receptor
endocytosis. Proc. Natl Acad. Sci.
USA 107(33), 1454114546
(2010).
314

Although the monoclonal antibody (mAb)like Herceptin has been used as a targeting agent for selective drug delivery to the
diseased cells, mechanisms of nanoparticlesmAb interactions with the target cells
and its effect on intracellular trafficking
and mechanism are still unknown. In this
article, cetuximab (C225), a monoclonal
anti-EGF receptor (EGFR) antibody, was
used to demonstrate the distinct patterning
Nanomedicine (2011) 6(2)

and dynamics of anti-EGFR antibody


cetuximab (C225) and the gold nanoparticle-conjugated C225-induced internalization of EGFR, as well as its effect on
intracellular trafficking and mechanism in
PANC-1 cells (primary cell line with high
EGFR expression), MiaPaca2 cells (primary cell line with low EGFR expression)
and AsPC-1 cells (metastatic cell line with
high EGFR expression).
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Research Highlights
In this article, confocal microscopy
tracking the C225-Cy3- and Au-C225Cy3-induced EGFR endocytosis demonstrated that nanoconjugation promoted
faster endocytosis of EGFR. In addition,
the pattern of uptake was quite different
between primary cancer cells and metastatic cancer cells. For PANC-1 cells and
MiaPaca2 cells 1h after the treatment,
EGFR was found completely perinuclear
in the Au-C225-Cy3 group, whereas
membrane staining could still be obtained
for the MiaPaca2 cells. Instead, Au-C225Cys induced notable endocytosis within
1h in the form of discrete puncta at the
membrane and cytosol for the AsPC-1
cells. By contrast, C225-Cy3 was unable to
induce significant EGFR endocytosis even
after 1h. The number of puncta was also
higher for Au-C225-Cy3, which indicated
enhanced clustering of EGFR and faster
endocytosis by nanoconjugatedC225.
To test the intracellular trafficking of
C225-induced EGFR internalization,
colocalization experiments were performed

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to determine C225-Cy3- and Au-C225Cy3-induced endocytosis in different


organelles, including early endosomes,
lysosomes, the Golgi complex and transferrin (representing the recycling compartment). The quantification data of
colocalization in different organelles indicated that conjugation to gold nanoparticles altered C225-induced intracellular
trafficking of EGFR.
Furthermore, the role of dyn-2 in C225induced endocytosis of EGFR was tested to
show the mechanism of this process. Dyn-2
is a signal transducing GTPase that has
been implicated in EGF-induced endocytosis of EGFR. The results demonstrated that
PANC-1 cells with high EGFR expression,
where C225-Cy3-induced EGFR endocytosis was dyn-2 dependent, switched to a
dyn-2-independent pathway upon nanoconjugation. On the other hand, AsPC-1
cells with similar levels of EGFR expression
as PANC-1 cells, where C225-Cy3-induced
EGFR endocytosis was dyn-2 independent, switched to a dyn2-dependent

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pathway with Au-C225-Cy3. However,


in MiaPaca2 cells with low EGFR expression, both C225-Cy3 and Au-C225-Cy3induced EGFR endocytosis remained
dyn-2-dependent. Thus, the regulation of
dynamin in EGFR endocytosis upon nanoconjugation may provide valuable insight
into the mechanism of C225 function.
In short, this article demonstrated that
the nanoconjugation cannot be simply
understood as an innocuous reaction
involved in merely attaching the targeting agent to the nanocarriers. Instead, it
may distinctly alter the cellular processes
at the molecular level, at least antibodyinduced receptor endocytosis. This article
provides useful information for reasonable
design of a nanocarrier-based nanomedicine for ta rgeted detection and treatment
of diseases.

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