Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85

Seminar article
New frontiers in nanotechnology for cancer treatment夡
Frank Alexisa,c, June-Wha Rheea,c, Jerome P. Richieb, Aleksandar F. Radovic-Morenoc,d,e,
Robert Langerc,d,e, Omid C. Farokhzada,c,*
a
Department of Anesthesiology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
b
Department of Urology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
c
MIT-Harvard Center for Cancer Nanotechnology Excellence, Cambridge, MA 02139, USA
d
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
e
Harvard-MIT Division of Health Sciences and Technology, Boston, MA 02115, USA

Abstract
Nanotechnology is a field of research at the crossroads of biology, chemistry, physics, engineering, and medicine. Design of multifunc-
tional nanoparticles capable of targeting cancer cells, delivering and releasing drugs in a regulated manner, and detecting cancer cells with
enormous specificity and sensitivity are just some examples of the potential application of nanotechnology to oncological diseases. In this
review we discuss the recent advances of cancer nanotechnology with particular attention to nanoparticle systems that are in clinical practice
or in various stages of development for cancer imaging and therapy. Published by Elsevier Inc.

Keywords: Cancer therapy; Cancer diagnosis; Nanocarriers; Targeting molecules

Introduction nents alone. Nanomaterials have a large surface area to

Cancer nanotechnology
Nanotechnology—the engineering and manufacturing of
materials using atomic and molecular components—is ex-
pected to benefit all branches of medicine, with oncology
being an early and the most notable beneficiary to date [1,2].
In its strictest definition from the National Nanotechnology
Initiative (NNI; http://www.nano.gov), nanotechnology re-
fers to structures roughly in the 1 to 100 nm size in at least
one dimension. To put this size range in perspective, a small
molecule, a virus, a bacterium, and the cross section of a
human hair are around 1 nm, 100 nm, 1,000 nm, and
100,000 nm, respectively. Nanotechnology more commonly
refers to structures that are up to several hundred nanome-
ters in size, and are developed by top-down or bottom-up
engineering of individual components (i.e., rational design)
[1,3–5]. The resulting nanomaterials may have functional
properties that are conferred through the precise assembly
of individual components but not by the individual compo-
夡
Supported by NIH grants CA119349 and EB003647.
* Corresponding author. Tel.: ⫹1-617-732-6093; fax: ⫹1-617-730-
2801.
E-mail address: ofarokhzad@partners.org (O.C. Farokhzad).
volume ratio and their physicochemical properties, such as
friction and interaction with other molecules, are distinct
from equivalent materials at a larger scale. The most com-
mon use of nanotechnology in medicine has been in the
areas of developing novel therapeutic and imaging modal-
ities that have the potential to outperform the current state of
the art in these areas. With the exponential introduction of
novel nanotechnology platforms for life science applications,
the potential application of nanotechnology in medicine ex-
tends significantly beyond these early uses. The National Can-
cer Institute in its 2004 commitment of $144 million toward
the development of novel nanotechnologies for improving can-
cer mortality (http://nano.cancer.gov) defined the path of op-
portunities in six areas including: (1) detection of molecular
changes responsible for disease pathogenesis; (2) disease di-
agnosis and imaging; (3) drug delivery and therapy; (4) mul-
tifunctional systems for combined therapeutic and diagnostic
applications; (5) vehicles to report the in vivo efficacy of a
therapeutic agent; and (6) nanoscale enabling technologies,
which will accelerate scientific discovery and basic research.
In this review, we will focus on the application of
nanotechnology to the development of smart drug deliv-
ery vehicles for cancer therapeutic applications. The most
common examples of these nanoscale delivery vehicles
(also referred to as nanocarriers) include polymeric nano-

1078-1439/08/$ – see front matter Published by Elsevier Inc.

doi:10.1016/j.urolonc.2007.03.017
 F. Alexis et al. / Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85
particles, dendrimers, nanoshells, liposomes, nucleic acid-
based nanoparticles, magnetic nanoparticles, and virus
nanoparticles [1]. Nanocarriers have the potential to im-
prove the therapeutic index of currently available drugs by
increasing drug efficacy, lowering drug toxicity, and achiev-
ing steady state therapeutic levels of drugs over an extended
period of time. Nanocarriers can also improve drug solubil-
ity and drug stability, allowing the development of poten-
tially effective new chemical entities that have been stalled
during the preclinical or clinical development because of
suboptimal pharmacokinetic or biochemical properties. Fi-
nally, nanocarriers may also facilitate the development of
multifunctional systems for targeted drug delivery [6,7],
combined therapies [8], or systems for simultaneous thera-
peutic and diagnostic applications. The development of the
first nanocarrier dates back approximately 40 years, when
the first example of a liposome was described [9,10]. Over
the past 2 decades an increasing number of nanoscale ve-
hicles with distinct physical and biochemical properties
have emerged for drug delivery applications. More recently,
the breakthrough potential of cancer nanotechnology is be-
Table 1
Nanoparticle platforms for drug delivery
Nanoparticle Material Structure
Polymeric Poly(lactide-co-glycolide)
Poly(lactide)
Poly(caprolactone)
Poly(orthoester)
Poly(anhydride)
Poly(beta-aminoester)
Liposome Doxil®/Caelyx®:
PEG-DSPE:HSPC/Cholesterol
(5:56:39)
DaunoXome®:
DSPC/Cholesterol (2:1)
Dendrimer Poly(amidoamine)
Branched Poly(ethylenimine)
Poly(peptide)
Polymeric nanoparticles encapsulate both hydrophilic and hydrophobic
small molecule and macromolecular drugs within a polymeric matrix.
Liposomes encapsulate hydrophilic and hydrophobic drugs within the
hollow liposome core and within their hydrophobic membrane, respec-
tively. Dendrimers are branched units with surface functional groups that
can be used to conjugate therapeutic or diagnostic payloads for subsequent
release.
75
coming increasingly recognized with several examples of
first generation nanocarriers approved by the FDA for ther-
apeutic (Abraxane [11], Doxil [12], DaunoXome [13]) and
diagnostic (Feridex [14]) applications (Table 1).
Targeted delivery of nanocarriers to tumors
The vascularity of tumors is highly heterogeneous, rang-
ing from areas of vascular necrosis to areas which are
densely vascularized in order to sustain the adequate supply
of oxygen and nutrients to the growing tumor. Tumor blood
vessels have several abnormalities compared to normal
blood vessels, including a high proportion of proliferating
endothelial cells with aberrant underlying basement mem-
brane, increased tortuosity of blood vessels, and a defi-
ciency in pericytes [15]. Tumor microvessels demonstrate
enhanced permeability, which is regulated in part by abnor-
mal secretion of vascular endothelium growth factor, bra-
dykinin, nitric oxide, prostaglandins, and matrix metallo-
proteinases [15]. The transport of macromolecules across
tumor microvasculature may occur through open interendo-
thelial junctions or transendothelial channels. The pore cut-
off size of these transport pathways in various models has
been estimated to be in the ⬍1 ␮m range, and in vivo
measurement of extravasation of liposomes into tumor
xenografts suggests a cutoff size in the 400 nm range [16].
In general, particle extravasation is inversely proportional to
size, and smaller particles (⬍200 nm size) would be most
effective for extravasating the tumor microvasculature
[16,17]. The tumor lymphatic system is also abnormal,
resulting in fluid retention in tumors and high interstitial
pressure with an outward convective interstitial fluid flow
[18]. This property is thought to promote tumor cell intra-
vasation, resulting in tumor metastasis and blockage of
nanocarrier extravasation from microvasculature into the
tumor interstitium. However, the lack of an intact lymphatic
system also results in retention of the nanocarriers in the
tumor interstitium since these particles are not readily
cleared from the interstitium. When taken together, the
leaky microvasculature and the lack of intact lymphatic
system results in enhanced permeation and retention (EPR)
effect and “passive” cancer targeting through the accumu-
lation of nanocarriers in the tumor at a higher concentration
that is present in the plasma and in other tissues [19]. The
release of drugs from nanocarriers in this case results in a
relatively higher intratumoral drug concentration translating
into enhanced tumor cytotoxicity. These nanocarriers may
be further modified for “active” cancer targeting by func-
tionalizing the surface of nanocarriers with ligands such as
antibodies, aptamers, peptides, or small molecules that rec-
ognize tumor-specific or tumor-associated antigens in the
tumor microenvironment. When nanocarriers are targeted to
the extracellular portion of transmembrane tumor antigens,
they may be specifically taken up by cancer cells through
receptor mediated endocytosis [20 –26]. The specific targeting,
intracellular uptake, and regulated therapeutic delivery of a
76 F. Alexis et al. / Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85

payload are properties that are derived through a rational de-
sign of nanocarriers. The application of nanotechnology to
cancer therapy, including the development of “smart” nano-
particles, is indeed an exciting and promising area of investi-
gation. In the following sections, we review some of the most
important breakthrough nanotechnology platforms for cancer
therapeutic applications.
Nanotechnology for cancer treatment
Biodegradable polymeric nanoparticles
Virtually every branch of medicine has been dramati-
cally impacted by controlled release polymer technologies
during the past four decades, including cardiology [27],
ophthalmology [28], endocrinology, [29] orthopedics, [30]
pain medicine [31], and oncology [32]. Several examples of
these systems in clinical practice today include Atridox,
Lupron Depot, Gliadel, Zoladex, Trelstar Depot, and San-
dostatin LAR. The annual worldwide market of controlled
release polymer systems, which extends beyond drug deliv-
ery, is now estimated at $60 billion, and these systems are
used by over 100 million people each year. “Controlled
release” occurs when a natural or synthetic polymer com-
bines with a drug in such a way that the drug is encapsulated
within the polymer system for subsequent release in a pre-
determined manner (Fig. 1). Polymeric drug delivery vehi-
cles can be designed as nanoparticles that release the en-
capsulated drugs through surface or bulk erosion, diffusion,
or swelling, followed by diffusion in a time or condition
dependent manner. The release of the active agent may be

Fig. 1. Schematic representation of nanoparticle targeting. (A) Nanoparticles are concentrated in the tumor interstitium via passive extravasation through the
leaky microvasculature shown as gaps in the endothelial layer (light orange). This process has been named the EPR effect. In this case the efficacy of
nanoparticles is largely mediated through the local release of the drug near the cancer cells. (B) Targeted nanoparticles similarly concentrate in the tumor
interstitium through the EPR effect but once there, nanoparticles are actively taken up by cancer cells after binding to their target antigens on the surface of
the cancer cells. In this case the drugs are released largely inside the cancer cells resulting in enhanced efficacy.
 F. Alexis et al. / Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85
constant over a long period, cyclic over a long period, or it
may be triggered by environmental or other external events
[33] such as changes in pH, [33–36] temperature, [37], or
the presence of an analyte such as glucose [38]. In general,
controlled-release polymer systems can provide drug levels
in an optimum range over a longer period of time compared
to other drug delivery methods. Thus it increases the effi-
cacy of the drug and maximizes patient compliance, while
enhancing the ability to use highly toxic, poorly soluble, or
relatively unstable drugs. The primary consideration of drug
delivery is to achieve more effective therapies while elim-
inating the potential for both under- and overdosing. Other
advantages of using controlled release delivery systems
include the maintenance of drug levels within a desired
range, the need for fewer administrations, optimal use of the
drug in question, and increased patient compliance. This is
particularly relevant to cancer therapy in which the effec-
tiveness of the treatment is directly related to the ability to
kill cancer cells while affecting as few healthy cells as
possible. By administering bolus doses of cytotoxic chemo-
therapeutic drugs, adverse effects are commonly observed
and these may be sufficiently intense to limit continuation of
the chemotherapy course.
Polymeric nanoparticles may represent the most effec-
tive nanocarriers for cancer chemotherapy [39,40]. These
nanoparticles can be surface-functionalized to target cancer
cells specifically and have a prolonged systemic circulating
half-life to enhance their therapeutic efficiency. The nano-
particle surface is usually sterically stabilized by grafting,
conjugating, or adsorbing hydrophilic polymers such as
polyethyleneglycol (PEG) to its surface [41,42]. Polymeric
nanoparticles can be readily formulated to deliver hydro-
philic or hydrophobic small drug molecules, and polymer
systems have been developed and used to deliver macro-
molecules such as proteins [43] and nucleic acids [44].
Biodegradable polymers are typically broken down into
individual monomers, which are metabolized and removed
from the body via normal metabolic pathways. The rate of
polymer degradation and subsequent drug release can be
controlled by modification of the polymer side chain, de-
velopment of novel polymers, or synthesis of copolymers.
This subject has been an active area of investigation by our
group and other investigators in academic and industry
laboratories for several decades [45– 48]. The result has
been an increasing arsenal of polymers with distinct encap-
sulation and release characteristics for a myriad of research,
industrial and clinical applications [49,50]. PGA and PLA
are common biocompatible polymers that are used for many
biomedical applications. PGA is more hydrophilic since it
lacks a methyl group and is more susceptible to hydrolysis,
making this polymer easily degradable. Alternatively, PLA
is relatively more stable in the body [51]. Through these
unique properties, polymers such as PLGA have been de-
rived that are made from both glycolic acid and lactic acid
components. The ability to change the ratio of these two
components of the polymer can then be used to alter the rate
77
of degradation. Using the PLA and PLGA polymer systems,
we have developed proof of concept docetaxel encapsulated
targeted nanoparticles, which target the prostate-specific
membrane antigen (PSMA) on the surface of prostate can-
cer cells and are engulfed by cells that express the PSMA
protein specifically and efficiently, in vitro and in vivo
[5,21,22]. We have shown that a single intratumoral injec-
tion of these particles results in tumor eradication in 5/7 of
the mice, with the remaining 2 mice having considerably
smaller tumor volumes as compared to controls [22]. These
targeted nanoparticles release their payload directly inside
the cancer cells, resulting in enhanced efficacy and decreased
systemic toxicity [22]. More recently, using the J591 antibody
which recognizes the PSMA protein, J591-targeted controlled
release nanoparticles have been developed for effective gene
delivery to prostate cancer cells [52,53]. Our group has also
developed HER2-targeted nanoparticles that may have util-
ity in the systemic therapy of breast, pancreatic, and ovarian
cancers (Fig. 2). More recently, our group and other inves-
tigators have begun to explore computational modeling and
screening strategies to identify optimized physicochemical
properties of nanoparticles (i.e., size, charge, hydrophilicity,
stability, drug release kinetics, and the density of targeting
molecules on nanoparticle surface) for cancer therapeutic
applications [54,55].
Dendrimers
Dendrimers represent a globular macromolecule defined
by core and branched units and surface groups [56]. With
well-controlled synthesis methods, new classes of dendrim-
ers are emerging for therapeutic and diagnostic applications.
A single dendrimer molecule can carry a therapeutic drug, a
diagnostic agent, and an active targeting molecule. Den-
drimers with a hydrophobic core and hydrophilic surface
groups are able to form micelles by hydrophobic/hydro-
philic self-assembly [57–59]. Although the rigidity and the
density of the branched units can affect the drug release rate
[60], much effort is devoted to the design of hybrid struc-
tures using pH or enzymatic sensitive linkages to disrupt
dendrimer micelles and trigger the site-specific release of
their payload. Taking advantage of the highly versatile
functionalities of dendrimers, hybrid structures of linear and
branched molecules (polymers or biomolecules) are being
investigated [61,62]. Biodegradable polyester dendrimers
based on 2,2-bis(hydroxymethyl)-propanoic acid monomers
have been developed for intracellular release of doxorubicin
after hydrolysis of hydrazone linkage [63]. The promising
properties of polyester dendrimers as a drug delivery system
has led to further studies based on tunable architectures and
molecular weights to optimize tumor accumulation [64,65].
The results show that higher molecular weight (ⱖ40 kDa)
molecules have a longer half-circulation time. The degree of
branching also affects the circulation time and renal clear-
ance of the drug. In a comparative study with Doxil in C-26
78 F. Alexis et al. / Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85

Fig. 2. (A) Schematic representation of a stealth targeted polymeric nanoparticle. (B) Scanning electron microscope (SEM) micrograph of PLGA
nanoparticles. The white represents 1 ␮m. (C) Comparative plasma drug concentration of polymeric controlled release drug delivery system vs. systemic
delivery of free drug. (D) Fluorescent micrograph of breast cancer cells treated with Her2 targeted nanoparticles loaded with rhodamine. The cell nuclei and
the actin cytoskeleton are stained with blue (DAPI), and green (Alexa-Flour phalloidin), respectively. The intracellular rhodamine-encapsulated Her2 targeted
nanoparticle bioconjugates are shown in red.

colon carcinoma-bearing mice, the polyester dendrimer-
drug conjugate shows similar efficacy to Doxil [66].
Biocompatible polyamidoamine (PAMAM) dendrimers
demonstrate a remarkable molecular monodispersity and
have been shown to be pH responsive. A variety of den-
drimers with different cores (diamino derivatives and eth-
ylenediamine) and surface functional groups (alcohol,
amine, succinic acid, and carboxylic acid) are commercially
available. More recently, multifunctional PAMAM den-
drimers with an imaging agent (fluorescein isothiocyanate
FITC), a cancer cell targeting molecule (folic acid) and a
therapeutic drug (Taxol) have been described with impres-
sive in vitro and in vivo results [67].
Nanoshells
Polymeric nanoshells (20 to 60 nm) of diblock copolymers
can assemble into a core/shell structure. These nanoshells can
be made by self-assembly of oppositely charged polymers
forming a thin multilayer structure. The characteristic of
nanoshells is defined by the layer-by-layer assembly of nano-
particles, allowing a high drug loading efficiency. Therefore,
the drug release rate is controlled by the properties of the
polymers and the diffusion coefficient through the polymeric
layer. In addition, the surface of the nanoshells can be easily
functionalized for targeting applications. pH-sensitive doxo-
rubicin encapsulated nanoshells have been synthesized us-
ing amphiphilic tercopolymerpoly(N-iso-propylacrylamide-
co-N,N-dimethylacrylamide-co-10-undecenoic acid) that
can trigger intracellular drug release at pH 6.6. Metallic
nanoshells (⬃20 nm) are characterized by a dielectric core
coated with a thin metallic shell to improve their biocompat-
ibility and optical absorption [68]. These particles possess a
highly tunable plasmon resonance mediated by the size of the
core and the thickness of the shell, which in turn determines
their absorbing and scattering properties over a broad range on
the spectrum from the near-ultraviolet to the mid-infrared [68].
Gold nanoshells have been developed for in vivo photothermal
therapy using near infrared light [69]. Similarly, thermally
sensitive polymeric hydrogels and optically active nanoshells
have been developed for the purpose of photothermally mod-
ulated drug delivery. Nanoshell particles with a magnetic core
(carbonyl iron) and a biodegradable poly(butylcyanoacrylate)
(PBCA) shell have also been developed for controlled release
of 5-fluorouracil [70]. The combination of magnetic nanoshells
and a drug encapsulated biodegradable polymer allows for
particle targeting to specific sites of the body in response to an
externally applied magnetic field.
 F. Alexis et al. / Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85
Liposomes
Liposomes form nanoparticles with an amphiphilic
unilamellar/multilamellar membrane of natural or synthetic
lipids [9]. Lipids are characterized by a hydrophilic head
group and a hydrophobic tail. Liposomes form lipid bilayers
through hydrophobic interactions and are able to carry hy-
drophilic and hydrophobic molecules simultaneously. The
hydrophobic and hydrophilic molecules can be encapsulated
into the lipid bilayer and hollow core respectively. This
unique property allows the formulation of a single system for
the combination therapy to treat cancer. Liposomes with mark-
edly prolonged circulation and enhanced tumor accumulation
have been developed by a series of modifications [9].
Liposomes have already been approved for routine uses
in medicine [9,71]. Doxorubicin encapsulated liposome
(Doxil) was the first to gain FDA approval in 1995 and has
potent antineoplastic activity against a wide range of human
cancers including Kaposi’s sarcoma [72] and ovarian cancer
[73]. The surface of Doxil (100 nm) is functionalized with
methoxypolyethylene glycol (MPEG, which provides a hy-
drophilic “stealth” coating, allowing Doxil to circulate in
the blood stream for a prolonged period of time [74]. The
clinical success of Doxil has led to numerous new liposome
formulations to be FDA approved, including DaunoXome
(daunorubicin liposomes), DepoDur (morphine liposomes),
and Ambisome (amphotericin B liposomes). DaunoXome, a
45 nm liposomal formulation, has been shown to be active
against Kaposi’s sarcoma and other tumors [75]. Despite the
clinical success of liposomes, these nanocarriers are limited
by suboptimal stability and drug release profiles in vivo.
Thus, efforts have been focused on the development of
stable and pH-sensitive liposomes that can unload the drug
in an acidic environment [76 –78].
Nucleic acid-based nanoparticles (DNA, RNAi, anti-sense)
Gene therapy refers to the transfer and expression of
genes for therapeutic applications in the target cells. In
contrast, RNAi [79] and anti-sense [80] therapies are
based on oligonucleotides that will shut down the gene
expression of a target gene involved in a pathway of the
disease. Aptamers are nucleic acid ligands that bind to
target proteins with high affinity and specificity, are anal-
ogous to antibodies, and exert a biological effect [81].
The greatest impact of these technologies is expected to
be in cancer therapy where there is a differentially ab-
normal pattern of gene or protein expression. DNA and
RNA macromolecules can be used as substrates for de-
veloping therapeutic and imaging nanocarriers. A multi-
valent DNA delivery vehicle (100 nm) was recently re-
ported for simultaneous targeted drug delivery, imaging,
and gene therapy [82]. Targeted multifunctional RNA
nanoparticles (25– 40 nm) have also been developed with
79
a trivalent RNA core, RNA aptamers for targeting, and
siRNAs for therapeutic effect [83].
Targeting molecule platforms
Antibodies or antibody fragments
Antibodies are the first macromolecular ligands used for
targeted delivery [84,85]. The use of monoclonal antibodies
(mAb) became widespread after the discovery of hybridoma
technology. Due to their inherent immunogenicity, murine
monoclonal antibodies were not suitable for clinical appli-
cations. Engineering antibody technologies led to the devel-
opment of chimeric humanized and fully humanized anti-
bodies. More recently, methods have been developed to
produce human immunoglobulins from transgenic mice
[86]. Combinatorial phage display libraries (⬃1010 different
antibody fragments) have also emerged as a powerful tool to
select novel protein ligands [87]. The latter approach relies
on multiple selection and screening parameters to select
human antibodies against specific cell types or antigens.
The selection process can be designed to improve the prop-
erties of the ligand such as stability, affinity, selectivity and
internalization. Most recently, this approach was used to
isolate cancer targeting antibodies using live cancer patients
in the selection process [88]. Antibody molecules show
good stability but are limited by their large hydrodynamic
diameter (⬃20 nm; Mw ⱖ 150 kDa) to diffuse into tumor
tissues [89,90]. Recent advances in protein engineering
have led to the development of single chain antibodies,
antibody fragments (Fab or scFv) and dibodies [85]. Antibody
fragments show less immune response and can be stabilized
with disulfide bond or chemically crosslinked [91].
There are several examples of FDA approved antibodies
in clinical practice today, including Rituxan—a chimeric
anti-CD20 antibody effective against CD20⫹ B-cell non-
Hodgkin’s lymphomas [92]. Other examples include anti-
HER2 Herceptin [93], anti-EGFR Erbitux [94], and anti-
VEGF Avastin [95]. These antibodies function by antago
nizing the signal transduction pathway that leads to un
controlled growth in cancer cells. Recent investigations [96]
have focused on exploiting the affinity of these antibodies
for their respective antigens to “arm” them with chemother-
apy drugs (doxorubicin [97], methotrexate [98], and cali-
cheamicin [99]), toxins, or radionucleotides [100]. Despite
much efforts, Mylotarg [99] is the only FDA approved
antibody-toxin conjugate in practice today. The payload and
the chemical conjugation of the drug to antibodies remain
the major limitations of chemoimmunoconjugates. Targeted
drug delivery carriers are being functionalized with antibod-
ies or antibody fragments to overcome some of these limi-
tations [20,101–103]. Furthermore, the conjugation of mul-
tiple antibodies to each nanocarrier enhances their avidity,
and nanocarriers can be surface functionalized with multiple
distinct antibodies to overcome tumor heterogeneity. Long-
80 F. Alexis et al. / Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85
circulating doxorubicin encapsulated immunoliposomes
conjugated to human HER2 antibodies have been developed
and evaluated in vivo [20]. After intravenous administra-
tion, nontargeted and targeted liposomes similarly accumu-
lated in the tumors; however, targeted liposomes had a signif-
icantly higher anti-tumor efficacy, presumably due to the
HER2 mediated internalization of targeted liposomes and in-
tracellular drug release vs. the nontargeted liposomes that re-
leased the drug locally into the extracellular space [20].
Peptides
Research using antibodies for targeting applications has
led to a better understanding of critical factors affecting
targeting. Peptides and antibody fragments have been de-
veloped to overcome some of the shortcomings of antibod-
ies, and several examples of these ligands are now under
clinical development. It is now well accepted that the bind-
ing affinity, stability, and the size of the ligand play a
critical role for successful targeting. Peptides are small,
synthetic molecules that can be manufactured in large quan-
tities with excellent quality control. Peptides are more stable
than antibodies and unlikely to be immunogenic. The dis-
covery of new peptide targeting domains has been success-
ful due to the development of peptide library screening
methods (e.g., phage display; ⬃1011 different peptides)
[88,104,105]. Display technologies are powerful tools to
isolate novel ligands for therapeutic and diagnostic applica-
tions. Twenty five years ago, Scott and Smith reported the
first peptide phage library [106]. Combinatorial peptide
libraries are defined by a large pool of random sequences or
based on a backbone sequence with random mutations.
Large libraries of linear or constrained peptides are cur-
rently available [107]. In general, circular stabilized or mul-
tivalent peptides show higher binding affinity due to en-
hanced rigidity and interaction with cell membrane
antigens. In most cases, short peptides are selected after
affinity screening of the library on a target protein, whole
cell, ex vivo tissues or live animals and even humans
[104,108 –110]. A large number of high affinity peptides
(Kd in uM–nM range) with potential for targeting applica-
tion have been isolated against a variety of cell surface
antigens [108,111–113]. Peptides that contain RGD (Arg-
Gly-Asp) domains can preferentially bind cells in tumor
microvasculature that express the ␣(V) ␤3 integrin
[109,114]. This strategy has been used experimentally to
deliver chemotherapy and radioactive molecules selectively
to tumors [115–117]. Moreover, cyclic RGD peptide (EMD
121974 or Cilengitide) is now under phase II clinical eval-
uation for the treatment of several tumors including andro-
gen independent prostate cancer and recurrent Glioblastoma
multiforme [118,119]. However, RGD sequences also act
as adhesive molecules and can non-specifically bind tissues
that also express its integrin complement. Integrin receptors
are also expressed on the cell membrane of macrophages
[120] and it is shown that RGD bioconjugates aggregate in
spleen and liver tissues due to macrophage clearance [121].
Ex-vivo selection of targeted peptide on biopsy specimens
have been developed and shown to successfully isolate
potent peptide ligands. In vivo phage library selection was
pioneered by Arap et al. [114] to further optimize the se-
lection parameters. The discovery of potent peptide se-
quences depends on the design of the selection protocols
and it is recognized that it is critical for the isolation of most
promising molecules. The ultimate conditions should be as
close as possible to the human physiological environment
and tumor structure. Thus, the in-vivo phage is designed to
select peptides that bind tumor vasculatures and to allow
distinction between common vascular cell receptors and
specific tissues’ receptor-binding peptides. Possible limita-
tions of using animal models that might not completely
represent the human vasculature profile have led to the
selection of peptide libraries directly on patients. This ap-
proach has already been reported for various cancers. Pep-
tide sequence binding to human interleukin (IL-11) receptor
on prostate cancers was characterized. However, the sur-
vival time was only extended by 20 days. The development
of selection protocols, ethical guidelines, and nanotechnolo-
gies will lead to the clinical translation of new imaging and
therapeutic technologies.
Other peptide-based targeting technologies have been
developed that provide several new platforms for generating
target specificity. Using the A-domain of various native
human receptors as a platform for modification, single-
chain proteins that can bind to multiple regions of target
molecules using multiple domains have been developed and
named avidity multimers or avimers [122]. In a proof of
concept study, an avimer specific for IL-6 (an acute phase
inflammatory mediator) was demonstrated to be non-immu-
nogenic in mice, as well as an inhibitor of IL-6 function in
vivo. More generally, this highlights the potential of this
class of ligand to antagonize proteins, which may ultimately
find application in cancer therapy. Functional, single-do-
main heavy chain antibodies, also known as nanobodies,
have been raised against cancer targets, which either antag-
onize receptor function [123] or deliver an enzyme for
prodrug activation [124], both for therapeutic benefit. Yet
another important strategy employing phage display tech-
nology used a 58 residue ␣-helical domain of staphylococ-
cal protein A as a platform to develop polypeptide targeting
ligands named affibodies [125]. Affibodies against a variety
of cancer-related targets have been developed and are now
commercially available, including: EGFR, HER2, and
transferrin.
Small molecule targeting
In general, small molecules are very attractive as target-
ing ligands due to their low cost and ease to conjugate with
drugs such as imaging probes (quantum dots, etc.) and
nanoparticles [126,127]. The small size of the targeting
ligand allows the functionalization of multiple molecules on
 F. Alexis et al. / Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85
single nanoparticles. In this regard, folic acid and sugar
molecules have been extensively used. The vitamin folic
acid is required for cell survival and folate receptors are
homogeneously over-expressed in many cancer cells
[128,129]. Folic acid receptors (FR) interact with high af-
finity folic acid conjugates and transport the conjugates via
receptor mediated endocytosis, before it recycles back to the
cell surface [130]. Thus, it is not surprising that folate-
targeted nanoparticles have shown to be effective in a num-
ber of tumors using liposomes [35,131–133] or polyplexed
nanosystems [134,135]. However, immunochemistry stud-
ies have shown the overexpression of folate receptors in
normal tissues like placenta and kidneys. This raises some
issues for possible clinical translation of folate bioconju-
gates. In addition, folate-targeted stealth nanoparticles need
to be carefully designed to avoid possible steric hindrance
of free PEG chains. Ideally, the folate ligand should be
accessible to enhance the specific binding of folate biocon-
jugates [136]. Cell surface lectins have been shown to be
overexpressed on numerous cancer cells [137,138] and to
internalize glycoconjugates via a receptor mediated endo-
cytosis mechanism. The presence of cell surface membrane
lectins, which recognize specific sugar molecules (lactose,
galactose, mannose) represents an interesting approach for
targeted delivery or imaging. However, to compensate for
the weak binding affinity of carbohydrates, multiple or
multivalent molecules should be conjugated to the nanocar-
rier. Targeting efficacy of galactosylated macromolecular
carriers were shown to depend on galactose ligand concen-
tration [139]. In addition, the conjugation of galactoside
residues with N-(2-hydroxypropyl) methacrylamide copol-
ymers (HPMA) resulted in a significant increase in three
different colon cancer cells [140]. Surprisingly, the trivalent
galactoside bioconjugates did not show higher binding af-
finity. Recently, Weissleder et al. [54] have demonstrated
that small molecules can change the biological affinity of
nanoparticles used for imaging specific tissues. Using a
high-throughput screening based on surface chemistry, 146
different small molecules (ⱕ500 Da) were conjugated to
nanoparticles and the results showed that very small mole-
cules can drastically affect the binding of nanoparticles to
different cell lines and pancreatic tumor xenografts.
Aptamers
Nucleic acid aptamers are single stranded DNA, RNA or
unnatural oligonucleotides that fold into unique structures
capable of binding to specific targets with high affinity and
specificity [141]. Nucleic acid aptamers are a novel class of
ligands [142] that are small (10 –20 kDa), non-immuno-
genic, easy to isolate, and exhibit high specificity and af-
finity for their target antigen, at best in the picomolar scale.
In addition, aptamers are synthetic molecules that can be
easily modified and scaled up for synthesis. The utility of
aptamers for possible clinical translation has been possible
due to chemical modification allowing a higher stability.
81
Systemic evolution of ligands by exponential enrichment
(SELEX) (⬃1015 different sequences) has been shown to be
very powerful for the selection of high affinity targeted
ligands, and multiple selection protocols are reviewed else-
where [143]. The unique antigen-aptamer binding mecha-
nism has led to the isolation of ligands with high binding
affinity. It was shown that aptamer-antigen interaction oc-
curs by a complex mechanism involving the folding of the
aptamer [144] on its antigen like a “paper clip”. In the short
time since Szostak and coworkers [145] and Tuerk and Gold
[146] independently described the ground-breaking meth-
odology for in vitro evolution of aptamers, this class of
ligands has emerged as an important arsenal in research and
clinical medicine. Aptamers offer the substantial advantage
of synthetic materials that can be produced in preparative
scale with high purity and reproduction. The consideration
of such favorable characteristics of aptamers has resulted in
their rapid progress into clinical practice. We began to
exploit this class of molecules for targeted delivery of con-
trolled drug releasing polymer vehicle. Recently we de-
scribed the first proof-of-concept drug delivery vehicles
utilizing aptamers for targeted delivery of drug encapsulated
nanoparticles [21], and subsequently showed the efficacy of
similarly designed nanoparticles against prostate cancer tu-
mors in vivo [22]. The efficacy study shows that a single
intratumoral injection of aptamer targeted nanoparticles
loaded with docetaxel allowed all the treated mice to sur-
vive more than three months in contrast to other controls.
More recently, we reported a novel strategy for targeted
doxorubicin delivery to cancer cells using an aptamer-Dox
physical conjugate [147]. Doxorubicin is known to interca-
late within the DNA strand due to the presence of flat
aromatic rings of this molecule. We demonstrated that
doxorubicin could be intercalated in aptamers and released
over a period of 6 hours and, most importantly, without
altering its affinity to bind to PSMA antigens.
Other strategies involving aptamers in cancer therapy
have included targeting the growth factors PDGF [148,149]
and VEGF [150] for delivery of therapeutic or diagnostic
agents, discovering new cancer-associated antigens using
cell-SELEX [151], and delivering toxins [152] or siRNA
[153].
Summary
The principal goals of cancer nanotechnology are to
develop safer yet more effective diagnostic and therapeutic
modalities for the fight against cancer. Drug encapsulated
targeted nanoparticles are a promising class of therapeutics
with the potential to improve efficacy and safety of cur-
rently available drugs. There are also opportunities to create
entirely novel therapeutics using nanotechnology. There is
an increasing arsenal of nanotechnology platforms under
evaluation including polymeric nanoparticles, liposomes,
nanoshells, dendrimers, and polynucleotide nanoparticles.
82 F. Alexis et al. / Urologic Oncology: Seminars and Original Investigations 26 (2008) 74 – 85
There are also increasing targeting molecule platforms that
have emerged for use in the engineering of nanocarriers.
These include antibody and antibody fragments, peptides,
aptamers, small molecules, and carbohydrates. The intrinsic
properties of the nanocarriers including the density of li-
gands on their surface might affect the treatment efficacy
and should be optimized. Targets within the vasculature are
readily accessible for bioconjugates to be internalized, but
physical and physiological barriers in solid tumors might
limit the diffusion of therapeutic nanocarriers to reach the
target cells. Each class of nanoparticles might be useful for
a particular application based on specific physiological en-
vironment and clinical requirements. Hence, multifunc-
tional nanocarriers are now emerging to combine radiother-
apy and chemotherapy, with or without imaging properties.
To move these technologies forward, novel approaches are
needed to accelerate the discovery process, such as synthe-
sis and screening automation. Future areas of nanotechnol-
ogy innovation for cancer treatment include the develop-
ment of bioresponsive and self-regulated delivery systems.
These are truly exciting times for medicine and, with con-
tinual support, medicine will continue to be a major bene-
ficiary of nanotechnology for years to come.
Acknowledgments
This work was supported by NIH/NCI CA119349, NIH/
NIBIB EB003647, and a development grant from the DFHCC
Prostate SPORE.
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