Combinatorial nanoparticles for cancer diagnosis and therapy.

Abstract

Nanotechnology when engineered together with biotechnology opens a fascinating field with applications in diverse areas such as drug targeting and delivery, medical imaging, biosensing, biomaterials and nanotechnology. Conjugating nanoparticles with biomolecules like QD-herceptin conjugates or QD-aptamer (Apt)-DOX conjugates provides many opportunities for improving many of the current challenges in cancer diagnosis and therapy. This paper reviews combinatorial nanoparticles designed and formulated for cancer imaging and therapy, including inorganic nanoparticles (quantum dots, iron oxide particles, gold nanoparticles and silica and carbon nanoparticles), polymeric nanoparticles (PLGA, PLGA-PEG, PAMAM), liposomes and lipid nanoparticles. These nanoparticles are multifunctional in nature and combine two or more functions like targeting, imaging and therapy. In this review, we have classified these combinatorial targeted nanoparticles into inorganic, polymeric and liposome based nanosystems.

Full text

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3714 Current Medicinal Chemistry, 2012, 19, 3714-3721

1875-533X/12 $58.00+.00
© 2012 Bentham Science Publishers
Combinatorial Nanoparticles for Cancer Diagnosis and Therapy
A. Mukerjee, A.P. Ranjan and J.K. Vishwanatha*
Department of Molecular Biology & Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences,
University of North Texas Health Science Center, Fort Worth, Texas, 76107, USA
Abstract: Nanotechnology when engineered together with biotechnology opens a fascinating field with applications in diverse areas such
as drug targeting and delivery, medical imaging, biosensing, biomaterials and nanotechnology. Conjugating nanoparticles with
biomolecules like QD-herceptin conjugates or QD-aptamer (Apt)-DOX conjugates provides many opportunities for improving many of
the current challenges in cancer diagnosis and therapy. This paper reviews combinatorial nanoparticles designed and formulated for
cancer imaging and therapy, including inorganic nanoparticles (quantum dots, iron oxide particles, gold nanoparticles and silica and
carbon nanoparticles), polymeric nanoparticles (PLGA, PLGA-PEG, PAMAM), liposomes and lipid nanoparticles. These nanoparticles
are multifunctional in nature and combine two or more functions like targeting, imaging and therapy. In this review, we have classified
these combinatorial targeted nanoparticles into inorganic, polymeric and liposome based nanosystems.
Keywords: Nanotechnology, cancer therapy, imaging, inorganic nanoparticles, polymeric nanoparticles, liposomes.
1. INTRODUCTION
Cancer remains one of the leading causes of death in the world.
Development of human cancer is a multi process event involving
various abnormalities at both genetic and cellular levels which
facilitates growth and progression of tumors. Despite advances in
our understanding of molecular and cancer biology, radiotherapy,
chemotherapy and conventional surgical procedures, the overall
survival rate of cancer patients have not improved significantly in
the past two decades [1]. There is an urgent need to develop novel
technologies for early detection and personalized treatment of
cancers in order to increase patient survival. Differences in the
expression of certain cellular receptors and/or proteins between
normal and tumor cells represent a great opportunity for targeting
nanoparticles to those surface molecules.
Recent advances in nanoscience and nanotechnology have led
to the development of combinatorial nanosystems. It is highly
desirable that nanoparticles can not only provide sensitive imaging
and selectively deliver anticancer drugs to tumor sites but also
specific targeting [2-5]. To achieve such specific targeting,
monoclonal antibodies seem like the ideal choice since they were
first shown in 1975 to be able to bind to specific tumor antigens [6]
but transitioning of these antibodies into tools for treatment of
cancer took a long time. Scientists over the world believe that these
antibody directed therapies for cancer targeting will lead to its
transition from clinic to its commercialization in the near future [7].
Also, these antibodies can themselves play a role in therapeutics
besides their ability to serve as carriers for drug delivery systems
for a more effective cancer therapy. These strategies take advantage
of the differences between a malignant cell and a normal cell like
uncontrolled proliferation, angiogenesis, tissue invasion and
metastasis [8].
Theranostic nanomedicine is emerging as a promising thera-
peutic strategy which combines therapy with diagnosis. This paper
reviews combinatorial nanoparticles designed and formulated for
cancer imaging and therapy, including inorganic nanoparticles,
polymeric nanoparticles and liposomes and lipid nanoparticles.
Figure 1 schematically illustrates some of the combinatorial
nanoparticle-systems. Combinatorial approach combines two or
more functions like targeting, imaging and therapy and can provide
a basic framework for fabricating specific nanosystems based on
factors such as the drug characteristics, targeting, tumor location,
and vascularity. This takes the benefit of the high capacity of

*Address correspondence to this author at the Department of Molecular Biology &
Immunology, Director, Institute for Cancer Research, Graduate School of Biomedical
Sciences, University of North Texas Health Science Center, Fort Worth, Texas 76107,
USA; Tel: 817-735-0477; Fax: 817-735-0243;
E-mail: Jamboor.vishwanatha@unthsc.edu
nanosystems to deliver drug molecules, targeting and/or imaging
agents. Recent literature suggests that such nanodelivery platforms
have shown promising potential in controlling stability, release
pattern, altering pharmacokinetic and pharmacodynamics profiles,
and lowering toxic effects of cancer therapeuticals [9, 10]. The
resulting nanosystems are capable of diagnosis, drug delivery and
monitoring of therapeutic response, are expected to play a
significant role in the genesis of the era of personalized medicine.
Some of the advantages of such combinatorial nanoparticles versus
mono-nanoparticles are outlined in Table 1.
2. COMBINATORIAL INORGANIC NANOPARTICLES
2.1. Quantum Dots (QDs)
The first report on using peptide conjugated to Quantum dot
(QD) to target receptors on lungs and blood vessels with exquisite
binding specificity was given by Akerman et al [11]. He showed
ZnS-capped CdSe QDs coated with a lung-targeting peptide could
accumulate in the lungs of mice after i.v. injection, whereas two
other peptide conjugated QDs could specifically direct them to
blood vessels or lymphatic vessels in tumors [11]. Park et al. [12]
co-encapsulated QDs and iron oxide nanoparticles along with
Doxorubicin (DOX), into PEGylated phospholipid micelles. These
conjugates were then attached with a tumor-homing peptide F3, and
were injected into an MDA-MB-435 xenograft model. Results
showed successful tumor targeting due to F3 peptide alongwith
optical and Magnetic resonance (MR) imaging modalities with
these particles.
Xiaohu Gao in 2004 demonstrated the development of
combinatorial nanoparticle probes based on semiconductor QDs for
cancer targeting and imaging in living animals [13]. Luminescent
QDs were encapsulated within an ABC triblock copolymer which
was further linked to tumor-targeting molecules like PSMA
antibody. In vivo targeting studies of human prostate cancer grown
in nude mice indicated that these combinatorial probes accumulated
at tumors both by the enhanced permeability of tumor sites and by
antibody based binding to cancer-specific cell surface marker
proteins. In another similar work, QDs-Herceptin conjugates were
synthesized by Nurunnabi et al. [14]. The CdTe/CdSe QDs were
coated with PEG-10,12-pentacosadiynoic acid (PEG-PCDA), which
was further crosslinked the coating shell by UV irradiation thereby
stabilizing it. These combinatorial nanoparticles were tested on an
MDA-MB-231 tumor model and results revealed efficient tumor
targeting rate and impressive therapeutic effects.
In an interesting study, a QD-aptamer (Apt)-DOX conjugate
was investigated by Bagalkot et al. [15], for its combinatorial use in
cancer imaging, therapy and therapy monitoring. In this study, A10
RNA aptamer was linked to the QD surface and DOX was

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Combinatorial Nanoparticles for Cancer Diagnosis and Therapy Current Medicinal Chemistry, 2012 Vol. 19, No. 22 3715





















Fig. (1). Schematic representation of various types of combinatorial nanoparticle-systems.

Table 1. Comparative Advantages of Combinatorial Nanoparticles Over Mono-Functional Nanoparticles.

Advantages Combinatorial Nanoparticles Mono Nanoparticles
Variation in payloads Multiple payloads of therapeutic and diagnostic agents Single payload
Simultaneous Diagnosis and
Treatment
Yes No
Simultaneous Targeting and
Therapy
Yes No
Therapy and Post Imaging Yes No
Modular Platform
Allows for screening different drug and imaging agent combinations within same
nanosystem
No such platform available with single load
Enhanced Targeting Allows the ability for synergistic combination
of passive and active targeting
Passive targeting in most cases
Personalized Therapy Ease in fabrication of nanosystems
for personalized therapy
Difficult to design personalized systems
Cancer Drug Resistance
Treatment
Multi drug formulation helps in treating drug resistance by either activating cells or
using non-resistant drug along with resistant drug
Single drug will not help in the case of drug
resistance
Design Traceable Therapeutic
Nanoparticles
Imaging moiety will provide traceability along with therapeutic efficacy Allows either therapeutic moiety or imaging agent
Design Activable Nanoparticles
for Imaging
Activable imaging capability by using different laser activation for different dye
combinations
Possible with some restrictions in case of imaging
based nanoparticles only

intercalated within the aptamer sequence. This system was used to
target PSMA. The results reported attenuation of fluorescence from
QD and DOX because of their interaction with DOX and RNA,
respectively, forming a quenched nanosystem. When such particles
were delivered into targeted tumor cells, DOX gradually got
released from the system, which initiated therapeutic functions and
thereby also recovered the QD fluorescence. Yuan et al. [16] coated
methotrexate (MTX) onto QD surfaces in order to induce
photoluminescence quenching. MTX was coated via simple
reversible physical adsorption. When such a system was delivered
in cells and MTX came in contact with DNA, it got released from
the surface of QD. This release of MTX led to a restoration of the
photoluminescence, which could be useful for imaging or
monitoring purposes.
2.2. Iron Oxide Particles
IONPs have unique magnetic properties which can be harnessed
for achieving improved accumulation under the effect of an external
magnetic field. This trait has been utilized by many researchers as a
targeting mechanism to improve drug delivery efficiency both in
animals [17, 18] and in humans [19, 20]. IONPs are flexible for
coupling with various drug molecules. Using this property, Zhang
et al. coupled an anti-cancer drug, MTX, on the surface of IONP
Liposome
Nanoparticle Nanocapsule
Inorganic?–Polymer?combination
Liposome?based
Polymer??based
Inorganic?based
Imaging?moiety/
Quantum?dot?
Therapeutic?moiety?1
Therapeutic?moiety?2
Polymer
Lipid?bilayer
Silica/iron?oxide/
Quantum?dots/gold
Ligand
Targeting?moiety
Inorganic?–Lipid?combination

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3716 Current Medicinal Chemistry, 2012 Vol. 19, No. 22
Mukerjee et al.
[21-23]. Results from the in vitro studies showed that the particles
accumulated in lysosomes within the cells. It was in the lysosomes
that MTX molecules were released due to the low pH and the
presence of proteases. Such nanoparticles served both purposes of
therapy and imaging. Hwu et al. [24] carried out similar study
where he coupled paclitaxel (PTX) to IONP surfaces through a
phosphodiester moiety at the (C-2?)-OH position. They reported an
average of 83 PTX molecules per nanoparticle and the release of
the PTX was found to be more effective when exposed to
phosphodiesterase. Further, Cheon et al. modified IONPs by using
meso-2,3- dimercaptosuccinic acid (DMSA) and used SMCC as the
crosslinker to attach Herceptin, an antibody against the HER2/ neu
receptor which is overexpressed in breast cancer, to the particles
[25, 26], where Herceptin provided the targeting modality. Several
research groups have been studying antibody conjugated inorganic
nanoparticles for the past few decades, especially magnetic
nanoparticles [27-34]. Gao et al developed superparamagnetic iron
particles 5–30 nm in size which could be functionalized through
surface coating with amphiphilic triblock polymers for providing
the functional groups necessary for conjugating tumor-targeting
antibodies [13]. Most of these combinatorial nanoparticles found
use in in vivo imaging as they retained properties of both antibody
and magnetic nanoparticles. In another report, magnetism-
engineered iron oxide (MEIO) nanoparticles were conjugated with
Herceptin. These multifunctional particles produced high sensitivity
imaging of HER2/neu due to in vivo targeting [35]. A more recent
study was published by the Sun group [36]. They prepared porous
IONPs by controlled oxidation and acid etching of Fe particles.
Cisplatin was then loaded into the particle-cavities, and Herceptin
was coupled to the particle surfaces to confer targeting specificity.
The resulting combinatorial system showed selective affinity to
ErbB2/Neu-positive breast cancer cells and a sustained cytotoxicity
attributable to the controlled release of cisplatin [36].
The major limitation of antibody conjugated to IO nanoparticles
is the large size of the intact antibody which hinders permeation of
these particles through the vasculature into tumor cells. The large
size of the antibody also poses problems for efficient conjugation to
the surface of IO nanoparticles. Peptides or biomolecules with
small molecular weight are more advantageous as are compatible in
size with the small IO nanoparticles and can be conjugated to
produce targeted IO nanoparticles. Much research has been carried
out in this area. As early as 1998, Soroceanu et al. started studying
peptide conjugation to IO nanoparticles. Chlorotoxin (Cltx) is a
peptide that specifically binds to MMP-2 on the surface of cells,
which is overexpressed in gliomas and plays a role in cancer
invasion [37-39]. Sun et al. conjugated Cltx to IO nanoparticles
using a covalently bound bifunctional PEG polymer. Results
showed 10-fold higher internalization of the Cltx conjugated IO
nanoparticles by 9L glioma cells than that of the unconjugated
nanoparticles. Further, in vivo MRI showed that the tumor contrast
enhancement was significantly higher in the mouse injected with
Cltx-targeted IO nanoparticles than in the mouse receiving
unconjugated nanoparticles [40].
Similarly, underglycosylated mucin-1 antigen (uMUC-1) is an
early tumor marker that is overexpressed on almost all human
epithelial cell adenocarcinomas. Moore et al. synthesized a peptide,
EPPT1, which distinctively recognizes uMUC-1 and conjugated it
to superparamagnetic iron oxide (SPIO) nanoparticles. For conju-
gation, they used the dextran coat on the crosslinked superparama-
gnetic iron oxide nanoparticles (CLIO). These combinatorial
nanoparticles showed a impressive results with a significant T2
signal reduction 24 hours post injection of targeted CLIO
nanoparticles in uMUC-1-positive LS174T tumors, while no
significant change was seen in uMUC-1-negative U87 tumors. [41].
In another study, LHRH conjugated SPIO nanoparticles were
synthesized by Leuschner et al. which was found to specifically
accumulate in primary tumor cells and metastatic cells. The uptake
of LHRH conjugated SPIO nanoparticles were determined to be 12-
fold higher than unconjugated SPIO nanoparticles in vitro. Their in
vivo data showed 7.5-fold higher accumulation in tumors and 11-
fold higher accumulation in metastatic lung cells for LHRH
conjugated as compared to nontargeted SPIO nanoparticles.
Another advantage of conjugating LHRH to SPIO nanoparticles
occurred due to the neutralizing effect of LHRH on SPIO which
resulted in increasing the circulation time. This study illustrated
clearly that LHRH-conjugated SPIO nanoparticles could be used as
MRI contrast agent to detect metastatic breast cancer cells in vivo
with high sensitivity [42].
Angiogenesis plays a critical role in the development of tumors.
?vß3 integrin is an ideal target for in vivo tumor imaging since ?vß3
integrin has been reported to be a marker of angiogenesis and its
level of expression correlates with tumor grade. To make
conjugated IO nanoparticles, Zhang and his group coated the
surface of IO nanoparticles with 3 aminopropyltrimetho-xysilane
(APTMS) which had functional amino groups. The Arg-Gly-Asp
(RGD) peptide binds to the ?vß3 integrin receptor. This peptide was
then conjugated to APTMS-coated USPIO nanoparticles to produce
combinatorial inorganic nanoparticles capable of specific targeting
and tumor imaging. Following systemic administration in nude
mice bearing tumors, these
demonstrated targeting capability to ?vß3 integrin positive sites
which could be detected and imaged by 1.5-T MR scanner [43].
RGD-USPIO nanoparticles
Another research group synthesized the tumor-homing peptide
CREKA (Cys-Arg-Glu-Lys-Ala) which has the property of forming
a distinct meshwork within the tumor stroma. CREKA was then
conjugated to SPIO nanoparticles and it was found that these
conjugated nanoparticles accumulated in both tumor vessels and
stroma, resulting in intravascular clotting in tumor blood vessels
which in turn attracted more nanoparticles into the tumorthereby
amplifying the targeting. The CREKA targeted-SPIO nanopar-ticles
possessed several advantages of high specificity for tumor homing,
enhanced MR imaging in tumor besides local embolism resulting in
physical blockade of tumor vessels. Further, this clotting caused by
CREKA-SPIO nanoparticles in tumor vessels could improve tumor
detection by optical imaging techniques [44].
The vitamin folic acid (FA) is a very good targeting agent since
its receptor is mostly overexpressed on the surface of many human
tumor cells. Choosing FA as the targeting agent has the advantage
as it is compatible in both organic and aqueous solvents which
makes it easy to couple with nanosystems and it lacks immuno-
genicity [45]. Sun et al. coated the surface of IO nanoparticles with
heterobifunctional PEG 600 and then coupled FA to the
nanoparticles through an amide linkage at the free terminus of PEG.
Their results showed 12-fold more FA-IO nanoparticles in folate
receptor-positive human cervical carcinoma HeLa cells than
nontargeted IO nanoparticles [46]. Another report illustrates that
SPIO-PEG-FA could target human nasopharyngeal epidermoid
carcinoma (KB) cells both in vitro and in vivo [47].
IONPs can by themselves play a dual role in imaging and
therapy as it can cause hyperthermia. The working concept is that in
an external alternating magnetic field, IONPs can convert
electromagnetic energy into heat [17, 18]. As tumor cells are more
vulnerable to elevated temperature than normal cells, this
characteristic property can be used very effectively for tumor
therapy [48, 17, 18]. In a study reported by Ito et al., phospholipid
coated IONPs were injected into a subcutaneous tumor model in
F344 rats, and were subjected to external alternating magnetic field
[18]. This resulted in raising the temperature of the tumor to more
than 43°C which in turn caused selective regression of tumor due to
the administered IONPs. To add a targeting functionality, an anti-
human MN antigen-specific antibody was chemically linked onto
IONP surfaces. These INOPs were administrated systemically into
tumor bearing mice. The particles showed high tumor uptake, due

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Combinatorial Nanoparticles for Cancer Diagnosis and Therapy Current Medicinal Chemistry, 2012 Vol. 19, No. 22 3717
to an antibody–antigen interaction, and induced efficient tumor
hyperthermia when exposed to an AMF [18, 49]. In another study
by Primo et al., a photodynamic therapeutic agent, Zn- Pc, was
loaded onto CoFe2O4 nanoparticles, which showed improved
hyperthermia effects as compared to IONPs [50]. When tested in
vitro with J774-A1 macrophage cells, a combined toxicity from Zn-
Pc and magneto-hyperthermia was reported.
Another area of research for engineering combinatorial
nanosystems included co-encapsulating drug molecules with IONPs
into polymeric matrices. Jain and group [51] loaded doxorubicin
(DOX) and paclitaxel, PTX, along with oleic acid coated IONPs,
into pluronic-stabilized polymeric nanoparticles. In a similar study,
Yu et al. encapsulated DOX into IONPs coated with anti-biofouling
polymer [52]. These DOX loaded nanoconjugates showed better
pharmacokinetics and therapeutic effects than DOX alone when
tested in a Lewis lung carcinoma xenograft model. Protein
molecules, such as human serum albumin (HSA) have also been
coated on IONPs [53]. Utilizing the excellent binding capacity of
HSA, many lipophilic pharmaceuticals could be loaded into such
nanoplatforms to yield theranostic agents.
2.4. Gold Nanoparticles
Another class of inorganic nanoparticles that find use in
photothermal therapy are gold nanoparticles. They have distinct
surface plasmon resonance which converts light into heat and kills
cancerous cells and hence can play a role as photothermal therapy.
Chen et al. reported that Au nanocages coated with PEG could
accumulate in a U87MG xenograft model and could increase the
tumor surface temperature to 54°C when it was exposed to Near
Infra Red light [54]. Further, in another study, Li attached [Nle4,D-
Phe7]?-MSH (NDP-MSH), a ?-melanocyte stimulating hormone
(MSH) analog, to gold nanoparticles and administrated such
nanosystems to a B16/F10 melanoma model [55]. Histology results
showed specific high accumulation of particles due to NDP-MSH in
the tumor. Efficient ablation of B16/F10 melanoma was confirmed
in the tumor exposed to laser illumination depicting validation of
photothermal therapy. In continuation of their study, they prepared
combinatorial nanoconjugates by conjugating folic acid on the
surface of Au nanoshells for tumor targeting. These combinatorial
particles could be used as light controllable siRNA carriers [56]
since the siRNA in here was prethiolated at the 5? end of the sense
chain and was conjugated to the particles surface via thiol–Au
interaction. Such nanoconjugates when uptaken by cells and
exposed to NIR light irradiation, lightinducible siRNA release and
subsequent NF-?B p65 downregulation (siRNA against p65) both
in vitro and in vivo was observed.
Prabaharan et al. synthesized a nanosystem that consisted of a
gold nanoparticle core with hydrophobic PASP inner shell, and a
hydrophilic, folate-conjugated PEG outer shell (PEG-OH/ FA) for
tumor targeting and drug delivery [57]. Further, DOX was
covalently conjugated onto the hydrophobic inner shell by acid-
cleavable hydrazone linkage. Such a combinatorial nanosystem
acheived both tumor targeting and therapy via an intracellular drug
release mechanism.
2.5. Silica and Carbon Nanoparticles
Silica nanoparticles are capable of encapsulating drug
molecules for efficient drug delivery. In a study, Roy et al.
encapsulated a hydrophobic photosensitizing anticancer drug, 2-
devinyl-2- (1-hexyloxyethyl)pyropheophorbide (HPPH), into silica
matrices [58]. It was found that the HPPH is more fluorescent in the
silica matrices than in the native form, and can efficiently kill
cancer cells when irradiated with a laser and hence function as a
theranostic agent. The same group then co-encapsulated HPPH and
a two- photon absorbing dye, 9,10 bis[4?-(4??-aminostyryl)
styryl]anthracene (BDSA), into silica nanoparticles [59]. Results
revealed that BDSA can efficiently up-convert the NIR light and
transfer the energy to HPPH within the particle itself to activate the
HPPH's PDT function. Silica nanoparticle can also be formulated in
a mesoporous form with controlled pore sizes which can be used to
trap small drug molecules via physical interactions [60, 61]. An
interesting modification has been researched wherein these
mesopores have been capped after drug loading to inhibit premature
drug release. The Lin group prepared mesoporous silica
nanoparticles loaded with PTX. The mesopores were subsequently
capped with Au NPs [62]. Further, this gold nanoparticle capping
was designed to be photolabile which could be uncapped to release
PTX molecules when photoirradiated. Other research groups have
exploited this mechanism based on activatable gatekeepers in QDs
[63], IONPs [64], courmarin [65] and diethylenetriamine [66]. Park
et al. published his work [67] on the preparation of luminescent
porous silicon nanoparticles (LPSiNPs) by HF etching of single-
crystal silicon wafers followed by ultrasonication and activation of
luminescence in an aqueous solution. The luminescence was
generated by quantum confinement effects. DOX was encapsulated
in these luminescent porous nanoparticles and their drug release and
cytotoxicity were studied in vitro.
The Dai group prepared theranostic nanoconjugates by
conjugating siRNA to phospholipid coated carbon nanotubes via a
disulfide bond, which was susceptible to enzymatic breakage in the
endolysosome [68]. These nanoconjugates showed high transfection
efficiency in human T cells and primary cells, which are difficult to
transfect [69]. The same group then used PTX as a therapeutic
agent and coupled it via a cleavable ester bond to the PEGylated
nanotube surface. This construct was tested in a murine 4T1 breast
cancer model and the results depicted a 10-fold increase in tumor
accumulation than PTX alone followed by improved tumor
suppression than clinically used Taxol [70]. They also reported the
coupling of other agents like Pt(IV) prodrug [71] PEGylated carbon
nanotubes to improve the pharmacokinetics and therapeutic effects.
3. COMBINATORIAL POLYMERIC NANOPARTICLES
Biocompatible and biodegradable polymers have been reported
to be very well suitable for targeted drug delivery. A new
multifunctional hybrid nanosystem has been reported by Yang and
his group. In this study, they combined magnetic nanocrystals,
anticancer drugs and biodegradable amphiphilic block copolymers,
all in one combinatorial nanosystem. Hydrophobic magnetic
nanocrystals (MnFe2O4) and DOX were simultaneously incor-
porated into poly(lactic-co-glycolic acid) (PLGA)-PEG-COOH by a
nanoemulsion method and then human epidermal growth factor
receptor 2 (HER) was conjugated to yield HER-nanosystem. They
showed sustained drug-release profiles owing to polymer
degradation. Results showed that these multifunctional hybrid
nanosystems were delivered in a target-specific manner to
overexpressed HER2/neu receptors on NIH3T6.7 cells in vivo. The
antibody-conjugated nanoparticles demonstrated efficient therapy
and ultrasensitive targeted detection by MRI in both in vitro and in
vivo models [72].
Ross and his group prepared a unique light-activated
theragnostic nanosystem [73]. These combinatorial nanoparticles
consisted of a polyacrylamide core and encapsulated a PDTagent,
Photofrin, iron oxide for MRI imaging, a F3-peptide for tumor
vascular homing. PEG was introduced to increase the circulation
time [73]. The particles were also fluorescently labeled with Alexa
Fluor 594 to determine intracellular uptake. Results indicated that,
F3-targeted nanoparticles were significantly internalized into
MDA-MB435 breast cancer cells, 4 hour post incubation. In MRI
study, the F3-targeted particles accumulated in the glioma (9L)
tumor within 2 hours as compared to the non-targeted particles
which got washed out of the tumor with no accumulation. In a
therapeutic study, F3-targeted nanoparticles brought about massive

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3718 Current Medicinal Chemistry, 2012 Vol. 19, No. 22
Mukerjee et al.
regional necrosis and a significant decrease of tumor volume.
Further, they improved the survival time more than either non-
targeted nanoparticles or just free Photofrin or laser irradiation.
More importantly, 60 days post-treatment, a complete eradication
of brain cancers was observed in three out of five animals.
Polymeric micelles are self-assembled nanoparticles from
amphiphilic block copolymers. They have unique characteristics of
high water-solubility, high drug loading capacity and low toxicity.
Using these delivery systems, Nasongkla et al. developed novel
combinatorial polymeric micelles where SPIO nanoparticles were
encapsulated within these micelles along with DOX, which could
be released through a pH-dependent mechanism. Since SPIO and
DOX are both hydrophobic in nature, encapsulating them within
micelles prevents nonspecific uptake by RES. Furthermore, the
cRGD ligand that can target ?vß3 integrins on tumor endothelial
cells was coupled to the micelle surface via a covalent thiol-
maleimide linkage. Once these combinatorial polymeric micelles
were internalized by target cells, DOX was released in cell nuclei.
This integrated micelle conjugate provided both targeted tumor
therapy and noninvasive imaging in vivo [74].
In another report on combinatorial polymeric nanosystems,
folate receptor targeted delivery of doxorubicin was described.
Polymeric micelles were synthesized from a self-assembled
poly(D,L-lactic-co-glycolic acid) (PLGA) and PEG diblock
copolymer. These nanosystems utilized the intrinsic fluorescence
properties of doxorubicin for imaging. The preparation of such
micelles included doxorubicin to be chemically conjugated to a
terminal end of PLGA. Folate was separately conjugated to a
terminal end of PEG and then the two diblock copolymers with
different functional moieties at their chains ends were physically
mixed with free base doxorubicin in an aqueous solution to form
mixed micelles with folate exposed on the micellar surface, while
doxorubicin was physically entrapped in the core of micelles [75].
In another report, micelles prepared using PEG-PCDA [10,12-
pentacosadiynoic acid) and
encapsulating near-IR quantum dot (QD) were prepared and such
system possessed properties of both imaging and specific targeting
[14].
PCDA-herceptin conjugates
An interesting work in combinatorial nanosystems was reported
by Medarova et al. [76]. In this study, thiolated siRNA was attached
to dextran particles using N-succinimidyl-3-(2-pyridyldithio)
propionate (SPDP) as a linking agent. To add more functionality,
the near infrared dye, Cy5.5, and myristoylated polyarginine
peptide (MPAP), a membrane translocation peptide, were attached
to the particle surface. The nanosystems were loaded with siRNA
that targets green fluorescence protein (GFP), and were then tested
in a mouse model bearing bilateral 9L-GFP and 9L-RFP tumors.
MRI and near-infrared fluorescence (NIRF) imaging were
performed, and results depicted appreciable accumulation of
particles in both tumors.
Another area of polymeric nanosystems are the dendrimers
which are repeated branched polymeric nanoparticles with a nearly
prefect three-dimensional structure that can be controlled in size,
shape, and terminal group functionality. These spherical
nanostructures have voids within them and can be synthesized by
polymerization with either divergent or convergent methods.
Fluorescein isothiocyanate entrapped polyamidoamine (PAMAM)
dendrimer conjugated to recombinant fibroblast growth factor-1 for
tumor targeting and imaging has been shown to be an effective
targeted delivery of chemotherapeutic drugs [77]. In another study,
PAMAM dendrimers entrapping 5-fluorouracil were synthesized.
Further, these dendrimers were coupled with folic acid on the
surface to achieve site-specific enhanced drug localization [78].
4. COMBINATORIAL LIPOSOMAL NANOPARTICLES
Liposome formulations are one of the commonly investigated
formulations for the incorporation of imaging, targeting and drug
delivery modalities [79]. They are vesicles composed of
amphiphilic phospholipids, which self-assemble into bilayers
having central aqueous space.
QDs modified with liopofectamine (a liposomal formulation)
have been successfully used as gene delivery vehicles [80] or with
other positively charged polymers. In one study, QDs were
encapsulated in poly(maleic-anhydride-alt-1-decene), and further
surface-modified with dimethylamino propylamine for imparting
positive charge [81]. Further, some carboxyls of these poly(maleic-
anhydride-alt-1-decene) coated QDs were converted to tertiary
amines with N,N-dimethylethylenediamine by Yezhelyev et al. The
resulting particles, held two functional groups on their surface
which facilitated various interactions that were highly responsive to
acidic endosome/ lysosome organelles. These QDs when used as
siRNA delivery vehicles showed a 10- to 20-fold increase in
silencing effect when compared with other common delivery
agents, such as lipofectamiane, JetPEI and TransIT [82]. For
labeling liposomes to be used as common contrast agents,
Gadolinium and QDs enclosed in the aqueous interior or chelated in
the liposomal bilayer have been used [83].
In a study, a mutant Raf gene which blocks endothelial
signaling and angiogenesis in response to multiple growth factors
was coupled to lipid-based cationic nanoparticles which was further
conjugated to an integrin avh-targeting ligand and tested in tumor-
bearing mice [84]. Results were compared for gene expression in
the tumor, lung, liver and heart for non-targeted particles, targeted
particles and targeted particles injected with excess soluble
targeting ligand.
Another research study used Dox, the commercial liposomal
doxorubicin formulation, to which the attached an antibody,
antibody F5, after modifying the liposomal surface with PEG. This
resulted in a coupled liposome system capable of targeting and
therapy. In vivo results in mice treated with F5-coupled Dox
showed a faster and greater regression in tumor volume as
compared to untargeted Dox [85]. To develop a strategy for
identification of the ideal ligands and to select internalizing
antibodies from phage libraries [86], this research group identified
two antibodies (F5 and C1) that bind to ErbB2, a growth factor that
is overexpressed in 20– 30% of human breast carcinomas and also
in other adenocarcinomas [85]. Another modification of Doxil was
made with the monoclonal nucleosome (NS)-specific 2C5 antibody
(mAb 2C5) that recognized many of the tumors by the tumor cell
surface-bound NSs. Specific targeting resulted in increased
accumulation followed by cytotoxicity of the liposomes towards
tumor cells [87].
CONCLUSION
In this article, we have reviewed the current status of
combinatorial multifunctional nanosystems as an emerging
powerful tool for cancer imaging and therapy. We have emphasized
the urgent need for the development of combinatorial approaches in
designing and synthesizing nanosystems capable of targeted drug
delivery for therapy and imaging. Combinatorial approaches have
several advantages over conventional methods in integrating
multiple components with varied properties into an ‘all in one’
nanoconjugate system. Although combinatorial nanoparticles seem
potential for drug and gene delivery, considerable challenges and
issues remain to be resolved like optimizing these multifunctional
nanosystems in terms of targeting and tissue accumulation.
Development of such multifunctional nanosystems using a

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Combinatorial Nanoparticles for Cancer Diagnosis and Therapy Current Medicinal Chemistry, 2012 Vol. 19, No. 22 3719
combinatorial approach will eventually turn out to be a more viable
and versatile strategy for the development of novel nanoplatforms
for successful cancer therapy and diagnosis.
FUTURE PERSPECTIVE
Major challenges remain for developing nanomaterials for
clinical diagnosis and therapy. These include minimizing toxicity,
comprehend how nanoparticles target certain tissues and factors,
studying how nanoparticles
microenvironments and finally developing a new generation of
medicine known as personalized theranostic medicine, quantifying
nanoparticles accumulation in a specific organ in vivo. Although
more research in interdisciplinary fields of pharmaceutical
engineers, biochemists, oncologists, clinicians is needed before
theranostic nanomedicine can be used in the clinic, the direction of
current research indicates that
nanoplatforms may transform and revolutionize the imaging,
diagnosis and treatment of cancer.
behave in biological
combinatorial theranostic
CONFLICT OF INTEREST
None declared.
ACKNOWLEDGEMENTS
This research was supported in part by grants from the
Department of Defense Breast Cancer Research Program
(BC075097) to Dr. Jamboor K. Vishwanatha and Susan G. Komen
Postdoctoral Fellowship (KG101213) to Dr. Anindita Mukerjee.
ABBREVIATIONS
APTMS = aminopropyltrimethoxysilane
CLSPIO = cross linked superparamagnetic iron oxide
Cltx = chlorotoxin
DMSA = dimercaptosuccinic acid
DOX = doxorubicin
FA = folic acid
HAS = human serum albumin
HPPH = 2-devinyl-2- (1-hexyloxyethyl) pyropheophorbide
IONPs = iron oxide nanoparticles
LHRH = luteinizing hormone-releasing hormone
MR = magnetic resonance
MTX = methotrexate
NS = nuclesome
PCDA = pentacosadiynoic acid
PSMA = Prostate specific marker antibody
PTX = paclitaxel
QD = quantum dot
SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-
1-carboxylate
SPIO = superparamagnetic iron oxide
u-MUC-1 = underglycosylated mucin-1
UV = ultraviolet
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Received: January 23, 2012

Revised: March 08, 2012 Accepted: March 16, 2012