Clinica Chimica Acta 436 (2014) 78–92

Contents lists available at ScienceDirect

Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

Targeted anticancer therapy: Overexpressed receptors

and nanotechnology
Mohd Javed Akhtar a,⁎, Maqusood Ahamed a, Hisham A. Alhadlaq a,b, Salman A. Alrokayan c, Sudhir Kumar d
a
King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia
b
Department of Medical Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
c
Department of Biochemistry, King Saud University, Riyadh 11451, Saudi Arabia
d
Department of Zoology, University of Lucknow, Lucknow 226007, India

a r t i c l e i n f o a b s t r a c t

Article history: Targeted delivery of anticancer drugs to cancer cells and tissues is a promising field due to its potential to spare
Received 12 February 2014 unaffected cells and tissues, but it has been a major challenge to achieve success in these therapeutic approaches.
Received in revised form 7 May 2014 Several innovative approaches to targeted drug delivery have been devised based on available knowledge in can-
Accepted 10 May 2014
cer biology and on technological advancements. To achieve the desired selectivity of drug delivery, nanotechnol-
Available online 15 May 2014
ogy has enabled researchers to design nanoparticles (NPs) to incorporate anticancer drugs and act as
Keywords:
nanocarriers. Recently, many receptor molecules known to be overexpressed in cancer have been explored as
Nanoparticles docking sites for the targeting of anticancer drugs. In principle, anticancer drugs can be concentrated specifically
Overexpressed receptors in cancer cells and tissues by conjugating drug-containing nanocarriers with ligands against these receptors. Sev-
Ligands eral mechanisms can be employed to induce triggered drug release in response to either endogenous trigger or
Anticancer drugs exogenous trigger so that the anticancer drug is only released upon reaching and preferentially accumulating in
Tumor microenvironment the tumor tissue. This review focuses on overexpressed receptors exploited in targeting drugs to cancerous tis-
sues and the tumor microenvironment. We briefly evaluate the structure and function of these receptor mole-
cules, emphasizing the elegant mechanisms by which certain characteristics of cancer can be exploited in
cancer treatment. After this discussion of receptors, we review their respective ligands and then the anticancer
drugs delivered by nanotechnology in preclinical models of cancer. Ligand-functionalized nanocarriers have de-
livered significantly higher amounts of anticancer drugs in many in vitro and in vivo models of cancer compared
to cancer models lacking such receptors or drug carrying nanocarriers devoid of ligand. This increased concentra-
tion of anticancer drug in the tumor site enabled by nanotechnology could have a major impact on the efficiency
of cancer treatment while reducing systemic side effects.
© 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2. Overexpressed receptors, their ligands, and drug delivery to cancer using nanotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.1. G protein-coupled receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.1.1. Bombesin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.1.2. Somatostatin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.1.3. Endothelin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.2. Integrin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.2.1. Integrin αvβ3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.2.2. Integrin α-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Abbreviations: Bn, bombesin; BnR, bombesin receptor; BR, biotin receptor; c(RGD-K), cyclic {Arginine-Glycine-Aspartic acid (RGD)} containing peptide; CA, cholic acid; DOX, doxoru-
bicin; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ETRB, endothelin receptor B; FA, folic acid; FGF, fibroblast growth factor; FGFR, fibroblast growth factor re-
ceptor; FR, folate receptor; FSH, follicle stimulating hormone; FSHR, follicle stimulating hormone receptor; NPs, nanoparticles; Nrp-1, neuropilin receptor-1; PEG, poly(ethylene glycol);
PLA, poly(lactic acid); PTMC, poly(trimethylene carbonate); PTX, paclitaxel; QDs, quantum dots; S1R, sigma receptor 1; S2R, sigma receptor 2; SRs, sigma receptors; SSTRs, somatostatin
receptors; Tf, transferrin; TfR, transferrin receptor.
⁎ Corresponding author at: King Abdullah Institute for Nanotechnology (KAIN), King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Tel.: +966 559981310 (mobile).
E-mail addresses: mjakhtar@ksu.edu.sa, mohd.j.akhtar@gmail.com (M.J. Akhtar).

http://dx.doi.org/10.1016/j.cca.2014.05.004
0009-8981/© 2014 Elsevier B.V. All rights reserved.
 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92 79

2.3. Folate receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

2.4. Transferrin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
2.5. Epidermal growth factor receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.6. Fibroblast growth factor receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.7. Sigma receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
2.8. Other overexpressed receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

1. Introduction
More than 10 million patients are diagnosed with new cases of can-
cer every year, and approximately 27 million new cases of cancer will
have been recorded by 2030 [1,2]. While cytotoxic chemotherapeutic
agents such as paclitaxel (PTX) and doxorubicin (DOX) effectively kill
cancer cells, they cannot distinguish cancer cells from normal cells.
This lack of selectivity leads to undesirable systemic toxicity when pa-
tients are exposed to the high dosages of cytotoxic agents required to
eradicate the tumor. Improving the selectivity of anticancer drug deliv-
ery to cancer cells and the tumor microenvironment while sparing nor-
mal cells and tissues is a major challenge in the effective treatment of
cancers of various tissues and organs. Marked differences are found in
cancer cells and tissues in terms of biochemical, molecular and physio-
logical features when compared with normal cells and tissues such as
differences in redox status, pH levels, expression of certain cell mem-
brane receptors, the leakiness of tumor tissues and the tumor vascula-
ture. Therefore, cancer stands out as a disease likely to benefit from
targeted drug delivery approaches exploiting these differences. These
differences between healthy and cancerous cells and tissues, or hall-
marks of cancer, have recently been reviewed [3].
Differences in normal and cancer biology are found at the level of
anatomy, biochemistry and molecular biology. The characteristic ana-
tomic features of tumor biology include the leakiness of blood vessels
and poor lymphatic drainage in tumor tissues. The morphology and
shape of blood endothelial cells are different in cancer vasculature due
to the presence of fenestrae between adjacent cells, and thus the lack
of contact inhibition. Most solid tumors, upon reaching a certain level
of growth, exhibit enhanced vascular permeability, ensuring a sufficient
supply of nutrients and oxygen to tumor tissues and outpacing the
growth of surrounding tissues. Blood vessels in tumors are often dilated
and convoluted, and compared with normal tissues, exhibit branching
patterns that feature excessive loops and arteriolar–venous shunts [4].
All these features enhance the permeability of blood vessels in tumors
compared with the vasculature in normal tissues, enabling the delivery
and accumulation of molecules and other substances in tumor tissues
that are generally not able to enter the vasculature in normal tissues.
This is called the enhanced permeability and retention (EPR) effect, a
phenomenon associated with the tumor vasculature. The net result of
the EPR effect for circulating molecules depends on their properties
including size, shape, charge, and polarity. The principal difference be-
tween EPR and passive localization lies in the characteristics of retention
and tissue clearance rather than uptake. Small drug molecules rapidly
penetrate into the tumor interstitial space, but in the absence of specific
binding to cellular proteins, drug is not retained and may be free to dif-
fuse out of the tissue back into the blood pool or the lymphatic system.
In contrast, macromolecules have smaller diffusion constants, reducing
the initial rate of tumor uptake but also tending to increase the half-life
of blood-pool circulation by enhancing tissue retention and decreasing
the rate of clearance [5]. Therefore, receptor–ligand interactions are
the most important aspect of active targeting of nanocarriers (or
nanoconjugates or macromolecules) and could be further potentiated
by EPR (Fig. 1). The biochemical conditions of acidity (lower pH value)
and hypoxia (lower concentration of oxygen) generally prevail in can-
cer cells and tumor tissues. The extracellular pH in tumor tissue is
slightly lower than that in normal tissue, and this difference has been
exploited to achieve pH-triggered drug release in tumor tissue [6]. A
diverse group of functional materials has been designed to accomplish
triggered drug release in response to other stimuli such as temperature
[7,8], redox potential [9], ultrasound [10], and light [11]. These mecha-
nisms of endogenously or exogenously triggered drug release [12] can
only be applied once the anticancer drug reaches and accumulates at
the desired site, i.e., tumor tissues. Molecular markers of cancer include
the differential expression of proteins residing in the cytosol, organelles
or membrane. The group of differentially expressed proteins includes
receptors that are specifically expressed or overexpressed in cancer
cells compared to normal cells. These overexpressed receptors have
provided important endogenous tools for exploitation in the active
targeting of drugs to cancer cells. Targeting nanocarriers to a particular
organ or tissue through the blood or lymph circulation is referred to
as primary targeting, while the accumulation around a cancer cell
is named secondary targeting and manipulating the uptake of
nanocarriers/drugs by cells and cellular compartments is known as ter-
tiary targeting [13]. This review focuses on overexpressed receptors
exploited for targeting drugs to cancer and the tumor microenviron-
ment (Table 1). We briefly evaluate the structure and function of
these receptor molecules, emphasizing the elegance of exploiting char-
acteristics of cancer in cancer treatment. After discussing these recep-
tors, their respective ligands are mentioned, followed by a review of
the anticancer drugs that have been delivered by nanotechnology in
preclinical models of cancer (Table 2).
2. Overexpressed receptors, their ligands, and drug delivery to
cancer using nanotechnology
Receptors overexpressed or specifically expressed in cancers of var-
ious tissues and cells are listed in Table 1. These receptors provide
unique opportunities to understand cancer biology and its treatment.
In attempts to treat cancer, overexpressed receptors are directly modu-
lated/inhibited by agents such as antibodies or antibody fragments, and
also by other small chemicals that directly bind these receptors and
block their activities. These treatment strategies thus block the consis-
tent unwanted stimulus for uncontrolled cell division, thus blocking
cancer progression. Other approaches to cancer therapy do not inten-
tionally interfere with receptor function, but rather exploit receptor
overexpression for the targeted delivery of effective anticancer drugs
that do not discriminate between cancer and normal cells. These
drugs can be guided by linking them to suitable ligands against such
overexpressed receptors. The field of nanotechnology has developed
the ability to synthesize an enormous variety of NPs, providing a plat-
form for guiding and carrying drugs to cancer cells and tumor tissues.
These NPs can be loaded with anticancer drugs and conjugated with li-
gands. Nanosized liposomes, dendrimers, micelles, metals, alloys, mix-
tures of inorganics and organics, and other materials have been
synthesized by nanotechnology for use in diverse applications such as
catalysis, sensing and medicine. NPs loaded with drug molecules and
conjugated with receptor ligands have been variously termed as nano-
conjugates, nano-formulations, nano-carriers, etc. Table 2 summarizes
some recent outcomes of the nanotechnology-based interventions in
the targeting of anticancer drugs in preclinical in vitro and in vivo
80 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92

Endothelial cells

Normal vasculature should allow very little Leaky tumour vasculature should allow
transport, if any, of nanoconjugatesfrom nanoconjugate transportation in either direction
circulation to tissues. with higher rate due to EPR effect.

No ligand -receptor Ligand-receptor interaction causing

interaction accumulation and retention of drug
within tumor microenvironment

Normal Unaffected Cancer Demise of

cell normal cell cell cancer cell

Fig. 1. Different biological responses mediated by receptor ligands and the resulting cellular fate in cancer vs. normal cells. Ligand decorated nanoconjugates would in principle gradually
concentrate within tumor tissues due to the higher leakage mediated by the EPR effect and the consequent entrapment caused by ligand binding with overexpressed receptors in cancer
cells.

models of cancer exploring overexpressed receptors and their respec-
tive ligands.
2.1. G protein-coupled receptors
Many tumor cells specifically express or overexpress G protein-
coupled receptors (GPCRs) compared with the original tissue they are
derived from. Researchers have recently begun to exploit this difference
in expression in the targeting of chemotherapeutics, radiodiagnostics
and radiotherapeutics to tumors [14]. A variety of external stimuli in-
cluding neurotransmitters, hormones, and phospholipids can activate
G-protein coupled receptors (GPCRs), transducing signals from the ex-
tracellular environment to the cytoplasm. GPCRs share the common
structure of a single polypeptide with seven membrane-spanning

Table 1
Description of overexpressed receptors widely used for selective drug delivery using receptor–ligand pairs in preclinical models of cancer.

Major receptor type Specific receptor(s) Overexpression in cancer cell types

G protein coupled receptors
(GPCRs)
Integrins
Folate receptors (FRs)
Transferrin receptors (TfRs)
Epidermal growth factor
receptor (EGFR)
Fibroblast growth factors
(FGFRs)
Sigma receptors (SRs)
Others
Bombesin receptor (BnR) Lung, prostate, breast, pancreatic, head/neck, colon, uterine, ovarian,
This family consists of three closely related proteins, based on their renal cell, glioblastomas, neuroblastomas, gastrointestinal carcinoids,
amino acid sequence homology — i) the gastrin-releasing peptide intestinal carcinoids, and bronchial carcinoids.
receptor (GRP receptor); ii) the neuromedin B receptor (NMB receptor)
and iii) the orphan receptor, BRS-3 with unknown ligands.
Somatostatins receptors (SSTRs) Small cell lung, neuroendocrine tumor, prostate cancer, breast cancer,
Five subtypes of SSTRs have been described so far termed as SSTR 1–5. colorectal carcinoma, gastric cancer, hepatocellular carcinoma
SSTR-2 is being widely used.
Endothelin receptors (ETRs) Melanoma tissues
Two highly homologous receptors — i) ETRA and ii) ETRB bind, but with
different affinities, to three closely related ligands, endothelins (ET;
ET-1, ET-2 and ET-3) each consisted of 21 amino acids.
Among several subtypes formed by various combinations of α and β Activated endothelial cells and tumor cells (such as U87MG
subunits, ανβ3 is of particular interest in selective drug targeting. glioblastoma cells), ovarian cancer cells.
FRα, FRβ and FRγ. The best studied, and widely used in selective drug Most tissues including breast cancer cells.
targeting, of these receptors is folate receptor-α (FRα), a cell surface
glycosyl phosphatidylinositol-anchored glycoprotein that can internal-
ize bound folates and folate-conjugated compounds via receptor-
mediated endocytosis.
Two types of receptors (ubiquitously expressed TfR1 and TfR2 Breast, ovary, and brain cancers such as glioma and glioblastomas.
restricted to hepatocytes) only have been described so far. The
transferrin receptor 2 (TfR2) shares a 45% identity and 66% similarity in
its extracellular domain with TfR1.
EGFR family consists of four members: EGFR (or ErbB1, HER1), ErbB2 Lung, breast, bladder, and ovarian cancers.
(HER2, neu in rodents), ErbB3 (HER3) and ErbB4 (HER4).
A hallmark of FGFRs is the presence of an acidic, serine-rich sequence in Breast, prostate, bladder, and gastric cancer
the linker between D1 and D2, termed the acid box.
The two sigma receptors – S1R and S2R – were distinguished classically Non-small cell lung carcinoma, prostate cancer, melanoma, and breast
on the basis of their binding affinity for pentazocine and guanidine- cancer.
DTG. Both bind pentazocine whereas only the latter binds with DTG.
Follicle stimulating hormone receptors (FSHRs) Ovarian surface epithelium
Biotin receptors (BRs) Leukemia
C-type lectin receptors (CLRs). Hepatocytes, dendritic cells, macrophages
Asialoglycoprotein receptor (ASGPR)
NRP-1 Human vascular cells
 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92 81

Table 2
Some recent outcomes of nanotechnology in the delivery of anticancer drugs in preclinical models of cancer exploiting overexpressed receptors and their respective ligands. Note that
specific names of nanocarriers, for example liposomes, may consist of the same or different building blocks/monomers. Similarly, micelles and other carriers might also differ in size,
shape, charges, composition, and other properties.

Specific Ligands/ligands analogues Nanoparticles/nanocarriers Anticancer drugs/agents Major outcome References

receptor(s)

BnR Peptide fragment called BN Liposome DOX Comparatively greater amount of DOX was delivered [27]
(7–14) to PC-3 cancer cells and xenografts expressing higher
BnR.
BnR BN (6–14) No nanocarrier Mitochondria-disrupting peptide, Greater cytotoxicity to various human cancer cells [31]
B28 (DU145, PC-3, MCF-7, HSF, MRC-5, PrSC)-expressing
BnR.
SSTR OCT Liposome DOX OCT enhanced DOX uptake in SMMC-7721 cells, and [45]
tumor of pancreas and melanoma of B16 tumor
bearing BALB/c mice.
SSTR OCT Liposome DOX Sterically stabilized liposomes (SSL) increased [46]
intracellular delivery of DOX in SSTR2-positive cells
through a mechanism of receptor-mediated endocy-
tosis.
SSTR OCT Micelles DOX OCT caused higher cytotoxicity to MCF-7 cells with [47]
high expression of SSTR. Nude mice bearing MCF-7
cancer xenografts exhibited enhanced anti-tumor
efficacy and decreased systemic toxicity.
ETRB mAB against N-terminal tail Antibody–drug conjugate Monomethylauristatin E (MMAE) ADC demonstrated remarkable efficacy against [56]
of ETRB (ADC) human melanoma cell lines and xenograft tumor
models in correlation with the levels of receptor
expression.
αvβ3 c(RGDfK) Liposome DOX This delivery method resulted in a 15-fold improve- [67]
ment in tumor and anti-metastatic activity in human
umbilical vein endothelial cells (HUVECs) expressing
high levels of αvβ3 when compared with free drug.
αvβ3 c(RGDyK) Iron oxide NPs coated with PEG Internalization potential testing Integrin-specific association between the ligand [68]
c(RGDyK)-iron oxide nanoparticle adducts and
U87MG glioblastoma cells.
αvβ3 c(RGDyK) Micelles PTX Presence of c(RGDyK) enhanced cytotoxicity by 2.5 [69]
folds in U87MG glioblastoma cells. Median survival
time was prolonged in mice bearing intracranial
U87MG tumor xenografts.
αvβ3 c(RGDyK) Micelles PTX and taxol Cellular uptake and, thus, cytotoxicity of c(RGDyK)- [70]
NP/PTX was found to be significantly higher than that
of NPs/PTX due to the integrin protein-mediated
endocytosis effect. Better bioavailability of PTX than
Taxol.
α-3 Peptide OA02 Micelles PTX Dramatically enhanced the uptake efficiency of these [72]
OA02-PEG-CA NPs in SKOV-3 and ES-2 ovarian cancer
cells via receptor-mediated endocytosis, but not in
α-3 integrin-negative K562 leukemia cells.
FR FA Liposome DOX Folate receptor targeted-liposomes loaded with [91]
doxorubicin (FA-L-DOX) inhibited proliferation of
nonfunctioning pituitary adenoma (NFPA) cells
FR FA Copolymeric NPs PTX NPs formulation has great advantages over the [92]
pristine drug, PTX, in achieving better therapeutic
effect in MCF-7 breast cancer cells.
FR FA Surface functionalized QDs – Established folate targeting of QDs in FR expressing [93]
MCF-7 cells.
FR FA Liposome DOX FR mediated endocytosis induced higher cytotoxicity [94]
against the 4T1 mouse mammary carcinoma cell line.
FR FA Liposome DOX Higher cytotoxicity in KB human carcinoma cells; [95]
greater tumor growth inhibition and almost a 50%
increase in life span of mice compared with mice
taking drug in absence of targeting.
FR FA Hybrid polymeric NPs PTX Folate significantly promoted drug-loaded NP's [96]
cellular uptake through folate receptor-mediated
endocytosis in FR overexpressing HeLa and glioma C6
cells as compared to no significant difference in FR-
mediated drug uptake in NIH 3T3 cells with normally
expressed folate receptors.
FR FA Liposome DOX More effective drug uptake in the KB and KB-V [97]
xenograft models, and in the J6456 model irrespec-
tive of administered routes.
TfR Transferrin Iron oxide NPs MRI Retention of Tf-SPIONs in cytoplasm of tumor glioma [116]
cells achieved
TfR Single-chain antibody Liposome p53-gemcitabine The combination treatment prolonged median [118]
fragment (TfRscFv) survival when compared with single drug treatment.
Also decreased tumor burden.
EGFR Peptide ligand (D4: Leu-Ala- Liposome Tested EGFR targeting potential D4 peptide conjugated liposomes efficiently entered [128]
Arg-Leu-Leu-Thr) in EGFR expressing cancer cells (H1299) and specifi-
cally accumulated in EGFR-expressing xenografts
tumor tissues.

(continued on next page)

82 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92

Table 2 (continued)
Specific Ligands/ligands analogues Nanoparticles/nanocarriers Anticancer drugs/agents Major outcome References
receptor(s)

HER2 mAB trastuzumab (TMAB) Human serum albumin (HSA) Methotrexate (MTX) The cytotoxicity of TMAB–MTX–HSA NPs on HER2 [129]
NPs positive cells was found to be significantly higher
than that for non-targeted MTX–HSA NPs and free
MTX.
EGFR Heptapeptide Liposome DOX Peptide-conjugated PLGA-PEG NPs loaded with DOX [131]
showed three-fold higher uptake than by peptide-
free PLGA-PEG NPs in a SKOV3 cell line with high
expression of epidermal growth factor receptor
EGFR Pentapeptide Lipid nanocarriers Tested EGFR targeting potential Higher internalization of pentapeptide ligand [132]
conjugated NPs in high EGFR expressing H1299 and
K562 cells.
FGFR2 Truncated (t) fibroblast Liposome DOX An enhanced antiproliferative activity was achieved [141]
growth factor (tbFGF) by co-delivering DOX and plasmid DNA with impor-
peptide tant a mutant gene simultaneously to the Lewis lung
carcinoma cells (LLC).
FGFR tbFGF Liposome PTX tbFGF-LPs-PTX significantly accumulated in tumor [142]
and prolonged the retention time in tumor-bearing
mice.
FGFR tbFGF Liposome PTX tbFGF, significantly enhanced cytotoxicity of PTX in [143]
FGFR expressing LL/2 cancer cells.
FGFR tbFGF Liposome PTX Higher accumulation of PTX loaded with PEGylated- [144]
tFGF nanoconjugates in tumor tissues expressing
FGFRs.
FGFR1 FGF1 Gold nanoparticles Irradiation FGF1 variant-AuNP conjugates was specifically inter- [145]
nalized only in cells expressing FGFRs. Cell viability
reduced significantly after irradiation with near-
infrared light whereas the proliferation potential of
cells lacking FGFRs is not affected.
S2R SV119 Liposome DOX DOX-loaded SV119 liposomes showed significantly [175]
higher cytotoxicity to DU-145 cells compared to the
DOX-liposomes without SV119.
S2R SV119 Gold nano-cages Tested targeting potential of SV119-ligand might be a promising tool in targeting [176]
SV119 certain tumor.
FSHR FSH33–53 peptide Liposome PTX FSH33–NP–PTX displayed stronger anti-proliferation [181]
and antitumor effects compared with free PTX or
NP-PTX both in vitro and in vivo.
BR Biotin Liposome PTX Results showed that the biotin conjugated NPs could [182]
improve the selective delivery of PTX into cancer cells
with overexpressed biotin receptors.
BR Biotin Taxoid (SB-T-1214) Excellent drug uptake and cytotoxicity in L1210FR [183]
leukemia cells.
BR Biotin Gold NNPs PTX Gold NPs surface-functionalized with PTX and biotin [184]
played a significant role in the diagnosis and therapy
of the cancer cells.
CLR GalNAc residue Liposome Targeting asialoglycoprotein Glycolipid conjugated liposome internalized [194]
receptor. significantly into hepatocytes by ASGPR-mediated
endocytosis.
CLRs Lectin Magnetic NPs (MNPs) PTX Five times higher uptake of PTX-MNPs conjugated [195]
with lectins in K562 leukemia cells as compared with
lectin receptors negative HEK 293 cell line.
NRP-1 CRGDK peptide Gold NPs p-12 (a peptide) CRGDK peptide increased intracellular uptake of gold [198]
NPs carrying therapeutic P12 peptide in NRP-1
receptor overexpressing MDA-MB-321 cells.

domains. Binding of specific agonists (or ligands) to these receptors
leads to the subsequent activation of heterotrimeric G-proteins (Gs,
Gi/Go, Gq/G11 and G12/G13), which in turn regulate the activity of
one or several effectors such as second-messenger-producing enzymes
or ion channels [15,16].
Here, we focus only on those GPCRs reported to be overexpressed in
various tumor cells, making them of interest to the field of cancer ther-
apy. A diverse array of ligands have been developed for drug targeting
against GPCRs. Bombesin and octreotide are among the major ligands
for a certain class of GPCRs used in the targeted delivery of anticancer
drugs by nanotechnology.
2.1.1. Bombesin receptors
Bombesin receptors (BnRs), also known as gastrin-releasing peptide
(GRP) receptors belonging to the GPCR superfamily, have been found to
be overexpressed in cell lines derived from several human tumor types,
including lung cancer, prostate cancer, breast cancer, pancreatic cancer,
head/neck cancer, colon cancer, uterine cancer, ovarian cancer, renal
cell cancers, glioblastomas, neuroblastomas, gastrointestinal carcinoids,
intestinal carcinoids, and bronchial carcinoids. Thus, there is a great deal
of interest in developing BnR-mediated agents to treat these tumors [17,
18]. The mammalian BnR family consists of three closely related G pro-
tein coupled receptors (GPCRs): the gastrin-releasing peptide receptor
(GRP receptor), whose native ligand is the 27 amino acid peptide GRP;
the neuromedin B receptor (NMB receptor), which mediates the action
of the 10 amino acid peptide NMB, and the orphan receptor BRS-3,
which shares 47–51% amino acid sequence homology with the GRP/
NMB receptors but still has an unknown ligand. Each of these receptors
and their ligands are widely distributed in both the central nervous sys-
tem and the peripheral tissues. Moreover, studies in animals suggest
that these receptors are involved in a broad range of physiological and
pathophysiological processes [19–22].
Numerous radiolabeled bombesin ligands (Bn) or their analogues
are currently undergoing investigation for applications in tumor
 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92
imaging and radiotherapy. A few non-radiolabeled ligand–drug com-
plexes constructed by the conjugation of Bn with chemotherapeutic
agents such as camptothecin, DOX, and PTX have successfully increased
the selectivity and efficacy of these drugs in preclinical studies [23–25].
Recently, several studies have used nanotechnology to exploit the spe-
cific overexpression of BnR to target anticancer drugs loaded with NPs
conjugated with Bn and/or Bn analogues. Accardo A et al. [26] have suc-
cessfully delivered more of the anticancer drug DOX to PC-3 cancer cells
using drug-containing liposomes conjugated with the BnR ligand
(7–14) peptide fragment called BN(7–14). The same study reported
significant cytotoxicity in PC-3 cells expressing high BnR as well as
greater inhibition of tumor growth in PC-3 xenograft-bearing mice
compared with liposome–DOX complexes without ligand. Previously,
radiolabeled liposomes containing the BN(7–14) peptide fragment
demonstrated that the peptide exposed on the liposome surface main-
tains the ability to selectively target aggregates of GRPR-expressing xe-
nografts [27]. Many studies have demonstrated that the BN(7–14)
fragment modified on its N-terminus with radiometal complexes for
diagnostic or therapeutic nuclear medicine applications preserves its af-
finity for these receptors [28–30]. Yang et al. [31] again proved the abil-
ity of BnR to facilitate targeted drug delivery to several tumor cells such
as human prostate cancer cells (DU145 and PC-3), human breast cancer
cells (MCF-7), human skin fibroblast cells (HSF), human lung fibroblast
cells (MRC-5), and human prostate stromal cells (PrSC) expressing BnR.
They have used the mitochondria-disrupting peptide B28 as an antitu-
mor agent together with the bombesin analogue BN(6–14), which con-
tains a bombesin receptor-binding motif for the targeting of B28 to
these tumor cells. B28 conjugated to BN(6–14) was far more cytotoxic
to tumor cells than unconjugated B28.
2.1.2. Somatostatin receptors
Somatostatin receptors (SSTRs), members of the superfamily of G-
protein coupled receptors (GPCRs), are widely expressed in a variety
of tumors and cancer cell lines, including small cell lung cancer [32],
neuroendocrine tumors [33], prostate cancer [34], breast cancer [35],
colorectal carcinoma [36], gastric cancer [37] and hepatocellular carci-
noma [38]. Not surprisingly, therefore, SSTRs have recently gained at-
tention due to the potential for targeting anticancer drugs to cancer
cells overexpressing SSTRs via suitable ligands against these receptors.
Physiologically, these receptors are involved in mediating signaling to
regulate the basic processes of secretion, cell division, proliferation
and apoptosis. The ligand for these receptors is the neuropeptide hor-
mone somatostatin (SST) [39,40]. Five subtypes of SSTRs have been de-
scribed so far, termed SSTR 1–5 [41]. SSTRs, especially SSTR subtype 2,
are found to be expressed at higher levels in many tumor cells and in tu-
moral blood vessels relative to normal tissues. The binding of somato-
statin (SST) to endogenous SSTRs is followed by the internalization of
SST, and several reports have shown that most hormone-secreting tis-
sue tumors have a high density of SSTRs [42].
Almost a decade ago, Huang C.M. et al. [43] reported the use of SSTRs
to target anticancer drugs to tumor cells by conjugating the anticancer
drug taxol with the SSTR ligand octreotide (OCT). SSTR-mediated
delivery of OCT-taxol conjugates triggered apoptosis and was exclusive-
ly toxic to SSTR-expressing human breast carcinoma (MCF-7) cells.
OCT-conjugated taxol was less toxic to cells expressing low levels of
SSTR compared with free taxol. These results suggested that OCT-
conjugated taxol demonstrated cell selectivity and might be used as a
targeting agent for cancer therapy. Thereafter, Shen et al. [44] evaluated
the anti-tumor effects of the anticancer drug PTX conjugated with the
SSTR ligand OCT in human lung epithelial adenocarcinoma (A549)
cells and human non-small-cell lung cancer (NSCLC) cells xenografted
into nude mice. The PTX–OCT conjugate, made up of two molecules of
PTX with one molecule of OCT, could enhance tumor growth inhibition
and reduce toxicity when compared with unconjugated PTX. The two
reports mentioned above did not use NPs to carry ligand–drug conju-
gates, but nanotechnology has since been applied to SSTR-mediated
83
anticancer drug delivery. Liposomes containing the anticancer drug
DOX with OCT ligand noticeably increased the uptake of DOX in
human hepatocellular carcinoma (SMMC-7721) cells and led to greater
cytotoxicity than liposomes without targeting ligands [45]. In contrast,
no significant difference in cytotoxicity occurred between drugs encap-
sulated in liposomes with or without OCT in CHO cells not expressing
SSTRs. Moreover, the study of the ex vivo fluorescence imaging of
BALB/c mouse tissues and the in vivo tissue distribution of B16 tumor-
bearing mice indicated that OCT caused a remarkable accumulation of
DOX in SSTRs expressing melanoma and pancreatic tumors compared
with the DOX-liposomal formulation without ligand [45]. Using the
same drug and ligand conjugated with novel sterically stabilized lipo-
somes (SSLs) composed of polyethylene glycol (OCT-SSL-DOX) led to
increased intracellular delivery of DOX in SSTR2-positive cells through
a mechanism of receptor-mediated endocytosis [46]. Compared to SSL,
OCT modification of SSL exhibited little effect on the physicochemical
properties of SSL. In summary, OCT-modified SSL might be a promising
system for the targeting of anticancer drugs in the treatment of SSTR2-
overexpressing cancers. Another study described efficient DOX
targeting and thus significantly higher cytotoxicity in MCF-7 human
breast cancer cells expressing SSTRs compared with normal human
lung fetal (WI-38) cells with no significant SSTR expression. In this
study, investigators constructed micelles of N-deoxycholic acid-O, N-
hydroxyethyl chitosan (DAHC) with good DOX loading capacity
(named DAHC-DOX). The in vivo administration of micelles to nude
mice bearing MCF-7 cancer xenografts confirmed that OPD (OCT–
PEG–deoxycholic acid)-DAHC micelles possessed a much higher
tumor-targeting capacity than the DAHC control and exhibited en-
hanced anti-tumor efficacy and decreased systemic toxicity. These re-
sults suggest that OPD-DAHC micelles might be a promising anti-
cancer drug delivery carrier for targeted cancer therapy [47].
Therapy with valproic acid and the SSTR2-targeting cytotoxic conju-
gate CPT–SST led to greater suppression of cervical cancer compared
with each agent alone. These findings provide a promising clinical op-
portunity for enhanced cancer therapy using combinations of Notch1-
activating agents (valproic acid) and SSTR2-targeting agents [48]. We
should expect more research in this area.
2.1.3. Endothelin receptors
The endothelin (ET) family of molecules (acting as ligands) is com-
posed of three polypeptides, ET-1, ET-2 and ET-3, each consisting of 21
amino acids that bind to two highly homologous G-coupled protein re-
ceptors (GCPRs), endothelin receptor A (ETRA) and endothelin receptor
B (ETRB or EDNRB), triggering a variety of signals according to cell type.
The three ETs are potent vasoconstricting peptides involved in the path-
ophysiology of various malignancies. ET-1, ET-2 and ET-3 are character-
ized by a single α-helix and two disulfide bridges, although the three
peptides are encoded by distinct genes. ETRB binds with all three ETs
with equal affinity, while ETRA binds ET-1 and ET-2 with a two-fold
higher affinity than the ET-3 [49–52]. ETRB has been reported to be
overexpressed in the vast majority of human melanoma tissue speci-
mens examined [53,54]. Differential endothelin receptor expression
and function has also been reported in rat myometrial and leiomyoma
ELT3 cells [55].
An antibody–drug conjugate demonstrated preclinical efficacy, even
in a model with a low level of ETRB expression [56]. The receptor ETRB is
particularly attractive as a target for antibody/ligand–drug conjugate
therapy due to its minimal expression on normal tissue, its cell surface
localization and its rapid endocytosis. However, we are not familiar
with any reports exploiting endothelin receptors in drug targeting
nanocarriers.
2.2. Integrin receptors
Among various integrins, αv (or αvβn) integrin receptors are found
to be highly expressed in activated endothelial cells and tumor cells
84 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92
(such as U87MG glioblastoma cells), but not in resting endothelial cells
or most normal organ systems. Therefore, these integrins represent po-
tential targets for tumor imaging and therapy [57]. Researchers are
currently investigating which integrins act as neoplastic markers. Up-
regulation of ανβ3 has been found to be tightly associated with a
wide range of cancer types, making it a broad-spectrum tumor marker
[58]. Similarly, another integrin, α-3, is also overexpressed in several
types of cancers, especially in ovarian cancer, breast cancer, and mela-
noma [59]. Before discussing the roles of ανβ3- and α-3 integrin recep-
tors in the targeted delivery of various anticancer drugs to cancer cells
and exploring their respective ligands, it is imperative to briefly review
the structure and function of integrins in normal biology. Integrins are
transmembrane receptors that bind extracellular matrix proteins or
other adhesion receptors on neighboring cells. Heterodimeric pairing
of integrin α and β subunits confers specificity of binding to one or
more substrates [60]. In particular, the αv subunit pairs with β1, β3,
β5, β6, and β8. While some pairings preferentially bind a single ligand
(αvβ5 for vitronectin), others recognize a number of ligands (αvβ3
binds vitronectin, fibronectin, vWF, tenascin, osteopontin, fibrillin,
fibrinogen, and thrombospondin). At least 24 distinct integrin heterodi-
mers are formed by the combination of 18 α subunits and 8 β subunits.
Because the integrins expressed on the surface of a cell will determine
whether it can adhere to and survive in a particular microenvironment,
the matching of integrins and ligands plays a key role in the regulation
of the sprouting ability of endothelial cells during angiogenesis, the
recruitment of inflammatory cells to sites in need of repair, and the in-
vasive potential of tumor cells [61,62]. The role of integrins in cell migra-
tion and invasion is one of their functions that has received the most
study in tumor biology, and this has been reviewed elsewhere [63,64].
Antagonists to some integrins such as cilengitide have shown promising
results in various phases of clinical trials, including for cancer therapy
[65]. Our focus is to review the utility of these well studied integrin re-
ceptors for targeted anticancer drug delivery in nanomedicine. Integrins
expressed by epithelial cells (including α6β4, α6β1, αvβ5, α2β1 and
α3β1) are generally retained in the tumor but at different levels.
Integrin expression also varies considerably between normal and
tumor tissues. Most notably, integrins αvβ3, α5β1 and αvβ6 are usually
expressed at low or undetectable levels in most adult epithelia, but can
be highly upregulated in some tumors. However, the expression levels
of some integrins decrease in tumor cells, potentially increasing tumor
cell dissemination. In fact, re-expression of α2β1 in breast cancer cells
reversed some of the malignant properties of those cells, suggesting
that α2β1 could function as a tumor suppressor. Several integrins that
might have important roles in cancer progression have now emerged
with great potential in targeted drug delivery. As expression of the
integrins αvβ3, αvβ5, α5β1, α6β4, α4β1 and αvβ6 is correlated with
disease progression in various tumor types, these integrins are the
most frequently studied in cancer [66].
2.2.1. Integrin αvβ3
Recent studies showed that the delivery of targeted NPs loaded with
DOX to the integrin αvβ3-positive tumor vasculature inhibited the
growth of metastases while eliminating the toxicity and weight loss as-
sociated with systemic administration of this drug [67]. This delivery
method resulted in a 15-fold improvement in tumor and anti-
metastatic activity when compared with administration of the free
drug. The preferential activity of these NPs on metastases suggests
that growing metastatic tumors may have a greater dependence on an-
giogenic vessels, making them more susceptible to integrin αvβ3-
targeted therapy. Chen et al. [68] coated iron oxide NPs with a
PEGylated amphiphilic triblock copolymer and then conjugated the
near-infrared fluorescent (NIRF) dye IRDye800 and a cyclic Arginine-
Glycine-Aspartic acid (RGD) containing peptide c(RGDyK) for integrin
αvβ3 targeting. In vitro binding assays confirmed the integrin-specific
association between the ligand c(RGDyK)-iron oxide nanoparticle ad-
ducts and U87MG glioblastoma cells. Successful tumor homing in vivo
was observed in a subcutaneous U87MG glioblastoma xenograft
model by both magnetic resonance imaging (MRI) and NIRF imaging.
The investigators achieved excellent tumor integrin targeting efficiency
and specificity. Another study also used the same ligand–receptor pair
[i.e., c(RGDyK)–αvβ3] in U87MG glioblastoma cells to deliver the anti-
cancer drug PTX, which was encapsulated in poly(ethylene glycol)-
block poly(lactic acid) micelles, resulting in a nanoscale formulation of
(c(RGDyK)–PEG–PLA–PTX). In vitro cytotoxicity studies proved that
the presence of c(RGDyK) led to a 2.5-fold enhancement in the anti-
glioblastoma cytotoxic efficacy. In an in vivo model, the c(RGDyK)–
PEG–PLA micelle accumulated in the subcutaneous and intracranial
tumor tissues, and the PTX-loaded micelle (c(RGDyK)–PEG–PLA–PTX)
exhibited the strongest tumor growth inhibition among the PTX formu-
lations studied here [69]. Other investigators prepared polymeric
micellar-like NPs (MNP) based on PEGylated poly(trimethylene carbon-
ate) (PEG-PTMC) conjugated with c(RGDyK) for active targeting to
integrin-rich U87MG cancer cells. A pharmacokinetic study in rats dem-
onstrated that the polymeric micellar NPs significantly enhanced the
bioavailability of PTX compared with Taxol. In vivo multispectral fluo-
rescent imaging indicated that c(RGDyK)-MNP/PTX exhibited high
specificity and efficiency in active tumor targeting [70].
2.2.2. Integrin α-3
Similarly, α-3 integrin is overexpressed in several types of cancers,
especially in ovarian cancer, breast cancer, and melanoma [59]. The
high affinity α-3 integrin-targeting peptide OA02 has been shown to
bind strongly to α-3 integrin-overexpressing ovarian cancer cells and
specifically target ovarian cancer xenografts (ES-2) in nude mice
when conjugated to near-infrared fluorescence dyes [71]. The overex-
pression of α-3 integrin on these ovarian cancer cells has been exploited
[72] as a promising pharmacologic target for selective drug delivery in
the treatment of these cancers. Conjugation of micellar NPs (PEG5k-
CA8 NPs) composed of polyethylene glycol (PEG) block dendritic cholic
acid (CA) copolymers with the OA02 peptide dramatically enhanced the
uptake and efficiency of these nanocarriers in SKOV-3 and ES-2 ovarian
cancer cells via receptor-mediated endocytosis, but uptake was not in-
creased in α-3 integrin negative K562 leukemia cells. Furthermore,
the in vivo biodistribution study reported that the OA02 peptide greatly
facilitated tumor localization and intracellular uptake of PEG5k-CA8 NPs
into ovarian cancer cells as validated in SKOV3-luc tumor-bearing mice
[72].
2.3. Folate receptors
Among cellular surface targets potentially suitable for use in drug
targeting, folate receptors (FRs) stand out as one of the most promising
and most investigated epithelial cancer markers. The FR constitutes a
useful target for tumor-specific drug delivery primarily because it is
up-regulated in many human carcinomas, including malignancies of
the ovary, brain, kidney, breast, colon, myeloid cells, and lung, while it
is expressed at low levels in normal cells and tissues [73–76]. FRs
(FRα, FRβ and FRγ) are cysteine-rich cell-surface glycoproteins that
bind folate with high affinity to mediate the cellular uptake of folate. Al-
though expressed at very low levels in most tissues, FRs, especially FR α,
are expressed at high levels in numerous cancers to meet the folate de-
mand of rapidly dividing cells under low folate conditions [77,78]. The
folate dependency of many tumors has been therapeutically and diag-
nostically exploited by the administration of anti-FRα antibodies,
high-affinity anti-folates [79,80], folate-based imaging agents and
folate-conjugated drugs and toxins [81–83]. The best studied isoform
of these receptors is FRα, a cell surface glycosyl phosphatidylinositol-
anchored glycoprotein that can internalize bound folates and folate-
conjugated compounds via receptor-mediated endocytosis [84]. Very
recently, its structural basis for the molecular recognition of folic acid
was reported [85]. FR α has a globular structure stabilized by eight di-
sulfide bonds and contains a deep open folate-binding pocket
 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92
comprised of residues that are conserved in all receptor subtypes. The
folate pteroate moiety is buried inside the receptor, whereas its gluta-
mate moiety is solvent-exposed and protrudes out of the pocket en-
trance, allowing for drug conjugation without adversely affecting FR α
binding affinity. The extensive interactions between the receptor and li-
gand readily explain the high folate-binding affinity of folate receptors
and provide a template for the design of more specific drugs targeting
the folate receptor system [85]. Sarcomas, lymphomas, and cancers of
the pancreas, testicles, bladder, prostate, and liver often do not show
elevated levels of folate receptors. When expressed in normal tissue, fo-
late receptors are restricted to the lungs, kidneys, placenta, and choroid
plexus; in these tissues, the receptors are limited to the apical surface of
polarized epithelia [86]. Folate or folic acid (FA), the FR ligand is a small
molecule (44 Da) stable over a broad range of temperatures and pH
values, inexpensive, and non-immunogenic, and it retains its ability to
bind to the folate receptor after conjugation with drugs or diagnostic
markers [87]. The overexpression of folate receptor in cancers of several
histologies relative to normal tissues, the low cost of folic acid (FA), and
the vast library of conjugation reactions available make it one of the
most used ligands for tumor-targeted drug delivery and tumor imaging
(reviewed in [88]). Folate is internalized into cells via a low-affinity re-
duced folate carrier protein or via high-affinity folate receptors.
Due to the elevated FR α expression in cancer cells, FR α has become
one of the most intensively investigated cellular surface antigens for the
targeted delivery of a variety of molecules, including imaging agents,
chemotherapeutic agents, oligodeoxynucleotides, and macromolecules.
Various types of drug carriers have been conjugated to folate such as
liposomes, lipid NPs, polymeric NPs, polymers, and micelles filled with
the active molecule [89]. Thus, solid tumors that are currently among
the most difficult to treat by classical therapeutic modalities may be
readily targeted with FA-linked therapeutics [73,90]. Folate receptor
targeted-liposomes loaded with DOX (FA-L-DOX) inhibited the prolifer-
ation of human non-functioning pituitary adenoma (NFPA) cells and
promoted apoptosis through the activation of caspase 8, caspase 9,
and caspase 3/7 more effectively than L-DOX (liposomes without FR
targeted but with DOX). Furthermore, FA-L-DOX also exerted greater
anti-invasive ability in NFPA cells than L-DOX through the suppres-
sion of the secretion of matrix metalloproteinase-2 and matrix
metalloproteinase-9. Addition of 1 mM free folic acid (to block FR-
mediated endocytosis of liposomes) significantly reduced the
pleotropic effects of FA-L-DOX in NFPA cells, suggesting that FRα
plays a critical role in mediating the antitumor effect of FA-L-DOX
[91]. Another study using nanotechnology showed that a two-
component copolymeric NP formulation has great advantages over
the pristine drug PTX, achieving a better therapeutic effect in MCF-7
breast cancer cells. These results further showed that the folate decora-
tion could promote significantly higher delivery of the drug into the cor-
responding cancer cells, enhancing therapeutic effect [92]. Later, the
same group prepared folate-decorated quantum dot NPs to improve
the imaging effects and reduce their side effects with surface function-
alized poly(lactide)–vitamin E–D-alpha-tocopheryl polyethylene glycol
succinate (PLA-TPGS) copolymer and vitamin E TPGS–carboxyl (TPGS-
COOH) copolymer designed to conjugate folate-NH2. This system pre-
sents the advantage of making the targeting effect adjustable. Much
higher internalization of the folate-decorated QD-loaded PLA-TPGS/
TPGS-COOH NPs was achieved in MCF-7 cells overexpressing folate re-
ceptors than in mouse NIH 3T3 fibroblast cells with low levels of folate
receptor expression. Compared with free QDs, the QDs conjugated with
PLA-TPGS/TPGS-COOH NPs showed lower in vitro cytotoxicity in both
MCF-7 and NIH 3T3 cells [93]. Prabaharan et al. [94] encapsulated
DOX into folate-conjugated amphiphilic hyperbranched block copoly-
meric micelles {a hydrophobic poly(L-lactide) inner shell and a hydro-
philic methoxy poly(ethylene glycol) outer surface}. The DOX-loaded
micelles provided an initial burst release (up to 4 h) followed by a
sustained release of the entrapped DOX over a period of approximately
40 h. Cellular uptake of the DOX-loaded micelles linked with FA was
85
found to be greater than that of the DOX-loaded micelles without FA
due to folate-receptor mediated endocytosis in the 4T1 mouse mamma-
ry carcinoma cell line. Further, in vitro degradation studies revealed that
the H40–PLA-b-MPEG/PEG–FA block copolymer hydrolytically degrad-
ed into polymer fragments within six weeks. Another study tested the
anticancer efficacy of free DOX and DOX bound with folate targeted li-
posome. DOX-liposome was superior to free DOX in two human
tumor models (KB, KB-V) and in one mouse ascitic tumor model (FR-
expressing J6456). The therapeutic effect was dose-dependent in the
KB model receiving DOX-liposome through the i.p. intra-cavitary
route and was schedule-dependent in the J6456 mouse model receiving
DOX-liposome via the intra-cavitary route. In some experiments,
however, toxic deaths aggravated by the folate-depleted diet were a
major confounding factor. While promising, this study indicates that
more sophistication is needed in selecting cancer type associated recep-
tors and ligands [95]. Another in vitro study [96] demonstrated signifi-
cant FR-mediated endocytosis of PTX-loaded micellar NPs in FR-
overexpressing HeLa and glioma C6 cells, compared with no significant
drug uptake in NIH 3T3 cells with lower folate receptor expression. In
their study, Riviere et al. [97] investigated the antitumor activity
of FA-linked liposome-DOX injected intravenously in mice bearing
KB tumor xenografts. In conclusion, systemically administered FA-
conjugated DOX-liposomes have demonstrated the potential to en-
hance the delivery of anticancer drugs in in vivo models.
2.4. Transferrin receptors
The efficient cellular uptake of transferrin (Tf) makes this pathway a
potential route for the delivery of anticancer drugs, proteins, and thera-
peutic genes primarily into proliferating malignant cells that overex-
press transferrin receptors (TfRs) [98–100]. Transferrin (Tf) is a single-
chain glycoprotein containing approximately 700 amino acids and is
one of the most widely used tumor targeting ligands in tumor cells ex-
pressing transferrin receptors (TfRs) more than other cells [101–103].
TfRs are divided into two subtypes, TfR1 and TfR2. TfR1, also known as
CD71, is found to be ubiquitously expressed at low levels in most nor-
mal human tissues, while TfR2, which is homologous to TfR1, is largely
restricted to hepatocytes [104,113]. Despite its ubiquitous expression,
TfR1 is expressed on malignant cells at levels several fold higher than
those in normal cells, and its expression can be correlated with tumor
stage or cancer progression [105–109]. TfR1 is found to be
overexpressed in several human carcinomas including breast, ovary,
and brain cancers such as glioma and glioblastomas [110–112]. This
high expression of the receptor on malignant cells, its ability to internal-
ize, and the necessity of iron for cancer cell proliferation make this re-
ceptor a widely accessible portal for the delivery of drugs into
malignant cells, and thus an attractive docking site for targeting antican-
cer drugs in cancer therapy. TfR1, a type-II transmembrane protein, is a
transmembrane homodimer that can bind up to two molecules of Tf
through its extracellular carboxyl end binding sites. Transferrin receptor
2 (TfR2) shares a 45% identity and 66% similarity in its extracellular do-
main with TfR1 [113]. These findings make the transferrin family of re-
ceptors potentially valuable cancer biomarkers as well. Moreover,
transferrin receptor and the receptor for insulin are highly expressed
by the endothelial cells forming the blood–brain barrier (BBB), provid-
ing a way for drugs to enter the nervous system [114,115]. As the ex-
pression of TfR2 is restricted to hepatocytes, TfR1 will be treated as
equivalent to TfR in the remainder of this review for the sake of clarity.
Jiang et al. [116] reported that transferrin-conjugated super-
paramagnetic iron oxide NPs (Tf-SPIONs) acted as promising con-
trast agents in targeted magnetic resonance imaging (MRI) for the
detection of glioma in the brains of rats. MRI showed obvious contrast
enhancement of brain glioma that could still be clearly observed even
48 h post injection due to the retention of Tf-SPIONs in the cytoplasm
of tumor cells, as proven by Prussian blue staining. A second factor
supporting TfR as an appropriate target in pancreatic cancer is that the
86 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92
TfR is recycled during internalization in rapidly dividing cancer cells,
thus improving the uptake of Tf-conjugated vectors [117]. Camp et al.
[118] have delivered human wild type p53 protein (SGT53) (in combi-
nation with anticancer gemcitabine, probably to enhance the anticancer
activity) bound with liposomal NPs, targeted to the transferrin receptor
by a single-chain antibody fragment (TfRscFv) in an in vivo metastatic
pancreatic cancer model. Compared with untreated mice harboring
metastatic tumors with a median survival of 20 days, SGT-53,
gemcitabine and SGT-53–gemcitabine complex increased the median
survival to 29, 30 and 37 days, respectively. The tumor targeting
liposomal-based SGT-53 nanoparticle is capable of sensitizing pancreat-
ic cancer to conventional chemotherapy in pancreatic cancer models.
This approach has the potential to be translated into a new, more effec-
tive therapy for pancreatic cancer. Further optimization is ongoing
moving towards a Phase 1B/2 clinical trial [118].
Using transferrin-conjugated NPs, however, Salvati et al. [119] have
found that proteins in the media can shield transferrin from binding to
both its receptors on cells and soluble transferrin receptors. Although
NPs continue to enter cells, the targeting specificity of transferrin is
lost. Their results suggest that when NPs are placed in a complex biolog-
ical environment, interaction with other proteins in the medium and
the formation of a protein corona can ‘screen’ the targeting molecules
on the surface of NPs and cause a loss of targeting specificity [119].
2.5. Epidermal growth factor receptors
Epidermal growth factor receptors (EGFRs) are important targets for
anticancer therapy. Special attention has been given to ligands that
interact with EGFRs. EGFRs are overexpressed in a wide variety of
human cancers including cancers of the lung, breast, bladder, and
ovary. EGFR expression has also been found to associate with various
features of advanced disease with poor prognosis [120]. Various EGFR-
targeting vectors and conjugates have been reported as delivery agents
of cytotoxic drugs, toxins, or radionuclides [121]. Moreover, it is an ‘in-
ternalizing’ receptor, indicating that following ligand–EGFR binding, the
ligand–receptor complex is actively endocytosed. Thus, directing
nanocarriers by linking them with ligands specific to EGFRs provides a
promising way to achieve receptor-mediated intracellular delivery of
the carrier with its anticancer cargo. The EGFR family consists of four
members: EGFR (ErbB1, HER1), ErbB2 (HER2, neu in rodents), ErbB3
(HER3) and ErbB4 (HER4). These structurally related receptors are
single chain transmembrane glycoproteins consisting of an extracellular
ligand-binding ectodomain, a transmembrane domain, a short
juxtamembrane section, a tyrosine kinase domain and a tyrosine-
containing C-terminal tail. Binding of soluble ligand to the ectodomain
of the receptor promotes homo- and hetero-dimer formation between
receptors. Receptor dimerization is essential for activation of the intra-
cellular tyrosine kinase (TK) domain and for the phosphorylation of the
C-terminal tail [122]. Phosphotyrosine residues then activate, either
directly or through adaptor proteins, downstream components of
signaling pathways including Ras/MAPK, PLCg1/PKC, PI(3)kinase/Akt,
and STAT pathways [123]. EGFR and ErbB4 can be thought of as fully
functional receptors with the ability to bind ligands as well as
autophosphorylate C-terminal tails through functional intracellular ty-
rosine kinase domains. ErbB2, however, is unique in that it has no
known ligand but is the preferred dimerization partner for other
EGFRs [124,125]. ErbB3 is also unique as it has no intrinsic tyrosine ki-
nase activity but can transduce signals through interactions with kinase
active receptors, namely EGFR, ErbB2, and ErbB4. Although all ErbB re-
ceptors have been localized to the nucleus, ErbB4 is notable for its abil-
ity to directly transduce extracellular signals to the nucleus through
liberation of the intracellular domain by a ligand-dependent dual prote-
ase cleavage of the receptor [126]. In essence, EGFRs bind ligands,
resulting in receptor homo- or hetero-dimerization followed by recep-
tor internalization (primarily via clathrin-mediated endocytosis) and
activation of the cytoplasmic tyrosine kinase domain [127].
In an initial study using EGFR to deliver a potential anticancer drug in
nanocarriers conjugated with a novel EGFR ligand called D4 peptide
(D4: Leu-Ala-Arg-Leu-Leu-Thr), liposomes accumulated in EGFR-
expressing xenograft tumor tissues up to 80 h after intravenous deliv-
ery, a marked contrast compared with the control group [128]. Another
study combined the somewhat less frequently used anticancer drug
methotrexate (MTX) with human serum albumin (HAS) NPs decorated
with trastuzumab (TMAB) molecules to form a nanoconjugate of
TMAB–MTX–HAS. TMAB–MTX–HAS achieved significantly higher up-
take and cytotoxicity in HER2 positive cells than non-targeted MTX–
HSA NPs and free MTX [129]. TMAB is a monoclonal antibody working
as ligand against HER2 receptors reported to be overexpressed in
20–30% of human breast cancers [130]. Other investigators prepared a
functional liposome of poly(D,L-lactide-co-glycolide)-poly(ethylene gly-
col) (PLGA-PEG) with an amino-active group for conjugation with a
peptide ligand (a heptapeptide). The peptide-conjugated PLGA-PEG
NPs loaded with DOX showed three-fold higher uptake than peptide-
free PLGA-PEG NPs in human ovarian carcinoma (SKOV3) cells with
high levels of EGFR expression [131]. Han et al. [132] found that
conjugation of a pentapeptide (AEYLR or Ala-Glu-Tyr-Leu-Arg) with
nanostructured lipid carriers caused increased cellular uptake of
nanocarriers in tumor cells expressing EGFR. Analyses with flow cytom-
etry and an internalization assay using human non-small-cell lung car-
cinoma (NCI-H1299) and human leukemia (K562) cells, with high EGFR
and no EGFR expression, respectively, indicated that FITC-AEYLR had
high EGFR targeting activity. Biotin-AEYLR specifically bound to
human EGFR, demonstrating high affinity for human non-small-cell
lung tumors. In a model of multidrug resistance (MTDR), EGFR binding
peptide (EBP) linked DOX was found to accumulate equally in DOX-
resistant (SW480/DOX) and non-resistant (SW480) cell lines of
human colon cancer, at higher levels than free, unconjugated DOX in
DOX-resistant cells [133].
2.6. Fibroblast growth factor receptors
Overexpression of fibroblast growth factor receptors (FGFRs) has
been reported in tumors of the breast, prostate, bladder, and gastric can-
cer, and has been associated with tumor progression and poor patient
prognosis [134,135]. There are four FGFR genes (FGFR1–FGFR4) that
encode receptors consisting of three extracellular immunoglobulin do-
mains (D1–D3), a single-pass transmembrane domain and a cytoplas-
mic tyrosine kinase (TK) domain [136]. A hallmark of FGFRs is the
presence of an acidic, serine-rich sequence in the linker between D1
and D2, termed the acid box. The D2–D3 fragment of the FGFR
ectodomain is necessary and sufficient for ligand binding and specificity,
whereas the D1 domain and the acid box are proposed to have roles in
receptor autoinhibition [137]. Depending on the localization and cancer
type, different types of FGFRs are overexpressed. For example, elevated
levels of FGFR1, FGFR2, and FGFR4 are commonly found in cancers of the
breast and prostate, whereas only FGFR2 overexpression is observed in
gastric cancers, and papillary thyroid carcinoma is limited to the overex-
pression of FGFR1 and FGFR3 only [138]. Moreover, a low level of FGFR
expression is found on the surface of non-cancerous cells in the vicinity
of the tumor. Due to their specific expression in various cancer types,
FGFRs represent potential therapeutic targets [139]. Fibroblast growth
factors (FGFs), ligands of FGFRs, exert their physiological roles through
FGFRs, regulating developmental pathways such as mesoderm pattern-
ing in the early embryo. The mammalian FGF family is composed of 18
ligands that elicit their actions through four (i.e., FGFR1, FGFR2, FGFR3,
and FGFR4) highly conserved transmembrane tyrosine kinase receptors
[140].
Xiao et al. [141] developed a novel cationic liposomal nanocarrier for
DOX which was further conjugated with truncated human basic fibro-
blast growth factor (tbFGF) peptide, a modified peptide containing
binding sites for the FGF2 receptor and part of heparin. This peptide
could effectively bind FGFR2 on the cell surface without stimulating
 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92
cell proliferation. As a result, conjugation of tbFGF led to an enhanced
anti-proliferative activity in mouse Lewis lung carcinoma (LLC) cells
by the synergistic actions of DOX and plasmid DNA (encoding the
phosphorylation-defective mouse survivin threonine 34 → alanine mu-
tant gene known as Msurvivin T34A plasmid) targeted in the same cells
by the same cationic liposome. The concentration of DOX in the co-
delivery system causing 50% cell killing was nearly 3-fold lower than
the concentration of free DOX responsible for the same cytotoxicity.
Furthermore, the co-delivery system suppressed tumor growth more
efficiently than either DOX or the Msurvivin T34A plasmid alone in
the Lewis lung carcinoma-bearing C57BL/6 mice. The co-delivery sys-
tem also caused a 15 day delay of tumor growth, longer than the other
treatment groups. In conclusion, this novel cationic liposome is an effi-
cient vector for the simultaneous delivery of drugs and DNA to the
same cells in vitro and in vivo. Another study compared the pharmacoki-
netics and tissue distribution of a novel truncated basic fibroblast
growth factor peptide-mediated cationic liposomal PTX (tbFGF-LPs-
PTX) with free PTX and cationic liposomal PTX (LPs-PTX) in tumor-
bearing mice. The data indicated that the concentration of tbFGF-LPs-
PTX significantly increased in the tumor and that the retention time
was prolonged [142]. Another group prepared biocompatible and bio-
degradable nanocarriers of cholesterol-block-poly(ethylene glycol)
(Chol-PEG2000-COOH) polymer with PTX linked with truncated bFGF
fragments (tbFGF). This nanocarrier significantly enhanced the cytotox-
icity caused by targeted PTX in FGFR expressing LL/2 (a modified Lewis
lung carcinoma) cells as measured by an MTT {3-(4,5-dimethyl thiazol-
2-yl)-2,5-diphenyl tetrazolium bromide} assay. Flow cytometry re-
vealed that the cellular uptake of rhodamine B, encapsulated in the
tbFGF conjugated micelles, was increased by 6.6-fold for HepG2
(human liver carcinoma), 6.2-fold for A549 (human lung adenocarcino-
ma), 2.9-fold for C26 (mouse colon adenocarcinoma) and 2.7-fold for
LL/2 (mouse Lewis lung carcinoma) cells, respectively, compared with
micelles without tbFGF. The fluorescence spectroscopy further demon-
strated that the tbFGF-conjugated micelles specifically bound to tumor
cells over-expressing FGFRs and then released rhodamine B into the cy-
toplasm [143]. Biodistribution studies in C57BL/6J mice bearing B16
melanoma revealed a higher accumulation of tFGF targeted PTX deliv-
ered by PEGylated liposomes in tumor tissue and organs containing
the mononuclear phagocyte system (liver and spleen), but a consider-
able decrease in other organs (heart, lung, and kidney) as compared
with PEGylated-liposome-PTX without tFGF and free PTX [144].
Szlachcic et al. [145] developed novel FGF1 variant-AuNP conjugates
found to be specifically internalized only in cells expressing FGFRs.
FGF1 coated AuNPs significantly reduced viability in cells expressing
FGFRs after irradiation with near-infrared light (down to 40% of control
cell viability), whereas the proliferation potential of cells lacking FGFRs
was not affected.
2.7. Sigma receptors
Researchers have recently become interested in exploring sigma re-
ceptor ligands for tumor imaging and targeted therapy. Sigma receptors
are overexpressed in a variety of human tumors including cancers of
non-small cell lung carcinoma (NSCLC), prostate, melanoma, and breast
[146–148]. Sigma receptor was initially proposed to represent a subtype
of opioid receptors [149]. These subtypes display different tissue distri-
butions and distinct physiologic and pharmacologic profiles in both the
central and peripheral nervous systems. Sigma-1 and -2 receptors were
classically distinguished on the basis of their binding affinity for [3H]-
(+)-pentazocine and [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). Both
sigma receptors bind pentazocine, while only the sigma-2 receptor
binds guanidine-DTG [150–152]. Although the sigma-1 receptor is a
well-characterized protein, the sigma-2 receptor protein has largely
remained elusive [153]. However, the novel sigma-2 receptor ligand
SW43 was reported to stabilize the progression of pancreatic cancer in
combination with gemcitabine [154]. The sigma-1 receptor (S1R), a
87
ligand regulated membrane protein chaperone of 25 kDa possessing
one putative transmembrane domain and an endoplasmic reticulum re-
tention signal, is involved in the ER stress response and inter-organelle
communication [155–157]. S1R is expressed primarily in the cerebral
cortex, hippocampus, and cerebellar Purkinje cells [158,159], and has
been proposed as a target for the treatment of central nervous system
diseases including amnesia, pain, schizophrenia, clinical depression,
Alzheimer's, stroke, and addiction [160,148]. S1R is primarily localized
to mitochondria-associated ER membranes [161,162], which are the
sites for mitochondrial bioenergetic regulation through the release of
ER calcium [163]. S1R activity is modulated by a number of endogenous
molecules such as N,N dimethyltryptamine, progesterone and sphingo-
sine, and exogenous molecules including opiates, antipsychotics,
antidepressants, antihistamines, phencyclidine-like compounds, β-
adrenergic receptor ligands, and cocaine [164–166]. Upon binding to
these molecules, activated S1R causes the dissociation of ankyrin from
the inositol triphosphate receptor (IP3R) [167], resulting in calcium re-
lease at the ER–mitochondrial interface. This released calcium is effi-
ciently taken up by mitochondria to increase energy production and
other stress responses [168].
Although the natural ligand for the sigma-2 receptor (S2R) and the
detailed structure of S2R itself remain unknown, this receptor has
been shown to be overexpressed in a variety of human tumors including
breast, pancreas, neuroblastoma, bladder, and lung. As such, various S2R
ligands have been extensively studied for their effectiveness in the
treatment of solid tumors due to their preferential uptake in proliferat-
ing cells [169]. Equally important is the notion that S2R ligands are rap-
idly internalized into cancer cells [170,171], including pancreatic cancer
which expresses the receptor at low levels [172]. Synthetic ligands to
this receptor could play an important role in cancer diagnosis, imaging,
and targeted drug delivery [173].
SV119, an S2R ligand, binds to pancreatic cancer cells and induces
target cell death in vitro and in vivo. When conjugated to SV119, small
molecular modulators known to interfere with intracellular pro-
survival pathways retained their ability to induce cell death, and their
efficiency was enhanced by the targeting effect of SV119. These findings
indicate the utility of S2R ligands to increase the tumor-specific delivery
of small molecules [174]. SV119 targeting of DOX-loaded liposomes to
S2R-overexpressing cells led to significantly higher uptake of SV119-
conjugated liposomes in cancer cells of human breast (MCF-7), human
prostate (PC-3, DU-145) and human lung (A549, 201T) lineages com-
pared to normal human bronchial (Beas-2B) cells. Thus, the SV119 li-
gand caused significantly higher cytotoxicity in DU-145 cells when
compared with the cytotoxicity induced by liposomal DOX without li-
gand [175]. Similarly, gold nano-cages functionalized with SV119
again demonstrated the promising ability of this ligand–receptor sys-
tem for cancer cell targeting [176]. These results suggest that the
SV119–S2R system might be an effective tool for targeting certain
tumors.
2.8. Other overexpressed receptors
Some less common receptors recently used for targeting drugs to
cancer cells are follicle-stimulating hormone receptors, biotin receptors,
C-type lectin receptors and neuropilin receptors. Overexpression of
follicle-stimulating hormone receptors (FSHRs) during ovarian cancer
makes FSHR a possible target against ovarian cancer. Follicle stimulating
hormone (FSH) is a glycoprotein consisting of α and β chains. Some
FSHR-binding domains in FSH have been identified including amino
acids 1 to 15, 33 to 53, 51 to 65, and 81 to 95 of the FSH β chain [177,
178]. The ovary is the target organ of FSH, which binds to the FSH recep-
tor (FSHR), a G protein-linked receptor. Overexpression of FSHR was
found on the ovarian surface epithelium and in some ovarian cancer
cell lines [179,180].
Zhang et al. [181] developed a ligand of FSHR called FSH33–53 that
was derived from 33 to 53 amino acids of the FSH β chain. Later,
88 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92
FSH33–53 was conjugated with NPs constructed from PEG-PLA [poly-
ethylene glycol-poly(lactic acid)] forming FSH33–NP complexes. The
anticancer drug PTX was chosen to test the targeting potential of
FSH33–NPs against ovarian cancer. The FSH33–NP–PTX nanoconjugates
displayed stronger anti-proliferation and antitumor effects compared
with free PTX or PTX-loaded NPs without FSH33 both in vitro and
in vivo. In summary, this novel FSH33–NP delivery system showed
very high selectivity and efficacy for FSHR-expressing tumor tissues.
Therefore, it has a strong potential to become a new therapeutic ap-
proach for patients with ovarian cancer.
Kim et al. [182] reported that biotin-conjugated poly(ethylene
oxide)/poly(ε-caprolactone) (PEG/PCL) amphiphilic block NPs carrying
PTX induced higher cytotoxicity in cancer cells than NPs carrying PTX
but not conjugated with biotin. These results showed that biotin-
conjugated NPs could improve the delivery of PTX into cancer cells via
interactions with overexpressed biotin receptors. Chen et al. [183]
have shown a good correlation between drug uptake and biotin
receptor expression. These investigators have conjugated second-
generation taxoid (SB-T-1214) as the cytotoxic agent with biotin and
reported excellent drug uptake and cytotoxicity in L1210FR leukemia
cells overexpressing biotin receptors compared with two biotin recep-
tor negative cell lines, L1210 leukemia cells and WI38 normal human
lung fibroblast cells. Other investigators have shown that gold NP
surface-functionalized with PTX and biotin are promising for applica-
tions in cancer diagnosis and therapy [184].
C-type lectin receptors (CLRs) have emerged with great potential
in cell-specific drug and gene delivery [185–187]. CLRs belong to a
large family of receptors that share a structurally homologous
carbohydrate-recognition domain and often bind to glycan structures
in a Ca2+-dependent manner [188], including the collectins, selectins,
lymphocyte lectins, and proteoglycans. Because of their endocytic prop-
erties, CLRs are suitable targets for cell-specific drug delivery [189]. In
addition to cell-specific drug delivery, CLRs may also be exploited to
modulate the functions of CLR-expressing cells such as endocytosis or
cell activation. In innate immunity, CLRs are mainly expressed by
antigen presenting cells such as dendritic cells and macrophages. They
serve as pattern-recognition receptors and bind to pathogen-associated
molecular patterns (PAMPs) [190], but may also sense self-antigens
released by damaged tissue or dead cells [191]. Hepatic lectin receptors
including asialoglycoprotein receptor (ASGPR) are capable of mediating
the endocytosis of bound ligands. Following recognition, bound ligands
are internalized via coated pits and the complex is then released into
endosomal compartments while the lectin receptor is recycled to the
cell surface for another round of endocytosis [192]. Thus, hepatic lectin
receptors represent promising targets to be exploited in liver-specific
drug and gene delivery. The human ASGPR, a type II transmembrane pro-
tein, consists of two subunits, H1 and H2, with the H1 subunit mediating
Size and stability of nanoparticle-ligand complex
determining its circulation time?
Nanoparticle’s drug release efficiency at
the desired site?
Ligand immunogenicity?
Nanoparticle’s loading efficiency with
anticancer drug?
Effect of bound ligand on its native affinity
with receptor?
Nanoparticle’s biocompatibility and
systemic toxicity?
Spatial and temporal variability in receptor overexpression?
Fig. 2. Major issues that need to be addressed in multidisciplinary research involving material sciences and life sciences.
Ca2+-dependent galactose/GalNAc recognition [193]. In a more recent
study, liposomes incorporated with synthetic glycolipids containing a
terminal GalNAc residue (acting as a ligand for ASGPR) were shown to
be internalized into hepatocytes by ASGPR-mediated endocytosis [194].
Singh et al. [195] reported five times higher uptake of PTX-loaded mag-
netic NPs (PTX-MNPs) conjugated with lectins in K562 leukemia cells
compared to lectin receptor-negative human embryonic kidney (HEK
293) cells.
Neuropilin receptors (NRPs) were initially considered to be
expressed in neuronal cells but were later found to be expressed in
many cell types such as myofibroblasts, endothelial cells and tumors.
The two NRPs (NRP-1 and NRP-2) expressed in vertebrates are trans-
membrane glycoproteins that show 44% homology at the amino acid
level. These receptors contain four distinct extracellular domains that
mediate ligand binding and a short cytoplasmic domain that lacks cata-
lytic activity [196]. NRP-1 is highly expressed in diverse tumor cell lines
including human neoplasms, and has potential implications in tumor
growth and vascularization [196,197]. CRGDK peptide, a ligand of the
NRP-1 receptor, increased the intracellular uptake of gold NPs carrying
the therapeutic P12 peptide (p12) in MDA-MB-321 human breast can-
cer cells overexpressing the NRP-1 receptor [198].
3. Conclusion
Targeting drugs to cancer cells and the tumor microenvironment is a
major challenge in the field of anticancer therapy. This article has
discussed strategies for achieving the specific delivery of anticancer
drugs to cancer cells and tissues by exploiting specific or overexpressed
receptors through the use of nanotechnology. Nanotechnology has en-
abled many options in manipulating nanocarriers based on proteins,
lipids and metals, which can be loaded with anticancer drugs and direct-
ed mostly to cancer cells by the attachment of moieties that recognize
cancer phenotypes. NPs ranging from lipids to metals are under explo-
ration as vehicles for anticancer drug delivery and as moieties guiding
drugs to tumor cells. Lipid-based NPs include polymers, micelles and li-
posomes. Note that lipid-based NPs are composed of the same blocks/
monomers, but their main structural differences lie in their three-
dimensional structures. Polymeric NPs are single, linear but folded poly-
mers made of the same or different blocks, whereas dendrimers are
composed of many macromolecules and are thus tree-like and
hyperbranched. Liposomes are well known vesicular structures com-
posed of lipid bilayers enclosing an aqueous core, whereas micelles
are composed of lipid monolayers without internal aqueous compart-
ments. All are colloidal structures with sizes ordered as polymeric NPs
( 10 nm), dendrimers (2–10 nm), micelles (10–100 nm) and liposomes
(100–200 nm) [199]. Polymeric NPs may be homopolymers (composed
of the same monomer) or heteropolymers (composed of more than two
NP nanoparticle
Anticancer drug molecules
Receptor ligands
Receptor
 M.J. Akhtar et al. / Clinica Chimica Acta 436 (2014) 78–92
different monomers). Liposomal NPs are mainly composed of PEG,
whereas micellar NPs are primarily based on PEG with another block
of cholic acid (CA) [72], poly(lactic acid) [69] and poly(trimethylene
carbonate) (PEG-PTMC) [70]. Instances of metal-based NPs include
iron oxide [116] and gold NPs [145,176,184,198]. Human serum albu-
min (HSA) NPs [129] and quantum dots are other nanoscale drug car-
riers [93]. Lipid-based NPs have been frequently used in anticancer
therapy as carriers of anticancer drugs, especially in targeted delivery
facilitated by overexpressed receptors. Physicochemical factors such as
the dimensions and stability of NPs, the ease of ligand conjugation, the
drug loading ability, and retention times all are likely to affect the suit-
ability of individual nanocarriers to varying degrees.
While nanotechnology is a highly promising technique for targeted
drug delivery to cancer cells and the tissue microenvironment, this ap-
proach will not achieve its potential success unless some technological
and toxicological challenges are addressed (Fig. 2). Several mechanisms
of triggered drug release can only be applied once the anticancer drug
reaches and accumulates at the tumor site. Major issues in nanocarrier
design include nanoparticle–drug loading efficiency, the overall stability
of nanoconjugates with attached ligands, optimal receptor–ligand inter-
actions, and the duration of expression of the targeted receptor. Nano-
particle toxicity and ligand immunogenicity are additional critical
factors that require proper attention in the engineering of clinically ef-
fective nanocarriers for the targeting of anticancer drugs.
Acknowledgment
This work was supported by the Research Chair of King Saud Univer-
sity on Drug Targeting and Treatment of Cancer using Nanoparticles.
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