Receptor-Targeted Surface Engineered Nanomaterials for Breast Cancer Imaging and Theranostic Applications

Abstract

Breast cancer is one of the most frequently diagnosed cancers in women and the major cause of worldwide cancer-related deaths among women. Various treatment strategies including conventional chemotherapy, immunotherapy, gene therapy, gene silencing and deliberately engineered nanomaterials for receptor mediated targeted delivery of anticancer drugs, antibodies, and small-molecule inhibitors, etc are being investigated by scientists to combat breast cancer. Smartly designed/fabricated nanomaterials are being explored to target breast cancer through enhanced permeation and retention effect; and also, being conjugated with suitable ligand for receptor-mediated endocytosis to target breast cancer for diagnostic, and theranostic applications. These receptor-targeted nanomedicines have shown efficacy to target specific tumor tissue/cells abstaining the healthy tissues/cells from cytotoxic effect of anticancer drug molecules. In the last few decades, theranostic nanomedicines have gained much attention among other nanoparticle systems due to their unique ability to deliver chemotherapeutic as well as diagnostic agents, simultaneously. Theranostic nanomaterials are emerging as novel paradigm with ability for concurrent delivery of imaging (with contrasting agents), targeting (with biomarkers), and anticancer therapeutics with one delivery system (as cancer theranostics) and can transpire as promising strategy to overcome various hurdles for effective management of breast cancer including its most aggressive form, triple-negative breast cancer.

Full text

Receptor-Targeted Surface-Engineered
Nanomaterials for Breast Cancer Imaging
and Theranostic Applications
Javed Ahmad,a,*,† Md. Rizwanullah,b,† Teeja Suthar,c,† Hassan A. Albarqi,a
Mohammad Zaki Ahmad,a Parameswara Rao Vuddanda,d
Mohammad Ahmed Khan,e & Keerti Jainc,*
aDepartment of Pharmaceutics, College of Pharmacy, Najran University, Najran, Saudi Arabia;
bDepartment of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia
Hamdard, New Delhi, India; cDepartment of Pharmaceutics, National Institute of Pharmaceutical
Education and Research (NIPER) – Raebareli, Lucknow, India; dResearch Centre for Topical
Drug Delivery and Toxicology (TDDT), University of Hertfordshire, Hertfordshire, United
Kingdom; eDepartment of Pharmacology, School of Pharmaceutical Education and Research,
Jamia Hamdard, New Delhi, India
*Address all correspondence to: Dr. Keerti Jain, Department of Pharmaceutics, National Institute of Pharmaceutical
Education and Research (NIPER) – Raebareli, Lucknow, India; Tel.: +91-522-2497903; Fax: +91-522-2497905,
E-mail: keertijain02@gmail.com; keertijain.02@niperraebareli.edu.in; or Dr. Javed Ahmad, Department
of Pharmaceutics, College of Pharmacy, Najran University, Najran, Saudi Arabia; Tel.: +966 17542 8744,
E-mail: jahmad18@gmail.com; jaahmed@nu.edu.sa
†These authors contributed equally to this work.
ABSTRACT: Breast cancer is one of the most frequently diagnosed cancers in women and the
major cause of worldwide cancer-related deaths among women. Various treatment strategies includ-
ing conventional chemotherapy, immunotherapy, gene therapy, gene silencing and deliberately en-
gineered nanomaterials for receptor mediated targeted delivery of anticancer drugs, antibodies, and
small-molecule inhibitors, etc are being investigated by scientists to combat breast cancer. Smartly
designed/fabricated nanomaterials are being explored to target breast cancer through enhanced per-
meation and retention effect; and also, being conjugated with suitable ligand for receptor-mediated
endocytosis to target breast cancer for diagnostic, and theranostic applications. These receptor-tar-
geted nanomedicines have shown efcacy to target specic tumor tissue/cells abstaining the healthy
tissues/cells from cytotoxic effect of anticancer drug molecules. In the last few decades, theranostic
nanomedicines have gained much attention among other nanoparticle systems due to their unique
ability to deliver chemotherapeutic as well as diagnostic agents, simultaneously. Theranostic nano-
materials are emerging as novel paradigm with ability for concurrent delivery of imaging (with con-
trasting agents), targeting (with biomarkers), and anticancer therapeutics with one delivery system (as
cancer theranostics) and can transpire as promising strategy to overcome various hurdles for effective
management of breast cancer including its most aggressive form, triple-negative breast cancer.
KEY WORDS: breast cancer, receptor, ligand, active targeting, imaging, nanoparticles
ABBREVIATIONS: BRBP1, brain metastatic breast cancer cell-(231-BR)-binding peptide; DOX, doxorubi-
cin; EGFR, epidermal growth factor receptor; EPR, enhanced permeability and retention; FA, folic acid; FR, fo-
late receptor; FSiNP, uorescent silica nanoparticles; HA, hyaluronic acid; HER2, human epidermal growth fac-
tor receptor 2; ICG, indocyanine green; IONPs, iron oxide nanoparticles; LHRH, luteinizing hormone-releasing
hormone; MDR, multidrug resistance; MMC, mouse mammary carcinoma; MRI, magnetic resonance imaging;
NIR, near-infrared; NPs, nanoparticles; PAMAM, poly(amidoamine); PA , photoacoustic; PT, photothermal;
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PAI , photoacoustic imaging; PAT, photoacoustic tomography; PEG, polyethylene glycol; PLA, polylactic acid;
PLGA, poly (D, L-lactide-co-glycolide); QDs, quantum dots; RGD, arginine-glycine-aspartic acid; scFv, single-
chain fragment variable; SPIONs, superparamagnetic iron oxide nanoparticles; Tf, transferrin; TfR, transfer-
rin receptors; TNBC, triple-negative breast cancer; TPGS, D-α-tocopheryl polyethylene glycol 1000 succinate;
VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor
I. INTRODUCTION
Breast cancer is one of the most common cancers in females, with an annual incidence
of about 2.3 million in 2020. It has been associated with the largest number of cancer-
related deaths in women and caused approximately 685,000 deaths, accounting for 15%
of all cancer-related mortality in females. It has been recently declared as the world’s
most prevalent cancer with a count of 7.8 million cases in the last ve years by the WHO
in March 2020.1 While the incidence rates of breast cancer are on the rise in almost every
region, the rates are relatively higher in the developed world.2 Recurrence is a major
concern in the treatment of breast cancer and patients with metastatic breast cancer show
a mean ve-year survival rate of 27%.3 The prognosis of breast cancer is dependent
upon the extent of expression of three receptors namely human epidermal growth factor
receptor-2 (HER2/Neu/ErbB2), estrogen and progesterone receptors.4 Heterogeneity in
their molecular expression in different solid tumor tissue makes it difcult to diagnose
and treat breast cancer. Triple-negative breast cancers (TNBC) which do not express any
of these targets, i.e., estrogen, progesterone, or HER2 receptors are the most challenging
to treat as these show a poor response to both HER2 targeted and hormonal therapies.5,6
Breast cancer therapy has advanced over time from monotherapy to a combinational ap-
proach. However, the development of multidrug resistance (MDR) has also emerged as
a huge challenge in the treatment of breast cancer. MDR to breast cancer occurs through
various complex mechanisms mediated by cellular and non-cellular pathways.7,8
Various advanced approaches such as nanotechnology, precision medicine, and three-
dimensional (3D) printing have recently emerged as an important area of interdisciplinary
research that has the potential to offer solutions for a variety of unresolved disease issues.9–15
Further, the advent of novel metallic, lipids, and polymeric nanoparticles (NPs), as
well as consequent smart design and development of targeting ligand-based NPs have
shown the utility of nanotechnology in the last two decades in the treatment of cancer
as well as other diseases including infectious diseases, cardiovascular and brain disor-
ders, etc.16–20 The main objective of nanocarrier-mediated anticancer drug delivery are,
to enhance the drug concentration in the cancerous tissue through active and passive
targeting, containment of drug during transit in the body, reducing its distribution in the
normal tissues through improvement in its pharmacokinetic prole, facilitate intrave-
nous administration by improving drug solubility, provide better stability and prevent
degradation, improve bioavailability and patient compliance, and to enhance cellular
uptake/intracellular delivery of anticancer agent.21–25
Nanotechnology-based carriers have been widely explored to reduce the dose of an-
ticancer therapeutics by preventing its off-target accumulation using active and passive
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targeting approaches. Engineered NPs can also be developed to vitalize the conven-
tional cancer therapeutic modalities.26,27 Multifunctional NPs have the unique strength
that they can carry multiple payloads including therapeutic and diagnostic agents or
their combination (i.e., theranostic) for the treatment as well as to evaluate the progress
of therapy,28 as depicted in Fig. 1. This parallel multifunctional property opens a new
horizon of opportunities in cancer diagnosis and therapy that have been conceptualized
recently.
Breast cancer is one of the major thrust areas for which various nanotechnological
therapeutic approaches are being explored and some of the NPs have shown promising
results and even reached the clinical stage for therapy of resistant breast cancer cases.
For example, several liposomal formulations of doxorubicin (DOX) have been designed
and developed to improve its therapeutic index without altering its efcacy.29 Thus, nan-
otechnology-based drug targeting approaches can be further explored to resolve long-
standing pitfalls in the treatment of breast cancer for a better prognosis of the therapy.
II. OVERVIEW AND SIGNIFICANCE OF DRUG TARGETING IN BREAST
CANCER
Targeted nanocarriers have special features that make them suitable for targeted delivery
of anticancer drugs by facilitating the accumulation of drugs via leaky vasculature in the
tumor. If the nanocarrier is decorated with a targeting ligand, it can enter the tumor cells
by endocytosis in an efcient manner.30 Intracellularly targeted nanocarriers also protect
the drug particles from substrate-specic efux pumps and prevent their expulsion.31
The drug targeting approach is classied into two major categories, i.e., passive and ac-
tive targeting approach (Fig. 2).
FIG. 1: Diagram of cancer-targeted theranostic nanomedicine
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Tumor vasculature has a very high number of proliferating endothelial cells and an
aberrant basement membrane. This leads to an increased vascular permeability and the
presentation of open fenestrations and inammatory processes on the vascular endothe-
lium. Further, tumor tissue also develops intermittent hypoxic areas.32,33 The mentioned
abnormal structural alterations provide an opportunity that is exploited by circulating
nanocarrier systems having a size range of 20–200 nm to extravasate and accumulate in
the tumor microenvironment. Further, due to the absence of lymphatic drainage in the
tumor, the accumulated nanocarriers are detained in tumor tissues leading to their pro-
longed retention at the target tissue, i.e., tumor. This phenomenon is called the “enhanced
permeability and retention (EPR) effect.”8,34–36 Thus, the abnormal vascular structure is
crucial for the EPR effect and drug targeting at the tissue level in cancer. The phenom-
enon of the EPR effect has been characterized in almost all types of cancers. The EPR
effect applies to and is utilized by almost all noncarriers and it is regarded as the gold
standard in designing cancer targeted drug delivery systems.37–39 Various factors affect
the EPR effect in solid tumors, including: (i) unique and abnormal tumor vasculature,
(ii) active angiogenesis and high vascular density, (iii) impaired lymphatic clearance,
and (iv) extensive production of vascular mediators that facilitate extravasation includ-
ing bradykinin, nitric oxide, carbon monoxide, heme oxygenase-1, vascular endothelial
growth factor (VEGF), prostaglandins and inammatory cytokines, collagenase (matrix
metalloproteinase), angiotensin-converting enzyme inhibitors, etc.40–43 The factors af-
fecting optimal EPR effect are schematically presented in Fig. 3.
The optimal EPR effect is obtained in the case of carriers that can evade the
immune system and can remain in circulation for a relatively longer duration. It
can lead to many fold increase in accumulation of the anticancer drug in tumors
FIG. 2: Diagram of passive and active targeting to tumor site depicting passive targeting of NPs
via EPR effect where NPs can pass through leaky vasculature of tumor blood vessels and accu-
mulates in tumor tissue, while in active targeting ligand conjugated NPs bind to receptors present
on tumor cells and enter tumor cell via receptor-mediated endocytosis. Figure reproduced from
Navya et al.26 under a Creative Commons license.
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compared with normal tissues.44 To achieve this fate a noncarrier needs to have three
ideal properties; rstly, its size should be in the range of 10–100 nm. In any case,
the size for extravasation should not exceed 400 nm. If the size is below 10 nm, it
will be ltered out by kidneys and if is above 100 nm, there is a probability of it
being trapped by the liver. Secondly, the carrier should have a neutral or anionic
charge to prevent its elimination by kidneys. Thirdly, it should bypass the trapping,
opsonization, and phagocytosis by the reticuloendothelial system.45 Opsonization
refers to an immune process, which utilizes proteins called opsonins, for tagging the
foreign nanocarriers which can be recognized and eliminated by phagocytes. Major
opsonins include immunoglubulins and complement components (C3, C4, and C5).
Opsonization is often unfavorable in active-targeting approaches as the adhered
opsonins masks the targeting ligands, resulting in a marked reduction in specic-
ity. The effective interaction between the opsonins and nanocarriers depends on the
size, surface charges and composition of the nanocarriers. Generally, hydropho-
bic and charged particles tend to be opsonized more easily in the bloodstream.46,47
Different properties of NPs, including size, surface properties, composition, etc
which affect their in vitro and in vivo performance including targeting ability in the
treatment of cancer are shown in Fig. 4.
The degree of tumor vascularization and angiogenesis may limit the passive target-
ing efciency of NPs and therefore, the extravasation will vary depending upon ana-
tomical location and type of tumor. Sometimes, solid tumors exhibit high uid pressure
in the interstitium which affects the uptake and distribution of nanocarriers.39,48 Thus,
a combination of high interstitial uid pressure and poor lymphatic drainage can be
FIG. 3: Schematic representation of factors affecting EPR effect
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correlated as the relation between EPR effect and size, i.e., there is more retention of
larger and long-circulating carriers than the smaller molecules.49,50
Active targeting involves the attachment of a ligand to the nanocarrier which can bind
to the receptors overexpressed by tumor cells or vasculature but not or less expressed
by normal cells of the body.51,52 Such targeted receptors should have a homogenous
distribution in the targeted tumor tissue. These targeting ligands could be non-antibody
peptides, antibody fragments, monoclonal antibodies, or endogenous ligands such as
folate, estrone, etc. Tumor penetration is affected by the binding afnity of the targeting
ligand to the binding site and higher afnity binding is observed in tumor vasculature
due to easy accessibility to the cells owing to the dynamic environment of systemic
circulation.37,53–55 The understanding of the difference in tumor microenvironment com-
pared with that of normal tissue has been utilized by researchers in designing better an-
ticancer therapies. Here, the differential expression of targeted receptors in normal and
cancerous cells has particular signicance in designing tumor-targeted nanocarriers for
delivery of anticancer drugs in breast cancer.56–58 The mechanism of receptor-mediated
targeting is depicted in Fig. 5.
III. RECEPTOR-MEDIATED TARGETING IN BREAST CANCER IMAGING
Imaging agents are crucial in the management of cancer attributed to their critical role
in screening and diagnosis of cancer which helps in therapy monitoring and planning
FIG. 4: Tunable physicochemical characteristics of cancer-targeted nanomedicine inuencing in
vitro/in vivo performance of loaded therapeutics/imaging agents to advance the therapy outcome
in breast cancer
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of current and future treatment strategies.59,60 In breast cancer, the degree of hormone-
receptor expression is directly related to the benecial effects offered by hormonal ther-
apy. For example, in a patient with overexpression of HER2 receptors, there are more
binding sites available for the monoclonal antibody (trastuzumab) to work.61,62 Although
immune-histochemistry has been the conventional method used to determine the degree
of hormone-receptor expression, its use in quantifying receptor expression is not wide-
spread.63 In addition, an attempt to detect different types of receptor proteins at once on
a single tumor specimen requires several steps.61,64 Receptor-targeted nanomedicines, on
the other hand, can quantify and prole several biomarkers more accurately and sensi-
tively, thus offering clear advantages over immune-histochemistry.65,66
Nanomedicines are being engineered as imaging and contrast agents for breast im-
aging techniques including magnetic resonance imaging (MRI),67–70 uorescence imag-
ing,71–74 ultrasound imaging,75–78 and photoacoustic tomography (PAT).79–82 In addition,
FIG. 5: Schematic representation of the mechanism of receptor-mediated targeted delivery
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nanoparticulate systems that can serve as multimodal imaging contrast agents are also
being developed and investigated.83–85 Various receptors which are overexpressed in
breast cancer are presented diagrammatically in Fig. 6.
A. MRI
MRI, a radiological technique, is widely used to analyze tissues. In the case of MRI,
rstly, hydrogen atoms present in tissues are excited by the application of the magnetic
eld of appropriate resonance frequency, then these excited hydrogen atoms return to
their equilibrium state while emitting a radiofrequency signal which is used to form the
image. The contrast difference among different tissues is attributed to the different rates
of relaxation of hydrogen atoms of different tissues. MRI offers high spatial resolution
without any ionizing radiation. Superparamagnetic iron oxide nanoparticles (SPIONs)
are the majorly used MRI contrast agents among various paramagnetic and superpara-
magnetic metals.86–89
Various nanocarriers apart from SPIONs has also been designed and investigated
by scientists for targeted imaging of breast tumors. Luteinizing hormone-releasing hor-
mone (LHRH) templated ferrosoferric oxide (Fe3O4) NPs has been investigated as MRI
agent for targeted diagnosis and treatment of breast tumors. The designed LHRH-Fe3O4
NPs showed promising results as a contrast agent to be used in the T2 eld as observed
in MRI experiments performed in female Balb/c mice.79 G3 decorated ankyrin repeat
protein and uorescein-5-maleimide multifunctional SPIONs (G3-5MF-SPIONs) have
been designed by Li and coworkers for MRI of breast tumors. The G3-5MF-SPIONs
showed selective binding (even in the presence of trastuzumab), long retention time,
signicant accumulation, and biodistribution in SK-BR-3 tumor-bearing female Balb/c
FIG. 6: Diagrammatic illustration of commonly overexpressed receptors in breast cancer. HER2,
human epidermal growth factor receptor 2; EGFR, epidermal growth factor receptor (HER1);
ER, estrogen receptor; VEGFR, vascular endothelial growth factor receptor; FR, folate receptor.
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nude mice. Additionally, G3-5MF-SPIONs showed the selectivity to image HER2-
positive breast tumors with remarkable T2 signal reduction after 24 h. These ndings
supported the promising credential of targeted nanomedicine in imaging breast cancer
by MRI.90
Ding et al. developed anti-HER2 single-chain antibody-conjugated iron oxide
nanoparticles (IONPs) as MRI agents. The NPs showed higher afnity toward HER2
overexpressing cells (NCI-N87), compared with HER2 lower expressing cells (SUIT2)
in in vitro cell lines studies. Further, these results were supported by in vivo studies with
NCI-N87 and SUIT2 tumor-bearing mice where a signicantly higher reduction in MRI
signals was observed with NCI-N87 tumors (19.3%) than SUIT2 tumors (6.2%) attrib-
uted to overexpression of HER2 receptors on NCI-N87 cells.91 In another study, Lee et
al. observed that hyaluronic acid (HA)-modied manganese ferrite (MnFe2O4) nanocrys-
tals enhanced the detection ability of MRI in CD44 expressing breast carcinoma cells
(MDA-MB-23 and MCF-7).92 Lim et al. also observed that hyaluronan-modied mag-
netic nanoclusters benet in MRI technique for the detection of CD44-overexpressed
breast cancer cells in MCF-7 and MDA-MB-231 cell lines as well as in MDA-MB-231
bearing female Balb/c nude mice. It was found that the developed nanoparticulate sys-
tem exhibited superior ability for targeted diagnosis of CD44-overexpressed breast can-
cer with excellent sensitivity for MRI.68
Meier et al. developed folic acid (FA) conjugated ultra-small SPIONs which
showed specic retention in imaging in various breast carcinoma cell lines including
MDA-MB-468, ZR-75-1, MDA-MB-435, MCF-7, SK-BR-3, and MDA-MB-231 as
well as in vivo imaging in folate receptors (FR) overexpressing cancer xenograft mice
model. The retention of FR-specic SPIONs led to a signicant negative enhancement
of FR expressed breast tumors on delayed magnetic resonance images at 24 hours post
injection.67 Similarly, Meng et al. found that LHRH-conjugated SPIONs exhibited much
greater efciency than that of unconjugated SPIONs by MRI technique in the detection
of LHRH overexpressed human breast cancer (Hs 578T) as well as breast cancer xeno-
grafts model in addition to metastases in the lungs of athymic nude mice.93 From the re-
search discussed above, it is clear that nanoconjugates systems investigated by scientists
for MRI of breast cancer have shown promising results and are encouraging enough for
scientists to further explore possible clinical applications of these nanomedicine-based
MRI contrast agents for detection of breast cancer to achieve better prognosis in cancer
management.
B. Fluorescence Imaging
Fluorescence spectroscopy is an important technique being used for years for imag-
ing in various elds related to biological and medical sciences such as biochemistry,
biotechnology, molecular biology, pharmaceutical technology, nanotechnology, and
nanomedicine, etc. Fluorescence spectroscopy can provide molecular specicity as
many biomolecules such as proteins, lipids, amino acids, drug molecules such as DOX
can inherently uoresce on excitation with UV-visible light and hence can be used as
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uorescent probes to label target molecules in uorescent imaging.94,95 In this spectros-
copy, the image is formed by photons emitted either from biomolecules having inherent
uorescence ability or from external uorescent active probes.96,97 It offers an inexpen-
sive imaging technique with suitable spatial resolution and is similarly sensitive to radio-
isotopes generally used in single-photon emission computed tomography (SPECT) and
positron emission tomography (PET).98–100 Limited tissue penetration with signicantly
high noise, interference from water molecules, light absorption by proteins, tissue scat-
tering, and autouorescence are the few limitations of uorescence microscopy. Some
of these challenges such as tissue scattering and autouorescence have been reduced to
some extent with tissue penetration to many centimeters by use of near-infrared (NIR)
light during in vivo imaging.101–103
Gold nanoclusters encapsulating protein are being explored for bioimaging and
biosensing applications in cancer due to their biocompatibility, relatively safe prole,
and signicant uorescing ability. Scientists have designed mannose conjugated bovine
serum albumin (BSA) encapsulated gold (Man-BSA-Au) nanoclusters for detection of
concanavalin A and imaging of breast tumor cells using uorescence imaging technique
(Fig. 7A). Man-BSA-Au nanoclusters showed bright red uorescence after incubation
with mannose receptor-expressing MDA-MB-231 cells for 1 h attributed to targeting
of nanoclusters to cancer cells mediated by mannose receptors, whereas cells incubated
with phosphate buffer and BSA encapsulated gold (BSA-Au) nanoclusters showed no
noticeable uorescence (Fig. 7B).73
Scientists have fabricated platinum nanocluster bio-nanoprobes with red emission
using polyamidoamine (PAMAM) dendrimer combined with Protein A (adapter), which
exhibited small size, low cytotoxicity and exceptional photostability for uorescence
imaging of HER2 in SK-BR-3 cells. These bio-nanoprobes exhibit minimal nonspecic
binding that allows highly sensitive optical imaging with visualization of deep anatomy
and increase in tissue penetration due to long-wavelength emission.74 Yamaguchi et al.
observed that technetium-99m (99mTc) and indocyanine green (ICG) labeled anti-HER2
antibody decorated PAMAM-coated silica NPs showed higher imaging efciency
with stronger NIR uorescent signals for HER2 positive SK-BR-3 cells than that in
HER2 negative MDA-MB-231 cells. The in vivo imaging results with SK-BR-3 and
MDA-MB-231 xenograft tumor mice model were also found in accordance with the in
vitro results.104
Quantum dots (QDs) have been exploited by scientists for spectrum analysis and
immunouorescence detection of epidermal growth factor receptors (EGFR). The out-
comes of this study showed the importance of EGFR in the prognosis of lymph node-
positive and HER2-positive invasive breast cancer via immunouorescent detection.63
Li et al. developed FA-functionalized two-photon absorbing 1,2-Distearoyl-sn-glycero-
3-phosphoethanolamine (DSPE)- polyethylene glycol (PEG) NPs with aggregation-
induced emission for specic targeting and imaging in MCF-7 cancer cells using a
two-photon uorescence microscope.105 Wu et al. described the targeted imaging potential
of arginine-glycine-aspartic acid (RGD) peptide-doped uorescent silica NPs (FSiNPs)
in athymic nude mice bearing the MDA-MB-231 tumor on intravenous injection. It
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exhibited a high and specic αvβ3 integrin expression level in the MDA-MB-231 tumor
due to the specic targeting potential of RGD peptide-doped FSiNPs and tumor uores-
cence reached maximum intensity after 1 h of injection.72
FIG. 7: (A) Schematic presentation of preparation of Man-BSA-Au nanoclusters for uores-
cence imaging of human breast cancer cells. (B) Confocal uorescence images of MDA-MB-231
cells after incubation with (a) PBS, (b) BSA-Au nanoclusters, and (c) Man-BSA-Au nanoclusters
for 1 h (excitation wavelength: 488 nm; scale bar: 20 μm) (reprinted from Sha et al.73 with per-
mission from Elsevier, copyright 2020).
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12 Ahmad et al.
Different nanomaterials including lipidic NPs, polymeric NPs, metallic NPs, QDs,
and dendrimers, etc have been engineered smartly by researchers for uorescent imag-
ing of breast cancer with promising results in preclinical studies in animals.
C. Ultrasound Imaging
Ultrasound imaging is widely used as a tomography technique for the clinical diagno-
sis of breast cancer. It can show minute internal details of organs and tissues attributed
to the use of sound waves of a shorter wavelength having frequencies of 2 MHz or
greater. Transmitting and receiving transducers are set to capture ultrasound wave in-
teractions.106 Ultrasound imaging is commonly used to diagnose various diseases via
imaging different internal organs, muscles, and tendons, and gives information related
to the structure, size, or presence of any abrasion, injury, or lesions in these areas. High
resolution with low cost, non-invasive procedure, and precise targetability of ultrasound
energy deposition are some of the advantages of ultrasound imaging.86,107
Xu et al. developed dual-targeted gold nanoshell poly (D,L-lactide-co-glycolide)
(PLGA) nanocapsules decorated with vascular endothelial growth factor receptor
(VEGFR)-2 and p53 antibodies for specic ultrasound molecular imaging of breast
cancer, which showed excellent biocompatibility. The results of in vivo studies in
the MCF-7 orthotopic mice model indicated efcient binding to cells overexpressing
VEGFR2 or p53 protein with no signs of organ damage.77 Polylactic acid (PLA) NPs
have been designed and evaluated for comparison of targeted ultrasound imaging of
HER2-overexpressing and HER2-negative breast cancer cells by scientists. In results,
HER2-overexpressing cells showed more staining after incubation with NPs/antibody
conjugates, whereas minimal staining was seen in HER2-negative cells, indicating di-
rect receptor binding of targeted NPs. The average gray matter of HER2-positive cells
was signicantly higher in NP-treated cells compared with the control shown in high-
resolution ultrasound images.76
In another study, Sakamoto et al. carried out the molecular analysis of breast cancer
using herceptin conjugated IONPs-based ultrasound imaging agents. The ultrasound im-
aging results supported the ability of herceptin decorated IONPs to differentiate between
HER2/neu positive SK-BR-3 cells as compared with the non-tagged NPs.75 These stud-
ies validated that conjugated NPs may have promising applications in receptor-targeted
non-invasive imaging of breast cancer.
D. Photoacoustic Imaging/Tomography
Photoacoustic imaging (PAI) or photoacoustic tomography (PAT) is an emerging non-
ionizing imaging tool combining high optical contrast and high ultrasound resolution.108
PAT makes use of pulsed laser light to generate light-induced acoustic signals.109 PAT
with nite element reconstruction has been used to image small nanometer-sized par-
ticles containing objects with high resolution.110 Photoacoustic provides in vivo morpho-
logical and functional information regarding the tumors within the surrounding tissue.
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Receptor-Targeted Surface-Engineered Nanomaterials 13
Photoacoustic (PA) with the use of targeted contrast agents is also capable of performing
in vivo molecular imaging, thus enabling further molecular and cellular characterization
of cancer.59,111,112
In vivo PAI studies using VEGF targeted, anti-VEGFR antibody-conjugated iron
platinum alloy NPs showed that with these NPs the PAI depth could be extended to more
than 5 cm in chicken breast tissues with the use of a laser energy density within the safe
limit. Further, it was observed that NPs were removed from the site of the tumor and
metabolized mainly by the liver supporting the safe use of this nanomedicine in early
detection and theragnosis of breast cancer with whole-body PAI.82 Balasundaram et al.
demonstrated that folate decorated NPs showed ~ 4-fold enhancement in the PA signal
in the MCF-7 breast cancer xenograft model compared with untargeted NPs, thus pro-
viding a better delineation from the surrounding tissue.113 Furthermore, Kanazaki et al.
developed a single-chain fragment variable (scFv) anti-HER2 moiety conjugated IONPs
for PAI studies. Their results demonstrated that anti-HER2 scFv-conjugated IONPs
showed high afnity and specic binding to HER2-expressing N87 tumor-bearing fe-
male Balb/c-nude mice and SUIT2 cells compared with untargeted NPs.114
Scientists have investigated breast cancer imaging techniques by combining high-
resolution NIR induced PAT using NIR dye-labeled with amino-terminal fragments of
urokinase plasminogen activator receptor-targeted magnetic IONPs in orthotopic mouse
mammary tumor model (mouse bearing mammary carcinoma cell line 4T1). Their re-
sults showed 4–10 fold amplication in PA signals in the tumor with receptor-targeted
NPs as compared with non-targeted ones.115 Wang et al., 2014 designed FR-targeted
ICG-loaded PLGA (ICG-PLGA) lipid NPs. It exhibited high targeting efciency for FR
and remarkable optical absorption in NIR wavelengths resulting in excellent PA signals
in in vitro as well as in vivo studies in MCF-7 bearing female Balb/c nude mice over
non-targeted ICG-PLGA lipid NPs.11 6 Furthermore, a strategically designed study using
HA decorated gold NPs for PAI showed that PA/photothermal (PT) signals from targeted
NPs in MDA-MB-231 cancer cells were 10- to 50-fold higher than untargeted NPs, and
the rate of ashing PA signals signicantly increased in Balb/c nude mice model.79
The use of receptor-targeted nanomedicines such as antibody-conjugated NPs, fo-
late, HA conjugated nanomaterials, etc have shown that the use of these ligand conju-
gated nanomedicines targeted toward a particular receptor overexpressed on tumor cells
can promisingly improve in vivo imaging efciency of PAI along with may provide
further details regarding the cellular and molecular characterization of tumor.
E. Multimodal Imaging
The intrinsic merits and demerits of each imaging technique are signicant and hardly
overcome by the improvement of imaging instrumentation alone. Multimodal imaging
includes the combined usage of various imaging tools such as MRI/uorescence imag-
ing and it has gathered signicant attention to advance the currently used techniques for
diagnosis of breast cancer. It is a powerful technique that provides more consistent and
accurate recognition of disease sites.117,118
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14 Ahmad et al.
Du et al., 2020 developed brain metastatic breast cancer cell-231-BR-binding pep-
tide (BRBP1)-functionalized ultra-small IONPs containing DiR uorescent dye for tar-
geted MR/NIR uorescent dual-modal detection of breast cancer brain metastasis. Their
results showed that the NPs exhibited a size of 10 nm with core-shell structure, high
relaxivity values, and efcient photon emission in vitro. Moreover, the NPs presented a
T2 contrast imaging effect and NIR uorescence signal enhancement. The MR/NIR u-
orescence signal of BRBP1-modied NPs in tumor tissue was signicantly enhanced as
compared with the control. The results showed that BRBP1-IONPs-mPEG NPs loaded
with DiR can be employed as an attractive targeting strategy for the detection of breast
cancer brain metastasis.85 Similarly, Han et al. developed CD44 monoclonal antibodies
coupled magnetic-uorescent iron oxide carbon hybrid nanomaterials which demon-
strated strong excitation wavelength-dependent uorescence in the blue-red region with
a quantum yield of 58.4%, and they displayed higher stability and T2 relaxivity than
simple IONPs. Moreover, there was signicant preferential uptake of the conjugates by
4T1 breast cancer cells as seen in biological transmission electron microscope imaging.
In addition, the developed CD44-hybrid nanomaterials were able to distinguish 4T1
cells from normal cells by virtue of its high binding afnity and specicity of the CD44
antibodies to the CD44 receptors on the surface of cancer cells.119
Herceptin-decorated paclitaxel-loaded ultrasmall superparamagnetic iron ox-
ide nanobubbles exhibited signicantly enhanced cytotoxic effects against HER2-
overexpressing SK-BR-3 breast carcinoma cells and signicantly lower cytotoxicity
against HER2-negative MDA-MB-231 breast carcinoma cells. In addition, the devel-
oped magnetic nanobubbles showed ability to enhance ultrasound, magnetic resonance,
and photoacoustics trimodal imaging.120 Hyaluronan-modied SPIONs were designed
for multimodal imaging of breast cancer which showed signicantly enhanced spe-
cic cellular uptake of HA-SPIONs in HA receptor-overexpressing MDA-MB-231
cells which was conrmed by uorescence imaging. In addition, HA-SPIONs demon-
strated signicant negative contrast enhancement on T2-weighted MR images of HA
receptor-overexpressing MDA-MB-231 breast tumor-bearing balb/c nude mice com-
pared with untargeted SPIONs.121 Kievit et al. developed herceptin decorated uores-
cent dye loaded multifunctional SPIONs that exhibited a 4-fold improvement in cellular
uptake and specicity to target the neu/HER2-overexpressing mouse mammary carci-
noma cells. It showed signicant contrast enhancement in magnetic resonance images
of breast tumors.83
These studies demonstrated that nanomedicine may advance the diagnosis of vari-
ous breast cancers by utilizing receptor-mediated targeting of imaging agents. The po-
tential of various receptor-targeted nanomedicines explored by different researchers/
investigators for the detection or imaging of breast cancer are summarized in Table 1.
IV. RECEPTOR-MEDIATED THERANOSTICS IN BREAST CANCER TREATMENT
NPs can be designed with some inherent properties to offer unique imaging and sur-
face functionalization ability. Also, NPs have optimum size, high surface area to volume
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TABLE 1: Receptor-targeted nanomedicines for detection/imaging of breast cancer
Imaging
technique
Types of
nanomedicine
Ligand Receptor targeted Breast cancer
model
Outcome Ref.
Magnetic
resonance
imaging
Fe3O4 NPs LHRH LHRH Human MCF-7
breast cancer cells
LHRH-Fe3O4 effectively accumulated in
the tumor region due to magnetic targeting.
It showed a good negative enhancement
effect that signicantly reduced the
intensity of T2 weighted images, allowing
it to be used as a contrast agent
70
Fe3O4-Au NPs Thiolated
AS1411
aptamer
Urokinase-type
plasminogen
activator (uPA)
4T1, mouse
mammary
carcinoma, and
HFFF-PI6, human
foreskin broblast
cell lines
–Developed nanoprobe produced strongly
darkened T2-weighted MR images with
4T1 cells, whereas brightened images with
HFFF-PI6 cells
–Specic targeting of 4T1 cells
overexpressing nucleolin on the cell surface
–Nanoprobe binds specically to breast
cancer cells and has very low accumulation
in normal cells.
69
MnFe2O4
nanocrystals
HA HA receptor In vitro/
MDA-MB-231 and
MCF-7 cells
Excellent MR imaging sensitivity to
diagnose CD44-overexpressing cancer cells
92
Magnetic
nanoclusters
Hyaluronan HA receptor In vitro/
MDA-MB-231
and MCF-7 breast
carcinoma cells
In vivo/Balb/c-Slc
nude mice bearing
MDA-MB-231
cancer cells
Nanoclusters showed excellent ability for
targeted detection of CD44-positive breast
carcinoma cells by showing excellent
sensitivity as MR imaging agents
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16 Ahmad et al.
TABLE 1: (continued)
Imaging
technique
Types of
nanomedicine
Ligand
Receptor targeted
Breast cancer
model
Outcome
Ref.
SPIONs FA FA receptor In vitro/
MDA-MB-231
In vivo/breast
cancer xenograft
in female athymic
Harlan
rats
–Specic retention in FR-positive
MDA-MB-231 cell line and in an FR-
positive human breast carcinoma xenograft
model in vivo
–Signicant negative enhancement of
FR-positive breast tumors on delayed MR
images at 24 hours post injection
67
SPIONs LHRH LHRH receptor In vitro- Hs 578T
cells
In vivo-breast
cancer xenografts
and lung
metastases of
athymic nude mice
–LHRH conjugated SPIONs exhibited
signicantly higher uptake in the cells than
that of the unconjugated SPIONs in both in
vitro and in vivo studies
–The enhanced uptake of intracellularly
accumulated LHRH conjugated SPIONs
provided T2 contrast enhancement leading
to improved spatial resolution in MRI by
classical T2 imaging
93
Fluorescence
imaging
Red-emitting
Platinum
nanoclusters
Protein A and
anti-HER2
antibody
HER2 SK-BR-3 breast
cancer cells
Platinum bio-nanoprobe binds to HER2-
overexpressing SK-BR-3 cells with a
higher afnity and selectivity
74
Mannose
functionalized
BSA Gold
Nanoclusters
Mannose
Concanavalin A on
Mannose receptors
Human
breast cancer
MDA-MB-231
cells,
HUVEC cells
Increased sensitivity and selectivity
of Man-BSA-Au nanoclusters for
determination of Concanavalin A on
mannose receptor
73
99mTc-
radiolabeled
nanosilica system
Trastuzumab HER2 Female Balb/c
nude mice
SK-BR-3 breast
cancer cells
–Radiolabeled system exhibited an
enhanced active targeting to the tumor,
compared with EPR-based passive
diffusion
–Signicantly enhanced accumulation of
SiNPs within the HER2 overexpressing
SK-BR-3 cells
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Receptor-Targeted Surface-Engineered Nanomaterials 17
TABLE 1: (continued)
DSPE-PEG NPs FA FA receptor
In vitro
/MCF-7
breast cancer cells
Two-photon uorescence images obtained
from the uorescence signal from
cytoplasm after incubation with developed
NPs clearly distinguished the MCF-7 breast
cancer cells from others
105
FSiNPs RGD peptide Integrin receptor In vitro/
MDA-MB-231 cell
Ex vivo/athymic
nude mice bearing
the MDA-MB-231
tumors
RGD peptide-conjugated FSiNPs were
able to image the specic αvβ3 integrin
expression level in the MDA-MB-231 cells
due to its special receptor-based targeting
effects
72
Ultrasound
imaging
Gold-Nanoshells
PLGA
Magnetic Hybrid
NPs
Anti-HER2
antibodies
HER2 Human breast
cancer SK-BR-3
cells
Conjugation with anti-HER2 antibodies led
to specic and sensitive binding with HER2
overexpressing SK-BR-3 breast cancer
cells.
Excellent contrast enhancement in vitro
78
Gold nano-
shelled PLGA
nanocapsules
Anti-VEGFR2
and anti-p53
antibodies
(DNCs)
Vascular endothelial
growth factor
receptor type 2
HUVECs, 4T1,
MCF-7, and
MDA-MB-231
breast cancer cells
MCF-7 cell bearing
Female Balb/c
nude mice
–DNCs exhibited high target specicities in
vitro in VEGFR2- and p53-overexpressing
cells compared with control cells
–Ultrasound imaging studies using novel
nano-sized, dual-targeted PLGA-Au
nanocapsules ultrasound contrast agents
allowed highly precise and reliable
detection of breast cancer in MCF-7
orthotopic mice model
77
Anti-EGFR
antibody-
conjugated gold
nanorods
Anti-HER2
antibody
EGFR EGFR-negative
MCF-7 cells
EGFR-positive
MDA-MB-231
cells
Mouse model of
human TNBC
Ultrasound-guided PAI using anti-
EGFR decorated gold nanorods showed
a very high sensitivity for the selective
visualization of EGFR-positive primary
breast tumors as well as Lymph Nodes
micrometastases
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18 Ahmad et al.
TABLE 1: (continued)
Imaging
technique
Types of
nanomedicine
Ligand
Receptor targeted
Breast cancer
model
Outcome
Ref.
PLA-NPs Anti-HER2
antibody
HER2 receptor In vitro/SK-BR-3
and MDA-MB-231
cell line
In high-resolution ultrasound B-mode
images, the average gray scale of the
HER2-positive cells (SK-BR-3 cell) was
consistently and signicantly higher
76
IONPs Herceptin HER2 receptor In vitro/SK-BR-3
human breast
cancer cells
Ultrasound imaging exhibited excellent
sensitivity and specicity in the detection
of HER2 overexpressed breast cancer cells
75
Photoacoustic
(PA) imaging
Iron-platinum NPs anti-VEGFR VEGF 4T1 breast cancer
cells
Accumulation in the tumor region,
Targeted delivery of NPs by EPR effect
82
Irradiated
nanodiamonds
Anti-HER2
peptide
HER2
4T1.2 breast cancer
cells
Female Balb/c
mice
–PA images demonstrated that
nanodiamonds signicantly accumulated in
breast tumors and traced the entire tumor in
less than 10 hours
–HER2 conjugation signicantly enhanced
the imaging of HER2-positive tumors
–The conjugation of PEGylated
nanodiamonds with anti-HER2 peptide
led to enhanced internalization of INDs
by HER2 positive tumor cells (4T1.2-neu)
and longer residence time in the region of
HER2 positive tumor
81
ICG conjugated
PLGA lipid NPs
FA FR In vitro/MCF-7
cells
In vivo/Female
Balb/c nude mice
bearing human
breast cancer
(MCF-7) cells
Developed NPs showed excellent optical
properties with reduced toxicity, enhanced
tumor-targeting capability, prolonged
circulation time, and signicantly improved
PA contrast for preclinical imaging
applications
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TABLE 1: (continued)
Gold nanorods Bombesin
(BBN)
Gastrin releasing
peptide (GRP)
receptor
Breast tumor-
bearing Balb/c
mice
T47D cells
In vitro studies showed a very selective
binding of PEG-BBN gold nanorods
towards breast cancer cells.
Biodistribution studies showed that after
the injection of PEG-BBN gold nanorods
conjugate (high uptake) and PEG gold
nanorods conjugate (low uptake) approved
the targeting ability of PEG-BBN gold
nanorods conjugate to breast cancer cells.
PEG-BBN gold nanorods is a reliable
vector for targeting GRP receptors over-
expressed in various cancers
125
IONPs NIR830 Urokinase
plasminogen
activator receptor
In vivo/Balb/c mice
bearing Mouse
mammary tumor
4T1 cells
In vivo studies in mice showed 4- and 10-
fold enhancement in PA signals of IONPs
compared with non-targeted IONP or
control Balb/c mice
115
Gold carbon
nanotubes
CD44
receptor-
specic
antibody
CD44 receptor
In vitro
/
MDA-MB-231
cells
In vivo/nude mice
–PA/PT signals from targeted
MDA-MB-231 cells were 10 to 50-fold
higher than those from unbound nanotubes
distributed randomly in suspension between
cells at relatively high laser uences of
300–500 mJ/cm2
–After subsequent injections of CD44-
decorated gold carbon nanotubes, the rate
of ashing PA signals increased to 46.7 ±
6.8 cells/min in nude mice
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TABLE 1: (continued)
Imaging
technique
Types of
nanomedicine
Ligand
Receptor targeted
Breast cancer
model
Outcome
Ref.
Multimodal
imaging
Ultra-small IONPs BRBP1
peptide
EGFR receptor Mice bearing 231-
BR xenografts
–Ultra-small IONPs offered a T2 contrast
imaging effect and enhanced NIR
uorescent signal
–The MR/NIR uorescence imaging signal
of BRBP1-modied NPs in tumor tissue
was signicantly enhanced as compared
with control NPs, which shows the
targeting ability of BRBP1 peptide
85
Natural magnetic
NPs with Gold
nanorods
Folate FA receptor In vitro
MDA-MB-231 cell
line
Mouse model
injected with breast
cancer cells
–Magnetotactic bacteria, natural magnetic
NPs, and natural magnetic NPs-gold
nanorods can be used for scanning as well
as in PA and PT imaging
–Signicant enhancement in PA signals
from natural magnetic NPs and natural
magnetic NPs-gold nanorods both in vitro
and in vivo
–Natural magnetic NPs-gold nanorods were
able to provide for real-time counting of
labeled cells, magnetic trapping of labeled
cells (e.g., CTCs), and killing of diseased
circulating cells
84
SPIONs Herceptin HER2 receptor In vitro/
MDA-MB-231
cells
In vivo/transgenic
breast cancer mice
–SPIONs showed up to 4-fold improved
cellular uptake and specic ability to
target neu/HER2 overexpressing mouse
mammary carcinoma (MMC) cells in vitro
and in vivo
–Signicant enhancement in the contrast
of SPIONs MR images in primary breast
tumors
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ratio, higher loading capacity for contrast agents, targeting ligands, and therapeutic mol-
ecules.126 Furthermore, many NPs have inherent imaging properties, which can further be
functionalized to become nanotheranostics. Theranostics refers to the integrated applica-
tions of diagnostic/ imaging agents and therapeutic moiety in one system. Theranostic
nanomedicines or nanotheranostics are multifunctional nanosystems designed for pre-
cise and personalized disease management.127 They consist of macromolecular materials/
polymers in which imaging and therapeutic agents can be absorbed, adsorbed, encapsu-
lated, entrapped, or conjugated for simultaneous imaging and therapy at the cellular and
molecular level.128 Ideal theranostic nanomedicines should have the following properties:
(i) safe for administration in humans, (ii) shows rapid and selective accumulation at the
target site, (iii) recognizes biochemical and morphologic characteristics of a disease, (iv)
able to deliver sufcient drug on-demand without damaging healthy organs, and (v) rap-
idly cleared from the body within hours or be biodegraded into nontoxic byproducts.129
Generally, theranostic nanomedicines can be fabricated in several ways: (i) thera-
peutic agents (e.g., anticancer drugs and photosensitizers) may be adsorbed, conjugated,
entrapped, or loaded, to existing NPs having intrinsic imaging abilities such as QDs,
IONPs, and gold nanocages; (ii) attaching the imaging agents, such as uorescent dyes,
optical or magnetic NPs, and various radioisotopes, to existing therapeutic NPs; (iii)
encapsulating both diagnosing and therapeutic agents together in different nanocarriers
such as polymeric NPs, porous silica NPs and ferritin nanocages; and (iv) some unique
NPs (e.g., porphysomes, 64Cu-CuS, and gold nanoshells or cages) with dual inherent im-
aging and therapeutic properties can also be engineered for theranostic applications.130,131
The surface of these nanocarriers can be modied with PEG and various target-
ing ligands to prolong the blood circulation half-life and to provide the active tumor-
targeting capability. Targeting ligands can be exploited to identify and selectively bind
to cell surface receptors overexpressed on tumor cells or surfaces. Various targeting
ligands including, antibodies, small peptides, aptamers, lectins, some proteins, or pro-
tein fragments can be used depending on the physicochemical properties of theranostic
nanocarrier or tumor of interest. In the last few decades, substantial efforts have been
made by researchers to design and evaluate different receptor-targeted nanomedicines
for simultaneous diagnosis and treatment of breast cancer with certain promising results;
however, some challenges have also been faced by scientists while designing targeted
theranostic nanomedicines. The following subsection of the article discusses different
receptor-targeted nanomedicines for theranostic applications in the breast cancer treat-
ment particularly in the invasive variants such as TNBC and deals with the challenges
encountered while designing and developing these theranostic nanomedicines.
A. HER2 Targeted Theranostics
HER2 receptors are overexpressed on breast cancer and have been explored by scientists
for targeted theranostic applications. Mechanistic approaches for exploring HER2 recep-
tors for targeted breast cancer theranostics are shown diagrammatically in Fig. 8. Recently,
Khaniabadi et al. investigated the theranostic potential of superparamagnetic Fe3O4 NPs
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decorated with protoporphyrin and trastuzumab for imaging and treatment of EGFR2-
overexpressing breast cancer. The results showed no signicant cytotoxicity after incubat-
ing the MCF-7 breast cancer cells under various Fe concentrations of NPs and theranostic
agents, whereas in vitro photothermal ablation of targeted NPs showed a reduction of 74%
in MCF-7 cells after 10 min at the highest Fe concentration.132 In another study, Choi et
al. developed IONPs and DOX-loaded multifunctional nanocarriers with an ability of in-
tegrated cancer-targeting via a HER2 monoclonal antibody for controlled delivery of an-
tineoplastic drug (DOX) as well as imaging agent (IONPs) for MRI and NIR uorescence
imaging. The results of in vitro studies demonstrated up to 5-fold higher cellular uptake and
signicantly higher cytotoxicity to HER2 overexpressing SK-BR-3 cells than HER2 nega-
tive MCF-7 cells, signifying HER2 receptor-based cancer targeting. In in vivo tumor xe-
nograft model, antibody-targeted nanocarriers exhibited signicantly higher uptake in the
cancerous cells and the size of the tumor signicantly reduced than the non-targeted ones.133
Parhi and Sahoo developed trastuzumab-conjugated rapamycin, and QDs-loaded
theranostic NPs which exhibited signicantly higher uptake in SK-BR-3 cells and
MDA-MB-231 cells compared with untargeted NPs. Targeted NPs showed ~ 3 and ~
1.5-fold higher toxicity to SK-BR-3 cells and MDA-MB-231 cells, respectively, com-
pared with untargeted NPs. The cellular uptake studies were performed using couma-
rin-6 dye and the results of cell uptake studies showed that confocal images indicated
a higher uorescence intensity in tumor spheroids treated with targeted NPs than that
treated with untargeted NPs (Fig. 9). Further, the therapeutic benets of targeted NPs
were explored at the molecular level and the results showed augmented downregulation
of mTOR signaling pathway thereby inducing further cell death.134
Kievit et al. developed multifunctional SPIONs for diagnosis and treatment of meta-
static breast cancer and evaluated in a FVB/N transgenic mouse model. They fabricated
uorescent dye-loaded NPs using a copolymer of chitosan and PEG for optical detection
FIG. 8: Diagrammatic representation of theranostic applications achieved with HER2 recep-
tor targeting, where QD: Quantum dots; Tmab-QD-rapa-NPs: Trastuzumab-conjugated rapamy-
cin and QDs-loaded theranostic NPs (reprinted from Parhi and Sahoo134 with permission from
Elsevier, copyright 2015)
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and labeled them with trastuzumab against the HER2 receptor. The results of cellular
uptake studies showed that trastuzumab-doped NPs exhibited a 4-fold higher cellular
uptake and could specically target HER2 overexpressing mouse breast cancer cells.
Also, the dye-coated NPs provided signicant enhancement in contrast of MR images
of primary breast tumors that were detected with MRI. Moreover, these NPs were able
FIG. 9: Qualitative intracellular uptake of coumarin-6, coumarin-6-NPs, and Tmab-coumarin-
6-NPs (50 ng/mL) in SKBR 3 cells and MDA-MB-231 cells by confocal microscopy after 2
hrs of incubation (reprinted from Parhi and Sahoo134 with permission from Elsevier, copyright
2015)
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to detect micrometastases by recognizing and tagging spontaneous micrometastases in
the lungs, livers, and bone marrow of these mice.83
In another study, Day et al. developed NIR resonant anti-HER2 antibody conjugated
gold-gold sulde NPs as theranostic agents for management of breast cancer via mul-
tiphoton microscopy and higher intensity photoablation. The study demonstrated that
anti-HER2 antibody-doped NPs could efciently bind to SK-BR-3 breast cancer cells.
Antibody conjugated NPs emitted luminescence upon excitation with a pulsed laser
resulted from a two-photon absorption process. At 1 mW laser power, Anti-HER2 anti-
body conjugated gold-gold sulde NPs could specically visualize SK-BR-3 cells and
cell death was induced upon increasing laser power to 50 mW followed by membrane
blebbing.135 Similarly, Yang et al. developed anti-HER2 antibody conjugated DOX-
loaded magnetic PLGA NPs for imaging and treatment of breast cancer. The in vitro
studies of anti-HER2 antibody conjugated DOX NPs using HER2/neu-overexpressing
broblast NIH3T6.7 cells and HER2-negative SK-BR-3 and MDA-MB-231 cell lines
demonstrated 87.4 times higher uorescence intensity than that of other cells. The mul-
timodal NPs demonstrated remarkable cancer cell afnity. Also, the NPs followed a
sustained release of loaded anticancer drugs for 3 weeks.136
HER2 receptors and HER2 targeting antibody, trastuzumab have been investigated
for targeted theranostic applications in cancer and have shown efcacy to be explored as
promising theranostic nanomedicines for treatment and imaging of breast cancer.
B. Folate-Targeted Theranostics
FR are highly overexpressed on different types of cancer cells including breast cancer
and have been exploited by scientists for targeted delivery of antineoplastic drugs, con-
trast agents as well as theranostic applications in cancer. Soleymani et al. fabricated fo-
late-targeted IONPs which showed a signicant reduction in the T2-weighted MR signal
intensity of breast tumors indicating the accumulation and retention of NPs in the tumor
tissue. Moreover, a signicant reduction in tumor progression was seen in the mice.137
In another study, Alibolandi et al. developed folate-decorated QDs and DOX-loaded
theranostic polymersomes for the imaging and treatment of breast cancer. The results
of uorescence microscopic and cytotoxicity studies showed that the folate-conjugated
DOX-QD NPs displayed signicantly higher cellular uptake and cytotoxicity in 4T1
and MCF-7 cells compared with untargeted NPs and pure drug solution. Fluorescence
imaging studies in Balb/c mice bearing 4T1 breast adenocarcinoma demonstrated that
the targeted NPs showed signicant accumulation at the tumor site after 6 h post intra-
venous injection. Also, acute toxicity studies showed no evidence of long-term harmful
histopathological and physiological changes in the treated animals. Moreover, the tar-
geted NPs demonstrated much better therapeutic efcacy in vivo compared with untar-
geted NPs and free drugs.138
FA and cisplatin prodrug conjugated gold nanoclusters showed that FA-conjugation
led to a signicant increase in the cellular uptake and cytotoxicity of cisplatin gold
nanoclusters in murine 4T1 breast cancer cells. Fluorescence imaging studies in 4T1
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tumor-bearing nude mouse showed that FA-cisplatin gold nanoclusters selectively ac-
cumulated in the 4T1 tumor cells and produced intense uorescence signals due to the
tumor-targeting effect of FA. In addition, FA-cisplatin gold nanoclusters showed sig-
nicant inhibition in the growth and lung metastasis of the orthotopically implanted
4T1 breast tumors.139 In another study, Heidari Majd et al. developed tamoxifen-loaded
FA-armed PEGylated MnFe2O4 NPs for imaging and treatment of the FR-positive breast
cancer cells. Fluorescence imaging and ow cytometry analyses revealed substan-
tial interaction of developed NPs with FR-overexpressing MCF-7 breast cancer cells.
Cytotoxicity studies demonstrated signicant inhibition in the growth of MCF-7 cells
treated with developed NPs.140 Muthu et al. also developed FR-targeted D-α-tocopheryl
polyethylene glycol 1000 succinate (TPGS) coated theranostic liposomes containing
docetaxel and QDs for diagnosis and therapy of breast cancer. Confocal laser micros-
copy study showed that folate-targeted liposomes exhibited a signicantly higher red
uorescence intensity and higher cell uptake in MCF-7 cells compared with untargeted
liposomes. Also, they observed a reduction in IC50 values of docetaxel-loaded TPGS
coated targeted and non-targeted liposomes by 97.58% and 83.64% compared with
Taxotere® after 24 h incubation with MCF-7 cells.141
FA targeting has shown promising outcomes in the treatment of breast cancer and
could be utilized for its potential theranostic applications in cancer.
C. CD44-Targeted Theranostics
CD44 receptors are overexpressed in cancer cells including breast cancer. HA is a prin-
cipal ligand for CD44 receptors and scientists have explored HA conjugated nanomed-
icines to achieve targeted delivery of therapeutic, diagnostic and theranostic agents.
Scientists have investigated HA-decorated, red emission cationic BSA protected gold
nanoclusters loaded with ICG and paclitaxel for chemo-photothermal therapy and nitric
oxide to modulate the tumor microenvironment and enhance the delivery of drug to the
cancer cells. The developed nanoclusters presented size-reducible ability in presence
of hyaluronidase and signicantly higher accumulation in breast cancer cells with the
homogenous intra-tumor distribution. Furthermore, their study showed a suppression of
95.3% in in situ tumor growth and 88.4% inhibition of lung metastasis growth.142
Similarly, Wang et al. developed pH-responsive HA prodrug micelles with aggre-
gation-induced emission properties by a chemical graft of biocompatible phosphoryl-
choline, DOX, and aggregation-induced emission uorogen tetraphenylene to the HA
backbone. The intracellular distribution of HA was observed by uorescence microscopy.
Flow cytometry and uorescent microscopy indicated that HA prodrug micelles were ef-
fectively internalized by MDA-MB-231 carcinoma cells due to HA-mediated endocyto-
sis. Also, the cytotoxicity results indicated that the prodrug micelles showed signicant
inhibition in the proliferation of cancerous cells. HA backbone could greatly enhance
the cellular internalization ability, furthermore, they observed a signicant inhibition
in the proliferation of cancer cells and higher cytotoxicity in MDA-MB-231 cells.143
Receptor-targeted theranostic nanomedicines based on CD44 receptor overexpression
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26 Ahmad et al.
on cancer cells have shown the ability for simultaneous targeted delivery of antineoplas-
tic drugs and imaging agents to cancer cells.
D. EGFR-Targeted Theranostics
EGFR receptors are overexpressed on breast cancer cells and scientists have explored
anti-EGFR antibodies and aptamers for targeted theranostic applications in breast can-
cer. Kim et al. designed aptamer-loaded lipid-based nanocarriers containing QDs and
siRNAs for imaging and treatment of TNBC. The hydrophobic QDs were incorporated
in the lipid bilayer of liposomes, and then liposomes containing QDs were complexed
with therapeutic siRNAs and functionalized with anti-EGFR aptamer for targeting
TNBC. The results of uorescence imaging studies showed enhanced delivery of de-
veloped nanocarriers to EGFR overexpressing cancerous cells and thus, more effective
gene silencing and enhanced tumor imaging compared with non-targeted nanocarriers.
Moreover, combinatorial therapy with Bcl-2 and PKC-ι siRNAs loaded into the anti-
EGFR QD lipid nanocarriers in tumor-bearing female Balb/c nude mice caused remark-
able inhibition in tumor growth and metastasis.144
In another study, Wang et al. developed anti-EGFR nanobody-decorated QDs based
theranostic micelles loaded with an antineoplastic drug, aminoavone for TNBC ther-
apy. The results of in vitro studies in EGFR overexpressing MDA-MB-468 cells showed
that anti-EGFR nanobody 7D12 conjugation resulted in an enhanced cellular uptake and
cytotoxicity of the QD-based micelles. Furthermore, there was a higher concentration of
targeted micelles in tumors as compared with non-targeted ones, leading to more effec-
tive tumor regression in an orthotopic TNBC xenograft mouse model. Also, they did not
observe any systemic toxicity with the treatments.145 From these studies, it could be con-
cluded that EGFR targeted nanomedicine could be explored promisingly for theranostic
applications in breast cancer.
E. Transferrin-Targeted Theranostics
Transferrin (Tf) functionalized nanomedicines have signicantly been explored for
targeted delivery of anticancer therapeutic agents. Scientists are also exploring Tf for
targeted theranostic applications in breast cancer. Tf-functionalized DOX-loaded lumi-
nescent blue copper (Tf-Cu) nanoclusters have been fabricated by Goswami et al. for
breast cancer theranostic applications. The results of Förster resonance energy transfer
within the DOX-loaded Tf-Cu nanoclusters showed striking red luminescence, wherein
the blue luminescence of Tf-Cu nanoclusters (donor) was quenched due to absorption
by DOX (acceptor). The blue luminescence of Tf-Cu nanoclusters was restored in the
cytoplasm of cancer cells upon Tf mediated internalization. Finally, the gradual release
of DOX from the nanoclusters led to the generation of red luminescence inside the
nucleus. Moreover, a confocal microscopy image of HeLa cells revealed successful in-
ternalization of Tf-Cu nanoclusters in the cells. The results of in vivo studies showed
superior targeting efciency of DOX-loaded Tf-Cu nanoclusters on transferrin receptor
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(TfR)–positive HeLa and MCF-7 cells compared with TfR-negative HEK-293 and 3T3-
L1 cells. Furthermore, the Combination index showed synergistic interaction of Tf-Cu
nanocluster and DOX upon treatment with DOX-loaded Tf-Cu nanoclusters. In vivo as-
sessment of the nanoclusters on TfR overexpressing Dalton’s lymphoma ascites-bearing
mice model showed signicant inhibition of tumor growth rendering prolonged survival
of the mice.146
In another study, Muthu et al. designed docetaxel and ultra-bright gold clusters
loaded Tf-tagged TPGS based theranostic micelles. Their results indicated that the mi-
celles showed signicantly higher cytotoxicity and cellular uptake along with the tar-
geted co-delivery, and enhanced theranostic activity compared with the non-targeted
micelles. Also, they observed a reduction in IC50 values of docetaxel-loaded TPGS based
non-targeted and targeted micelles by 15.31- and 71.73-fold as compared with that of
Taxotere® after 24 h incubation with MDA-MB-231-luc breast cancer cells, respectively.
Thus, they proposed that the developed micelles could be an attractive system for the
co-delivery of poorly soluble anticancer drugs and imaging agents to TfR overexpress-
ing carcinomas.147 Tf-conjugated micelles, nanoclusters and other nanomedicines have
shown promising potential for targeted delivery of antineoplastic drugs and simultane-
ous imaging of TfR overexpressing carcinoma.
The potential of receptor-targeted theranostic nanomedicines explored for breast
cancer is summarized in Table 2. Systematic research on ligand conjugated nanomedi-
cines for receptor-mediated targeting of breast cancer for theranostic applications may
result in a promising outcome and will surely aid in the clinical management of breast
cancers including its most invasive variant TNBC.
V. CHALLENGES IN DEVELOPMENT OF BREAST CANCER THERANOSTICS
AND FUTURE PERSPECTIVES
Major challenges in developing theranostic nanomedicines include (i) developing
simple, controllable, and reproducible methods for synthesis of nanomedicines; (ii)
insufcient batch-to-batch reproducibility, low yield, and variable physicochemical
characteristics; (iii) understanding the in vivo biochemical mechanisms through which
nanomedicines function (i.e., how nanomedicines target certain tissues and factors that
affect the release of drugs from nanocarriers); (iv) understanding the biodistribution of
NPs in vivo; (v) identifying the transformation and metabolic pathways; (vi) understand-
ing the chronic toxicity of nanotheranostics in vivo; (vii) stringent regulatory and safety
guidelines for timely and effective translation of theranostics to market. Finally, to bring
nanomedicine to the clinic for the effective diagnosis and treatment of human diseases,
we need multidisciplinary knowledge and techniques from pharmaceutical scientists,
nanochemists, nanophysics, nanotoxicologists, clinical doctors, and so forth to construct
safe and effective nanotheranostics.154,155
The high heterogeneity of breast cancers poses a big challenge for its successful
prognosis and treatment, which could be resolved by selectively targeting the theranostic
agents to cancer cells. Although, development of an effective theranostic nanomedicine
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TABLE 2: Receptor-targeted theranostic nanomedicines for breast cancer treatment
Receptor
targeted
Targeting
ligand
Type of
nanomedicines
Type of
imaging
Imaging agent
Therapeutic
agent
Breast cancer
model
Outcome
Ref.
HER2 Trastuzumab SPIONs MRI Protoporphyrin IONPs MCF-7 breast
cancer cell line
In vitro photothermal
ablation of IONP-
protoporphyrin-
trastuzumab revealed
a 74% reduction in
MCF-7 cells after
10 min exposure
at the highest Fe
concentration
132
Anti-HER2
antibodies
Gold nanoshell
PLGA hybrid
nanocapsules
Ultrasound/
MRI
Peruorooctyl
bromide
DOX and
SPIONs
HER2-positive
SK-BR-3 cells
and HER2-
negative
MDA-MB-231
cells
–Signicant
photothermal
cytotoxicity
Signicantly enhanced
anti-tumor effect
148
Herceptin Graphene QDs Fluorescence Graphene QDs DOX HER2-negative
MCF-7 breast
cancer cells,
HER2-positive
BT-474 breast
cancer cells
Signicant
accumulation of
nanocarriers in the
cancer cells,
Internalization into the
cells via endocytosis
149
Trastuzumab
Lipid NPs
Fluorescence
QDs
Rapamycin
SK-BR-3 cells,
MDA-MB-231
cells
signicantly higher
cellular uptake, higher
uorescence intensity
in tumor spheroid,
~ 3 and ~ 1.5-fold
higher toxicity to
SK-BR-3 cells and
MDA-MB-231 cells,
respectively
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TABLE 2: (continued)
Herceptin IONPs MRI and
Fluorescence
IONPs DOX
In vitro
/
SK-BR-3 and
MCF-7 breast
cancer cells
In vivo/
Athymic nude
mice bearing
SK-BR-3 cancer
cells
–5 fold higher cellular
uptake
–Signicantly
enhanced tumor
regression in vitro and
in vivo
133
Herceptin Mesoporous
silica NPs
Ultrasound Mesoporous
silica NPs
Mesoporous
silica NPs
In vitro/
SK-BR-3 and
MDA-MB-231
breast cancer
cells
–Signicantly higher
cellular uptake
–Signicant
enhancement in
ultrasound image
contrast and enhanced
breast tumor-specic
toxicity
150
Trastuzumab SPIONs MRI Fluorescent
dye
SPIONs In vivo/MMC
cells
In vivo/FVB/N
transgenic
mouse model
–4-fold higher cellular
uptake
–Signicant contrast
enhancement in MR
images of primary
breast tumors in vitro
and in vivo
83
Herceptin Magnetic PLGA
NPs
MRI Magnetic
nanocrystals
DOX In vitro/
NIH3T6.7,
SK-BR-3, and
MDA-MB-231
cancer cells
–87.4 times greater
uorescent intensity
–Signicantly higher
cancer cell afnity
–Signicantly higher
cellular uptake
–Excellent tumor
growth retardation both
in vitro and in vivo
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TABLE 2: (continued)
Receptor
targeted
Targeting
ligand
Type of
nanomedicines
Type of
imaging
Imaging agent
Therapeutic
agent
Breast cancer
model
Outcome
Ref.
Folic acid FA Fe3O4 NPs MRI IONPs IONPs MC4-L2 cells,
Female Balb/c
mice
–The NPs were
signicantly
accumulated and
retained within the
tumor tissues.
–MRI experiments
showed a signicant
decrease in T2-
weighted MR signal
intensity of breast
tumors indicating the
accumulation and
retention of NPs in the
tumor tissue in vivo.
–Reduction in tumor
progression
137
FA Nanocomposite Fluorescence Graphene
oxide
manganese-
doped zinc
sulde QDs
DOX Breast cancer
cell line
MDA-MB-231
(FA receptor
positive) and
NIH-3T3
cell line (FA
receptor
negative)
–FA functionalization
improves the selectivity
for discriminating
positive FR cancer
cells.
–Signicant reduction
in the toxicity of the
nanocomposite
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TABLE 2: (continued)
FA IONPs MRI IONPs DOX Breast cancer
cell lines,
i.e., MCF-7,
BT549, and
MD-MBA-231
Female Balb/c
nude mice
–FA conjugated NPs
showed the strongest
cytotoxicity against
breast cancer cells,
compared with
non-targeted NPs,
and caused cellular
apoptosis.
–Suppression in in vivo
tumor growth in mice,
No signs of toxicity
against healthy organs
152
FA PEGylated
magnetic NPs
MRI Magnetic NPs Tamoxifen In vitro/MCF-7
breast cancer
cell line
–Signicantly enhanced
cellular uptake
–Signicant inhibition in
growth of MCF-7 cells
140
FA TPGS micelles Fluorescence QDs Docetaxel MCF-7 breast
cancer cells
–Signicantly higher
red uorescence
intensity and higher
cellular uptake
–Signicantly improved
cytotoxicity in vitro
141
CD44 HA Cationic BSA
protected gold
nanoclusters
PAI ICG Paclitaxel 4T1, A549, and
RAW 246.7
Cells
Female Balb/c
mice
–Signicantly
enhanced cellular
uptake
–Suppression in in situ
tumor growth by 95.3%
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TABLE 2: (continued)
Receptor
targeted
Targeting
ligand
Type of
nanomedicines
Type of
imaging
Imaging agent
Therapeutic
agent
Breast cancer
model
Outcome
Ref.
HA HA prodrug
micelles
Fluorescence Aggregation-
induced
emission
uorogen
tetraphenylene
DOX CD44-
overexpressing
MDA-MB-231
cell line CD44
negative
NIH3T3 cell
line
–Flow cytometry and
uorescent microscopy
indicated that HA
prodrug micelles were
effectively internalized
by MDA-MB-231 cells
due to HA-mediated
endocytosis
–Signicant inhibition
of the proliferation of
cancer cells
–Signicantly enhanced
cytotoxicity against
MDA-MB-231 cells
143
EGFR Anti-EGFR
aptamer
Lipid
nanocarriers
Fluorescence QDs siRNAs EGFR positive
MDA-MB-231
and EGFR
negative
MDA-MB-453
cell lines,
Tumor-bearing
female Balb/c
nude mice
–Enhanced delivery to
target cancer cells
–Signicant reduction
in target gene
expression
–Inhibition of tumor
growth and metastasis
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TABLE 2: (continued)
Anti-EGFR
Nanobody
Micelles Fluorescence QDs Aminoavone EGFR
overexpressing
MDA-MB-468
TNBC cells
MDA-MB-468
breast cancer
xenograft mouse
model
–Nb-conjugated
micelles accumulated
in tumors at higher
concentrations, leading
to more effective tumor
regression in the mouse
model
–No systemic toxicity
observed
–Enhanced cellular
uptake and increased
cytotoxicity
145
Transferrin Tf Nanoclusters Fluorescence luminescent
blue copper
DOX TfR positive
HeLa,
TfR positive
MCF-7 cells,
TfR partially
positive HEK-
293 and
TfR negative
Mouse embryo
broblast cell
lines
–Tf-CuNC-DOX-NPs
showed excellent
targeting efciency
on TfR overexpressed
cells (HeLa and MCF-
7) as compared with
the less TfR expressed
cells (HEK-293 and
3T3-L1)
–Confocal microscopy
images of HeLa cells
revealed successful
internalization of Tf-Cu
nanoclusters in the
cells
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TABLE 2: (continued)
Receptor
targeted
Targeting
ligand
Type of
nanomedicines
Type of
imaging
Imaging agent Therapeutic
agent
Breast cancer
model
Outcome Ref.
Tf
TPGS micelles
Fluorescence
Ultra-
bright gold
nanoclusters
Docetaxel
In vitro/MDA-
MB-231-luc
and NIH-3T3
broblast cells
In vivo/female
CB-17 mice
bearing MDA-
MB-231-luc
cells induced
tumor
Signicantly higher
cellular uptake and
theranostic effect
4.6-fold reduction in
IC50 value compared
with non-targeted
micelles
147
Tf PEGylated gold
NPs
PAI Fluorescein
isothiocyanate
Gold NPs In vitro/Hs578T
cancer cells
–6-fold higher cellular
uptake in breast cancer
cells
–Reduced in laser
power effective for
therapy from 1600 W/
cm2 to 7 W/cm2, which
is more than two orders
of magnitude lower
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with targeting propensity toward breast cancer cells may encounter many challenges,
yet it will be benecial to develop these nanomedicines as the conventional techniques
used for diagnosis and management of breast cancer are not efcient enough to image
the breast tumors completely with minute detailing. Further, targeted theranostic medi-
cines, which can be sorted out using novel techniques and strategies, give a unique op-
portunity to maximize the accumulation of anticancer therapeutics to cancer cells which
could result in an improved therapeutic index with a signicant reduction in toxicity
which is one of the major hurdles in the treatment of cancer along with the development
of resistance to chemotherapeutic agents.
Cancer nanotechnology has advanced over the last two decades to reach a stage
where the novel approach of “cancer theranostics” has been conceptualized and devel-
oped for the diagnosis and therapy of breast cancer. It provides a unique combination
of drug and a diagnostic probe having controlled release and receptor-mediated target-
ing potential. Theranostics offers an effective and patient-friendly alternative. Various
polymeric, lipid-based and inorganic NPs have been considered for this approach. They
have shown therapeutically signicant and noteworthy results in various investigations.
However, the majority of theranostic applications in breast cancer have only been dem-
onstrated at the pre-clinical level. In addition, due to nanoscale sizing, complex syn-
thetic pathways can alter the physicochemical properties and consequently become a
source of toxicity at the cellular or subcellular level. Various investigations related to
the same have established the potential of nanomaterials for biochemical and physi-
ological alterations. Therefore, a correlation needs to be established between their ef-
cacy and safety. The study of in vitro/in vivo correlation has also become a challenge
due to the non-availability of suitable tools. Thus, there is an unmet need to match the
changing paradigm of breast cancer therapy using receptor-mediated nanomedicine with
advanced bio-pharmaceutical and toxicity assessment tools.
VI. CONCLUSION
Breast cancer is emerging as one of the most frequently diagnosed cancer in women in
developed and developing countries. Management of breast cancer is a difcult task if
not diagnosed at an early stage. Further, the most aggressive form of breast cancer, i.e.,
TNBS is very difcult to manage due to metastasis which is further worsened by poor
prognosis. Receptor-targeted nanomedicines are being explored to augment the selec-
tivity of drug delivery to cancer cells. With the help of suitable ligand and engineering
technology, promising nanomedicines may be designed to target the therapeutic, imag-
ing agents, or both simultaneously to cancer cells. The result of ongoing studies and
studies done in the last few decades are strongly supporting the candidature of receptor-
targeted nanomedicines for targeted delivery of therapeutic and diagnostic moieties to
breast cancer with an improved therapeutic index.
Further nanomedicine-based approaches are also being explored for theranostic ap-
plications in breast cancer with promising results which have been obtained in research
studies conducted in the last decade. These nanomedicines based theranostic approaches
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have revealed an ability to deliver the therapeutic agents to breast cancer, selectively, to
enhance the efcacy of cancer therapy with simultaneous imaging of cancer microen-
vironment for better prognosis and management of breast cancer. So, from the result of
these investigations, it could be expected that continuous and coherent investigation in
this direction may help in developing promising therapeutic approaches based on nano-
medicines for targeted delivery of diagnostic agents, therapeutic moieties, and theranos-
tic modalities to breast cancer, which may get approval for clinical use.
ACKNOWLEDGMENTS
T.S. and K.J. are thankful to National Institute of Pharmaceutical Education and Research
(NIPER), Raebareli, for providing the facilities to write this manuscript. Author, Dr.
Keerti Jain, acknowledges Indian Council of Medical Research (ICMR), New Delhi
for the nancial support in the form of ICMR Extramural Research Project (Project
ID: 2020-4686; Ref. No. 5/13/34/2020/NCD-III). The NIPER Raebareli communication
number for this manuscript is NIPER-R/Communication/177.
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