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Recent advances in nanomedicine have shown that dramatic improvements in nanoparticle therapeutics and diagnostics can be achieved through the use of disease specific targeting ligands. Although immunoglobulins have successfully been employed for the generation of actively targeted nanoparticles, their use is often hampered by the suboptimal characteristics of the resulting complexes. Emerging data suggest that a switch in focus from full antibodies to antibody derived fragments could help to alleviate these problems and expand the potential of antibody–nanoparticle conjugates as biomedical tools. This review aims to highlight how antibody derived fragments have been utilised to overcome both fundamental and practical issues encountered during the design and application of antibody–targeted nanoparticles.
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Antibody fragments as nanoparticle targeting
ligands: a step in the right direction
Daniel A. Richards,*Antoine Maruani and Vijay Chudasama*
Recent advances in nanomedicine have shown that dramatic improvements in nanoparticle therapeutics
and diagnostics can be achieved through the use of disease specic targeting ligands. Although
immunoglobulins have successfully been employed for the generation of actively targeted nanoparticles,
their use is often hampered by the suboptimal characteristics of the resulting complexes. Emerging data
suggest that a switch in focus from full antibodies to antibody derived fragments could help to alleviate
these problems and expand the potential of antibodynanoparticle conjugates as biomedical tools. This
review aims to highlight how antibody derived fragments have been utilised to overcome both
fundamental and practical issues encountered during the design and application of antibodytargeted
1. Introduction
The last twenty years have seen a rapid, and accelerating,
increase in the use of nanoparticles for biomedical applica-
tions. From a conceptual standpoint it is not dicult to
understand why; various nanoparticles are now at a stage of
being tuneable, functionalisable and biocompatible vehicles
that can safely transport large quantities of cargo through the
body. This enables the delivery of entities at concentrations
signicantly higher than traditional methods.
This factor, in
combination with the ease in which the surface of nanoparticles
can be decorated with high anity disease-specic targeting
ligands to enhance selective delivery, means that they have
a plethora of downstream therapeutic and diagnostic applica-
tions. A large variety of chemical and biological molecules have
been explored for this enhanced targeting purpose, including:
novel small molecules, sugars, fatty acids, proteins, peptides,
antibodies, and aptamers.
Of these, antibody based targeting
ligands have become incredibly popular due to their unique in
vivo properties and high target specicities.
Whilst the
contributions of other targeting ligands should not be ignored,
this review focuses on the use of antibodies, or more specically
their associated fragments, as targeting ligands for nano-
particle-based therapeutic and diagnostic tools. To ensure
broad accessibility of the review content, a brief overview of
Dr Daniel Richards began his
studies at the University Of York,
obtaining an MChem in 2011
with his thesis focused on the
study of metalhalogen
exchange reactions in nitrogen
containing heterocycles. He
subsequently joined the lab of Dr
James Baker at University
College London (UCL) to study
for his PhD, focusing on the
development and application of
novel biocompatible photo-
chemical reactions. He is currently working as a postdoctoral
fellow in the group of Dr Vijay Chudasama, developing novel
methods for the selective functionalisation of nanoparticles.
Dr Antoine Maruani obtained
his Master's degree in Chemistry
from ´
Ecole Normale Sup´
of Lyon (France) in 2011. He
then joined Prof. Stephen Cad-
dick's group at University
College London (UK) where he
obtained his PhD in 2015 with
his thesis focusing on the site-
selective dual modication of
proteins. He is currently working
as a postdoctoral fellow under
the supervision of Dr Vijay
Chudasama on the development of novel methodologies for
Department of Chemistry, University College London, 20 Gordon Street, London,
WC1H 0AJ, UK. E-mail:;; Tel:
+44 (0)207 679 2077
Cite this: Chem. Sci.,2017,8,63
Received 31st May 2016
Accepted 5th September 2016
DOI: 10.1039/c6sc02403c
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,6377 | 63
common nanoparticle (Section 2.1) and antibody (Section 2.2)
scaolds used in this context will be given.
2. Antibodydecorated nanoparticles
2.1 Nanoparticle structure
When designing nanoparticleantibody conjugates for
biomedical applications several considerations regarding the
structure of the nanoparticle are important. The nanoparticle
must be biologically inert, stable under physiological condi-
tions, move freely through the body, securely encapsulate
chemical entities (where applicable), and contain a surface
which is easily conjugated to the desired targeting antibody. In
the case of therapeutics, it is also important to consider the
mechanism by which the nanoparticle vehicle will release cargo
and whether this will be compatible with other aspects of the
overall construct. The most successful approaches strike a deli-
cate balance between the properties of the nanoparticle, the
targeting antibody, and where appropriate the encapsulated
cargo. Fortunately, a great deal of research has been done on the
design and modication of nanoparticles over the last 20 years,
providing a rich pool of work from which suitable vehicles can
be selected for antibody conjugation. Nanocarriers can be
broadly categorised as organic or inorganic,and each of these
will be discussed in turn (Fig. 1, Table 1).
2.1.1 Organic nanoparticles
Liposomes. Liposomal nanoparticles were rst developed near
the genesis of nanomedicine and have since become one of the
most widely utilised vehicles for encapsulating chemical
payloads, with several formulations having gained FDA
They comprise natural lipids with polar and non-
polar components which self-assemble into colloidal particles.
Whilst early liposomal nanoparticles suered from issues of
stability and rapid clearance, the introduction of surface
ligands such as polyethylene glycol (PEG) chains has helped to
address these drawbacks.
The main advantages of liposomal
nanoparticles created from state-of-the-art technologies lie in
their excellent biocompatibility, ease of synthesis/functionali-
sation, and their ability to safely encapsulate a variety of small
However, they are limited by a high level of
sensitivity to structural change(s) and have demonstrated
highly specic cargo-dependency, thus decreasing their
universal appeal and broad applicability.
Polymeric micelles. Polymeric micelles consist of a core of
aggregated hydrophobic polymers surrounded by hydrophilic
polymeric chains. Their small size and hydrophilic nature allow
them to avoid uptake by the reticuloendothelial system,
signicantly increasing their circulation time.
Their hydro-
philic exterior also allows polymeric micelles to eectively
and safely encapsulate very hydrophobic drugs for safe trans-
port through the body.
As with liposomal nanoparticles,
polymeric micelles also demonstrate excellent biocompati-
However, poorly controlled release proles of encapsu-
lated cargo, and a high sensitivity to structural change(s), mean
that there is still signicant scope for improvement.
Polymeric nanoparticles. Polymeric nanoparticles can be
further categorised as either nanospheres or nanocapsules.
Nanospheres consist of a solid polymer matrix which is able to
encapsulate hydrophobic drugs, whilst nanocapsules contain
an aqueous hydrophilic core that is more amenable to the
loading of hydrophilic payloads such as DNA/RNA.
payload exibility increases the versatility of polymeric nano-
particles, making them attractive candidates as nanocarriers.
Additionally, it has been shown that the release rates of
encapsulated payloads are constant and proceed on clinically
relevant time scales.
Nonetheless, despite these favourable
characteristics, polymeric nanoparticles are not simple to purify
and do not store well, making them a poor choice for applica-
tions that require large scale production.
Dendrimers. Dendrimers are branched polymer complexes
generated through highly controlled successive polymerisation
steps. This leads to a nanoparticle which consists of an initiator
core contained within branched polymer chains. These polymer
chains are generally synthetic, although examples that employ
natural polymers such as sugars and amino acids have been
Their highly regulated synthesis enables excellent
control over shape and size important parameters for medical
They also display excellent solubility and have
been shown to be non-immunogenic.
Whilst dendrimers have
several excellent qualities, research into their use in the
biomedical eld is still early stage. Further studies to establish
their biocompatibility and toxicity are ongoing and will be
pivotal to their further application.
2.1.2 Inorganic nanoparticles
Iron oxide nanoparticles. Iron oxide nanoparticles generally
consist of an iron oxide (typically Fe
) core surrounded
by a dextran coating to improve the physical properties of the
complex. The application of these nanoparticles commonly
centres on their innate magnetic properties, which allow them
to act as excellent MRI contrast agents and tools for thera-
peutic magnetic hypothermia.
This dual functionality has
led to superparamagnetic iron oxide nanoparticles (SPIONS)
being used as theranostic tools, i.e. chemical entities which
Dr Vijay Chudasama obtained
his MSci degree and PhD from
University College London in
2008 and 2011, respectively.
Following post-doctoral studies
under the supervision of Prof.
Stephen Caddick, Vijay obtained
a Ramsay Memorial Fellowship.
During this time, he was made
Technical Director of a biotech-
nology spin-out (ThioLogics). In
April 2015, he was awarded
a lectureship at UCL for him to
focus on the research areas of aerobic CH activation and various
aspects of Chemical Biology. Vijay's research has recently been
highlighted by Forbes, Scientic American, CNN, Nature Chemistry
and the Royal Society of Chemistry.
Hybrid organicinorganic particles will not be focused on in this review.
64 |Chem. Sci.,2017,8,6377 This journal is © The Royal Society of Chemistry 2017
Chemical Science Perspective
display both therapeutic and diagnostic properties. However,
the lack of a spacious coreor any porous space leads to low
loading volumes,
an issue for many applications. Whilst the
generation of hybrid iron oxide/polymer-based nanoparticles
has gone some way towards addressing these issues, the
current situation is not ideal.
Fig. 1 Pictorial representation of dierent types of nanoparticles used in biomedical applications.
Table 1 A table summarising the dierent types of nanoparticles with focus on material used, cargo attachment, and their various advantages &
Nanoparticle Material(s) Cargo attachment Advantages Disadvantages
Liposomes Self-assembling lipid
Encapsulated within the
hydrophilic core
Easily synthesised,
biocompatible, high
internal loading
Highly sensitive to
structural changes and
nature of payload
Hydrophobic polymer core
surrounded by hydrophilic
polymeric chains
Encapsulated within the
hydrophobic core
Small, biocompatible, able
to incorporate highly
hydrophobic cargo
Highly sensitive to
structural changes, poor
release proles
Solid hydrophobic polymer
matrix with optional
aqueous core
Embedded in the polymer
matrix or within the core
High loading capacity,
exible loading
capabilities, reliable
release proles
Dicult to purify and poor
store properties
Dendrimers Highly branched polymer
Embedded in the polymer
Highly soluble, non-
immunogenic, high
loading capacity,
controlled synthesis
Lacking data on toxicity and
Iron oxide
Iron oxide core surrounded
by biocompatible coating
Attached to the surface/
surface coating
Innate magnetic properties No internal loading capacity
Solid gold particles
typically coated with PEG
Attached to the surface/
surface coating
Innate optical and
photothermal properties
No internal loading
capacity, poor
biocompatibility and
Mesopores surrounded by
a silica framework
Encapsulated within the
High loading capacity,
good biodegradability
Issues with physiological
stability, rapid clearance
Graphite arranged in either
a sheet or cylindrical
Attached to the carbon
Innate optical and
electrical properties, high
surface loading capacities
Poor biodegradability,
organ accumulation
Quantum dots Typically a cadmium
selenide core with a zinc
selenide cap
Attached to the surface/
surface coating
Innate optical properties,
high extinction coecients
No internal loading
capacity, potential toxicity
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Perspective Chemical Science
Gold nanoparticles. Gold nanoparticles have been extensively
studied for use in biomedical applications due to their inter-
esting size dependent physicochemical and optical properties.
For example, their ability to produce heat upon absorbance of
near-infrared light has been explored for use in photothermal
therapy, whilst the ability to enhance optical processes such as
absorbance and uorescence has led to widespread use in
the eld of biosensors and imaging agents.
However, their
non-hollow structure precludes internal loading,
and they also
tend to suer from poor biodegradation and questionable
Mesoporous silica nanoparticles. Mesoporous silica nano-
particles (MSNs) consist of mesopores (250 nm pores) sur-
rounded by a silica framework. These nanoparticles have
a high surface area to volume ratio which aords them
a large loading capacity. MSNs have also demonstrated good
biocompatibility and biodegradability, desirable features for
biomedical purposes.
clearance rates signicantly restrict the use of MSNs from
certain applications.
Carbon nanoparticles. Carbon nanoparticles, such as carbon
nanotubes, comprise a single layer of graphite in either a sheet
or cylindrical conformation. Excellent loading capacities,
unique optical and electrical properties, and low synthetic costs
make them promising candidates for several applications,
especially imaging and diagnostics.
Unfortunately, issues of
poor biodegradability,
pulmonary damage,
and undesir-
able organ accumulation
have hindered the adoption of
carbon based nanoparticles for in vivo applications.
Quantum dots. Quantum dots (QDs) most commonly consist
of a cadmium selenide core with a zinc selenide cap, although
many other combinations exist. QDs emit bright colours and
also display size dependent optical properties, making them
ideal for imaging or biosensing technologies.
Whilst potential
toxicity issues have to-date limited their utility in vivo, recent
advances are helping to overcome these remaining hurdles.
2.2 Antibody structure and function
Antibodies, or immunoglobulins (Ig), are large glycoproteins
found in all vertebrate life forms. These essential proteins are
involved in several key processes within the immune system
including complement dependent cytotoxicity (CDC), opsonisa-
tion, phagocytosis, and antibody-dependent cytotoxicity (ADCC).
To date, ve major classes of immunoglobulin have been
discovered, IgA, IgD, IgE, IgG and IgM, each characterised by
unique structural characteristics. IgGs represent the dominant
class of human immunoglobulins and can be further divided
into four sub-types; IgG1, IgG2, IgG3 and IgG4. Although the IgG
sub-types show signicant sequence variation in key regions,
they share a common overall structure. IgG antibodies consist of
four protein chains; two identical ca. 25 kDa light chains (i.e.
L-subscript) and two identical ca. 50 kDa heavy (i.e. H-subscript)
chains. These chains contain multiple domains which are
characterised by their degree of sequence variability. The
N-termini of the chains converge in the variable domain (V) to
form the antigen-binding region. Further from the terminus, the
structure becomes more conserved, leading to the area being
designated the constant region (C). The heavy and light chains
are held together by several interchain disulde bonds and
considerable non-covalent interactions to form a Y-shaped
structure. The overall structure can be broadly divided into two
distinct segments; the fragment antigen-binding (Fab) region
and the fragment crystallisable (Fc) section. Fabs can be further
divided into variable (Fv, V
) and constant (C
) regions
(Fig. 2).
2.2.1 Antibody fragments. In addition to being integral to
the function of parent immunoglobulins, the individual protein
domains of antibodies can be isolated or expressed and have
found extended use in biomedical research. Through careful
and precise disassembly of a full antibody, researchers have
been able to isolate and individually employ the Fab, Fab0,
, and Fv regions of antibodies to great eect (Fig. 2).
Additionally, advancements in protein engineering and
expression have allowed for the generation of novel classes of
antibody fragments such as the ScFv, ds-Fv, ds-ScFv, single
domain antibodies (sdAb), and diabodies (Fig. 2).
All of
these antibody fragments retain at least one antigen-binding
region, meaning that the function of active targeting is still
present. These individual fragments have then been exploited
as part of nanoparticleantibody fragment conjugates, leading
to several interesting studies of the use of these targeting
ligands for selective nanoparticle delivery. Given recent
advancements in phage display techniques for the generation of
antibody derived fragments,
a surge in interest in their use as
targeting ligands is unsurprising. Several excellent reviews have
been written on the design, production, and applications of
antibody fragments, with focus on their merits relative to whole
and as such this will not be covered in
detail in this review.
2.3 Nanoparticleantibody fragment conjugates
Antibodies function by targeting specic antigens that are
expressed only on the surface of diseased cells, or heavily
overexpressed on these cells relative to healthy cells. As these
antigens are present solely, or majorly, on the surface of the
target diseased cells, antibodies can conceptually be exploited
to courier nanoparticles (and also their cargo) through the body
and enable selective delivery/targeting. Whilst this approach
was rst conceptualised in the early 1980's, practical and
theoretical limitations at the time (e.g. insucient methods for
generating and evaluating antibodydecorated nanoparticles)
prevented signicant progress in the area. Advancements in
both antibody expression techniques and nanoparticle design
over the past few decades have enabled a more thorough
exploration of nanoparticleantibody conjugates, which has
resulted in a rapid expansion of the eld. Early developments
focused almost entirely on using full antibodies as targeting
ligands, primarily due to the wealth of available information on
both their generation and modication. However several issues
associated with the use of full antibody ligands, such as
rapid elimination,
poor stability,
lower than expected ecacy,
soon came to light and these
66 |Chem. Sci.,2017,8,6377 This journal is © The Royal Society of Chemistry 2017
Chemical Science Perspective
are being increasingly emphasised/supported by emerging data.
A signicant amount research has now been published on the
use of antibody fragments to address both fundamental and
practical issues encountered during the use of whole immu-
noglobulins. In addition to being less immunogenic, the small
size of antibody fragments allows for higher loading capacities
and superior orientation of targeting ligands, leading to overall
improvements in ecacy (Fig. 3).
In view of the above advantages, it is anticipated that the use
of antibody fragments as directing ligands for nanoparticle
targeting will increase signicantly over the next few years.
Whilst several excellent reviews have been written on the use of
targeted nanoparticles in biomedicine, with a few focusing on
the subject of antibodies as targeting ligands,
very few
specically highlight and accurately detail work on nano-
particleantibody fragment conjugates. This short review aims
Fig. 2 Graphic representations of whole antibody (IgG1) and various fragments.
Fig. 3 Graphic representations comparing whole antibody and antibody fragment (Fab0) targeting ligands for nanocarriers.
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,6377 | 67
Perspective Chemical Science
to introduce the area, with particular emphasis on recent
developments in the generation and application of nano-
particleantibody fragment conjugates for biomedical uses.
3. Generating nanoparticleantibody
fragment complexes
During the design of nanoparticleantibody fragment
complexes important consideration must be given to the
method by which the two entities are attached. The antibody
fragment needs to be conjugated to the nanoparticle in a way
that causes minimal perturbation to the shape, size, and func-
tionality of both the nanoparticle and the antibody fragment
itself. Additionally, the linker between the two should be stable,
biocompatible, non-toxic, and facile to install. Fortunately,
a great deal of work on the installation of functional chemical
moieties on both nanoparticles and antibody fragments has
been carried out. Moreover, attempts to utilise these chemis-
tries to functionalise nanoparticles with antibody fragments
have been largely successful, as will be discussed in more detail
3.1 Modication of antibody fragments
Modications of antibody fragments largely centre on exploit-
ing the innate chemical reactivity of the natural amino acids on
the backbone of each protein. The amino acids most commonly
used as sites for modication include lysine, cysteine, and
glutamic/aspartic acid, as they can be functionalised using well-
established chemistries. Initially, lysine was a popular target for
modication as it could be readily conjugated, however, the
high abundance of this amino acid on the surface of many
proteins means that it is hard to control conjugation, resulting
in random functionalisation and a heterogeneous mixture of
antibody fragment products post-conjugation. More recently,
site-selective methods which exploit the natural structure of
antibody fragments, such as the hinge thiols of Fab0fragments,
or utilise amino acids incorporated through site-directed
mutagenesis, have been successfully employed; this has resul-
ted in far more homogeneous and better characterised conju-
gates. Antibody modication (including antibody fragments)
has maintained a healthy research focus for several decades
now, largely due to the rapid development of the antibodydrug
conjugate eld. This has resulted in a rich toolbox of chemical
reactions which enable facile, site-selective modication whilst
avoiding negative eects on the function of the protein. Several
excellent reviews have been written on this subject, so it will not
be covered in depth here.
However, Fig. 4 highlights some of
the most common methods employed for functionalising anti-
body fragments for subsequent attachment to nanoparticles.
3.2 Modication of nanoparticle surfaces
Nanoparticle surface modication techniques can be broadly
separated in two main categories: (i) covalent and (ii) non-
covalent. Covalent modications involve the incorporation
of a chemical functional group that can subsequently attach
covalently to a targeting ligand. In contrast, non-covalent
technologies involve the incorporation of a functionality that
can interact either (i) intermolecularly or (ii) by physisorption
with a ligand. For decorating nanoparticles with antibodies,
covalent methods are preferred as they provide greater in vivo
Moreover, covalent methods also allow for greater
control over the position and orientation of the attached anti-
body fragment, especially when combined with a site-selectively
modied antibody fragment itself. Methods for incorporating
an assortment of functional groups onto the surfaces of various
nanoparticles have been reported, including amines, carboxylic
acids, alcohols, thiols, azides, alkynes, aldehydes, and mal-
eimides. Subsequent modication of these groups can further
expand the reactivity prole of the nanoparticle, leading to
a large selection of functional handles which can be paired with
complimentary groups on the desired antibody ligand (Fig. 5).
Several reviews have been written on the incorporation and
utilisation of chemical functionality on nanoparticles,
including a comprehensive overview by Sapsford et al.
these advances, non-specic interactions of antibody ligands
with nanoparticle surfaces remains an issue, and methods for
distinguishing specic interactions from non-specic interac-
tions are lacking. These issues can be particularly problematic
when site-specic or oriented conjugation of an antibody frag-
ment is desired.
4. Nanoparticleantibody fragment
conjugates in biomedicine
4.1 As therapeutic agents
The ability to safely encapsulate a cocktail of toxic chemicals
and deliver them selectively remains a long standing goal for
medicine. To this end nanoparticleantibody conjugates have
shown great potential and indeed several promising candidates
have entered clinical trials (Table 2). Interestingly, the majority
of these candidates utilise antibody fragments as the targeting
ligand, highlighting a preference over full-length antibodies for
therapeutic applications. This preference is indicative of the
advantages provided by the use of smaller, less immunogenic
antibody-derived targeting ligands. However, it is important to
note that in the cases exemplied in Table 2, side-by-side
comparisons to whole immunoglobulins were not made, or at
least the data was not published.
Nonetheless, a lack of clarity regarding the advantages and
disadvantages of whole mAb compared with antibody frag-
ments for therapeutic purposes was, at least to some extent,
addressed by Cheng and Allen.
During the design of lipo-
somes which could selectively target B-cell malignancies with
encapsulated doxorubicin (Stealth® immunoliposomes, SIL),
they compared the in vivo eectiveness of doxorubicin bearing
liposomes targeted with HD-37 mAb, HD-37-Fab0and a HD-37-
ScFv against the B-cell antigen CD19.
The targeting ligands
were attached to the protein using maleimidethiol conjuga-
tion techniques, natively in the case of the Fab0and ScFv
and via lysine thiolation in the case of the whole antibody.
In vitro binding assays revealed no signicant dierence in
CD19 binding between HD-37-mAb and HD-37-ScFv targeted
68 |Chem. Sci.,2017,8,6377 This journal is © The Royal Society of Chemistry 2017
Chemical Science Perspective
liposomes, however, a steep improvement in binding was
observed for HD-37-Fab0. Interestingly the HD-37-ScFv tar-
geted liposome proved the most selective for CD19
cells with the mAb being the worst performer over both
studies. Drastic dierences were also noticed in vivo,withthe
HD-37-mAb targeted liposome being rapidly cleared (0.41 mL
) due to Fc-mediated uptake into the liver and spleen in
comparison to the fragment conjugates (0.10 mL h
for the
Fab and 0.12 mL h
for the ScFv). Of the fragment-decorated
liposomes HD-37-ScFv cleared slightlyquicker,possiblydueto
His-tag/c-myc tag mediated uptake into the liver. The culmi-
nation of these eects is an improved mean survival rate of
Fig. 4 Schematic representations of common ways in which antibody fragments are modied.
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,6377 | 69
Perspective Chemical Science
mice treated with HD-37-Fab0targeted doxorubicin liposomes
when compared to HD-37-mAb and HD-37-ScFv targeted
doxorubicin liposomes. Although the presence of the His and
c-myc tags caveat the results of the HD-37 ScFv targeted lipo-
some due to increased clearance rates, this work clearly
demonstrated the dierences between using full mAb and
antibody fragments as targeting ligands for nanoparticles. It
also provided early evidence for advantages in using smaller
fragments that do not contain the Fc region. These results
corroborated previous work by Allen which showed that a Fab0
conjugated liposome outperformed a full mAb conjugated
liposome due to increased circulation time.
4.1.1 Targeted delivery of small molecule drugs. In addi-
tion to the clinical examples mentioned above, a plethora of
preclinical nanoparticleantibody fragment conjugates exist for
the targeted delivery of cytotoxic payloads.
Manjappa et al.
used an anti-neuropilin (NRP) Fab0targeted liposome contain-
ing docetaxel to simultaneously target both solid tumours and
the surrounding microvasculature.
The anti-NRP Fab0was
conjugated to the liposome via surface PEG-maleimide groups,
resulting in a site-specic thioether bridge. This allowed the
targeting fragments to be arranged in a desirable orientation,
an approach that is not possible with a full antibody. By taking
this approach the group obtained promising results, with the
targeted liposome showing the greatest degree of suppression
on both tumour volume and microvessel density when
compared to controls.
Whilst the majority of nanoparticleantibody fragment drug
delivery systems utilise lipid-based nanoparticles (Table 2), the
last few years have seen an increased exploration of non-lipo-
somal nanoparticleantibody conjugates for cytotoxic drug
delivery. Work by Ahn et al. showed that anti-tissue factor (TF)
Fab0targeted polymeric micelles loaded with dichloro(1,2-dia-
minocyclohexane)platinum(II) displayed greater selectivity for
the cellular target, increased internalisation rate, and aorded
signicant retardation of tumour growth when compared to
non-targeted polymeric micelles or free drug alone.
By utilis-
ing a selective maleimidethiol reaction to attach their Fab0
ligand, the group were able to exert delicate control over the
conjugation and introduce a single Fab0per micelle. This
allowed for the installation of targeting capabilities whilst
causing minimal perturbation to the nanoparticle properties,
an advantage for moving forward into the clinic.
Further to this, Xiangbao et al. successfully used an anti-
VEGFR ScFv targeted polyethylene glycolpolylactic acid
(PEGPLA) polymersome containing As
as the cytotoxic
Despite the use of suboptimal non-specic lysine
NHS ester conjugation techniques to attach the ScFv ligand,
their approach yielded improved selectivity and decreased
tumour volume, resulting in far greater mean survival rates
when compared to the non-targeted nanoparticles and free drug
controls. It is expected that controlled orientation of the ScFvs
would yield even better results.
Proof of principle research by Quarta et al. has demonstrated
the tumour targeting capability of iron oxide nanoparticles
conjugated to anti-folate receptor antibody (AFRA) Fab frag-
The group chose the Fab fragment over the full anti-
body in order to minimise any increase in the diameter of the
resulting conjugate and thus increase internalisation rate and
stability. The ARFA Fab had been previously expressed to
contain a hinge region with a single glutathione protected
cysteine residue that could be used to conjugate to the mal-
eimide coated nanoparticle aer reductive deprotection. Inter-
estingly, the group employed TCEP for the deprotection,
Fig. 5 Graphical representation of common functional ligands
attached to the surface of a nanoparticle.
Table 2 A list of nanoparticleantibody conjugates currently undergoing clinical trials. Adapted from tables previously published by Van der Meel
et al.
and Goodall et al.
For details on individual therapeutics see references contained within these reviews
Name NP type Target Ligand Bioactive compound Indication Phase
SGT-53 Lipid Transferrin receptor Anti-transferrin receptor ScFv p53 DNA Solid tumours Ib
SGT-94 Lipid Transferrin receptor Anti-transferrin receptor ScFv RB94 DNA Solid tumours I
C225-ILS-Dox Lipid EGFR Cetuximab Fab Doxorubicin Solid tumours I
derived mini-cell
EGFR Bispecic monoclonal
antibody (mAb)
Paclitaxel Solid tumours II
MM-302 Lipid HER2 Anti-HER ScFv Doxorubicin Breast cancer I
Lipovaxin-MM Lipid Dendritic cell CD209 dAb Melanoma
antigens + IFNg
Melanoma vaccine I
MCC-465 Lipid Uncharacterised (GAH) Anti-GAH F(ab0)
Doxorubicin Metastatic stomach
Anti-EGFR ILs-Dox Lipid EGFR Cetuximab Fab Doxorubicin Solid tumours I
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Chemical Science Perspective
a reducing agent known to cleave the interchain heavy-light
disulde bond of the Fab fragment. This would enable cysteine
residues on both chains to react independently with the nano-
particle, potentially decreasing the control oered through the
specic introduction of the hinge cysteine, although this it is
appreciated that all liberated thiols are distal from the binding
site. Whilst no cytotoxic compounds were delivered in this
preliminary study, the group did demonstrate excellent in
vivo stability, along with dramatically increased selectivity for
aFR-expressing tumours when compared to non-targeted
controls. Thus, whilst this approach is still in its relative
infancy, it shows promise as a way of utilising inorganic iron
oxide nanoparticles to deliver cytotoxic payloads for the treat-
ment of ovarian cancer.
Other early stage research has explored the use of bispecic
ScFv and SdAb targeted liposomes, and have demonstrated
a clear advantage in the use of both bispecic ScFv and SdAb
fragments as targeting ligands for liposomal nanoparticles.
4.1.2 Targeted gene therapy. Gene therapy relies on the
selective delivery of nucleic acids to the cytoplasm or nucleus of
a target cell. The delivered gene is then able to replicate within
the cell and elicit its desired therapeutic eect. Whilst the
majority of therapeutic nanomedicine is focused on the delivery
of cytotoxic drugs, increasing eort is being spent on devel-
oping nanoparticleantibody conjugates for targeted gene
Indeed, two of the eight nanoparticleantibody
conjugates currently in clinical trials utilise specic DNA
strands as their payload (SGT-53 and SGT-94, Table 1). Nano-
particle-based gene delivery was partially covered by Zhang
et al.
and Li et al.,
however with little focus on the details of
the antibody-directed approaches, as will be discussed here.
Recently, Katakowski et al. showed that liposomes contain-
ing small interfering RNA (siRNA) could be targeted at dendritic
cells using anti-DEC205 ScFv fragments, with in vivo results
demonstrating improved gene silencing.
Their targeting ScFv
was conjugated to the nanoparticle via a C-terminal cysteine
introduced using site-directed mutagenesis, allowing conjuga-
tion to occur distal to the binding region so as to minimise any
deleterious eects on binding. The authors note that in
unpublished preliminary data they were unable to utilise full
anti-DEC205 antibody for the same purpose, and highlight the
risks of proceeding to the clinic with full mAb targeted
In addition to this, early in vitro work by Okamoto et al.
suggests siRNA containing liposomes targeted to heparin-
binding epidermal growth factor (HB-EGF) using anti-HB-EGF
Fab0fragments could provide eective treatment for breast
Similarly, Laroui et al. demonstrated eective treat-
ment of colitis through the delivery of TNF-asiRNA encapsu-
lated within F4/80 Fab0targeted PEGPLA polymersomes.
group found that Fab0targeted TNF-asiRNA containing nano-
particles granted a greater reduction in all symptoms of colonic
inammation when compared to the non-targeted controls. In
both studies the Fab0fragment was site-specically conjugated
to the nanoparticle via the hinge region using maleimidethiol
chemistry, highlighting the emerging prevalence of this
approach for conjugating antibody fragments to nanoparticles.
Further to the above examples, work carried out at Sun Yat-
sen University has pioneered the use of ScFv targeted super-
paramagnetic iron oxide nanoparticles (SPIONS) as MRI visible
siRNA delivery vectors.
One study demonstrated the appli-
cability of this approach towards the treatment of neuroblas-
toma tumours, with in vivo data suggesting signicant gene
silencing and subsequent tumour suppression.
Early data
suggests a similar approach could be utilised for the treatment
and imaging of gastric cancer.
These studies show that
delivery of nucleotides is not limited to organic nanoparticles,
and that the innate physical properties of inorganic nano-
particles can grant signicant benets.
4.1.3 Magnetic eld therapy. Within the connes of tar-
geted nanomedicine, magnetic eld therapy relies on the
localised induction of heat to a cell through the use of targeted
nanoparticles which respond thermally to the application of an
alternating magnetic eld. Utilisation of targeting ligands, such
as antibodies, has enabled nanoparticles to localise at a tumour
site, and upon application of an alternating magnetic eld
cause heating which destroys the proximal diseased cells
(Fig. 6).
Whilst liposomal nanoparticles are preferred for drug
and gene delivery, the inherent superparamagnetic properties
of iron oxide nanoparticles (SPIONs) have led to their predom-
inant usage in this area. The idea of using antibodies to direct
magnetic nanoparticles was explored extensively by Gerald and
Sally DeNardo in the mid- to late-2000s,
and signicant
progress has been made ever since. Whilst most of this devel-
opment has focused on the use of full antibodies as targeting
ligands, with optimisation more focused on the nanoparticle
some preliminary work has demonstrated advantages
in the use of antibody fragments in this context. For example,
early work by Shinkai et al. showed eective use of a Fab0
of antibody G250 to deliver a magnetoliposome to MN-antigen
presenting cells.
Application of an alternating magnetic
eld to this complex resulted in tumour suppression and
almost doubled the mean survival rates of mice when compared
to negative controls. An excellent paper by Cui et al. also
exemplied the theranosticutility of targeted SPIONs
via the application of an anti-prostate specic antigen
(PSA) ScFv-decorated uorescent magnetic nanoparticle.
combining the uorescent payload with the superparamagnetic
Fig. 6 A graphical representation of actively targeted nanoparticle
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,6377 | 71
Perspective Chemical Science
properties of the iron oxide nanoparticle, the group were able to
track delivery in vivo using uorescence and magnetic reso-
nance imaging, as well as initiate cell death through the
application of an alternating magnetic eld. This approach
aorded a substantial increase in lifespan in diseased mice
when compared to controls. It is worth noting that the ScFv was
conjugated to the nanoparticle via non-specic reactions
between nucleophilic amino acid residues and surface-bound
glutaraldehyde linkers, leading to uncontrolled surface load-
ings and orientation. Thus, it is possible that these results could
be improved through the use of a more controlled conjugation
Towards the end of their studies into magnetic eld therapy,
Gerald and Sally DeNardo published work in which the full mAb
was abandoned in favour of a di-ScFv ligand, which was
attached in a highly oriented fashion via a carefully introduced
cysteine residue.
Whilst the SPION-ScFv showed greatly
increased accumulation at the tumour site in vivo,ecacy of the
hyperthermic properties of the nanoparticle was not explored.
Similar work by Yang et al. showed that magnetic iron oxide
nanoparticles can be selectively targeted towards the EGFR
using an anti-EGFR ScFv ligand, showing promise as a treat-
ment for various EGFR presenting cancers.
The results discussed above clearly demonstrate the
advanced capabilities of nanoparticleantibody fragment
conjugates for chemotherapy. It is anticipated that the trend
of using antibody fragments could also provide benets in
other areas of nanomedicine, e.g. targeted immunotherapy
through the activation of cell receptors such as Death Receptor
5 (DR-5).
4.2 As imaging agents
Conceptually, targeted nanoparticles provide a myriad of
benets for in vivo imaging of cellular targets. The generous
loading capacity of most particles enables the site-selective
delivery of large quantities of imaging agent, increasing signal-
to-noise ratio, and/or the nanoparticle surface itself can oen be
tailored to provide intrinsic imaging functionality, as is the case
with SPIONs, gold nanoparticles or quantum dots. Early work in
the use of antibodydecorated nanoparticles for imaging
applications encountered problems due to specic accumula-
tion, with the limiting step found to be extravasation of the
nanoparticles from the vasculature, rather than cell
Whilst this is also a problem for therapeutic
nanoparticles, it is more apparent for imaging applications
where the utility is highly dependent on achieving high reso-
lution between the target site and the background. In an
attempt to tackle this problem, recent work has focused on the
use of smaller antibody fragments as targeting ligands. By uti-
lising smaller antibody fragments, which do not contain the Fc
region, overall circulation times and subsequent tumour accu-
mulation rates can be increased greatly.
4.2.1 Targeted optical imaging agents. Antibody fragment
decorated nanoparticles can be employed as optical imaging
agents either by: (i) encapsulation of certain small molecules;
(ii) incorporation of highly uorescent compounds onto the
nanoparticle or targeting antibody; or (iii) the use of innately
uorescent materials to construct the nanoparticle itself. An
excellent example of the former approach is shown in a study by
Fiandra et al. which compared the use of antibody fragments to
the parent full antibody for imaging HER2 positive tumours.
Iron oxide nanoparticles modied with a uorescent dye were
targeted towards HER2 cells using full trastuzumab, trastuzu-
mab half antibody (consisting of a single heavy chain and
a single light chain), or a trastuzumab derived ScFv. Ex vivo
results suggested a signicant improvement in tumour accu-
mulation for the half antibody and the ScFvdecorated nano-
particles when compared to the full antibody targeted
nanoparticles. Importantly, each of the targeted nanoparticles
showed at least a 30-fold increase in uorescence when
compared to the non-targeted control. It should be noted that
dierent conjugation techniques were used for the dierent
ligands; both the half antibody and the ScFv were attached via
thiol selective covalent disulde formation, whereas the full
antibody was attached via stable non-covalent protein A anity
interactions. These results further demonstrate the benets of
actively targeted nanoparticles for imaging tumours, and the
importance of ligand choice.
Another excellent example is provided by the work of R¨
et al. who used a self-quenching near-infrared dye incorporated
inside a ScFvdecorated liposome to image broblast activation
protein alpha (FAP) expressing cells. Application of a self-
quenching uorophore ensured signicant uorescence was
only observed aer intra-cellular degradation of the liposome
post-FAP cell binding. This approach led to a signicant
increase in the signal-to-noise ratio of the ScFvdecorated
liposomes when compared to the non-targeted controls in vivo.
The authors specify their decision to utilise an ScFv rather than
a whole mAb was driven by potential immunogenic concerns.
Exploiting inherently uorescent nanoparticles such as gold
nanoparticles or quantum dots is more widely utilised, likely
due to their relatively large extinction coecients and resis-
tance to photobleaching. Several excellent examples of antibody
fragment-decorated approaches exist. As way of an example, Xu
et al. showed that anti-GRP78 ScFv-conjugated quantum dots
can be tracked in vivo using uorescence imaging.
A similar
approach was used by Balalaeva et al. to image breast cancer in
Other groups are currently exploring the use of an anti-
CEA sdAb conjugated quantum dot for imaging CEA expressing
cancer cells, with initial results showing great promise.
Use of an sdAb allowed for highly orientated attachment of the
targeting ligand through an engineered cysteine residue, greatly
increasing avidity. The superiority of their sdAb is supported by
recent results comparing the sdAb ligand with a full antibody
analogue; the study demonstrated a dramatic increase in
sensitivity when the smaller targeting ligand was employed.
In both cases lysine residues on the targeting antibody ligands
were modied with D-biotin using NHS ester chemistry, allow-
ing the ligands to be attached to the quantum dot using the
highly stable biotinstreptavidin interaction. Whilst this non-
covalent approach is not ideal, it allowed the researchers to
utilise the same coupling strategy for both ligands and thus
gain a fairer comparison of their sdAb against the full antibody.
72 |Chem. Sci.,2017,8,6377 This journal is © The Royal Society of Chemistry 2017
Chemical Science Perspective
4.2.2 Targeted nuclear imaging agents. Nanoparticles have
also been employed with great success in the selective delivery
of radionuclides for imaging techniques such as positron
emission tomography (PET) and single photo emission
computed tomography (SPECT).
The selective delivery of
radionuclides can also have a desirable therapeutic eect,
allowing targeted nanoparticles loaded with radionuclides to
act as successful theranostic tools.
Whilst a multitude of
examples exist in which full antibodydecorated nanoparticles
have been utilised for this purpose, less work has been carried
out using smaller antibody-based fragments.
there is movement towards this area and a few notable exam-
ples are outlined below.
Chen et al. utilised a highly functionalised mesoporous silica
nanoparticle (MSN) to successfully image tumour vasculature in
vivo using a multimodal approach which employed both PET
and optical imaging techniques.
To target the nanoparticles,
the group attached a Fab fragment targeted against CD10,
a vascular-specic marker for tumour angiogenesis, and
demonstrated a signicant improvement in both PET and
uorescence imaging resolution in vivo compared to non-tar-
geted controls.
Work by Hoang et al. has utilised
In-labelled block copol-
ymer micelles conjugated to trastuzumab Fab to image HER2
positive cell lines in vitro using SPECT/CT.
In addition to
the trastuzumab Fab targeting ligand the group incorporated
nuclear localisation signal (NLS) peptides onto the surface of
their nanoparticle, leading to eective nuclear translocalisation
aer initial HER2 mediated internalisation. More recently, this
approach was demonstrated in vivo, with signicant benets in
tumour accumulation, cellular uptake, and nuclear uptake being
reported, when compared to non-targeted controls. Tumour
uptake studies indicate the nanoparticles functionalised with
both extra-cellular (trastuzumab Fab) and intra-cellular (NLS
peptides) targeting ligands outperformed the nanoparticles tar-
geted using trastuzumab Fab alone, indicating post-internal-
isation nuclear translocation could be benecial.
4.2.3 Targeted MRI agents. A great deal of eort has been
put into exploring the use of nanoparticles as contrast agents for
magnetic resonance imaging (MRI). Although the majority of
this work has focused on the use of innately magnetic nano-
particles such as SPIONS and carbon nanotubes, organic nano-
particles have also found some use due to their ability to safely
encapsulate existing MRI contrast agents.
Actively targeted
approaches have gained popularity in recent years, with anti-
body-derived ligands showing particular promise.
An early
example of the use of an antibody fragment to target a magnetic
nanoparticle was provided by Yang et al., who used an anti-EGFR
ScFv to selectively deliver iron oxide nanoparticles to EGFR-
expressing cancer cells.
In vivo results showed signicant
improvement in MRI contrast when ScFv targeted iron oxide
nanoparticles were compared to non-targeted controls. The
group utilised non-selective lysineNHS ester chemistry to attach
the ScFv, so it is likely that further improvements could be
achieved through the use of a more controlled conjugation
strategy. Vigor et al. utilised a similar approach to target their
SPIONs towards CEA expressing cells.
By attaching an anti-
CEA ScFv fragment to the surface of their SPION the group were
able to demonstrate excellent target specicity and MRI contrast
in vitro when compared to non-targeted controls. More recently,
Alric et al. showed that an anti-HER2 ScFv could be eectively
employed to trac PEG coated SPIONS to HER2 expressing
These targeted SPIONS maintained binding anity and
demonstrated increased cellular uptake when compared to non-
targeted controls. It should be noted that the authors explicitly
employ a small ScFv and site-selective maleimidethiol coupling
to achieve optimal orientation, cause minimal perturbation to
nanoparticle size, and avoid any problems associated with the
employment of full antibodies.
4.3 As immunoassays
The impact of nanoparticles on biomedicine is perhaps most
pronounced in the eld of immunoassays and diagnostics. The
varied optical, physical, and electrochemical properties of nano-
particles present a wide range of observable outputs which can be
exploited for the detection of disease biomarkers. The in vitro
nature of diagnostic tools eliminates the negative impact of
the suboptimal in vivo properties found with many inorganic
nanoparticles (e.g. toxicity, bioaccumulation), allowing their
full potential to be more readily realised. To date, nanoparticle
full antibody conjugates have found use in immunoassays based
on uorescence,
orster resonance energy transfer (FRET),
catalytic redox reactions,
surface plasmon resonance (SPR),
surface-enhanced Raman (SER),
and surface electrochem-
amongst many others (Fig. 7).
Examples of
nanoparticleantibody fragment conjugates are less abundant;
this may be as a result of the relative infancy of the eld and mAb
immunogenicity no longer being an issue. However, recent
reports suggest that signicant gains can still be obtained through
a switch in focus from full antibodies to antibody fragments,
some of which are described below.
4.3.1 Fluorescence/FRET immunoassays. Optical immu-
noassays rely on colourimetric or uorescence-based reporter
molecules for the detection of the target analyte. These assays
are oen simple and require relatively basic equipment to
interpret, an advantage for the design of point of care/point of
demand (POC/POD) diagnostic devices. A simple example of
this is provided by Anderson et al., who utilised sdAbQD
conjugates in an immunoassay for the detection of ricin.
group exploited the uorescence of the quantum dot as
a reporter in a sandwich assay, observing limits of detection
comparable with traditional uorescent dyes. In the same
study, the group showed that the same sdAbQD conjugate
could be used in a surface plasmon resonance (SPR) assay,
achieving a 10-fold increase in sensitivity compared to the sdAb
alone. Thus the group were able to utilise their sdAbQD
conjugate in a dual-detection capacity, exploiting both the
optical and physical properties of the quantum dot. Interest-
ingly, and in support of controlled antibody fragment orienta-
tion, the group attached the ScFv to their quantum via
a selectively introduced His-tag, exploiting the interaction with
the zinc ions on the surface of the quantum dots.
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,6377 | 73
Perspective Chemical Science
Further to the above, Wegner et al. have employed the FRET
capabilities of QDs in their sandwich immunoassays to great
Their assays rely on an antigen-mediated FRET
coupling between a QD conjugated reporter antibody and
a terbium-labelled capture antibody. The group compared full
antibody, F(ab0)
, and Fab fragments as targeting ligands for
their QDantibody conjugates in an immunoassay for prostate
specic antigen (PSA). In each case, non-specic conjugation
techniques were employed. It was found that the QDFab
signicantly outperformed the QDfull antibody, achieving
a 5-fold increase in sensitivity for PSA in serum samples. The
authors attributed this to a combination of decreased distance
between the FRET pairs and improved orientation of the Fab on
the surface of the QD. The group utilised a similar assay for the
detection of EGFR in serum, achieving comparable success
when employing a QDnanobody construct as their reporter
These solution based assays hold advantages over
the more traditional surface based assays as they do not require
immobilisation of the capture antibody onto a surface. This
increases eciency and practicality, whilst eliminating poten-
tial inaccuracies brought about by non-specic sticking of the
nanoparticles to the plate.
4.3.2 LSPR immunoassays. Localised surface plasmon
resonance (LSPR) relies on changes occurring on the surface of
a nanoparticle upon successful binding of a disease marker to
a surface-immobilised targeting ligand. In the case of LSPR
immunoassays, binding of the antigen to the antibody ligand
results in small changes in the dielectric eld surrounding the
conjugated magnetic nanoparticle. This changes the frequency
of the surface plasmon produced during interaction of the
particle with electromagnetic radiation, a phenomenon which
can be measured with great accuracy.
Byun et al. showed
that LSPR could be used to detect C-reactive protein (CRP),
a protein used as a biomarker for inammatory diseases.
group utilised a gold nanorod conjugated to an ScFv via
a selective cysteine residue and were able to detect CRP in
serum at concentrations lower than 1 ng mL
. The authors
note that a conscious decision was made to use the small ScFv
rather than a whole antibody as LSPR eects are more
pronounced when the antigenantibody interaction occurs
closer to the surface of the nanoparticle. The smaller size of the
ScFv compared to the full antibody helped to achieve this.
4.3.3 SER immunoassays. SER immunoassays exploit the
observed amplication of the Raman scattering prole of a system
Fig. 7 Various designs of immunoassay ranging from surface based, FRET and lateral ow assays to LSPR, SERS and electrochemical biosensing.
74 |Chem. Sci.,2017,8,6377 This journal is © The Royal Society of Chemistry 2017
Chemical Science Perspective
upon binding of a disease marker. Typical SER immunoassay
systems involve a metallic nanoparticle which has been func-
tionalised with both an antibody capture ligand and a sensitive
Raman reporter molecule. DierencesintheRamanspectra
before and aer binding of the antigen can be used to quantify the
amount of antigen present. This technique has been shown to be
highly sensitive, allowing single molecules to be detected in
certain cases.
Bishnoi et al. exploited this successfully to
generate an immunoassay against a protein implicated in retinal
The group used lysineNHS ester chemistry to conju-
gate the Fab fragment of their expressed antibody to the surface of
a gold nanoparticle which had been pre-functionalised with the
Raman reporter p-mercaptoaniline. Using this approach, a linear
relationship between retinal lysate concentration and the Raman
signal was observed, with negative controls producing only
negligible eects on the signal. Similarly, Qian et al. were able to
successfully exploit SERS to detect the presence of EGFR on the
surface of human cells in vitro using a gold nanoparticleScFv
Whist it is appreciated that the work performed was
not strictly an immunoassay, the results suggest that that an
EGFR immunoassay based on SERS could be readily developed.
4.3.4 Electrochemical immunoassays. Electrochemical
immunoassays utilise the electronic or electrochemical prop-
erties of inorganic nanoparticles to determine antigen binding
to surface-bound antibody/antibody fragment ligands. In
a typical set up a conductive nanoparticle, such as a carbon
nanotube, is conjugated to a capture antibody ligand. Binding
of the antigen to the antibody causes minute changes in the
electrical environment on the surface of the nanotube, altering
the electrical conductance and thus producing a quantiable
signal. Electrochemical techniques have been found to be
highly sensitive, robust, and easy to use. Through combination
with microuidic cells, electrochemical immunoassays have
been fabricated into full integrated immunosensors for point of
care applications.
Lo et al. employed the use of an electrochemical immuno-
assay for the detection of CEA. By immobilising an anti-CEA
ScFv onto the surface of nickel coated carbon nanotubes the
group were able to demonstrate a quantiable dierence in
electrical conductivity before and aer incubation with the
disease marker.
This approach provided a detection limit of
10 ng mL
, a 10-fold increase in sensitivity compared to a near
identical study where a full antibody against CEA was
The authors attribute this increased sensitivity to
the smaller size of the ScFv and its orientation on the nano-
particle through a selective interaction between the nickel
coating and the His tag on the ScFv. When this selectivity was
removed through the introduction of multiple chelating sites,
a nullication of the activity was observed, thus demonstrating
the importance of oriented immobilisation. More recently,
Lerner et al. utilised a carbon nanotube to design an immu-
noassay for the detection of osteopontin (OPN), a disease
marker for prostate cancer.
The group attached an anti-OPN
ScFv to a carbon nanotube and were able to detect OPN in
serum samples at concentrations as low as 1 pg mL
, a detec-
tion limit three orders of magnitude lower than commercial
ELISA assays against the same marker.
5. Conclusions and future outlook
Whilst traditional nanoparticlefull antibody conjugates have
proven to be eective tools for both therapeutic and research
purposes, limitations resulting from the use of whole immu-
noglobulins briey plateaued progress in the area. However,
a switch in focus to antibody-based fragments, both natural
and engineered, is leading to a positive step-shiin progress. It
is clear from the evidence presented in this review that anti-
body fragments have great potential as targeting ligands for
nanoparticle based therapeutics, diagnostics and bioassays,
with the resulting constructs demonstrating greater selectivity,
superior antigen binding, and more favourable pharmacoki-
netic properties.
It seems we are now at a stage where we are ne-tuning how
the antibody fragment is specically connected to the nano-
particle; as exemplied, the choice of conjugation technique
plays an important role in the properties of the resulting
nanoparticleantibody fragment conjugate with more controlled
chemistries consistently providing superior results. The
marriage of site-selective conjugation strategies with the unique
properties and smaller size of antibody fragments allows for the
installation of highly oriented targeting ligands, a clear advan-
tage for selectivity, in vivo tolerance and binding anity. We
predict that the future in this eld will see a continuation in the
trend towards antibody fragment based targeting ligands being
installed via increasingly selective and controlled chemistries;
potentially providing access to hitherto unexplored applications
for antibody targeted nanoparticles.
We gratefully acknowledge EPSRC (EP/M01792X/1) and i-sense
EPSRC IRC in Early Warning Sensing Systems for Infectious
Diseases (EP/K031953/1) for funding AM and DAR, respectively.
Certain images in Fig. 3, 6 and 7 were obtained from http://
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Perspective Chemical Science
... 160,161 Owing to the extraordinarily high specificity and affinity to tumor-associated cell surface antigens, with dissociation constants in the nanomolar to the sub-picomolar range, antibodies, and their derivatives have become the most well-known and efficient ligands for targeted delivery of nanoparticles to cancer cells over the past decades. 162,163 Currently, these antibody-relevant targeting agents can be broadly categorized into three groups: monoclonal antibodies (mAbs), antibody fragments, and bispecific antibodies. ...
... Conjugation of nanoparticles carrying chemo-/radiotherapeutic agents to a monoclonal antibody that binds to a target expressed exclusively on tumor cells can create a guided missile for precise delivery of the toxic payloads to the tumor tissue, which will not only improve treatments but will also reduce side effects. 162,163,165 Epidermal growth factor receptors (EGFR), human epidermal growth factor receptor-2 (HER2), and prostate-specific membrane antigen (PSMA) are three representative targets that have been extensively investigated in mAbfunctionalized nanoparticles for cancer therapy. 166 EGFR exhibits increased expression in various solid tumors, including non-small cell lung cancer, breast cancer, ovarian cancer, as well as head and neck squamous cell carcinoma. ...
... To circumvent this issue, a series of antibody fragments that retain at least one antigen-binding region were proposed as targeting moieties for selective nanoparticle delivery. 162 For example, by means of selective enzymatic cleavage, an intact antibody can be cleaved into several different fragments including antigen-binding fragment (Fab), Fab', and F(ab') 2 . In addition, with the advent of genetic engineering and phage display techniques, a variety of engineered antibody fragments, such as the single-chain variable fragment (scFv), single domain antibody (sdAb), and diabody have been developed and exploited as targeting ligands. ...
Full-text available
Cancer remains a highly lethal disease in the world. Currently, either conventional cancer therapies or modern immunotherapies are non-tumor-targeted therapeutic approaches that cannot accurately distinguish malignant cells from healthy ones, giving rise to multiple undesired side effects. Recent advances in nanotechnology, accompanied by our growing understanding of cancer biology and nano-bio interactions, have led to the development of a series of nanocarriers, which aim to improve the therapeutic efficacy while reducing off-target toxicity of the encapsulated anticancer agents through tumor tissue-, cell-, or organelle-specific targeting. However, the vast majority of nanocarriers do not possess hierarchical targeting capability, and their therapeutic indices are often compromised by either poor tumor accumulation, inefficient cellular internalization, or inaccurate subcellular localization. This Review outlines current and prospective strategies in the design of tumor tissue-, cell-, and organelle-targeted cancer nanomedicines, and highlights the latest progress in hierarchical targeting technologies that can dynamically integrate these three different stages of static tumor targeting to maximize therapeutic outcomes. Finally, we briefly discuss the current challenges and future opportunities for the clinical translation of cancer nanomedicines.
... In addition, the LNPs can be noncovalently coated with targeting antibodies via a recombinant lipoprotein ( named ASSET ) that is incorporated into siRNA-loaded LNP and interacts with the antibody Fc domain [ 79 ]. Several studies have also developed the use of antibody fragments instead of whole immunoglobulins in order to reduce immunogenicity, increase loading capacities and, thereby, improve the efficacy [ 80 ]. This methodology has been used for anti-cancer and siRNA therapies [ 81 ]. ...
Full-text available
The successful employment of messenger ribonucleic acid (mRNA) as vaccine therapy for the prevention of COVID-19 infection has spotlighted the attention of scientific community on the potential clinical application of these molecules as innovative and alternative therapeutic approaches in different fields of medicine. As therapy, mRNAs may be advantageous due to their unique biological properties to target almost any genetic component within the cell, many of which may be unreachable using other pharmacological/therapeutic approaches and to encode any proteins and peptides without the need of their transport into the nuclei of the target cells. Additionally, these molecules may be rapidly designed/produced and clinically tested. Once the chemistry of the RNA and its delivery system are optimized, the cost of developing novel variants of these medications for new selected clinical disorders is significantly reduced. However, although potentially useful as new therapeutic weapons against several kidney diseases, the complex architecture of kidney and the inability of nanoparticles that accommodate oligonucleotides to cross the integral glomerular filtration barrier have largely decreased their potential employment in nephrology. However, in the next few years, the technical improvements in mRNA that increase translational efficiency, modulate innate and adaptive immunogenicity, and increase their delivery at the site of action will overcome these limitations. Therefore, this review has the scope to summarize the key strengths of these RNA-based therapies and illustrate potential future directions and challenges of this promising technology for widespread therapeutic use in nephrology.
... It was used as a drug delivery compound because it controls drug release for a prolonged period. It also has the ability to deliver proteins, peptides, and DNA transporters in gene therapy to its potential in recruiting disabled members [4][5][6]. ...
Full-text available
This study includes two parts, and the first was the preparation of the Zn(II)complex by reacting N-[4-(5-{(Z)-[(5-oxo-2-sulfanyl-4,5-dihydro-1H-imidazol-1-yl)imino]methyl}furan-2-yl)phenyl]acetamide with ZnCl2. The complex was characterized by using microscopic analysis such as UV-Vis spectrum, LC-MS, FTIR spectrophotometer, measurements of conductivity, magnetic susceptibility, and atomic absorption. The second part was the preparation of the ZnO nanoparticles by dissolving the Zn(II) complex in HNO3 and HCl and its use as a drug transporter to treat leukemia. FSEM, TEM, and XRD were examined for the characterization of ZnO nanoparticles that will be used in the synthesis of most medicines and drugs in the future.
... However, due to antibodies' bulkiness, it is difficult to maintain their functional geometric orientation on the membrane surface during particle coating. To overcome this challenge, smaller antibody fragments can be used to enhance spatially selective conjugation with lipid anchors [168]. In addition to their supporting role as anchors for ligand attachment, lipids can serve functional roles by providing responsive properties to stimuli such as light, hypoxia, or pH. ...
Full-text available
Achieving precise cancer theranostics necessitates the rational design of smart nanosystems that ensure high biological safety and minimize non-specific interactions with normal tissues. In this regard, “bioinspired” membrane-coated nanosystems have emerged as a promising approach, providing a versatile platform for the development of next-generation smart nanosystems. This review article presents an in-depth investigation into the potential of these nanosystems for targeted cancer theranostics, encompassing key aspects such as cell membrane sources, isolation techniques, nanoparticle core selection, approaches for coating nanoparticle cores with the cell membrane, and characterization methods. Moreover, this review underscores strategies employed to enhance the multi-functionality of these nanosystems, including lipid insertion, membrane hybridization, metabolic engineering, and genetic modification. Additionally, the applications of these bioinspired nanosystems in cancer diagnosis and therapeutics are discussed, along with the recent advances in this field. Through a comprehensive exploration of membrane-coated nanosystems, this review provides valuable insights into their potential for precise cancer theranostics
... Nanoparticles (NP) are promising agents mostly for intramacrophage or phagosome antimicrobial action, since their construction implies the design of specific targets for their controlled release, so that large but not excessive drug concentrations can be administered (Gharatape et al., 2016). Some previous studies reported the use of polymers modified on their surface with biomacromolecules or antibodies for the recognition of proteins expressed only by tumor cells, or release conditions in slightly acidic environments that are characteristic of cancer cells (Richards et al., 2017;Marques et al., 2020). NPs can also enter infected macrophages and release AMPs to exert their action, especially when they do not have cell-penetrating characteristics (Tenland et al., 2019;Meng et al., 2023). ...
Full-text available
Tuberculosis and lung cancer are, in many cases, correlated diseases that can be confused because they have similar symptoms. Many meta-analyses have proven that there is a greater chance of developing lung cancer in patients who have active pulmonary tuberculosis. It is, therefore, important to monitor the patient for a long time after recovery and search for combined therapies that can treat both diseases, as well as face the great problem of drug resistance. Peptides are molecules derived from the breakdown of proteins, and the membranolytic class is already being studied. It has been proposed that these molecules destabilize cellular homeostasis, performing a dual antimicrobial and anticancer function and offering several possibilities of adaptation for adequate delivery and action. In this review, we focus on two important reason for the use of multifunctional peptides or peptides, namely the double activity and no harmful effects on humans. We review some of the main antimicrobial and anti-inflammatory bioactive peptides and highlight four that have anti-tuberculosis and anti-cancer activity, which may contribute to obtaining drugs with this dual functionality.
... A major advantage of the various Fabs is that linker engineering is not required, which saves time and resources. Fabs are typically conjugated as targeting ligands for therapeutic or diagnostic tools [20]. ...
Full-text available
Attached to proteins, lipids, or forming long, complex chains, glycans represent the most versatile post-translational modification in nature and surround all human cells. Unique glycan structures are monitored by the immune system and differentiate self from non-self and healthy from malignant cells. Aberrant glycosylations, termed tumour-associated carbohydrate antigens (TACAs), are a hallmark of cancer and are correlated with all aspects of cancer biology. Therefore, TACAs represent attractive targets for monoclonal antibodies for cancer diagnosis and therapy. However, due to the thick and dense glycocalyx as well as the tumour micro-environment, conventional antibodies often suffer from restricted access and limited effectiveness in vivo. To overcome this issue, many small antibody fragments have come forth, showing similar affinity with better efficiency than their full-length counterparts. Here we review small antibody fragments against specific glycans on tumour cells and highlight their advantages over conventional antibodies.
Full-text available
We developed a carcinoembryonic antigen (CEA) conjugated polymer nanoparticle (CPN510-CEA-Af) probe to target CEA-expressing CRC cells in vitro. Its efficacy was evaluated in 2D and 3D cultures of LS174T, LoVo,...
Non-small cell lung cancer (NSCLC) has a long history of defying traditional cytotoxic treatment. Significant advancements in biotechnology, cancer biology, and immunotherapy have provided new insights that have altered the landscape for the management of NSCLC, clearing the way for a new era of pharmaceuticals in the form of monoclonal antibodies and their fragments. Antibody fragments are superior to monoclonal antibodies because of their small size, which allows them to penetrate cells and tissues effectively. When combined with functional nanocarriers, antibody fragments can target cancer cells while offering improved efficacy and fewer off-target effects. We discuss current topics of interest including anti-CTLA-4 mAbs, Talactoferrin alfa (TLF), and the CYFRA 21-1 biomarker, with brief insights into its novel detection system.
Full-text available
One of the most important classes of proteins in terms of drug targets is cell surface membrane proteins, and yet it is a challenging set of proteins for generating high-quality affinity reagents. In this review, we focus on the use of phage libraries, which display antibody fragments, for generating recombinant antibodies to membrane proteins. Such affinity reagents generally have high specificity and affinity for their targets. They have been used for cell staining, for promoting protein crystallization to solve three-dimensional structures, for diagnostics, and for treating diseases as therapeutics. We cover publications on this topic from the past 10 years, with a focus on the various formats of membrane proteins for affinity selection and the diverse affinity selection strategies used. Lastly, we discuss the challenges faced in this field and provide possible directions for future efforts.
Full-text available
Carbon nanotubes (CNTs) are cylindrical sheets of hexagonally ordered carbon atoms, giving tubes with diameters on the order of a few nanometers and lengths typically in the micrometer range. They may be single- or multiwalled (SWCNTs and MWCNTs respectively). Since the seminal report of their synthesis in 1991, CNTs have fascinated scientists of all stripes. Physicists have been intrigued by their electrical, thermal, and vibrational potential. Materials scientists have worked on integrating them into ultrastrong composites and electronic devices, while chemists have been fascinated by the effects of curvature on reactivity and have developed new synthesis and purification techniques. However, to date no large-scale, real-life biotechnological CNT breakthrough has been industrially adopted and it is proving difficult to justify taking these materials forward into the clinic. We believe that these challenges are not the end of the story, but that a viable carbon nanotube biotechnology is one in which the unique properties of nanotubes bring about an effect that would be otherwise impossible. In this Outlook, we therefore seek to reframe the field by highlighting those biological applications in which the singular properties of CNTs provide some entirely new activity or biological effect as a pointer to "what could be".
Full-text available
Surface-enhanced Raman scattering (SERS) has enabled the detection of pathogens and disease markers at extremely low levels. This review examines the potential impact of SERS in addressing unmet needs in pathogen diagnostics both in a traditional clinical setting and in the point of care (POC) arena. It begins by describing the strengths and weaknesses of today's diagnostics technologies in order to set a contextual stage for an overview which highlights a few of the many recent developments using SERS in biodefense, human and animal health, and monitoring food and water safety. These sections are followed by discussions of the challenges for the translation of these developments to POC settings, including the performance attributes and metrics for quantification of analytical and clinical figures of merit (e.g., limit of detection and clinical accuracy), and the pathways for large-scale test validation and the build-out of instrumentation and tests kits for POC deployment.
Colloidal gold is undoubtedly one of the most extensively studied nanomaterials, with 1000s of different protocols currently available to synthesise gold nanoparticles (AuNPs). While developments in the synthesis of AuNPs have progressed rapidly in recent years, our understanding of their biological impact, with particular respect to the effect of shape, size, surface characteristics and aggregation states, has struggled to keep pace. It is generally agreed that when AuNPs are exposed to biological systems, these parameters directly influence their pharmacokinetic and pharmacodynamic properties by influencing AuNPs distribution, circulation time, metabolism and excretion in biological systems. However, the rules governing these properties, and the science behind them, are poorly understood. Therefore, a systematic understanding of the implications of these variables at the nano-bio interface has recently become a topic of major interest. This Review Article attempts to ignite a discussion around the influence of different physico-chemical parameters on biological activity of AuNPs, while focussing on critical aspects of cellular interactions, uptake and cytotoxicity. The review also discusses emerging trends in AuNP uptake and toxicity that are leading to technological advances through AuNP-based therapy, diagnostics and imaging.
In the present study, we describe the synthesis and characterization of new generation of cancer-targeting magnetic nanoprobes: superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylene glycol (PEG) shell functionalized with recombinant anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. An anti-HER2 scFv with terminal cysteine (scFv 4D5-Cys) has been rationally engineered in order to favor its orientation- and site-directed covalent conjugation to the polymeric surface of PEGylated SPIONs. Optimization of scFv and nanoparticles production allowed to obtain well-characterized SPIONs-PEG–scFv nanoparticles carrying ∼7 fragments per nanoparticle, having a hydrodynamic diameter of ca. 86 nm and nearly neutral surface. The nanoprobes-scFv capability to recognize the HER2 protein has been confirmed by enzyme-linked immunosorbent assay (ELISA). Compared to non-targeted PEGylated SPIONs, the SPIONs–PEG–scFv nanoprobes showed an enhanced binding to HER2-overexpressing cells (SK-BR3) in vitro as it was shown by immunofluorescence. Finally, ICP-AES measurements shown that in 1 hour the uptake of SPIONs–PEG–scFv in HER2-overexpressing cells is 2.1 times greater than non-targeted PEGylated SPIONs. Therefore, both due to their physico-chemical characteristics and the immunotargeting of HER2-positive breast cancer cells, the SPIONs–PEG–scFv appear as promising nanoplatforms for future applications in theranostic treatment of cancers.