Antibody fragments as nanoparticle targeting ligands: a step in the right direction

Abstract

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.

Full text

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 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.
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 difficult 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
signicantly higher than traditional methods.
1
This factor, in
combination with the ease in which the surface of nanoparticles
can be decorated with high affinity disease-specic 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.
1–7
Of these, antibody based targeting
ligands have become incredibly popular due to their unique in
vivo properties and high target specicities.
8–11
Whilst the
contributions of other targeting ligands should not be ignored,
this review focuses on the use of antibodies, or more specically
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 metal–halogen
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´
erieure
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 modication of
proteins. He is currently working
as a postdoctoral fellow under
the supervision of Dr Vijay
Chudasama on the development of novel methodologies for
bioconjugation.
Department of Chemistry, University College London, 20 Gordon Street, London,
WC1H 0AJ, UK. E-mail: daniel.richards.11@ucl.ac.uk; v.chudasama@ucl.ac.uk; 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
www.rsc.org/chemicalscience
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,63–77 | 63
Chemical
Science
PERSPECTIVE

common nanoparticle (Section 2.1) and antibody (Section 2.2)
scaffolds used in this context will be given.
2. Antibody–decorated nanoparticles
2.1 Nanoparticle structure
When designing nanoparticle–antibody 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 modication 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).
4
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
approval.
12
They comprise natural lipids with polar and non-
polar components which self-assemble into colloidal particles.
Whilst early liposomal nanoparticles suffered from issues of
stability and rapid clearance, the introduction of surface
ligands such as polyethylene glycol (PEG) chains has helped to
address these drawbacks.
12,13
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
molecules.
4,6,14
However, they are limited by a high level of
sensitivity to structural change(s) and have demonstrated
highly specic cargo-dependency, thus decreasing their
universal appeal and broad applicability.
6,14
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,
signicantly increasing their circulation time.
15
Their hydro-
philic exterior also allows polymeric micelles to effectively
and safely encapsulate very hydrophobic drugs for safe trans-
port through the body.
16
As with liposomal nanoparticles,
polymeric micelles also demonstrate excellent biocompati-
bility.
17
However, poorly controlled release proles of encapsu-
lated cargo, and a high sensitivity to structural change(s), mean
that there is still signicant scope for improvement.
4
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.
10
This
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.
6
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.
18
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
reported.
19
Their highly regulated synthesis enables excellent
control over shape and size –important parameters for medical
applications.
20
They also display excellent solubility and have
been shown to be non-immunogenic.
21
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
3
O
4
) 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.
22,23
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 C–H activation and various
aspects of Chemical Biology. Vijay's research has recently been
highlighted by Forbes, Scientic American, CNN, Nature Chemistry
and the Royal Society of Chemistry.
†Hybrid organic–inorganic particles will not be focused on in this review.
64 |Chem. Sci.,2017,8,63–77 This journal is © The Royal Society of Chemistry 2017
Chemical Science Perspective

display both therapeutic and diagnostic properties. However,
the lack of a spacious “core”or any porous space leads to low
loading volumes,
24
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.
23
Fig. 1 Pictorial representation of different types of nanoparticles used in biomedical applications.
Table 1 A table summarising the different types of nanoparticles with focus on material used, cargo attachment, and their various advantages &
disadvantages
Nanoparticle Material(s) Cargo attachment Advantages Disadvantages
Liposomes Self-assembling lipid
bilayer
Encapsulated within the
hydrophilic core
Easily synthesised,
biocompatible, high
internal loading
Highly sensitive to
structural changes and
nature of payload
Polymeric
micelles
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 proles
Polymeric
nanospheres/
nanocapsules
Solid hydrophobic polymer
matrix with optional
aqueous core
(nanocapsule)
Embedded in the polymer
matrix or within the core
High loading capacity,
exible loading
capabilities, reliable
release proles
Difficult to purify and poor
store properties
Dendrimers Highly branched polymer
matrix
Embedded in the polymer
branches
Highly soluble, non-
immunogenic, high
loading capacity,
controlled synthesis
Lacking data on toxicity and
biocompatibility
Iron oxide
nanoparticles
Iron oxide core surrounded
by biocompatible coating
Attached to the surface/
surface coating
Innate magnetic properties No internal loading capacity
Gold
nanoparticles
Solid gold particles
typically coated with PEG
chains
Attached to the surface/
surface coating
Innate optical and
photothermal properties
No internal loading
capacity, poor
biocompatibility and
biodegradability
Mesoporous
silica
nanoparticles
Mesopores surrounded by
a silica framework
Encapsulated within the
mesopores
High loading capacity,
good biodegradability
Issues with physiological
stability, rapid clearance
rates
Carbon
nanoparticles
Graphite arranged in either
a sheet or cylindrical
conformation
Attached to the carbon
backbone
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 coefficients
No internal loading
capacity, potential toxicity
issues
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,63–77 | 65
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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.
25,26
However, their
non-hollow structure precludes internal loading,
4
and they also
tend to suffer from poor biodegradation and questionable
biocompatibility.
27–29
Mesoporous silica nanoparticles. Mesoporous silica nano-
particles (MSNs) consist of mesopores (2–50 nm pores) sur-
rounded by a silica framework. These nanoparticles have
a high surface area to volume ratio which affords them
a large loading capacity. MSNs have also demonstrated good
biocompatibility and biodegradability, desirable features for
biomedical purposes.
30,31
However,stabilityissuesandrapid
clearance rates signicantly restrict the use of MSNs from
certain applications.
32–34
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.
35,36
Unfortunately, issues of
poor biodegradability,
37
pulmonary damage,
29,38
and undesir-
able organ accumulation
29,39,40
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.
41
Whilst potential
toxicity issues have to-date limited their utility in vivo, recent
advances are helping to overcome these remaining hurdles.
41–43
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 signicant 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 disulde 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
H/L
) and constant (C
H/L
) regions
(Fig. 2).
44
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,
F(ab0)
2
, and Fv regions of antibodies to great effect (Fig. 2).
45
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).
46–50
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 nanoparticle–antibody 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,
51
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
immunoglobulins,
52–57
and as such this will not be covered in
detail in this review.
2.3 Nanoparticle–antibody fragment conjugates
Antibodies function by targeting specic 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. insufficient methods for
generating and evaluating antibody–decorated nanoparticles)
prevented signicant 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 nanoparticle–antibody 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 modication. However several issues
associated with the use of full antibody ligands, such as
immunogenicity,
9
rapid elimination,
58
poor stability,
59,60
and
lower than expected efficacy,
1,6,8,61
soon came to light and these
66 |Chem. Sci.,2017,8,63–77 This journal is © The Royal Society of Chemistry 2017
Chemical Science Perspective

are being increasingly emphasised/supported by emerging data.
A signicant 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 efficacy (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 signicantly over the next few years.
9
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,
8,9
very few
specically highlight and accurately detail work on nano-
particle–antibody 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.,2017,8,63–77 | 67
Perspective Chemical Science

to introduce the area, with particular emphasis on recent
developments in the generation and application of nano-
particle–antibody fragment conjugates for biomedical uses.
3. Generating nanoparticle–antibody
fragment complexes
During the design of nanoparticle–antibody 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
below.
3.1 Modication of antibody fragments
Modications 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 modication include lysine, cysteine, and
glutamic/aspartic acid, as they can be functionalised using well-
established chemistries. Initially, lysine was a popular target for
modication 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 modication (including antibody fragments)
has maintained a healthy research focus for several decades
now, largely due to the rapid development of the antibody–drug
conjugate eld. This has resulted in a rich toolbox of chemical
reactions which enable facile, site-selective modication whilst
avoiding negative effects on the function of the protein. Several
excellent reviews have been written on this subject, so it will not
be covered in depth here.
62–65
However, Fig. 4 highlights some of
the most common methods employed for functionalising anti-
body fragments for subsequent attachment to nanoparticles.
3.2 Modication of nanoparticle surfaces
Nanoparticle surface modication techniques can be broadly
separated in two main categories: (i) covalent and (ii) non-
covalent. Covalent modications 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
stability.
8
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
modied 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 modication of these groups can further
expand the reactivity prole 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,
4,8,18,66,67
including a comprehensive overview by Sapsford et al.
68
Despite
these advances, non-specic interactions of antibody ligands
with nanoparticle surfaces remains an issue, and methods for
distinguishing specic interactions from non-specic interac-
tions are lacking. These issues can be particularly problematic
when site-specic or oriented conjugation of an antibody frag-
ment is desired.
4. Nanoparticle–antibody 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 nanoparticle–antibody 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 exemplied 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.
69
During the design of lipo-
somes which could selectively target B-cell malignancies with
encapsulated doxorubicin (Stealth® immunoliposomes, SIL),
they compared the in vivo effectiveness of doxorubicin bearing
liposomes targeted with HD-37 mAb, HD-37-Fab0and a HD-37-
ScFv against the B-cell antigen CD19.
69
The targeting ligands
were attached to the protein using maleimide–thiol 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 signicant difference in
CD19 binding between HD-37-mAb and HD-37-ScFv targeted
68 |Chem. Sci.,2017,8,63–77 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
+
over
CD19

cells with the mAb being the worst performer over both
studies. Drastic differences were also noticed in vivo,withthe
HD-37-mAb targeted liposome being rapidly cleared (0.41 mL
h
1
) due to Fc-mediated uptake into the liver and spleen in
comparison to the fragment conjugates (0.10 mL h
1
for the
Fab and 0.12 mL h
1
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 effects is an improved mean survival rate of
Fig. 4 Schematic representations of common ways in which antibody fragments are modified.
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,63–77 | 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 differences 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.
70
4.1.1 Targeted delivery of small molecule drugs. In addi-
tion to the clinical examples mentioned above, a plethora of
preclinical nanoparticle–antibody fragment conjugates exist for
the targeted delivery of cytotoxic payloads.
9,71
Manjappa et al.
used an anti-neuropilin (NRP) Fab0targeted liposome contain-
ing docetaxel to simultaneously target both solid tumours and
the surrounding microvasculature.
72
The anti-NRP Fab0was
conjugated to the liposome via surface PEG-maleimide groups,
resulting in a site-specic 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 nanoparticle–antibody fragment drug
delivery systems utilise lipid-based nanoparticles (Table 2), the
last few years have seen an increased exploration of non-lipo-
somal nanoparticle–antibody 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 afforded
signicant retardation of tumour growth when compared to
non-targeted polymeric micelles or free drug alone.
74
By utilis-
ing a selective maleimide–thiol 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 glycol–polylactic acid
(PEG–PLA) polymersome containing As
2
O
3
as the cytotoxic
payload.
75
Despite the use of suboptimal non-specic 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-
ments.
76
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 aer 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 nanoparticle–antibody conjugates currently undergoing clinical trials. Adapted from tables previously published by Van der Meel
et al.
73
and Goodall et al.
11
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
Erbitux-EDVs
pac
Bacterially
derived mini-cell
EGFR Bispecic 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)
2
Doxorubicin Metastatic stomach
cancer
I
Anti-EGFR ILs-Dox Lipid EGFR Cetuximab Fab Doxorubicin Solid tumours I
70 |Chem. Sci.,2017,8,63–77 This journal is © The Royal Society of Chemistry 2017
Chemical Science Perspective

a reducing agent known to cleave the interchain heavy-light
disulde bond of the Fab fragment. This would enable cysteine
residues on both chains to react independently with the nano-
particle, potentially decreasing the control offered through the
specic 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 bispecic
ScFv and SdAb targeted liposomes, and have demonstrated
a clear advantage in the use of both bispecic ScFv and SdAb
fragments as targeting ligands for liposomal nanoparticles.
77,78
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 effect. Whilst the
majority of therapeutic nanomedicine is focused on the delivery
of cytotoxic drugs, increasing effort is being spent on devel-
oping nanoparticle–antibody conjugates for targeted gene
therapy.
4,79,80
Indeed, two of the eight nanoparticle–antibody
conjugates currently in clinical trials utilise specic DNA
strands as their payload (SGT-53 and SGT-94, Table 1). Nano-
particle-based gene delivery was partially covered by Zhang
et al.
79
and Li et al.,
81
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.
82
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 effects 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
nanoparticles.
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 effective treatment for breast
cancer.
83
Similarly, Laroui et al. demonstrated effective treat-
ment of colitis through the delivery of TNF-asiRNA encapsu-
lated within F4/80 Fab0targeted PEG–PLA polymersomes.
84
The
group found that Fab0targeted TNF-asiRNA containing nano-
particles granted a greater reduction in all symptoms of colonic
inammation when compared to the non-targeted controls. In
both studies the Fab0fragment was site-specically conjugated
to the nanoparticle via the hinge region using maleimide–thiol
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.
85,86
One study demonstrated the appli-
cability of this approach towards the treatment of neuroblas-
toma tumours, with in vivo data suggesting signicant gene
silencing and subsequent tumour suppression.
85
Early data
suggests a similar approach could be utilised for the treatment
and imaging of gastric cancer.
86
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 signicant benets.
4.1.3 Magnetic eld therapy. Within the connes 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).
87
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,
88–91
and signicant
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
side,
92–95
some preliminary work has demonstrated advantages
in the use of antibody fragments in this context. For example,
early work by Shinkai et al. showed effective use of a Fab0
of antibody G250 to deliver a magnetoliposome to MN-antigen
presenting cells.
96
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
exemplied the “theranostic”utility of targeted SPIONs
via the application of an anti-prostate specic antigen
(PSA) ScFv-decorated uorescent magnetic nanoparticle.
97
By
combining the uorescent payload with the superparamagnetic
Fig. 6 A graphical representation of actively targeted nanoparticle
therapeutics.
This journal is © The Royal Society of Chemistry 2017 Chem. Sci.,2017,8,63–77 | 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
afforded 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-specic 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
method.
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.
98
Whilst the SPION-ScFv showed greatly
increased accumulation at the tumour site in vivo,efficacy 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.
99
The results discussed above clearly demonstrate the
advanced capabilities of nanoparticle–antibody fragment
conjugates for chemotherapy. It is anticipated that the trend
of using antibody fragments could also provide benets in
other areas of nanomedicine, e.g. targeted immunotherapy
through the activation of cell receptors such as Death Receptor
5 (DR-5).
100,101
4.2 As imaging agents
Conceptually, targeted nanoparticles provide a myriad of
benets 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 oen be
tailored to provide intrinsic imaging functionality, as is the case
with SPIONs, gold nanoparticles or quantum dots. Early work in
the use of antibody–decorated nanoparticles for imaging
applications encountered problems due to specic accumula-
tion, with the limiting step found to be extravasation of the
nanoparticles from the vasculature, rather than cell
binding.
1,4,7,41,93,102
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.
103
Iron oxide nanoparticles modied 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 signicant improvement in tumour accu-
mulation for the half antibody and the ScFv–decorated 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
different conjugation techniques were used for the different
ligands; both the half antibody and the ScFv were attached via
thiol selective covalent disulde formation, whereas the full
antibody was attached via stable non-covalent protein A affinity
interactions. These results further demonstrate the benets of
actively targeted nanoparticles for imaging tumours, and the
importance of ligand choice.
Another excellent example is provided by the work of R¨
uger
et al. who used a self-quenching near-infrared dye incorporated
inside a ScFv–decorated liposome to image broblast activation
protein alpha (FAP) expressing cells. Application of a self-
quenching uorophore ensured signicant uorescence was
only observed aer intra-cellular degradation of the liposome
post-FAP cell binding. This approach led to a signicant
increase in the signal-to-noise ratio of the ScFv–decorated
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.
104
Exploiting inherently uorescent nanoparticles such as gold
nanoparticles or quantum dots is more widely utilised, likely
due to their relatively large extinction coefficients 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.
105
A similar
approach was used by Balalaeva et al. to image breast cancer in
vivo.
106
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.
107–109
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.
110
In both cases lysine residues on the targeting antibody ligands
were modied with D-biotin using NHS ester chemistry, allow-
ing the ligands to be attached to the quantum dot using the
highly stable biotin–streptavidin 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.
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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).
111,112
The selective delivery of
radionuclides can also have a desirable therapeutic effect,
allowing targeted nanoparticles loaded with radionuclides to
act as successful theranostic tools.
113,114
Whilst a multitude of
examples exist in which full antibody–decorated nanoparticles
have been utilised for this purpose, less work has been carried
out using smaller antibody-based fragments.
115,116
Nonetheless,
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.
117
To target the nanoparticles,
the group attached a Fab fragment targeted against CD10,
a vascular-specic marker for tumour angiogenesis, and
demonstrated a signicant improvement in both PET and
uorescence imaging resolution in vivo compared to non-tar-
geted controls.
Work by Hoang et al. has utilised
111
In-labelled block copol-
ymer micelles conjugated to trastuzumab Fab to image HER2
positive cell lines in vitro using SPECT/CT.
118
In addition to
the trastuzumab Fab targeting ligand the group incorporated
nuclear localisation signal (NLS) peptides onto the surface of
their nanoparticle, leading to effective nuclear translocalisation
aer initial HER2 mediated internalisation. More recently, this
approach was demonstrated in vivo, with signicant benets 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 benecial.
119
4.2.3 Targeted MRI agents. A great deal of effort 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.
120
Actively targeted
approaches have gained popularity in recent years, with anti-
body-derived ligands showing particular promise.
121
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.
99
In vivo results showed signicant
improvement in MRI contrast when ScFv targeted iron oxide
nanoparticles were compared to non-targeted controls. The
group utilised non-selective lysine–NHS 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.
122
By attaching an anti-
CEA ScFv fragment to the surface of their SPION the group were
able to demonstrate excellent target specicity and MRI contrast
in vitro when compared to non-targeted controls. More recently,
Alric et al. showed that an anti-HER2 ScFv could be effectively
employed to traffic PEG coated SPIONS to HER2 expressing
cells.
123
These targeted SPIONS maintained binding affinity 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 maleimide–thiol 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,
124
F¨
orster resonance energy transfer (FRET),
125
catalytic redox reactions,
126,127
surface plasmon resonance (SPR),
128
surface-enhanced Raman (SER),
128,129
and surface electrochem-
istry,
130,131
amongst many others (Fig. 7).
124,132,133
Examples of
nanoparticle–antibody 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 signicant 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 oen 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 sdAb–QD
conjugates in an immunoassay for the detection of ricin.
134
The
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 sdAb–QD 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 sdAb–QD
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.,2017,8,63–77 | 73
Perspective Chemical Science

Further to the above, Wegner et al. have employed the FRET
capabilities of QDs in their sandwich immunoassays to great
effect.
135
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)
2
, and Fab fragments as targeting ligands for
their QD–antibody conjugates in an immunoassay for prostate
specic antigen (PSA). In each case, non-specic conjugation
techniques were employed. It was found that the QD–Fab
signicantly outperformed the QD–full 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 QD–nanobody construct as their reporter
molecule.
136
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 efficiency and practicality, whilst eliminating poten-
tial inaccuracies brought about by non-specic 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.
133,137
Byun et al. showed
that LSPR could be used to detect C-reactive protein (CRP),
a protein used as a biomarker for inammatory diseases.
138
The
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
1
. The authors
note that a conscious decision was made to use the small ScFv
rather than a whole antibody as LSPR effects are more
pronounced when the antigen–antibody 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 amplication of the Raman scattering prole of a system
Fig. 7 Various designs of immunoassay ranging from surface based, FRET and lateral flow assays to LSPR, SERS and electrochemical biosensing.
74 |Chem. Sci.,2017,8,63–77 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. DifferencesintheRamanspectra
before and aer 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.
133,137,139,140
Bishnoi et al. exploited this successfully to
generate an immunoassay against a protein implicated in retinal
damage.
141
The group used lysine–NHS 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 effects 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 nanoparticle–ScFv
conjugate.
142
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 quantiable
signal. Electrochemical techniques have been found to be
highly sensitive, robust, and easy to use. Through combination
with microuidic cells, electrochemical immunoassays have
been fabricated into full integrated immunosensors for point of
care applications.
130,133,143
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 quantiable difference in
electrical conductivity before and aer incubation with the
disease marker.
144
This approach provided a detection limit of
10 ng mL
1
, a 10-fold increase in sensitivity compared to a near
identical study where a full antibody against CEA was
employed.
145
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 nullication 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.
146
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
1
, a detec-
tion limit three orders of magnitude lower than commercial
ELISA assays against the same marker.
5. Conclusions and future outlook
Whilst traditional nanoparticle–full antibody conjugates have
proven to be effective tools for both therapeutic and research
purposes, limitations resulting from the use of whole immu-
noglobulins briey plateaued progress in the area. However,
a switch in focus to antibody-based fragments, both natural
and engineered, is leading to a positive step-shiin 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 specically connected to the nano-
particle; as exemplied, the choice of conjugation technique
plays an important role in the properties of the resulting
nanoparticle–antibody 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 affinity. 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.
Acknowledgements
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://
www.freepik.com.
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