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Design and Applications of Protein-Cage-Based Nanomaterials

Article  in  Chemistry - An Asian Journal · July 2016

DOI: 10.1002/asia.201600769

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CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article

Title: Design and applications of protein cage-based nanomaterials

Authors: Yu Zhang; Maziar Soleymani Ardejani; Brendan Patrick Orner

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gital Object Identifier (DOI) given below. The VoR will be published online in Early
View as soon as possible and may be different to this Accepted Article as a result
of editing. Readers should obtain the VoR from the journal website shown below
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for the content of this Accepted Article.

To be cited as: Chem. Asian J. 10.1002/asia.201600769

Link to VoR: http://dx.doi.org/10.1002/asia.201600769

A Journal of A sister journal of Angewandte Chemie

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Chemistry - An Asian Journal 10.1002/asia.201600769

FOCUS REVIEW

Design and applications of protein cage-based nanomaterials

Yu Zhang [a]*, Maziar S. Ardejani [b] and Brendan P. Orner [c]*
Chemistry - An Asian Journal 10.1002/asia.201600769

FOCUS REVIEW

Abstract: Materials science is beginning to focus on reaction vessels with multiple potential opportunities for
biotemplation, and in support of that trend, it is realized that nanotechnology that have been extensively explored.[10] Protein
protein cages, proteins that assemble from multiple monomers cages vary in size from several to hundreds of nanometers and
into architectures with hollow interiors, can instill a number of in shape from spherical to nanotube-like structures, and these
unique advantages to nanomaterials. In addition, the structural characteristics can directly impact the properties of resulting
and functional plasticity of many protein cage systems permit templated materials.
their engineering for specific applications. In this review, the
most commonly used viral and non-viral protein cages, which
exhibit a wide diversity of size, functionality, and chemical and
thermal stabilities, are described. Moreover, how they have been
exploited for nanomaterial and nanotechnology applications is
summarized.

Introduction

The synthesis of materials with nanoscale structure has

drawn extensive attention due to these material’s emergent
properties and applications in physics and biotechnology. Taking
inspiration from naturally evolved processes, the exploitation of
biological templates is an emerging trend in the fabrication of
nanomaterials, and it has been reported that material size,
shape, and morphology can be controlled by interactions
between biomolecule and inorganic components.[1] Proteins and
Figure 1. Structural comparison of protein assemblies used to build nanoscale
peptides, due to their ready availability and large structural and
materials. All structures are shown to scale. The colors are added to highlight
functional diversity, especially have high utility for achieving
the morphological units, and are not necessarily indicative of different protein
control in the synthesis of materials.[2] Moreover, biotemplate-
sequences. All the structures are generated using UCSF Chimera.
directed syntheses have the potential to be more ‘green’ than
traditional methods due to the mild reaction conditions, such as
The shell of protein cages, usually 2 ~ 5 nm thick, separates
lower temperature, near neutral pH, and the fact that they often
the interior volume from the exterior and often contains selective
employ aqueous reaction solutions usually required for ensuring
or non-selective pores. Protein cages therefore have three
bio-molecule stability.
distinct surfaces: the inner surface that projects into the central
Many of the biotemplates that have been exploited for the
cavity, the outer surface that faces the exterior of the cage and
synthesis of nanomaterials are assemblies made up of
the inter-subunit surfaces that form the protein-protein interfaces
structurally simpler components. Nature uses self-assembly to
in the assembled cage.[11] (Figure 2) The interior cavities of
generate a wide diversity of large, complex, and often highly
these cages offer the potential to package and deliver novel
symmetric protein architectures with a minimum of synthetic
genetic material or to encapsulate other particles. Through either
remuneration,[3] and self-assembling protein systems form a
chemical modification or a genetic-based approach, each of
variety of supramolecular structures with a wide diversity of
these surfaces has potential plasticity to facilitate a protein cage
biological functions and properties. Moreover, nature provides
be the basis for the design of novel functional nanostructures.
many proteins that assemble into cage-like architectures,
In this review, we will outline the most commonly used viral
including virus,[4] bacteriophage,[5] vault,[6] heat shock,[7]
and non-viral protein cages, and how they have been recently
chaperonin[8] and ferritin proteins.[9] (Figure 1)
applied to the growing field of nanomaterials and
Protein cages are proteins that adopt structures enclosing a
nanotechnology with the focus primarily on the interior and
central volume, and they can act as robust, size-constrained
exterior surfaces.

[a] Dr. Y. Zhang

Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals
College of Chemical Engineering
Nanjing Forestry University
Nanjing 210037 (P. R. China)
E-mail: yuzhang@njfu.edu.cn
[b] Dr. M. S. Ardejani
Department of Chemistry, The Scripps Research Institute,
La Jolla, CA 92037, United States
[c] Dr.B. P. Orner
Department of Chemistry, King’s College London
London SE1 1DB, United Kingdom
E-mail: brendan.orner@kcl.ac.uk
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Figure 2. Schematic representation of the three surfaces of protein cages,
each of which can be modified to impart function by design. Reproduced from
reference [11]. Copyright 2007 WILEY-VCH Verlag.
1 Viruses and bacteriophage
Viral and bacteriophage capsids are among the major protein
cage classes and are the primary components of the most
abundant biological entities on earth.[12] With a variety of shapes
and sizes ranging from 20-750 nm, capsids are emerging
platforms for synthetic manipulation with a range of applications
from materials to medicine.[13] In this section, the viruses and
bacteriophages that are most widely used as biocontainers are
introduced and their recent applications are described.
1.1 Cowpea Chlorotic Mottle Virus
Icosahedral virus capsids are round proteinaceous particles
with icosahedral symmetry in the arrangement of their
assembled subunits (known as capsomers). A geometric
icosahedron with 532-point symmetry is comprised of twenty
equilateral triangle facets fused into a closed shell around twelve
vertices.[14] Icosahedral capsids require at least sixty identical
subunits as each facet is generally composed of three
capsomers. In 1962 Caspar and Klug proposed the concept of
quasi-equivalence to describe how larger viruses with
icosahedral symmetry are built up with multiples of sixty
subunits.[15] Many icosahedral viruses can self-assemble into
non-infectious biocontainers devoid of genomic material either
naturally or as a consequence of genetic manipulation.
One icosahedral virus that has been frequently used in
nanomaterials applications is cowpea chlorotic mottle virus
(CCMV) and this example will be used in this review to introduce
common approaches that have been, or could easily be, applied
across many protein cages. This virus is a member of the
bromovirus group of the Biromoviriedae family.[16] Its capsid is
composed of 180 identical copies of the capsomer (20 kD) that
form an icosahedral shell with 28 nm and 24 nm outer and inner
diameters respectively. The CCMV genome contains three
10.1002/asia.201600769
unique, single-stranded RNA polymers inside the shell and the
highly negatively charged RNA is thought to interact strongly
with the positively charged interior capsid surface. The
generation of CCMV in vitro is straightforward, as the Pichia
pastoris heterologous expression system can be use to generate
capsomers that assemble spontaneously, and this has facilitated
the engineering of the cage. For example, complementary
interactions can be designed across the protein-protein
interfaces to generate a stabilized capsid.
(Interior) CCMV was the first icosahedral virus that was shown
to reassemble in vitro into an infectious particle.[17] The
Dr. Yu received her B.S (2004) from China
University of Mining and Technology and M.
S (2007) in Chemistry from Xiamen
University. She completed her PhD in 2012
from Nanyang Technological University under
supervision of Dr. Brendan Orner. Now she is
an associate professor at Nanjing Forestry
University. Her current research focuses on
the design and preparation of functional
nanomaterials based on protein assemblies.
Dr. Maziar S. Ardejani graduated from Sharif
University of Technology with a B.Sc. in
chemistry in 2015. He receiced his M.Sc. and
Ph.D. from Nanyang Technological
University. He has received postdoctoral
training from King’s College London and
currenly is a research associate at The
Scripps Research Institute. His research
revolves around the molecular self-assembly
and its applications.
Dr. Brendan Orner received his B. S. (1995)
from Haverford College where he conducted
research with Professor Terry Newirth. He
completed his M.S. in 1998 at the University
of Pittsburgh and his PhD in 2001 at Yale
University both under the mentorship of ((Author Portrait))
Professor Andrew Hamilton. He conducted
postdoctoral research at University of
Wisconsin with Professor Laura Kiessling.
He started his independent career as an
assistant professor at Nanyang Technological
University in 2006 and moved his lab to the
UK in 2012. He currently is a senior lecturer
at King’s College London.
highly positive interior surface is due to the presence of basic
residues (arginine and lysine) that interact with the negatively
charged genetic RNA and these interactions are essential during
the normal assembly process in vivo.
Douglas and Young exploited the positively charged interior of
CCMV to direct the nucleation of polyoxometalate mineralization
(tungstates H2W12O4210–, molybdates (NH4)xMoOy, and
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vanadates V10O286–) inside the empty cage.[18] They also
reported that by altering the charge of the interior surface from
positive to negative (by glutamic acid substitution of the
internally projecting basic residues), the mineralization of metal
oxide particles (Fe2O3, Fe3O4, Co2O3) can be achieved without
disrupting cage assembly.[19] Furthermore, after some additional
protein engineering, CCMV has also been used as a biotemplate
for the potentiated reduction and directed synthesis of gold
nanoparticles.[20]
A remarkable and highly useful property of CCMV is that its
capsid can undergo pH and metal ion-dependent structural
“gating”, which can be exploited to control the acquisition and
release of guest cargos.[16, 21] This gating involves an increase in
size of up to 10% and the formation of sixty, 2 nm pores in the
capsid at pH above 6.5 or with the forced removal of divalent
metal ions.[21] (Figure 3A) The gating allows the protein to be
loaded for some delivery applications without requiring
reassembly.
In vitro reassembly can also be used to encapsulate larger
cargo inside the CCMV capsid. For example, this approach was
used to package the 40-kDa horseradish peroxidase (HRP)
enzyme within CCMV.[22] Once reassembled, pH dependent
capsid gating influences access of substrate to the active,
encapsulated enzyme and subsequent release of product.
Engineered nanoreactors like these, especially if designed to
incorporate multiple enzymes in a multi-step metabolic pathway,
could have great utility in small molecule synthesis and in aiding
in the fundamental understanding of interactions between
enzymes and metabolites on single enzyme scales.
Using a similar capsid reassembly strategy, Cornelissen et al.
generated a construct consisting of multiple enhanced green
fluorescent proteins (EGFP) encapsulated within a CCMV
capsid by employing heterodimeric coiled-coil peptide fusions.[23]
Through genetic modification, the complementary coild-coil pair,
K-coil (KIAALKE)3 and E-coil (EIAALEK)3, were fused to the N-
terminus of the CCMV capsid protein and the C-terminus of
EGFP, respectively. Noncovalent heterodimerization of the two
coiled-coil elements brings the unassembled capsid proteins
together with the EGFP and adjusting the pH assembles the
capsid. Depending on the concentration and ratio of EGFP-
capsid protein conjugate and the wild type capsid protein, up to
fifteen EGFP proteins can be encapsulated per assembled
capsid. (Figure 3B) The two strategies of pore gatting and
reassembly have been implemented in other protein cage
systems for nanomaterials applications.
10.1002/asia.201600769
Figure 3. The pH-dependent gating of CCMV and a capsid reassembly
strategy to afford guest cargo encapsulation. (A) Unswollen and swollen forms
of cowpea chloriotic mosaic virus (CCMV). The swollen form has sixty, 2 nm
pores that are pH-gated. (B) The capsid of CCMV can be engineered to
assemble around EGFP. Reprinted from reference [21，23]. Copyright 2009
American Chemical Society.
(Exterior) The exterior surface of icosahedral virus capsids
provides the opportunity for multivalent display of ligands in
defined orientation with respect to each other and CCMV has
been employed as a robust platform in this direction. There are a
total of 540 lysines and 560 carboxylates in wild type CCMV that
have been demonstrated to be reactive with isothiocyanate or
malonate presenting dyes respectively.[24] Although the native
cysteines are not reactive toward chemical modification, reactive
thiol groups can be introduced through mutagenesis. These
conjugation approaches have been used to attach a wide variety
of ligands to the exterior surface of CCMV, including small
peptides, biotin, fluorophores, photosensitizers, and even intact
IgG antibodies.[24-25] In fact, Comellas-Aragones et al. were able
to incorporate two different synthetic polymers onto two different
surfaces of the capsid. First polyethylene glycol (PEG) was
conjugated through an NHS ester and lysines on the virus
exterior. After disassembly and removal of viral RNA,
reassembly of the conjugate was inhibited probably due to steric
interactions between the PEG chains. However, addition of
anonic polystyrene sulfonate (PSS) resulted in assembly
effectively generating a CCMV capsid encapsualting PSS and
presenting PEG on its exterior.[26] (Figure 4) It is conceivable that
along with enhancing the generation of materials, multivalently
presented ligands can be exploited for the directed delivery of
encapsulated materials as part of biomedical applications or for
providing the geometric and chemically specific interactions that
would be required in the design of higher order nanostructure.
Figure 4. Schematic pathway toward a PSS-CCMV-PEG particle. The
polyethylene glycol (PEG) chains were attached through lysine residues on
the outer surface of the CCMV capsid. The fluorescein-functionalized PEG
chains induced irreversible dissociation of the virus due to steric interference
between the protein subunits and subsequent release of the viral RNA.
Incubation of the PEG–CCMV conjugate with the anionic polystyrene sulfonate
(PSS) resulted in formation of stability-enhanced reassembled PSS-CCMV-
PEG particles. Reprinted from reference [26]. Copyright 2009 American
Chemical Society.
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1.2 Cowpea Mosaic Virus
Cowpea mosaic virus (CPMV) is a plant virus of the
comovirus group. CPMV capsids are icosahedral, 30 nm in
diameter, and assembled from sixty large and sixty small subunit
proteins. The small capsid proteins form the twelve, pentameric
caps at the 5-fold symmetry axes, and the large capsid proteins
trimerize at the twenty, 3-fold axes of symmetry.[10a] The highly
symmetric and heteromeric nature of the capsid provides a
remarkable opportunity to differentially introduce multiple types
of functionality with control over both the spatial distribution and
the degree of multivalency. With discrete shape and size, and a
high degree of symmetry and polyvalency, plant viruses such as
CPMV, can provide versatility to a wide range of
bionanomaterials. Moreover, CPMV can remain intact at
temperatures up to 60 °C for an hour and at pH 4.0 or 9.0 at
room temperature for at least two days. With a variety of
appealing properties, CPMV has been extensively investigated
as a robust, monodisperse nano-building block has been
extensively investigated.
(Interior) Nucleic acid-free CCMV capsids can be obtained by
in vitro assembly after isolation from infected plant material,
however this method can be impeded by the fact that the
disassembled protein monomers are only soluble in the
presence of denaturants. To overcome this problem, Saunders
and coworkers managed to obtain empty CPMV capsids in
abundance by co-expressing in plants the coat protein precursor
along with the 24K proteinase. In this method, the proteinase
processes the coat protein precursor into large and small
proteins which can efficiently co-assemble into empty capsid.[27]
Such empty capsids can be used to encapsulate a wide range of
cargo, such as metals, fluorescent dyes or drugs.[28] For
example, without forming protein conjugates, Aljabali et al.
succefully loaded cobalt metal and iron oxide using sodium
borohydide as the reductant within the interior of empty CPMV
capsid obtained through Sauders’ methodology.[28a] Steinmetz
and coworkers systematically examined the capacity of empty
CPMV particles to encapsulate a wide variety of molecules.
Through targeting reactive cysteines, fluorophores, biotin affinity
tags, poly(ethylene glycol) (PEG) and various peptides were
selectively displayed on the interior surface.[29] The in vitro and
in vivo properties of many of these protein conjugates were
investigated in tissue culture and a preclinical mouse model as
the foundation for their developmemt in biomedical applications.
(Exterior) Through the genetic or synthetic introduction of
chemical functional groups onto the protein subunits, the
derivatization of the CPMV capsid exterior has been adopted as
a strategy to utilize the virus capsid as a nanoscale building
block as part of larger, designed architectures.[30] By controlling
the degree of multivalency and spatial distribution of conjugation
through carboxylate and ammonium groups in glutamate,
aspartate, and lysine side chains, well-established chemistry has
been used to attach various dyes,[30a] polymers,[31] and
antigens,[32] and to crosslink the exterior of CPMV for various
applications.[33]
One capsid exterior conjugation strategy, due to its ubiquity
and power, is worth specific mention, and it has been adopted
10.1002/asia.201600769
extensively in the CPMV system. The Huisgen reaction (also
known as “click chemistry”), is a highly specific orthogonal
method that allows single-site conjugation in large
biomacromolecules.[34] In this reaction, alkynes and azides react
in a [3+2] cycloaddition to form a triazol with little cross reactivity
to other functional groups found in proteins and nucleic acids.
This reaction has been developed to operate catalytically,[35] with
photoactivation[36] and with strained and electron-poor
alkynes.[37] To effectuate the Huisgen reaction on the CPMV
exterior, lysine and cysteine residues have been modified into
either alkynes or azides via conventional conjugation strategies,
and various challenging substrates including complex sugars,
peptides, PEG and the iron carrier protein transferrin have been
conjugated to the capsid surface.[31] Even hydrophobic fullerenes
have been attached to capsids enhancing the fullerene water
solubility and bioavailability in HeLa cells.[38] (Figure 5) Possible
developments of these strategies for anti-tumor therapeutic
devices will be discussed below.
Figure 5. The mutilvalent derivatization of CPMV and bacteriophage Qβ
capsids with multiple fullerenes. The well characterized fullerene, PCBA ([6,6]-
phenyl-C61-butyric acid) was activated as the N-hydroxysuccinimate ester
(NHS) and coupled to solvent-exposed Lys residues on exterior of a virus
capsid (in this figure depicted as an icosahedron viewed down a C2 symmetry
axis). Reprinted from reference [38]. Copyright 2009 American Chemical
Society.
CPMV capsids have also been used as polyvalent platforms
for the presentation of full-length proteins. One strategy for doing
this began by genetically converting four of the five outer surface
lysine residues in the monomer to arginine and introducing a
cysteine residue on the surface of CPMV capsid. The remaining
lysine or the introduced cysteine was conjugated to various
proteins, including T4 lysozyme, the LRR domain of internalin B
and the Intron 8 gene product of the HER2 tyrosine kinase
receptor, activated as NHS esters.[39] In every example, the
protein structure and biological activity was preserved, opening
the door for biomedical applications including vaccines and
antiviral therapeutics.
(Applications) CPMV has been extensively investigated as a
template for new materials, expecially in drug delivery and
medical imageing applications. For example, to overcome the
dose-limiting non-specific toxicities of Cr3+, which can selectively
inhibit the proliferation of high glucose-induced human aortic
smooth muscle cells (HASMC) in vitro, chromium chloride was
loaded into the CPMV interior cavity, and the resulting particles
significantly reduced glucose-induced HASMC proliferation and
presented anti-atherogenic effects under hyperglycemic
conditions.[40]
A number of approaches have been explored to direct CPMV
toward in vivo applications. To understand the fate of CPMV
after intravenous injection, Manchester and coworkers tracked
Chemistry - An Asian Journal
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the fluorescently labeled virus in a murine model,[41] and the
intact capsids were detected in a number of organs suggesting
that CPMV could serve as a multi-tissue delivery vehicle. In this
direction, some work has been undertaken to target CPMV
specifically to tumor cells, and both the Finn and Manchester
groups reported the chemical attachment of folic acid to the
capsid using the copper-catalyzed Huisgen reaction.[42] Not only
was folic acid-conjugated CPMV able to efficiently target the
cell-surface folate receptors on cancer cells, the study also
showed that higher density loading of targeting ligands on
CPMV might not be necessary for efficient delivery.
Imaging is another in vivo application toward which CPMV
capsids have been steered. For example, fluorescent dyes were
displayed in a multivalent fashion on the outer surface of the
CPMV capsid, and these conjugates were used to image
vasculature and blood flow to a depth of up to 500 um in living
mouse and chick embryos.[43] Moreover, CPMV capsids have
also been used as magnetic resonance imaging (MRI) contrast
agents. Both the Finn and Manchester groups modified azide-
displaying viruses with an alkyne-modified DOTA
(tetraazacyclododecane tetraacetic acid) ligand via the Huisgen
reaction.[44] After loading with gadolinium, the agent shows
increased T1 relaxivity relative to free Gd (DOTA) complexes in
solution. These reagents, which were determined to be nontoxic
in vivo, were subsequently used for the MRI imaging of live mice.
[45]
1.3 Bacteriophage MS2 and P22
In analogy to the work done with eukaryotic viruses, phage
(viruses that infect bacteria) capsids have also been employed
as the basis for nanomaterials.[46] Both bacteriophage MS2,
which encapsulates single stranded RNA, and P22, which is a
double stranded DNA phage, have icosahedral capsids that
have been used for a variety of applications. Bacteriophage MS2
which infects E. coli, self-assembles from 180 copies of identical
monomers into a capsid that is 27 nm in diameter with thirty-two,
2 nm wide pores that provide access to the capsid’s interior
space.[47] It exhibits high stability in a wide range of temperatures,
pHs, and organic solvents, suggesting that it could be easily
applied to materials and biomedical applications. [48] The
Salmonella bacteriophage P22 assembles from 420 copies of a
coat protein with the assistance from an approximately 300
amino acid residue scaffolding protein[49] which can be removed
after assembly by heating to 65 °C and subsequent treatment
with the protease thrombin.[50] Moreover, the fact that the
structure of the P22 capsid is reversibly responsive to
temperature,[51] suggests that it could act as a dynamic platform
for a number of applications.
(Interior) P22 possesses a relatively large inner cavity that can
be loaded with a wide variety of cargo directed to a diverse set
of applications.[52] For example, Douglas and co-workers
employed P22 for the size constrained synthesis of Fe 2O3
nanoparticles by fusing a polyanionic peptide to the scaffolding
protein to thereby conceptually mimic the charged interior cavity
of ferritin.[53] A similar strategy was also used to synthesize
photoactive, small diameter, TiO2 nanoparticles. By using fusions
10.1002/asia.201600769
displaying specific biomineralization peptides, both the anatase
and rutile mineral forms of TiO2 could be generated.[54]
Furthermore, by utilizing metal-ligand coordination chemistry,
various guest molecules such as diphan can be encapsulated to
the interior of P22 capsid.[55] In this approach, cysteine residues
were genetically introduced onto the capsid interior surface,
followed by chemical modification with metal ligand-presenting
5-iodoacetamido-1,10-phenanthroline, the coordination by
various metal complexes. By attaching small molecules to
exogenously introduced ligands for the metal complex, the
loading of cargo inside the P22 cage could be achieved. This
approach, performed under relatively mild conditions, could be
used as a versatile and general strategy for introducing a wide
variety of molecules into protein cage nanoparticles.
Taking advantage of the interaction between the coat and
scaffolding proteins, Douglas and coworkers packaged an
exogenous protein, alcohol dehydrogenase D, efficiently into the
P22 interior space.[56] (Figure 6) This research showed that
encapsulation of AdhD inside P22 affected the enzyme’s kinetic
parameters in comparison with the free enzyme, thereby
underlining the considerations involved in using modified protein
cages as catalytic reaction vessels. Similarly, cytochrome P450
(CYP) was encapsulated inside the capsid and the enzyme’s
stability towards protease degradation and acidic pH was
enhanced.[57] This work suggests the potential of these
biocatalytic virus-like particles as delivery vehicles for clinically
relvant enzymatic activity in biomedical applications.
Figure 6. Encapsulation of active alcohol dehydrogenase D (AdhD) inside
assembled P22 capsids. The co-assembly of bacteriophage coat protein (blue)
and AdhD (red) is enabled through the fusion of a capsid scaffold protein
(yellow) to the AdhD. Reprinted from reference [56]. Copyright 2012 American
Chemical Society.
(Exterior) Modification of the exterior surface of
bacteriophage particles makes it possible to control how these
protein assemblies interact with their environment. Francis and
coworkers used periodate chemistry to attach sixty copies of
phenylene diamine-substituted nucleic acid aptamers, selective
for cell-specific surface receptors, to the outside of genome-free
MS2 capsids that were modified with aniline functional
groups.[52b] The cell delivery of proof of principle fluorescent dye
was enhanced due to the multivalent effect. (Figure 7) Using a
similar concept, Ashley et al. reported the selective delivery of
chemically diverse therapeutic and imaging agents to human
hepatocellular carcinoma (HCC) by using MS2 modified with a
targeting peptide that binds to HCC cell surface receptors. [58]
Chemistry - An Asian Journal
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Decoration protein (Dec) has been used to demonstrate the
feasibility of presenting large proteins on the surface of P22
capsid in a modular, fully folded, and functional manner.[59] The
successful assembly of the fusion resulted in a displayed protein
that maintained its function and a P22 capsid that was minimally
altered, highlighting the broad potential for similar presentation
strategies.
Figure 7. Covalent loading of functional cargo at the interior surface of the
MS2 bacteriophage capsid and exterior surface modification with cell-specific
nucleic acid aptamers (depicted as red sqiggles). Reprinted from reference
[52b]. Copyright 2009 American Chemical Society.
(Applications) Bacteriophage P22 capsids can also serve as
nanocontainers for therapeutic applications. Kelly et al.
developed a peptide drug delivery system using P22
bacteriophage which is modified to serve as a tunable
nanocontainer for the packaging and applied ring opening
metathesis polymerization (ROMP) reaction to trigger
disassembly of the capsid and release of bioactive peptides
under physiological conditions.[60]
Gd3+-based MR contrast agents have drawn strong interest in
recent years. Site selective initiation of atom transfer radical
polymerization (ATRP) reactions has been recently established
for high-density fabrication and delivery of Gd. The Douglas
research group reported the use of ATRP to make addressable
polymer networks within the interior cavity of P22 capsid. A 2-
aminoethyl methacrylate (AEMA) network was used as a
scaffold for the attachment of Gd-based MRI contrast agents,
through reaction with the primary amine groups of the
poly(AEMA). Substantial increase in the degree of labeling per
capsid was achieved compared to those of other virus-like
particles.[61] Similarly, MS2 has also been applied as a scaffold
for the generation of MR contrast agents.[62] In this case, the
protein cage was conjugated with Gd chelates on either its
internal or external surfaces. Datta et al. labeled the MS2 with
TREN-bis(HOPO)-terephthalamide ligand, either at the interior
of the capsid through tyrosine residues or on the exterior
through lysine residues, by functionalization with an aldehyde,
followed by conjugation with the ligand via oxime condensation.
The externally modified MS2 was measured to have an iron
relaxivity of 307 mM-1s-1, while that of the internally modified
MS2 was 416 mM-1s-1, a 5-fold increase compared to small
molecule Gd chelates. This result suggests that there are
significant advantages to using the internal surface capsids for
contrast agent attachment, which fortuitously leaves the exterior
surface available for tissue-targeting group modifications.
Recently, MS2 cages encapsulating a Au nanoparticle (AuNP)
inside and bearing fluorescent dyes on the outer surface have
been fabricated for energy transfer studies. The dye-AuNP
distance could be tuned with DNA linkers of different lengths,
and distance-dependent fluorescence intensity enhancement
10.1002/asia.201600769
was observed. [63] This work demonstrates the utility of viral
capsids as essential components of complex systems that have
the potential to be used to understand fundamental physics.
1.4 Tobacco Mosaic Virus
Tobacco mosaic virus (TMV), also known as tobamovirus,
was the first virus particle to be isolated and has been studied
intensively as a model system for diverse investigations in the
materials and analytical sciences.[64] The TMV capsid is a 300
nm long rod with an 18 nm outer diameter. Its rod shape results
from a regular helical array of 2130 identical protein subunits
which encloses the single stranded RNA genome. [65] Due to its
unique anisotropic shape and remarkable stability, TMV has
been used as the template of choice for protein-based rod
shaped materials synthesis.
The capsid of TMV undergoes a reversible state change from
micron-length rods at pH 5.5 to double disc morphologies in
phosphate buffer at pH 7.0.[66] Through chemical and genetic
modification of the inner and outer surfaces, the assembled
capsids have been used as templates to grow metal and metal
oxide nanowires, liquid crystals, and coated conductive
nanowires.
(Interior) A variety of metals including Cu, Pt, Ag, Au, Ni, and
Co have been deposited in the narrow cavity of the rod-shaped
TMV particle.[67] In addition, metal alloy nanowires with lengths
up to 100 nm and 4 nm diameter composed of CoPt, CoPt 3 and
FePt3 have been synthesized inside TMV capsids. [68] However,
metal deposition on the inner surface of TMV can be difficult due
to weak initiation. To enhance nucleation, Kobayashi et al.
genetically modified the capsid to introduce the positively
charged amino acid lysine on the inner surface and were able to
generate 3 nm aligned Pt and Co nanoparticles inside the
virus.[69] Interestingly, control experiments with unmodified TMV
resulted in internalized nanowires instead of discrete
nanoparticles, emphasizing the importance of appropriately
presented interior surface charge.
In a similar vein, Wang et al. achieved site-specific nucleation
of gold nanoparticles at sulfhydryl groups arrayed on the inner
surface of a TMV nanotube generated by point mutation of a key
threonine to cysteine.[70] (Figure 8) The strong interaction
between the sulfhydryl group and gold secures the site-selective
chemisorption, nucleation, and growth of Au seeds within the
genetically modified TMV nanotubes.
Figure 8. Schematic illustration of the site-specific biomineralization of gold
nanostructures inside TMV protein nanotubes. The mutated cysteines arrayed
on the interior surface of T103C-TMV can specifically bind gold ions which
initiate growth of Au nanoparticle chains or Au nanorods in the confined cavity
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of the nanotubes. Reprinted from reference [70b]. Copyright 2009 American
Chemical Society.
(Exterior) The exterior surface of TMV has also been exploited
for the generation of nanomaterials. The structure of the coat
protein monomer reveals three potential regions for chemical
modification on the exterior surface of the assembled capsid, the
N-terminus, the C-terminus and the 63–66 loop region.[71] Harris
and coworkers inserted two cysteine residues at the TMV coat
protein N-terminus and the resulting assembled capsid
facilitated the ready deposition of Au, Ag, Pd, and Au-Pd
clusters onto the cysteine-modified template with enhanced
density and stability in comparison to the wild-type TMV
template.[72] Similarly, the exterior exposed C-terminus has also
been utilized, by introducing His-tag metal-binding peptide, to
create protein nanorods that could bind gold nanoparticles
uniformly.[73] Demir et al. introduced a single lysine by point
mutation at the C-terminus, which can be selectively coupled to
small moieties such as FITC and biotin using NHS chemistry for
the production of a variety of chemospecific nano-tubular
materials.[74]
Chemical modification can enhance the performance of the
exterior of TMV to act as a template. Culver and coworkers
investigated the coating of TMV with silica to both enhance
template stability and increase its affinity for metal ions, and
various metal nanoparticles including Au, Ag, Pt and Pd were
successfully deposited on the silica coated exterior surface of
the TMV capsid.[75]
Other nanomaterials including silicon nanotubes, TiO2 and
Al2O3 nanotubes, and Cu nanorods and nanowires have been
generated at different lengths ranging from 300 nm to microns
on the outer surface of wild type or modified TMV. [76] Such
biological-based materials may find applications in
nanoelectronics, sensing, or cancer therapy.
As a large array of unnatural amino acids can be incorporated,
site specifically, into proteins by using a nonsense codon and
orthogonal tRNA-synthetase pairs, this methodology should
prove powerful when applied to TMV. Wu et al. developed an
effective method for site-specific and high yield modification of
TMV coat protein using amber codon suppression and Huisgen
reaction.[77] Biotin molecules were successfully conjugated on
the coat protein and the resulting TMV nanoparticles self-
assembled into large disk-like and normal rod-like structures
under different conditions.
(Applications) Through precise genetic and chemical
modification at defined positions on its capsid, TMV can be
directed toward multiple applications, most notably those
involving cell-targeted imaging, and the generation of reagents
for influencing immunology.[78] For example, Yin et al. produced
TMV capsids conjugated to tumor-associated carbohydrate
antigens (TACAs) through Huisgen chemistry. [79] These particles
were able to elicit an immune response and the resulting high
titer IgGs were strongly reactive to the antigen when presented
on cancer cells. Interestingly, it was found that the position and
presentation of the immobilized antigen on the capsid had great
influence on the strength of the resulting immune responses.
10.1002/asia.201600769
In the realm of analytical chemistry, Wang and coworkers
used TMV as a template to develop a thin film sensor for the
detection of volatile organic compounds. [80] Oligoaniline, a small
molecule that is electroactive, has found application in
electronics and sensing, and in this example, oligoaniline motifs
were chemically conjugated to the outer surface of TMV capsids.
Due to the high-density distribution of oligoaniline on the TMV
surface and good processing ability of modified TMV, the virus
was readily fabricated into a thin film by directly drop coating
onto a glass substrate. Upon integration of the glass substrate
into a prototypical device, the virus-based thin film exhibited
good sensitivity and selectivity toward ethanol and methanol
vapor.
2. Non-viral protein cages
2.1 Ferritins
Ferritins, one of the most widely studied and utilized family of
cage proteins play an in vivo role in iron homeostasis and ferric
oxide storage[81] and have highly conserved biochemical and
structural properties. Structurally, mini-ferritins are formed from
twelve identical monomer subunits which assemble into ~ 9 nm
outer diameter tetrahedrally symmetric cages with 5 nm inner
diameter cavities that can store around 500 Fe atoms.
Conversely, maxi-ferritins are made up of twenty-four identical or
similar monomers that form an octahedrally symmetric cage with
~ 12 and 8 nm outer and inner diameters, respectively, and the
maxi-ferritin cavity can typically accommodate up to ~ 4500 Fe
atoms.
(Interior) Due to their small cavity and distinct symmetries,
ferritins have been utilized as the basis of a wide variety of
inorganic materials with unique electronic, magnetic and optical
properties. In addition, these protein cages have been exploited
as size-constrained reaction vessels to synthesize various
nanoparticles including metals, oxides, hydroxides, carbonates
and semi-conductors[12, 82]. Therefore, multiple methods have
been developed to mineralize nanoparticles using ferritins [83].
Many of these strategies involved the engineering of the interior
of the ferritin cavity with functionality that facilitates nanoparticle
formation. For example, Kramer et al. fused a peptide sequence
to the internally projecting C-terminus of human L-chain ferritin.
This peptide has been shown to bind specifically to silver
nanoparticles.[84] Thus, the assembled protein cage was used for
the generation of silver nanocrystals with tight
polydispersities.[85] Embracing an alternative approach, Fan et al.
developed a two-step process to mineralize gold nanoparticles
inside native horse spleen apoferritin. [86] This strategy bypassed
the necessity of engineering additional functional groups onto
the ferritin interior surface either through chemical or molecular
biology means and thus should be prove to be a strategy
applicable to protein cages in general. [87]
(Exterior) The exterior surface of ferritins can be engineered
to project functionality by modifying positions near the N-
terminus.[88] For example, Wang and coworkers
chemoselectively modified horse spleen apoferritin via acylation
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of N-terminal lysine residues followed by a series of other
conjugation reactions, including the Huisgen reaction. The
resulting conjugate was used to template ATRP to generate
ferritin shells grafted to oligo ethylene glycol methacrylate-
polymers, a material that is soluble in dichloromethane. [89]
The highly symmetric nature of protein cages is
advantageous for the formation of isotropic hierarchal structures,
however, breaking this functional symmetry is essential for the
topological control required for applications relying on
anisotropic assembly or polarized multicomponent presentation.
Using a masking/unmasking strategy on a solid support
presenting two differentially activated thiol groups, Kang et al.
generated Janus-like miniferritins dual functionalized with both a
fluorescent and an affinity label.[90] This process was initiated
through the genetic modification of exterior surface residues of
Listeria innocua DNA binding protein from starved cells (LiDPS).
Functional groups were differentiated spatially on the protein
cage exterior, and hierarchical toposelective assembly was
achieved through a layer-by-layer strategy.
(Applications) Because of ferritin’s native iron mineralization
activity, these proteins have drawn considerable interest in
magnetic imaging applications. For example, human ferritins
have been employed as diagnostic MRI agents to detect
vascular macrophages and Uchida et al. engineered dual
functional human H-chain with genetically modified cell-specific
targeting peptides on the exterior surface and a synthetic iron
oxide (magnetite) nanoparticle within the interior cavity. [91]
Ferritin has also been well utilized for drug delivery. For
example, the anticancer drug cisplatin was encapsulated into the
horse spleen apoferritin cage and the cellular uptake of this
species was found to be increased by four-fold over that of free
cisplatin presumably a result of enhanced endocytosis.[92] In
addition, the anticancer drug doxorubicin and a zinc-
phthalocyanine-based photosensitizer have both been delivered
from the interior of apoferritin cages with high success.[93] The
Yan group demonstrated that native H-ferritin nanocages could
tumor-specifically deliver high doses of doxorubicin (DOX)
without the conjugation of a targeting ligand. Presumably, the
enhanced delivery was facilitated by the overexpression of
transferrin receptor 1 in tumor cells.
Because of their unique small size compared to other protein
cages, the metallic nanoparticles generated inside ferritins are
expected to have a higher surface area to mass ratio and thus
there is much interest in applying them to catalysis. For example
Pd nanoclusters synthesized inside an apoferritin cage were
used as hydrogenation catalysts.[94] Building upon this, Au/Pd
core-shell and alloy nanoparticles were also prepared inside
ferritin resulting in material that delivered a two- to four-fold
increase in catalytic activity.[95]
Metal nanoparticles prepared in ferritin cages can mimic the
activities of enzymes, such as catalases and peroxidases, that
control in vivo oxidative stress.[96] Exhibiting higher peroxidase
activity than monometallic particles, bimetallic Fe-Pt
nanoparticles have been constructed, and the improved catalytic
activity may result from a synergic effect between Fe and Pt. [97]
Capitalizing on this concept, Yan and coworkers synthesized
iron oxide nanoparticles inside ferritin and demonstrated that
10.1002/asia.201600769
they could be used for specific imaging of cancer cells. The
ferritin itself provides selective targeting for cancer cells that
overexpress transferrin receptors and the magnetoferritin
nanoparticles catalyze the oxidation of peroxidase substrates in
the presence of H2O2 to produce a colored response that is used
to visualize the cancer tissue.[98]
2.2 Small heat shock proteins
Small heat shock protein (sHsp), originally isolated from the
hyperthermophilic archeaon M. jannaschii, assembles into a
protein cage with symmetry similar to that of maxiferritin. The
cage of sHsp is made up of 24 monomer subunits and has a 12
nm exterior diameter and 6.5 nm interior cavity[10c]. However,
diverging from the structure of maxiferritin, sHsp has large pores
(3 nm diameter), which facilitate exchange between inside and
outside of the cage.[99]
Similar to other nanocage proteins, sHsp has been used as a
delivery vehicle for imaging and therapeutic agents. [100] For
example, Uchida et al. engineered LyP-1, a nine-residue peptide
which targets tumor-associated macrophages, onto the exterior
surface of sHsp, and this cage was used to deliver the imaging
agent Cy5.5 encapsulated inside the central cavity. The resulting
conjugate allowed visualization of macrophage-rich murine
carotid lesions by fluorescence imaging. [101] In a related example,
an sHsp cage, genetically modified through the C-terminus to
display externally the neuropilin 1-
 binding peptide (iRGD),
which is known to interact with integrin and neuropilin-1
receptors on pancreatic cancer cells, and loaded internally with
the cytotoxic drug OSU03012, was shown to be selectively
cytoxic to pancreatic cancer cells.[102]
The protein cage sHsp has also been directed toward hybrid
materials. For example, to generate artificial hydrogenase
activity, Pt clusters were introduced into the interior cavity to act
as sites for the reduction of H+ to H2.[103] In another example
Douglas, Young, and coworkers synthesized a cross-linked
branched dendritic polymer using the Huisgen reaction and
initiation by cysteine residues within the interior cavity of
sHsp.[104] Protein cages encapsulating the branched polymer
maintain their native shape and size distribution but are
remarkably more stable. (Figure 9)
Figure 9. Fabrication of a dendritic polymer inside small heat shock protein
(sHsp). (A) A cartoon of a protein cage filled with a branched polymer that can
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be used to load drugs, imaging agents, or other functional components. (B) A
cutaway view of the HspG41C interior cavity highlighting exposed cysteine
residues (red). (C) Scheme for sequential synthesis of the dendritic polymer.
Generation numbers are indicated at the bottom. Reprinted from reference
[104]. Copyright 2009 American Chemical Society.
2.3 Chaperonins
Chaperonins are a class of hollow, cylindrical protein
assemblies that catalyze the native folding of other proteins.
Many chaperonins are double-ring structures with 14-18
subunits, depending on the organism of origin. Based on
sequence comparisons, chaperonins can be organized into two
groups with canonical representatives. Group I assembled from
14 subunits has structural homology to Escherichia coli GroEL,
whereas group II has 16 or 18 subunits and the widely
considered examples are thermosomes and rosettasomes,
respectively, both of archaeal origin.[105] Composed of two
stacked supramolecular protein rings, chaperonin proteins open
and close through the binding and hydrolysis of ATP. As a
protein cage that operates uniquely as a conformational switch,
the ATP-driven chaperonins have been utilized to conditionally
encapsulate and release drugs and semiconducting
nanoparticles.[106]
There has been much recent work to manipulate the
conditionality of chaperonins. For example, Kortemme and
colleagues demonstrated that group II chaperonins could be
designed to be light activated through the placement of
photoswitchable azobenzene-based functionality at a key hinge
region. Photoisomerization of the azobenzene drives large-scale
conformational changes of the protein assembly. [107] Recently,
derivatives of azobenzene that use different colors of light to
achieve enhanced conversion have been developed, and these,
could provide improved photocontrol in similar chaperonin-based
systems.[108] In principle, the coupling of photoisomerization to
other cooperatively self-assembling proteins could lead to a wide
variety of novel photocontrolled devices.[109]
The chaperonin GroEL has been recently developed into
photoconfigurable nanotubes. First, six cysteine residues were
genetically inserted onto the rim of the barrel-shaped protein,
and these were chemically conjugated with the protochromic
ionic molecule merocyanin which can bind Mg2+ and
photoisomerize into a non-polar spiropyran that does not bind
divalent cations. Thus, in the presence of Mg2+, the GroEL
assembles into nanotubes, however, upon photoexcitation,
these tubes disassemble.[110] Placing superparamagnetic iron
oxide nanoparticles (SNPs) inside the cavity of this engineered
chaperonin provides an additional level of structural control.
When the SNP-containing chaperonin nanotubes are subjected
to a 0.5 T magnetic field, they assemble laterally into thick
bundles. These bundles disperse into nanotubes upon removal
of the field.[111] (Figure 10)
10.1002/asia.201600769
Figure 10. Light- and magnetic field-switchable 1-D arrays of
superparamagnetic nanoparticles encased in chaperonin protein cages.
Superparamagnetic nanoparticles (SNPs) were encapsulated by GroEL
chaperonins which are stitched together with Mg2+ cations bound through
merocynanine (MC) which is conjugated to the rim of the GroEL. Light
photoisomerizes the MC so it changes conformation, releasing the cations and
dispersing the arrays. The arrays can also be reversibly formed into bundles
through the application of a magnetic field. Reprinted from reference [111].
Copyright 2015 American Chemical Society.
3 Two- and three-dimentional supra-
assemblies of protein cages
It is widely recognized that the next generation of materials
engineering will focus on the simultaneous manipulation of
structure across multiple length scales with a high degree of
specificity. For example, the two and three-dimensional
assembly of nanoparticles has drawn significant interest
because these higher order structures could exhibit collective
behaviors and properties beyond those of the individual
particles.[112] With regard to the use of biomolecules, much of the
early focus in building up two- and three-dimensional structures
has relied on DNA and peptides as building blocks and affinity
elements.[113] However, folded proteins have been under-
exploited.[114]
Protein cages, as discussed in the earlier sections of this
review, have been employed in the generation of various
nanomaterials as size constrained reaction vessels and as
platforms for functional design. However, the fabrication of
higher-order hierarchical structures from protein cages has
become an active research area only recently. Protein cages
possess many key features that make them desirable building
blocks for the engineering of hierarchical suprastructure. Among
these are: 1) Their size and structure are homogeneous; 2)
Their central cavities provide the ability to transport and
sequester various cargo, and the variety in volume and shape of
this cavity across the family add to their attractiveness.
Moreover the cavities can act as excellent templates for the in
situ synthesis of nanoparticles or quantum dots; 3) A variety of
functionality can be incorporated onto the various surfaces of the
individual cages, either through bioconjugate chemistry or
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molecular biology, thereby enhancing their properties. Moreover,
protein cage-guided formation of nanoparticle supralattices
provides a biocompatible platform that will facilitate the
development of advanced delivery and sensing applications in
biological systems.[115]
To construct two- and three-dimensional architectures from
protein cage building blocks, two general strategies have been
developed. The first utilizes direct physical interaction, such as
external surface electrostatics, between the protein cages, and
the second employs either covalent or non-covalent linker
functionality.[112, 116]
The Kostianien group has extensively utilized a supra-
assembly strategy based on surface electrostatics. For example,
lattices were assembled between positively charged gold
nanoparticles and protein cages that displayed surface negative
charge. It is possible to generate face centered cubic lattices if
the protein cage is CCMV with encapsulated RNA or simple
cubic lattices if the cage is ferritin filled with magnetic iron oxide
nanoparticles. It is thought that these materials will have
applications in delivery or imaging, respectively.[116a] (Figure 11)
In another example, the same lab explored lattices formed
between apoferritin protein cages displaying negative surface
charges and poly (amidoamine) dendrimers (PAMAM) which
present positively charged surfaces. It was found that the
morphology of the resulting lattices could be rationally tuned by
either altering the concentration of salt which shields the
Coulomibic attraction between the particles or the effective size
of the dendrimer with respect to that of the ferritin. [116b] In a third
example, body centered cubic lattices were assembled between
negative-presenting CCMV cages and positively charged avidin
proteins. Because CCMV presents its acidic residues in
icosahedrally symmetric patches and avidin its basic patches
with tetrahedral symmetry, it was thought that the lattice
morphology is controlled by the geometric display of the charge,
thus adding an additional layer of control. Moreover, the
resulting material could be functionalized with compounds
conjugated with biotin, a specific and high affinity ligand for
avidin.[117] (Figure 12)
Figure 11. Cryo-TEM images of superlattices of CCMV with encapsulated
gold nanoparticles viewed along [100], [110] and [111] zone axes (scale bar
50 nm, inset is image Fourier transform). Reprinted from reference [116a].
Copyright 2012 American Chemical Society.
Virus capsid-based, three-dimensional crystals provide a
porous, highly organized scaffold around which an inorganic
matrix can be assembled. Work in this direction has been
performed in the solid phase to generate hybrid materials on a
larger scale. For example, it is known that the capsid of CPMV
crystallizes into a body centered cubic morphology with large
10.1002/asia.201600769
nanoscopic channels between the virus particles. The catalytic
precipitation of palladium can be achieved within these channels
by soaking CPMV crystals in buffers containing Pd (II) and Pt (II)
and relying on specific binding of the cations to basic residues
on the virus exterior surface. [118]
While a strategy to generate three-dimensional supra-
assembled lattices exploiting protein cage surface charge
involves relatively straightforward design principles and provides
a surprising degree of morphological control, a linker-focused
strategy contributes opportunities to optimize and tune additional
parameters, such as interaction strength, distance between
cages, specificity, and conformational flexibility.
Figure 12. Pre-and post-functionalization approaches of CCMV-avidin
crystals. Electrostatically assembled CCMV (acidic)/avidin (basic) co-crystals
can be functionalized selectively with biotin-tagged functional units. Method1:
Pre-functionalization of avidin with the biotinylated functional unit followed by
addition of the virus particles. Method 2: Assembly of the crystals followed by
post-functionalization with the biotinylated agent. Reprinted from reference
[117]. Copyright 2014 American Chemical Society.
One linker-based strategy involves the “capsid decoration
protein” (Dec) from phage L which is know to specifically bind to
sites of three-fold symmetry on the P22 capsid. Using this
knowledge, Douglas and coworkers generated linkers of
dimerized Dec or of ferritin displaying multiple copies of Dec on
its external surface. These linkers were not only used to stich
together lattices of the P22 protein cage, but, along with wild-
type Dec, were employed as part of a strategy to build up
hierarchical protein cage materials layer-by-layer.[112]
Nucleic acids can introduce the robust logic of Watson-Crick
interactions to highly specific linkers for the supra-assembly of
protein cages. For example CPMV cages were first chemically
conjugated to DNA through a lysine or cysteine. These
conjugates could assemble with cages displaying complimentary
DNA strands into supra-assembles whose stability was
dependent on the degree of strand overlap and that could be
broken up through the addition of unconjugated, competing
DNA.[119] Using a similar strategy, Fin, Park, and coworkers
created a non-compact, diamond-type lattice made up of
alternating DNA-presenting gold nanoparticles and
complementary DNA-functionalized Qβ phage capsids.
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Interestingly, the size of the nanoparticles could be varied
without changing the lattice morphology if the DNA linker length
was changed accordingly.[120] The use of nucleic acids as linkers
to build up hierarchical structures based on proteins cages has
the additional advantage that it can be coupled to DNA origami
structures which can then be employed to scaffold the cages
with precise nanoscale positioning.[121] For example, origami tiles
presenting oligonucleotide strands have been used to arrange
MS2 phage capsids chemically conjugated with complimentary
single stranded DNA. These tiles can also be stapled together
into arrays with nanoscale structural control.[122]
Another linker strategy to generate protein cage arrays has
relied on metal coordination complexes. For example a
thermophillic mini-ferritin that was decorated with phenathroline
ligands could form amorphous assemblies in the presence of
iron (II). [123]
Branched dendric polymers[124] can act as multivalent linkers
to stitch together assembles of protein cages. For example
polycationic dendrimers were used to “glue” together CCMV
capsids presenting that present anionic surface charges.
Because photolytically degradable dendrons were employed, the
virus particles could be released from the arrays by treatment
with light. A very similar strategy generated light dependent, face
centered cubic lattices of ferritin. Interestingly if the ferritin
cages were loaded with iron oxide magnetic nanoparticles, the
magnetic properties of the material could be influenced by the
supra-assembly state of the ferritin cages.[125] The ability to tune
magnetic properties of nanoparticles by assembling them into
ordered superstructure and to reverse the assembly process by
external stimuli will surely facilitate the development of a variety
of novel practical applications.
Conclusion
This review concisely summarized the viral and non-viral
protein cages commonly employed as templates for the
generation of nanomaterials and described how these materials
have been directed toward multiple applications. The
exploitation of new bio-based templates and novel pathways for
nanomaterial synthesis has drawn tremendous attention over
the past decades. The use of protein cage architectures of viral
and non-viral origin provides a number of unique advantages.
They are biological in origin and thus amenable to genetic
manipulation and large-scale production. Furthermore, they can
be incorporated into nanostructured architectures with functions
not available by conventional material synthesis. Thus bio-
conjugated protein cage architectures can facilitate various
biomedical applications such as imaging and the delivery of bio-
active molecules.
Acknowledgements
Financial support from National Natural Science Fundation of
China (No. 31200564), Natural Science Foundation of Jiangsu
Higher Education of China (16KJB180009), Top-notch Academic
10.1002/asia.201600769
Programs Project of Jiangsu Higher Education Institutions
(TAPP), a Marie Curie CIG (PCIG13-GA-2013-618538), and
BPO’s personal salary.
Keywords: protein cages • viruses • protein design •
nanoparticles • nanomaterials
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Chemistry - An Asian Journal 10.1002/asia.201600769

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Entry for the Table of Contents

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Protein cages, assembling from Yu Zhang*, Maziar S. Ardejani and
multiple monomers into architechtures Brendan P. Orner*
with hollow interiors, can instill a
number of unique advantages to Page No. – Page No.
nanomaterials. In this review, the most
Design and Applications of Protein
commonly used viral and non-viral
Cage-Based Nanomaterials
protein cages that exhibit a wide
diversity of size, functionality, and
chemical and thermal properties are
described. Moreover, how they have
been expoited for nanomaterial and
nanotechnology applications is
summarized.
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