Summary

Introduction:
From a long time scientists have been deliberately trying to mimic nature to reach the ultimate
innovation. Being inspired by hierarchical structures in nature with characteristic colors and exquisite
optical properties, such as blue-faced monkeys, bird skins and beetle exocuticles, the current study of
Chung et al investigated the optical and photonic properties of the self-templated supramolecular
liquid crystal structures of M13 bacteriophage. The self-templated structures exhibited very different
polarizing and other optical properties. Depending on the hierarchical organization of the filaments,
some displayed iridescence, and others were colored, which could be attributed to the same type of
light scattering that results in the bright skin coloration, a nice example of mimicking the nature.
Moreover, by genetically engineering the virus particles to express peptides that mediate the growth
of soft and hard tissues, the viral films could be used as scaffolds for tissue growth, suggesting a very
real potential for applications in regenerative biomedicine.
Materials and Methods:
The Bacteriophage M13 was used as a building block because of its helical, homochiral and
nanofibrous shape akin to collagen. It also has monodispersity and the ability to display multiple
functional motifs. Glass and gold-coated silicon were used as substrate.
Figure 1: a) nematic phase with orientational order, b) cholesteric phase composed of twisted
nematic layers in which the director axis rotates through the layer normal to form periodic pitch, P, c)
smectic phase with both positional and orientational orders. a, b and c structures are transformed to
d) nematic orthogonal twist, e) cholesteric helical ribbon, and f) smectic helicoidal nanofilament,
respectively.
The approach to generate hierarchically structured virus involved allowing the virus to self-
assemble into a thin film on a substrate. The process essentially involved dipping the substrate into a
bath of virus in saline and then pulling it out again at precisely defined speeds. Varying the speed at
which the support was pulled out and changing the virus concentration allowed to control the liquids
viscosity, surface tension, and rate of evaporation during the film growth process. The interplay
between all these parameters determined how the virus particles assembled. At low concentrations
the phage particles formed nematically ordered bundles of fibers. Periodic changes in particle flux to
the meniscus due to stick-slip motion meant that these were arranged on the glass support as
regularly spaced, alternating ridge (stick region) and groove (slip region) stripes lying perpendicular to
the pulling direction. As the virus solution concentration and pulling speed were increased, the self-
assembly became more complex, resulting in the bundles within each ridge first taking on a helical
structure and then as the concentration was increased further, a flat, helical ribbon-like structure. At
the highest virus concentration the deposited films formed periodic structures with even more levels
of hierarchical order and formed the ramen-noodle-like smectic helicoidal nanofilaments (SHNs)
(Fig. 1).
Various parameters that affect the kinetics and thermodynamics of the assembly are phage
concentration, pulling speed, ionic concentration, phage surface chemistry, and substrate surface
properties which can be used to tune the structures. For example by varying the phage concentration
(C
p
) and pulling speed (s
p
) nematic orthogonal twists (C
p
=0.21.5mgml
1
), cholesteric helical ribbons
(C
p
=0.250.5mgml
1
, s
p
=1020 m min
1
) and smectic helicoidal nanofilaments (C
p
=46mgml
1
)
were produced. Increasing phage concentration increased the width, height and interspacing of the
patterns, whereas increasing the pulling speed decreased the height and interspacing. The meniscus
forces, the friction forces and the meniscus shapes were affected by variation in each parameter.
These forces in turn induced the formation of a variety of chiral self-templated structures. Regardless
of the inherent homochirality of M13 phage, the higher-order handedness of the structures can be
controlled by external perturbations.
Results and Discussions:
As stated before, of particular interest was the finding that the self-templated structures
exhibited very different polarizing and other optical properties, especially the highly complex ramen-
noodle-like structures that could bend light in a manner not observed before in nature or in other
engineered materials. At faster pulling speeds, narrower phage fibers were formed with a shorter-
wavelength color. Color pattern changes were observed in response to different incident light angles
when illuminated unidirectionally with a white LED (Fig. 2a). The observed colors changed very little
when viewed under omni-directional illumination regardless of viewing direction (Fig. 2b). Similar
structural color generation often occurs in mammalian and avian skins.
Also by alternating the speed of film growth between slow and fast optical filters were made
composed of alternating bands of SHN and nematically aligned structures. On gold, reflection of
incident light was selectively on or off depending on the direction of illumination (orange arrows).On
glass, the films selectively filtered the three letters CAL underneath, depending on the direction of
illumination (Fig. 2c).


Figure 2: Optical properties of the self-templated supramolecular structures
Because hierarchically organized extracellular matrices act as templates that provide the
physical and biochemical cues for controlling the formation of soft and hard tissues, the researchers
looked to see whether their virus-based materials could have similar properties. The phage particles
were engineered to display peptides that promote cell adhesion and bone mineral formation and then
a b
c
generated fabricated films having alternating SHN and nematic hierarchical structures as tissue-
guiding matrices.


Figure 3: Growth of biomimetic soft and hard tissue on the self-templated structures
It was found that cells cultured on the films recognized the underlying microstructures and
their bodies and internal actin filament networks parallel to the long axes of the phage fibers on
nematic regions but perpendicular to them on SHN regions (Fig. 3a, b). When SHN films were then
treated with a solution containing calcium and phosphate ions, they acted as templates for the
biomineralization of calcium phosphate, resulting in the production of tooth-enamel-like organic-
inorganic composites (Fig. 3c). As can be seen from the figure, Youngs modulus of the film is
increased after biomineralization process. Thus the aspects of tissue formation were recapitulated by
using self-templated materials composed of genetically engineered phages displaying specific
functional motifs.
Conclusions:
This work has demonstrated that self-templating assembly strategies which occur in nature
can be mimicked using a synthetic system to control the growth of phage-based films. It shows the
development of a physical process that allows a harmless virus to mimic the way single
macromolecules such as collagen self-assemble into diverse and complex functional chiral
structures. It also provides insight into how the kinetics during the conversion of helical
macromolecules into higher-ordered structures in nature is controlled by environmental factors and
how these same factors can be used to control the assembly artificially. Development of advanced
structural, optical, and biomedical materials is possible by further improvement and tuning of
synthetic self-templating systems.







a
b
c
Motivation
In various fields of science and technology such as electro-optics, photonics, sensors, artificial
muscles and even in laboratory modeling of the early universe [1], liquid crystals are widely used. But
even these applications are nowhere near as complex and important as the use of liquid crystals in
biological systems, how they underpin the organization of both soft and hard tissues or in the special
case where they show distinct color in certain tissue even in the absence of color pigments. Needless
to say, natures self-templated supramolecular material assemblies surpass the functional and
structural complexity achievable by current top-down and bottom-up fabrication methods. Chung et al
[2] in their study of Biomimetic self-templating supramolecular structures borrow an ingenious trick
from biology to produce highly structured biomimetic materials from liquid crystals which provides
insight into the chemistry of nature at nanometer scale. Their discovery of how to fine-tune the
assembly of such complex structures from identical building blocks could help to accelerate the large-
scale fabrication of new functional materials.
This paper eventually deals with various branches of chemistry at nanometer scale such as
supramolecular chemistry, self assembly, liquid crystals, biomineralization etc. The simplest liquid
crystals are typically rod-like molecules that spontaneously orient along a common direction while
flowing under shear or gravity

[1]. The chirality (handedness) of such molecules and their various
degrees of positional ordering introduce a plethora of different phases that have unique combinations
of order and fluidity. Biological tissues often resemble crystalline structures or liquid-crystal flow
patterns frozen in time [3], but the physical origins of their organization are not well understood. The
synthetic templating of biomimetic structures by this interesting study [2] provides valuable insight into
these processes.

Figure 4: Self-assembly of liquid-crystal structures
The intriguing thing about this study is how simple their approach is. Chung et al. have grown
films in a single-step process in which substrates are dipped in and out of suspensions of rod-like
particles (phage viruses) in water. In Fig. 4a it can be seen that when a glass substrate is withdrawn
from a suspension, rapid water evaporation (red arrows) occurs at the contact line formed at the
interface of the suspension, the substrate and the surrounding air. This evaporation, coupled with the
need for the contact angle between the surface of the water and the substrate to be maintained as
the substrate is withdrawn, causes capillary flow (deep blue arrows) that replenishes water at the
contact line and carries particles to the meniscus. Particles concentrated at the meniscus
spontaneously form a nematic liquid crystal in which the particles align with the contact line (Fig. 4b).
Subsequently in fig. 4c, interactions between highly concentrated particles promote their assembly
into twisted, helical structures. The wedge-shaped geometry of the suspension just below the contact
line causes the particles to form features like arches (red lines). Finally in Fig. 4d, the discontinuous
receding of the contact line as the substrate is withdrawn (green arrow), and rapid water evaporation
at the line, promote the mechanical imprinting of self-organized liquid-crystal patterns into the film that
forms as particles are deposited on the substrate.
It is fascinating how the authors [2] show that this process can yield biomimetic films that have
pigment-free structural colors. These structured films are of interest for use as reflectors and filters,
for providing periodic patterns for applications in unconventional diffraction gratings, and for
controlling mechanical properties of artificial materials that resemble those of tissues found in nature.
Although in conventional liquid crystals, such as those used in mobile-phone displays, these
structures would possibly be unstable in the absence of external fields, relaxing to the ground state of
the corresponding phases. For this reason further development is essentially needed.
This type of biomimetic self-templating could extend the scope of application of many powerful
fabrication techniques based on liquid-crystal self-organization and dip-coating [4-6]. As liquid-crystal
behavior is not restricted to rod-shaped particles, new liquid-crystal phases may be discovered in
dispersions of particles that have low symmetry. It will be of great interest, both from a fundamental
viewpoint and for potential applications, to explore how the approach taken by Chung et al can be
extended to dispersions of particles of varying composition, shape, rigidity and size, as well as to
composite dispersions comprising different types of particles.
Another specific part of the paper, that I feel needs more research focus, is the prospect of
biomineralized M13 bacteriophage in the field of regenerative biomedicine. Although after the
biomineralization process the phage showed significant improvement in its Youngs modulus, which
the authors claimed to be a tooth enamel like substance, the final value of the Youngs modulus is
only around 300kPa. Whereas the actual Youngs modulus of tooth enamel is 83GPa and even a
rubber band has a greater Youngs modulus (10-100MPa) than the biomineralized phage! So this is
an issue which I feel demands more room for research, improvement and explanation.
As an ending remark to my motivation to choose this paper, I would like to add that the
biomimetic self-templating process is powerful and versatile by its own simplicity. It has the potential
to be used to engineer tissues to acquire sophisticated structure, to investigate links between liquid-
crystal self-organization and morphogenesis [7] and to fabricate metamaterials, photonic crystals and
organic photovoltaics, adding to an already impressive list of applications.

References:
[1] Chaikin, P. M. and Lubensky, T. C. Principles of Condensed Matter Physics (Cambridge Univ.
Press, 2000).
[2] Chung, W.-J. et al Nature 478, 364368 (2011).
[3] Cowin, S. C. J. Non-Newton. Fluid Mech. 119, 155162 (2004).
[4] Fan, H. et al Science 304, 567571 (2004).
[5] Lu, Y. et al Nature 389, 364368 (1997).
[6] Brinker, C. J., Lu, Y., Sellinger, A. and Fan, H. Adv. Mater. 11, 579585 (1999).
[7] Bourgine, P. and Lesne, A. Morphogenesis: Origins of Patterns and Shapes (Springer, 2010).










Potential Nanotoxicity Issue
The rapid expansion of nanotechnology promises to have great benefits for society, yet there is
increasing concern that human and environmental exposure to engineered nanomaterials may result
in significant adverse effects. That is why the field of nanotoxicology has emerged to deal with effects
and potential risks of particulate structures <100 nm in size, growing significantly over the past
decade from long-standing foundations of well established knowledge on the toxicology of fibrous and
non-fibrous particles and the interactions of viruses with cells. Discoveries of nanoparticle (NP)-
specific concepts of toxicology related to their small size and large specific surface area go back to
the early parts of the past century, although the distinctive biological effects and kinetics of NP were
not recognized until the last decade of the past century. Today, the propensity of NP to cross cell
barriers, enter cells and interact with subcellular structures is well established, as is the induction of
oxidative stress as a major mechanism of nanoparticle effects. In addition to the significance of small
size and surface area of NP, uncovering the impact of many other physico-chemical characteristics
in particular NP surface properties for initiating effects in the mammalian organism and the
environment is now an active area of research.
In our current study we are dealing with M13 virus. It has a 6kb circular single stranded DNA
genome. This filamentous bacteriophage has a length of ~880 nm and a diameter of 6.5 nm [1]. It
infects E. coli, but rather than killing host cells it just slows growth. This virus has been studied for its
potential uses in nanostructures and nanotechnology. Although nanotoxicology mainly deals with
non-living nanoparticles, in some sense, these nanoparticles may resemble viruses (18-500 nm
structures), not only in size but also with respect to interactions with cells [2]. Indeed, accumulated
knowledge gleaned from virology research over the last 100 years might be considered a valuable
basis for todays research on the interactions of nanoparticles with cells and the living organism. For
instance the mechanism by which virus causes the tobacco mosaic (TM) disease in plants [3] or the
nose to brain translocation of 30 nm poliovirus via the olfactory neuron in chimpanzees [4]. With the
advance of transmission electron microscopy (TEM) and X-ray diffraction, it was possible to
determine the fine structure of viruses. Since then, there is accumulating information on viral
mechanisms for entering cells and their interactions with subcellular structures [5] including viral entry
into cells via clathrinmediated or caveolae-mediated pathways, their intracellular endosomal and
caveosomal movement and entry into the cell-nucleus via 39 nm nuclear pore complex [6]. Indeed,
viral capsids are considered a valuable tool or carrier (Trojan horses) for targeted delivery of drug
molecules for pharmaceutical and materials applications [7], and the propensity of engineered
nanoparticles to endocytose and transcytose appears to be due to similar mechanisms.
In general phages are considered to be very safe for therapeutic use. So far only a very few
side-effects have been reported in the patients undergoing phage therapy; those that were seen
seemed directly associated with the therapeutic process [8]. So in conclusion, it can be said that
nanotoxicity issue in case of M13 bacteriophage seems to be insignificant if we consider the toxicity
caused by other nanoparticles such as carbon NPs (SWCNT, MWCNT, fullerine), metal NPs etc. [9]


References:
[1] Day, L. A., Marzee, C. J., Reisberg, S. A. and Casadevall, A., Biophys. Chem. 17, 509539
(1988).
[2] Oberdrster, G., Oberdrster, E., and Oberdrster, J., Environ Health Perspect 113,823-839
(2005).
[3] Beijerinck M. J., Phytopath. Class. 65 (7), 3-21 (1942).
[4] Bodian D., Howe H. A., Experimental Studies on Intraneural Spread of Poliomyelitis Virus, 248-
267 (The Johns Hopkins Press 1941).
[5] Smith A. E., and Helenius A., Science, 304, 237-242 (2004).
[6] Pante N., and Kann M., Molec. Biol. Cell. 13, 425-434 (2002).
[7] Douglas T., and Young M., Science, 312, 873-875 (2006).
[8] http://www.phages.org/PhageInfo.html
[9] Lewinski, N., Colvin, V., and Drezek, R., Small, 4(1), 2649 (2008).




Questions:
1. What are the driving forces behind the formation of the phage film?
2. What is the reason behind the chirality of the produced film and what kind of chiral properties
are shown by them?
3. What is the reason behind the distinct optical properties shown by the phage films?
4. How well is the comparison between biomineralized phage and tooth enamel?
5. How do you expect the self-templating supramolecular structures to be changed for different
shape, size and composition of nanoparticles?