Protein Nanotubes-Future of Nanobiotechnology
Jyotsna
R., Malavikha Rajiv Moorthy,
A. Neeha Dev
Department of Chemical Engineering, SSN
College of Engineering, Kalavakkam
*Corresponding Author E-mail:- neehadev@gmail.com
ABSTRACT
Nanobiotechnology is that branch of nanotechnology that deals with biological and biochemical applications or uses. The future
of technology resides on the vast potential applications of the field of nanotechnology.
Nano- systems in biology, the most complex and highly
functional nano-scale materials and machines have been invented by nature. Proteins and nucleic
acids, and other naturally occurring molecules (polymers) regulate and control biological systems with incredible precision. Recent research has opened up the avenues for the use
of protein nanotubes as the intriguing alternative to
carbon nanotubes due to its immense biocompatibility and
much lesser cytotoxicity. Generation of PNTs are fields for research themselves. Flagellin-based PNTs were synthesised
using a FliC-thioredoxin
fusion protein, denoted FliTrx. The flagellin subunit FliC has also
been utilized as a potential vector for liposome-based drug delivery. [1] T4P are
polymers of a single monomeric type IV pilin (PilA) subunit, a protein comprised
of a four-stranded antiparallel β-sheet wrapped around
one end of a long α-helix. It was this engineered pilin
monomer that was observed to assemble into PNTs. PNTs can be generated using a template-assisted
assembly which can provide a means of patterned PNT assembly followed by removal
of the template layer resulting in free PNT. Alumina templates or human serum albumin
can be used for a layer by layer approach. [1] Several studies highlight the potential
applications of PNTs including targeted drug delivery systems, tissue-engineering
scaffolds and biosensing devices. Further research is being carried out to characterize
the PNTs, and explore its potential use as biosensors and bionanowires.
The development of protein-based nanotubes for biologically
based nanosystems is receiving increased interest due
to their richness in structural diversity, adaptability through protein engineering
approaches and inherent biocompatibility.
KEYWORDS: Proteins, PNT, Biocompatibilty,
Flagellin-based PNTs, Pilin, Template-assisted
assembly, Bionanosensors
INTRODUCTION:
Nanobiotechnology is that branch of nanotechnology that deals
with biological and biochemical applications or uses. It deals with the study of
existing elements of living organisms and nature to fabricate new nano-devices. It bridges areas in physics, chemistry, and biology
and is a testament to the new areas of interdisciplinary science that are becoming
dominant in the twenty-first century.
OBJECTIVES
Applying
nanotools to relevant medical/biological problems and
refining these applications.
Developing
new tools for the medical and biological fields.
Imaging
of native biomolecules, biological membranes, and
tissues
Use of
cantilever array sensors and the application of nanophotonics
for manipulating molecular processes in living cells.
A NEW STRIDE IN THE FIELD OF NANOBIOTECHNOLOGY
The use of microorganisms to synthesize functional
nanoparticles has been of great interest. This approach
has become an attractive focus in current green bionanotechnology
research towards sustainable development. [2][4]
Fig.1.The evolution of Nanoscience through the ages
THE PROMISE OF PROTEINS
Proteins possess many properties that are valued in nanostructured materials: their function is template-encoded,
they can self-assemble into complex supramolecular arrays,
and they can mediate specific interactions with diverse substrates and other macromolecules.
Protein nanotubes, in particular, hold the promise of
ease of functionalization, intrinsic biocompatibility,
and modular molecular recognition. These properties have been difficult to achieve
with carbon or inorganically derived nanotubes.[1]
CNTs Vs PNTs
Peptide and protein based nanotubes can be assembled utilizing both template and non-template
assembly mechanisms under milder conditions (ambient temperature, physiological
pH), and provide a readily customizable system via modern protein engineering methods
PNTs, being composed of biomolecules themselves,
are much more biocompatible. The biodegradability of CNTs also poses a question
when it comes to biomedical applications. The lack of solubility of CNTs in aqueous
solutions is also one such discrepancy.Wormlike, filamentous nanoparticles are better
than spherical ones at avoiding immune responses allowing for longer circulation
times due to the difficulty of macrophages have adjusting tertiary and/or quaternary
structure to engulf such elongated particles. Peptide and protein-based nanotubes will likely have applications as drug-delivery vehicles
as their relatively large inner cavity and high surface areas would enable them
to transport drug molecules, nucleic acids or antigens to targeted cell surface.
One such PNT developed by the biologists at the University of California, Sant Barbara has been
shown in Fig.2.
Fig.2 "Smart" bionanotubes. Lipid protein nanotubes
made of microtuble protein (made of tubulin protein subunits shown as red-blue-yellow-green objects)
[6]
(a)
(b)
Fig.3 Flagella-based
PNTs. (a) Fluorescent microscopic image of self-assembled FliTrx
PNTs (scale bar = 10 μm). b) Atomic force
microscopy image of mineralized FliTrx PNTs. Thirteen
PNTs were generated in a layer-by-layer method separating layers of FliTrx protein containing an engineered glutamate-aspartate (negatively charged) surface-exposed peptide loop
with layers of calcium carbonate (Ca2CO3). [1]
(c)
Fig.4 Pilin-derived PNTs. (a) The
structure of the monomeric K122-4 pilin
(PDB ID 1QVE), 54 and model of the K122-4 pilin-derived
PNTs. (b) Transmission electron microscope image of solution-oligomerized pilin-derived PNTs.
(c) Atomic force microscopy image of pilin-derived
PNTs from a self-assembled alkylthiol monolayer on a
Au(III) surface. [1]
MICROORGANISM:THE
GENESIS OF PNTs
A diverse group of protein nanotube systems has emerged in recent years. The bulk of research
in this area makes use of naturally occurring protein scaffolds such as viral capsids, amyloid protein, actin, tubulin, pilin, and flagella.. Safety-modified
versions of pathogenic viruses or bacteria like flagella based PNTs can deposit
genes and induce production of anti-cancer agents upon administration to tumors.
[1]
FLAGELLA BASED PNTs - [1]
Flagella, fiber-like structures produced
by bacteria for cellular motility, are structurally composed of three general multi-protein
components: a proton gradient-driven motor complex, a joint structure, and long
helical fiber. Native flagella
are 1015 μm in length with inner and outer diameters
of 23 nm and 1225 nm, respectively. It was synthesised
self-assembly of a flagellin-based PNT using a FliC-thioredoxin fusion protein, denoted FliTrx. The FliTrx construct fused
109 thioredoxin residues between Gly-243 and Ala-352 of
FliC such that the thioredoxin
active site was readily accessible by several loop peptides designed to be presented
on the PNT surface. FliTrx PNTs
were observed through fluorescence microscopy to
form 410 μm bundles. FliTrx-based
PNTs are used in sensing and nanoelectronics applications
because of their ability to coordinate with metals like Al, Au, Cd. The flagellin
subunit FliC has also been utilized as a potential vector
for liposome-based drug delivery.Fig.3 shows
PILIN BASED PNTs [1][2]
Type IV Pili (T4P)
are flexible hair-like structures produced at the
poles of many gram-negative bacteria. Native T4P are 6 nm in diameter and have a
length of up to several micrometers. T4P are polymers of a single monomeric type IV pilin (PilA) subunit, a protein comprised of a four-stranded antiparallel β-sheet wrapped around one end of a long α-helix. A C-terminal disulfide-bound
loop region is the receptor-binding domain (RBD) that mediates interactions with
cellular receptors and abiotic surfaces.N-terminal
portion of the α-helix is utilized as an hydrophobic
oligomerization domain in native T4P.Removal of the exposed
N-terminal region of the α-helix results in a highly soluble pilin monomer that retains the antigenic and receptor-binding
characteristics of the native protein. It was this engineered pilin monomer that was observed to assemble into PNTs that are
similar in morphology and diameter (∼6 nm) to native T4P.PNT assembly is triggered
in the presence of hydrophobic compounds. Pilin-based PNTs may be useful in a variety of nanotechnology applications
including biosensing, bioseparations,
and bionanoelectronics. Pili
are acting as biological nanowires with potential applications
in microbial-based fuel cells. Pili-based nanowires have been shown to have long-range metallic-like conductivity
and to impart super capacitor behavior. [1]
TEMPLATE ASSEMBLED PNTs [1]
PNTs can be generated using a template-assisted assembly which can
provide a means of patterned PNT assembly followed by removal of the template layer
resulting in free PNT A layer-by-layer approach to prepare PNTs composed of two
proteins, bovine serum albumin (BSA), and lyophilized hemoglobin from bovine erythrocytes,
from glutaraldehyde (GA)- functionalized alumina membranes.
The use of the alumina template allowed for uniform PNT length, while pore size
and PNT outer diameters could be tailored based upon the alumina support or number
of layering steps, respectively. In another
layer-by-layer assembly, human serum albumin (HSA) PNTs were generated by alternating
layers of HSA with poly-l-arginine (PLA) from an etched
polycarbonate membrane based on the difference in overall net charge between HSA
(negative) and PLA (positive). The resultant HSA/PLA nanotubes
were of were able to bind magnetite (Fe3O4) on their outer surface enabling the
magnetic capture of the Fe3O4-HSA/PLA nanotubes. The magnetic
capture of Fe3O4-HSA/PLA nanotubes present an promising avenue for bioseparation,
the efficient nanoscale
separation, and recovery of biomolecules from complex
mixtures, and an important component of any nanomedical
regiment.
INNOVATIVE
CHALLENGES IN THE FIELD OF BIOLOGY
New molecular
imaging techniques
Quantitative
analytical tools
Physical
model of the cell as a machine
Better
ex-vivo tests and improvement in current laboratory techniques and
Better
drug delivery systems
POTENTIAL APPLICATIONS OF PNTs
Peptide and protein-based nanotubes present unique alternatives to CNT scaffolds for new
nanomedical and bionanotechnological
applications. The in-built assembly characteristics of flagella,
pili, and viral coat proteins are examples of self- assembling
nanosystems that show distinct promise in the design and
development of bionanosystems for nanomedicine,
drug delivery, bionanowiring, etc. In addition, template-assisted
PNT generation of layered PNTs presents opportunities to develop multifunc- tional PNTs that may be
targeted to a specific cellular location through
surface-exposed epitopes.New bionano
approaches to the treatment of disease as well as novel implantable devices such
as bio-based computing, molecular wires, and integrated biosensors. [1][2][3]
FEW SPECIFIC APPLICATIONS
Drug delivery
It refers to the method,
and route through which a therapeutic is administered. Due to the cytotoxicity and questions arising on the biocompatibility of
CNTs, PNTs are proving to be a better alternative for the same. The PNTs have a
huge surface area and a large inner cavity. Also, their structures prove to be in
favour of their use in drug delivery systems. The wormlike, filamentous nature of
the protein nanotube is also partly able to avoid an immune
response, if used in biomedical applications, since the long structures are difficult
for macrophages to engulf. In the future therefore, the protein coat and hollow
cavity of PNTs may allow the transport of smaller molecules, such as drugs, to their
targets on the cell surface.
Biosensors
Biosensor is a device
that uses a living organism or biological molecules, esp. enzymes or antibodies,
to detect the presence of chemicals. Protein based nanomaterials
that respond to the biological environment thus have the potential as biosensors.
CONCLUSION:
The development of protein-based nanotubes
for biologically based nanosystems is receiving
increased interest due to their richness in structural diversity, adaptability
through protein engineering approaches and inherent biocompatibility.There
lies a mammoth potential in the PNTs which when wisely utilised would bring
about miracles. Further research is being carried over all over the world to
tap its inherent capabilities.
REFERENCES:
1.
Anna Petrov and Gerald F Audette,
Peptide and protein based nanotubes for nanobiotechnology, WIREs Nanomed Nanobiotechnol 2012.
2.
Anna Pertov, Gerald F Audette,
Stephanie Lombardo, Fibril mediated oligomerisation
of pilin derived protein nanotubes,
Journal of Nanobiotechnology
3.
Sungho Choi and Kurt Geckler, Carbon nanotube meets protein, Nanotechnology journal Small.
4.
Qu X, Lu G, Tshuchida
E, Komastu T, Protein nanotube
comprised of an alternate layer-by-layer assembly using polycation
as an electrostatic glue, PubMed.
5.
N. R. Reshma and U. Gayathri, Biological Agents for Delivery of Therapeutic
Genes, Research Journal of Engineering and Technology 2013, Vol
4, Issue-4
6.
Image by Peter Allen, Article on Smart bio-nanotubes developed, may help in drug delivery, NanoNews Digest, August 2, 2005.
Received on 10.09.2013 Accepted on 28.09.2013
© Asian Pharma Press
All Right Reserved
Asian J. Pharm. Tech. 2013; Vol. 3: Issue
4, Pg 195-199