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 PNT’s 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 10–15 μm in length with inner and outer diameters of 2–3 nm and 12–25 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 4–10 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