Formation Well Ordered Nanostructures By Self Assembly Process Biology Essay

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The formation of well-ordered nanostructures by self-assembly process by a variety of building blocks, both organic and inorganic have drawn much attention owing to their potential for application in biology as well as chemistry. Among the organic building blocks, peptides are the most promising ones due to their biocompatibility and chemical diversity which resembles with proteins. By inspiring from the protein assembly in biological systems, various peptide building blocks can be made using several amino acids. Here, we describe recent advances in peptide self-assembly and their applications in various fields.


Nanometer-sized structures have attracted tremendous attention owing to their potential for diverse application ranging from nanotechnology to biotechnology. In biological systems, sub-micron sized nano-objects are generally much smaller than most cells, but other subcellular components (proteins and DNA), cellular organelles, and microorganisms are comparable size with them. Biological objects with submicron size can be considered as 'biological nanostructures' as compared to 'synthetic nanostructures'.

Self-assembly is a spontaneous organization of disordered molecular units into ordered structures as a consequence of specific, local interactions among the components themselves (Lehn 2002). The formation of most biological nanostructures is driven by the self-assembly process, e.g., the formation of cell membranes upon self-assembly of phospholipids, DNA double helix formation through specific hydrogen bonding interactions, and the folding of a polypeptide chain to form protein tertiary or quaternary structure. The most simple and widely known self-assembled structures in a biological system is the lipid membrane structure. The cell membrane is composed of lipid bilayers which are arranged such a way that their hydrocarbon tails face one another to form a hydrophobic core, while their hydrophilic head groups face the aqueous solutions on either side of the membrane.

J. -M, Lehn, Proc. Natl. Acad. Sci USA, 2002, 99, 4763-

Self-assembly at the nanoscale is becoming increasingly important for the design of novel supramolecular structures in the field of nanotechnology and nanomedicine. The self-assembly process is mediated through non-covalent interactions including vander waaals interaction, electrostatic interaction, hydrogen bonds and stacking interactions. Recently, there is increasing interest in the fabrication of new materials using natural building blocks such as oligonucleotides, oligosaccharides, phospholipids, proteins and peptides (Whitesides 1991). Among them peptides drawn much attention due to their simple structure, their chemical diversity, richness of shapes, relative chemical and physical stability and easy to synthesize in large amounts. Also, peptides have been known as a very useful building block for creating self-assembling nanostructures for medical applications due to their intrinsic biocompatibility and biodegradability. There are 20 natural amino acids that are used in the synthesis of peptides and proteins in biological cells. All of them are L configuration in nature and chiral except glycine. Basic structures of all amino acids are same and only difference in the adjacent groups attached with chiral carbon. Depending upon the amino acids sequence peptide can form different structures. The versatile design of the peptides, in combination with their ability to adopt specific secondary structures, provides a unique platform for the design of materials with controllable structural features at the nanoscale.

Peptides structures were used as major building blocks for the assembly of nano-ordered structures more than a decade ago. A number of peptide-based building blocks, including cyclic peptides, dendritic peptides, amphiphilic peptides, surfactant-like oligopeptides, copolypeptides, and aromatic dipeptides, have been designed and developed for making supramolecular structures and the exploration of their possible applications in biology and nanotechnology. The simplest peptide block for the self-assembly is generally considered as diphenylalanine peptide (L-Phe-L-Phe, FF). It can self-assemble into a tubular structure with a long persistence length (~ 100 um) mediated by hydrogen bonding as well as π - π stacking of aromatic residues (Reches 2004).

Ghadiri and co-workers were the first to describe a new class of organic nanotubes based on rationally designed cyclic peptides. These peptides formed extended β-sheet-like structures, and stacked on top of each other to form hollow and extended cylinders (Ghadiri 1993, 1994).

Polypeptide self-assembly into amyloid fibrils is a natural process which is associated with many human medical disorders (……………….). This self-assembly properties of peptides have been exploited for the formation of bio-inspired nanoassemblies, including nanospheres, nanotubes, nanofibers, nanobelts and hydrogels at the nanoscale order (…………..).

Tubular nanostructures

Nanotubes are particularly interesting because of their potential application in different fields such as molecular separation and transport, catalysis, optics, electronics, chemotherapy and drug delivery (Martin 2003). Over the past few decades, researchers have made significant progress in covalently bonded nanostructures. Currently, noncovalent nanotubes have drawn attention due to their signifinact advantages including easy synthesis, high efficiency, control in diameter size and self-organization. Several methods have been reported so far for the preparation of these noncovalently assembled nanotubes using different structures including helical, hollow bundles of rod-like units and stacked rings. Among these methods, the self-assembling peptide nanotubes formed by stacking cyclic peptides and stabilized by hydrogen bonds have attracted attention because of the potential ease in changing the structural and functional properties. For this kind of self-assembly, peptides should have particular orientation, where amino acid side chains would be directed toward outward and the carbonyl and the amino groups of the amide backbone would be directed perpendicular to the ring so that they can participate in intermolecular hydrogen bonding.

The pioneer works on peptide nanostructures were the cyclic peptide nanotubes with the following sequence: cyclo [-(D-Ala-Glu-D-Ala-Gln)2-], which were developed by Ghadiri and co-workers. They used alternating D- and L-amino acids in making the cyclic octapeptide to form a planar ring that could be self-assembled through hydrogen bonding to form nanotube structures of a desired diameter. The proton triggered self-assembly resulted numerous ring-shaped peptides subunits interacting through an extensive network of hydrogen bonds to form nanotube structures. They hypothesized that cyclic peptides with an even number of alternating D- and L- amino acids could adopt a low-energy ring shaped flat conformationin in which all backbone amide groups lie approximately perpendicular to the plane of the structure. At the same time, subunits can stack in an antiparallel fashion and participate in backbone-backbone intermolecular hydrogen bonding to produce a contiguous β-sheet structure. At alkaline pH, the large repulsive intermolecular electrostatic interactions between the negatively charged carboxylate side chains disfavors ring stacking. In contrast, controlled acidification of alkaline peptide solution triggers the spontaneous self-assembly of peptide subunits into rod-shaped crystals which is an organized bundle of hundreds of tightly packed nanotubes. Here, intersubunit hydrogen bonding provides the main driving force in the assembly process. The internal diameter of the nanotubes ranges between 7-8 Å and can be controlled by changing the number of amino acids in the peptide sequence (Ghadiri 1993, Hartgerink 1996). Various applications of these peptide nanotubes have been demonstrated in different fields. Because of the unique structural features of these cyclic peptides, when they are placed in such a condition that favors hydrogen bonding, for example, adsorption onto lipid membranes, they can stack to form hollow, β-sheet like tubular structures that are open-ended, presenting the amino acid side chains on the outside surface of the ensemble. Therefore, rationally designed cyclic D, L-α-peptides may be able to selectively target and self-assemble in bacterial membranes and exhibit antibacterial activity by increasing the membrane permeability. Some of the amphiphilic cyclic peptides show antibacterial activity. So, these can be used as antibiotic agents (Fernandez-Lopez 2001). These cyclic peptides decorated with appropriate hydrophobic surface residues form transmembrane channel structures by spontaneous self-assembly on incorporating a sufficient concentration of the peptide monomer in lipid bilayers. The authors suggested that not only the hydrophobic surface characteristic of the channel-forming peptide is an important factor, but also the peptide must be able to participate in extended hydrogen-bonded stacking interactions to produce channel structures long enough to span the lipid bilayer. The design of transmembrane channel structures with larger pore diameters has potential application in 'molecular' transport across lipid bilayers and can be used as a vehicle for drug delivery into living cells in antisense and gene therapy applications (Ghadiri 1994).

Another cyclic peptide that was reported to self-assemble into tubular structures is the Lanreotide octapeptide, which is synthesized as a growth hormone inhibitor (Valery 2003). Lanreotide can self-assemble into nanotubes of viral capsid-like dimension. The nanotube walls are built up from helicoidal filaments, formed by peptide dimer building blocks self-assembled into antiparallel β-sheets through an alternating pattern of the aliphatic and aromatic aminoacid residues. .

C. R. Martin and P. Kohli, Nat Rev Drug discovery, 2003, 2, 29-

Peptide amphiphiles

Peptide amphiphiles (PA) consist of oligo-peptides that are modified with a hydrophobic alkyl tail to form molecules with distinctly hydrophobic and hydrophilic ends, similar to lipids. Phospholipid membrane in the biological systems also consists of a hydrophobic tail and a hydrophilic head. They self-assemble into organized structures in water through hydrophobic interations. Peptide amphiphiles have the combination of structural features of amphiphilic surfactants and the functions of bioactive peptides. These molecules generally assemble in water into high aspect ratio nanofibers under specific solution conditions (pH, ionic strength, and temperature). It has been demonstrated that stable nanofibers can be made by mixing two oppositely charged PA molecules to promote close interaction (Behanna 2005). A broad class of amphiphilic molecules have been used to create organized biomaterials by Stupp and co-workers. Artificial 3D scaffolds consist of nanofiber networks formed by the aggregation of the amphiphilic molecule IKVAV, and this process is triggered by the addition of neural progenitor cell suspensions to the aqueous solution (Silva 2004). The cells were encapsulated in vitro within the 3D network of nanofiber scaffold which induced very rapid differentiation of cells into neurons. To understand the drug or protein carrier capabilities and biological signaling applications for peptide amphiphilic structures, it is imperative to determine the nature of the interior structure and assess the encapsulation ability of the assembled structure toward the small molecules. Fluorescence techniques offer powerful tools to investigate the structure and dynamics of self-assembled macromolecular system (Tovar 2005). PAs bearing fluorophores (tryptophan or pyrene) when self-assembled driven by low pH, experience differing degrees of solvent exposure and quencher accessibility. Stern-Volmer analysis of fluorescent intensity changes upon the addition of a fluorescence quenching agent should reveal the extent of small molecule access within the nanostructures. One-dimensional (1D) nanostructures have drawn attention among researchers for their applications in biomaterials (Nagarkar 2008). Many alkylated peptide amphiphiles have been reported to self-assemble into 1D cylindrical nanofibers. PA molecule that contain charged amino acid sequence covalently attached to a hydrophobic alkyl chain typically self-assemble into nanofibers as a result of hydrophobic collapse of the alkyl chains and β-sheet formation by the peptide segments. The formation of nonspherical supramolecular aggregates with controlled dimensions has been demonstrated using a rigid-rod dumbbell-shaped template that limits the 1D self-assembly of PA molecule (Bull 2008). Atomic Force Microscope (AFM) and Transmission Electron Microscope (TEM) data reveal a dramatic change in the form of the PA supramolecular aggregate upon the addition of the dumbbell shape template from high-aspect-ratio nanofibers to small, nearly monodisperse nanostructures. This technique has potential to control the delivery of therapeutic molecules in supramolecular form.

Alternating tetrapeptide sequence containing hydrophobic and negatively charged residues (V and E) and alkyl segments with 16 carbons self-assemble into 1D nanostructures that lose all curvature and grow laterally to create nanobelts (Cui 2009, Figure). Peptides with alternating hydrophobic and hydrophilic amino acids are known to have a strong tendency to form β-sheet structures (Dong 2007). The structural motif of VEVE, when adopting an extended β-strand conformation, flips the hydrophilic and hydrophobic side chains to the opposite sides of the peptide backbone. Tapping-mode atomic force microscopy (AFM) imaging reveals the flat, beltlike morphology of the nanostructures formed in aqueous solution over the course of 2 weeks. Disruption of alternating hydrophobic and hydrophilic amino acid sequence by replacing the VEVE peptide segment with a structural motif of VVEE resulted cylindrical nanofibers under the exact same conditions. This indicates the nanobelt flat architecture is associated with highly effective packing among β sheets with peptides containing alternating sequence. Self-assembly of PAs into nanofibers is also triggered by light (Lee 2008). To induce the self-assembly of the pH-responsive PAs inside liposomes, a photoacid generator (PAG) was introduced to lower the pH upon exposure to light. Encapsulation of PAs within the aqueous interior of liposomes was done simply by using a PA solution to hydrate a phospholipid film. The encapsulation and light-triggered PA self-assembly were confirmed by confocal fluorescent microscopy, SEM, FTIR and CD. This method can enable the preparation of nanofiber bundles to be delivered as packed nanofibers within the interior of liposomes for use in targeting specific tissues or tumors.

PA molecule incorporating photocleavable groups between hydrophobic and peptide domains has been designed by Muraoka et al (Muraoka 2008). This PA molecule contains a palmitoyl tail, a 2-nitrobenzyl group, and an oligopeptide segment GV3A3E3, in which the 2-nitrobenzyl group is covalently linked to the N-terminal amide of the glycine residue and can be cleaved by irradiation at 350 nm.


Amphiphilic oligopeptides containing various ratio of hydrophilic to hydrophobic block length can self-assemble into vesicular structures in aqueos solution at neutral pH. It is also demonstrated that hydrophilic compounds could be encapsulated within the peptide vesicles and therefore it may be useful as drug delivery systems (Van hell 2007). These peptides were recombinantly produced in bacteria as an alternative to solid phase synthesis. Charged, amphiphilic block copolypeptides form stable vesicles and micelles in aqueous solution. Aqueous self-assembly of poly(L-lysine)-b-poly(L-leucine) and poly(glutamic acid)-b-poly(L-leucine) block copolypeptides resulted unilamellar vesicles (Holowka 2005). To check the integrity of the vesicle membrane, a water soluble cargo, Texas Red labeled dextran was encapsulated during vesicle assembly and after dialysis resulting vesicles were found to contain reasonable amounts of encapsulated dextran. Though positively charged polypeptides are relatively unstable whereas negatively charged polypeptides vesicles are quite stable. The polypeptide consists of zwitterionic diblock copolypeptides poly(L-glutamic acid)-b-poly (L-lysine) (PGA-b-PLys), which was synthesized by sequential ring- opening polymerization of the corresponding α-amino acid N-carboxyanhydrides (Rodriguez-Hernandez 2005). Upon neutralization of the polypeptide block, it changed from a random coil conformation into a neutral and α-helical structure (rod). At acidic pH, the PGA block is neutralized , which resulted the vesicle in the core while the PLys block forms the shell. In contrast, under basic conditions, the protonated PLys block is transformed into neutral and insoluble -NH2 groups, forming the core of the aggregates. It was believed that the vesicle formation was related to the systematic presence of the polypeptide in a rodlike conformation in the hydrophobic part of the membrane, inducing a low interfacial curvature and as a result a hollow structure. Elastin-like polypeptides (ELPs) in a linear AB diblock architecture can be triggered to self-assemble into a spherical micelle by a small increase of temperature to between 37 and 42 0 C, the temperature range approved for clinical application of hyperthermia (Dreher 2008). The size if the micelle is controlled by both the length of the copolymer and hydrophilic-hydrophobic block ratio.


In biological systems, self-assembly of peptides and proteins into fibrillar structure formation is very common phenomena. Amyloid fibrils, associated with a large number of diseases, are key example of protein self-assembly into nano-fibrillar structures (Nelson 2005). The accumulation of amyloid fibrils is a common characteristic of various diseases including Alzheimer's disease, Type II diabetes, Parkinson's diseases and many others. Moreover, self-assembled nanofibrils from natural or artificial peptides show remarkable potential for application in bionanotechnology. These fibrils exhibit a typical β-sheet conformation. Amyloid fibrils are formed in most cases by polypeptides of 30-40 amino acids or longer. Recently it is observed that short peptide fragments, for example, tetra- to hexapeptides, to form amyloid fibrils that show all the typical biophysical and structural properties of amyloid fibrils. Also, they can serve as an excellent model system for studying amyloid fibrils formation and biological self-assembly processes. For example, short peptides fragments like NFGAIL, FGAIL can form fibrillar structures.

Then the researchers were motivated to find the minimum peptide fragment which can form amyloid fibrils. It was found that aromatic residues have key roles in the process of amyloid formation. Diphenylalanine, the core recognition motif of the Alzheimer's β-amyloid polypeptide was very interesting one since it has been seen that larger peptides and conjugated organic molecules that contain this motif inhibits fibrils formation by Aβ. Actually, FF leads the process of self-assembly in the molecule. In fact, dipeptide containing FF can undergo self-assembly into long and stiff nanotubes through an association of hydrogen bonding and π- π stacking. (Cherny 2008)

These peptide nanotubes are significantly rigid and potentially useful in electrochemical biosensing (Yemini 2005). Interestingly, it is found that 9-fluorenylmethoxycarbonyl (Fmoc) protected diphenyalanine (Fmoc-FF) self-assembles into nanofibrils in aqueous solution which leads to a hydrogel formation. (Jayawarna 2006, Mahler 2006). The rheological behavior of this hydrogel exhibits similar characteristics of solid-like gels materials. This hydrogel is significantly stable at wide range of temperatures and pH values including extreme acidic conditions. Thus these hydrogels can be more useful compared to other peptide hydrogels in drug delivery and 3D cell culture. Recent molecular model shows that in this gel peptides are aligned in an antiparallel β-sheet fashion and adjacent sheetsare interlocked through lateral π- π interactions, which results to the formation of a cylindrical structure (Smith 2008).

Later modification of this dipeptide by acetylation of the N-terminal amine and amidation of the C-terminal carboxyl showed the formation of nanotube structures of similar properties. This indicates that the role of aromatic moieties, rather than the charged one, in the formation of peptide nanotubes (Reches 2005).

Further extension of this study showed that diphenylglycine, a similar analogue and the simplest aromatic peptide, formed nanospheres. These nanostructures are quite stable under extreme chemical conditions. The unusual stability of the peptide nanostructures is exteremly useful for their use in (bio) organic and/or inorganic nanoscale fabrications process, including optic and electron-beam lithographic protocols. Interestingly, the introduction of a thiol group into FF peptide can alter the self-assembly properties of the resultant building blocks, e.g., cysteine-diphenylalanine tripeptide (CFF) self-assembles into spherical vesicles rather than into nanotubes (Reches 2004).