Amphiphilic Peptide Nano Structures

Amphiphilic peptides can self-assemble into nano-structures such as fibers and vesicles

Peptides that self-assemble, can be used as building blocks for nanoscience, to form controlled fibrils, vesicles and potentially as carriers for therapeutical drugs!

Amphiphilic peptides have been shown to form defined nanostructures such as molecular wires, well defined nanotubes as well as nanovesicles. Advances made in the synthesis of amphiphilic peptides during the past few years, allows for their use as scaffolds for the synthesis of defined nanometer structures. Extensive studies of biological systems on the molecular level in the 20th century revealed that molecular self-assembly is a fundamental process in all living systems. Specifically, proteins that form the primary components of the molecular machinery that allows life to exist have a vast array of resources available to allow them to self-assemble. However, it also has been observed that the aberration of protein self-assembly is often the cause of severe, progressive human diseases that can have a serious social impact. Small organic molecules as well as peptides possessing both, hydrophilic and hydrophobic moieties can bind other peptides or protein molecules through hydrogen bonds and hydrophobic interactions and are behind the driving forces for molecular self-assembly.

Amphiphilic peptides are peptides that contain structurally defined hydrophobic and hydrophilic regions within their amino acid sequence which are often spaced in defined blocks along their sequence length.

In cells and in cell-cell surface interactions and recognition the extracellular micro and nano-environments play fundamental roles in controlling cellular behavior. Biomolecular self-assembly techniques useful for the synthesis of multifunctional nano-structured materials have the potential to provide bioactive micro- and nano-environments for the optimization of biomaterial performance such as the incorporation of specific biomolecules by which specific cellular functions can be stimulated. For example, recent tissue engineering strategies have aimed to design surface properties critical for the development of bioactive devices that elicit appropriate cellular responses and induce lineage-specific stem-cell differentiation. Furthermore, more recent research has revealed that different neurodegenerative disorders such as Huntington’s, Alzheimer’s, and spongiform encephalopathy diseases, have in common the presence of insoluble protein aggregates. Generally these aggregates are termed “amyloid” and it was shown that they share several physicochemical features such as a fibrillar morphology, a predominantly beta-sheet secondary structure, birefringence upon staining with the dye Congo red, insolubility in common solvents and detergents, and protease resistance. Birefringence is a property of a material which refractive index depends on the polarization and propagation direction of light. Stacking interactions and conformational constrains can play key roles in the formation of fibrils. Furthermore, protein-protein and peptide-peptide interactions that result in self-assembly of peptides to form fibrils can be influenced and modulated by the nature of side chains of natural biomolecules. In the year 2000 Jayakumar et al. synthesized a novel dodecyl amine dipeptide derivative that contained a long alkyl chain. This amphiphilic peptide which structure and energy minimized molecular model is shown below can form multilamillar “oninon” type vesicles.

Conductance and 90° light scattering measurements together with cryogenic transmission electron microscopy based experiments allowed the group to determine critical vesicular concentrations that are characteristic for micelle and vesicle formation. A model of the “onion” vesicle is shown above.
Hartgerink et al. in 2001 designed an amphiphilic peptide that self-assembled in a pH-induced manner. This peptide allows the formation of reversible nanofibers as well as the formation of stable cross-linked structures. Furthermore, the researchers report that, the fibers are able to mineralize hydroxyapatite after cross-linking to form a composite material. The alignment was shown to occur along the long axis of the fibers similar to the alignment found in collagen fibrils and hydroxyapatite crystals in bone. Hopefully in the near future this or a similar approach may be useful for the design of biomaterials that can help an individual to regenerate lost bone.

(Above) Figure A shows the chemical structure of the amphiphilic peptide illustrating its key structural features. Region 1 was designed to contain a long alkyl tail that adds the hydrophobic character to the molecule which combined with the peptide region makes the molecule amphiphilic.

. Region 2 contains four consecutive cysteine residues that when oxidized can form disulfide bonds to polymerize the self-assembled structure. A flexible linker containing three glycines was added to region 3. This region provides flexibility to the hydrophilic head group. Region 4 contains a single phosphorylated serine residue that was added to allow for the molecule to interact strongly with calcium ions and help to direct mineralization of hydroxyapatite. Region 5 displays the cell adhesion ligand RGD to the outer surface of the vesicle. (B) The molecular model of the amphiphilic peptide shows the overall conical shape of the molecule including the narrow hydrophobic tail to the bulkier peptide region. Color scheme: C, black; H, white; O, red; N, blue; P, cyan; S, yellow. (C) The schematic of the self-assembly of the peptide molecules into a cylindrical micelle is depicted as well.

The researchers used existing knowledge of amphiphile self-assembly to design this amphiphilic peptide. The two acidic amino acids, phosphoserine and aspartic acid, are abundant in proteins present in mineralized tissues and have been shown to initiate hydroxyapatite growth. The material self-assembles below pH 4 and disassembles again when the pH is brought back to neutral using KOH. However, the oxidized fibers were found to be stable to alkaline solutions. Furthermore, this behavior could be reversed by treatment of the cross-linked vesicle with DTT.
Santoso et al. in 2002 published a review of different amphiphilic peptide motifs that can be used for scaffolds to form nanometer structures such as molecular wires and mineralization of hydroxyapatite crystals in a particular orientation. They also report on the design of a new class of short and simple surfactant-like peptides that self-assemble into well-defined nanotubes and nanovesicles. The amphiphilic peptide from Jayakumar et al.’s group was also included in this report as well as the peptide synthesized by Hartgerink et al.

The peptide which structure and molecular model is shown to the left is a bis(N-α-amido-glycyl-glycyine)-1,7-heptane dicarboxylate. This peptide is an example of a dicarboxylic oligopeptide bola-amphiphile consisting of partially of natural amino acids. This type of peptides self assemble in a pH dependant manner. The general feature of this type of peptides is the presence of both hydrophopic and hydrophilic regions in the same molecule that allows the peptides to self-assemble. Bolaamphiphiles sometimes called bolaform surfactants, bolaphiles, or alpha-omega-type surfactants are amphiphilic molecules that have hydrophilic groups at both ends of a sufficiently long hydrophobic hydrocarbon chain. The introduction of a second head-group makes the molecule more soluble in water, and increases the critical micelle concentration (cmc) as well. However, the aggregation number is decreased. The morphology of bolaamphiphilic aggregates can include nano-spheres, cylinders, disks, and vesicles.

The V6D2 peptide illustrated above to the right contains six hydrophobic valines and one hydrophilic aspartic acid at the carboxyterminus of the peptide and is made up of L-amino acids. It can self-assemble into tubes of homogeneous diameter. If the hydrophobic tail is lengthened, the size distribution becomes more poly disperse. This poly disparity is a result of the greater freedom the monomers have to pack in different arrangements.
In 2002 Hartgerink et al. added more functionality to their list of designed amphiphilic peptides. Two new modes of self-assembly were added, the drying on surfaces and the addition of divalent ions such as calcium. All reported amphiphilic peptides self-assembled into one-dimensional fibers.
Silva et al. reported in 2004 that the incorporation of sequences that are known to direct cell differentiation such as the pentapeptide epitope, IKAV, found in laminin, can self-assemble into nanofibers. Furthermore, the nanofiber scaffold containing the laminin epitope induced a very rapid differentiation of cells into neurons but discouraged the development into astrocytes. These results indicate that nanofibers can be used to present bioactive epitopes to cells.

Since Haberland and Reynolds in 1975 reported on the nature of the interaction of the AI polypeptide chain from human high density serum lipoprotein with L-α -Palmitoyl lyso-phosphatidylcholine the number of publications reporting the use, design and investigations of amphiphilic peptides has grown dramatically. This is illustrated in the graphic to the right were the increase in the number of published papers is shown as bars increasing in size per year. This article is not indented to exhaustively cover the whole collection of published papers reporting on the nature of amphiphilic peptides rather only a few selected examples and reviews will be covered here.

A review article published by Mart et al. in 2006 reported on the design and use of “Peptide-based stimuli-responsive biomaterials.” The review covered the design and synthesis of these materials and listed the nature of forces governing the molecular interactions of these peptides during self-assembly as well. The diversity of naturally occurring peptides allows for rational incorporation of non-covalent interactions such as electrostatic (acidic and basic amino acids), hydrophobic, π-stacking (aromatic amino acids), hydrogen bonding (polar amino acids) as well as covalent (disulfide) bonds and steric contributions (strand directing amino acids) into the peptide molecules. Furthermore, the review covered peptide based responsive systems made up of helices and coil-coils, β-sheets, β-hairpins, amphiphiles, aromatic interactions, and elastin-like polymers. Even a molecular motor based on the interaction of kinesin with a coiled-coil system is covered.

Stephen Mann in 2009 reviewed the chemical processes that govern equilibrium and non-equilibrium self-assembly approaches to allow the synthetic construction of discrete hybrid inorganic-organic nano-objects and higher-level nanostructured molecular networks. This report includes a list of various nano-objects such as nano-fibers, vesicles, nanobodies and others, including a few based on peptides and selected protein sequence motifs.

Zhou et al. in 2009 report the design of a novel amphiphilic peptide, Ac-RADAGAGARADAGAGA- NH2, that was able to stabilize pyrene. This designed peptide formed a colloidal suspension by encapsulating pyrene inside the peptide–pyrene complex. The researchers used egg phosphatidylcholine (EPC) vesicles to mimic cell bilayer membranes. They found that pyrene was released from the peptide coating into the EPC vesicles by mixing the colloidal suspension with EPC vesicles. The release could be followed by steady fluorescence spectra as a function of time. The peptide was designed after the poly-GA (where G is Glycine, and A is Alanine) motif which is a prevalent conserved motif in silk protein, particularly in silk produced by the silk moth Bombyx mori.

Samuel Stupp’s group (Zhang et al., 2010) reported on the physico-chemistry of the self-assembly pathway that leads to aligned monodomain gels. The researchers used the amphiphilic peptide containing the sequence V3A3E3(CO2H) together with a C16 alkyl tail. Scanning electron microscopy (SEM) small-angle X-ray scattering (SAXS), quick-freeze/deep-etch (QFDE) transmission electron microscopy (TEM) and light-microscopy was used to study the morphology of the nanofiber bundles in the gel.

Cavalli et al. in 2010 published a critical review about amphiphilic peptides and their cross-disciplinary role in nanoscience. In this review the researchers elaborate on the use of peptides as molecular building blocks useful for bottom-up fabrication of supramolecular structures based on self-assembly and their potential for applications in fields such as biotechnology, bioengineering and biomedicine. They discussed the main categories of peptide-based amphiphiles by highlighting relevant examples such as building blocks used for the construction of nanometer-scale assembled structures made out of amino acid containing amphiphilic peptides, long chains alkylated and/or acylated peptides, peptide-phospholipid conjugates, and peptide-based copolymers. General synthetic and protein engineering strategies as well as applications for peptide-based nanostructures were also discussed in this review.

Gudlur et al. in 2012 report the design and synthesis of branching amphiphilic peptides that spontaneously form aqueous-filled nanovesicles and that can be used for cargo delivery. By combining an oligolysine tail segment (K = 5) with two strong β-forming hydrophobic sequences the researchers designed a peptide with a branched arrangement of the hydrophobic sequences that they showed to be essential for assembly. The hydrophobic sequences used in this design can undergo pH dependent aggregation and adhesive qualities when flanked by oligo-lysine segments (i.e. KKKFLIVIKKK). The addition of two of these sequences to a lysine residue generated a branched sequence that mimicked certain properties of phospholipids such as the ability to form bilayer like structures that can fuse under certain conditions and increase in size.

Finally, the coworkers Ko and Kim studied peptides that can self-assemble into amyloid fibers. The two researches created peptides containing two copies of KFFE, a simple four-residue amyloidogenic domain and connected the two peptide blocks with GS-rich linker sequences of different lengths but similar physicochemical properties. Their experimental results indicate that aggregation occurred most rapidly when KFFE domains were connected by a linker of an intermediate length. The researchers reported that their findings were consistent with estimated entropic contribution of a linker length toward the formation of partially structured intermediates along the aggregation pathway. The inclusion of a relatively short linker was found to inhibit formation of aggregates with mature fibril morphology. The researcher conclude that the results demonstrate that the intramolecular distance between amyloidogenic domains is an important yet overlooked factor affecting amyloid aggregation. This could have implications for further research in the study of amyloid aggregate formation.

In conclusion, it has become apparent that naturally occurring amino acids and peptide motifs can be used as building blocks to synthesize peptide based nano-structures such as nano-fibers, nano-fibrils and nano-vesicles. However, the potential of these synthetic strategies reported here for the design and synthesis of new types of biomaterials useful for applications in biotechnology, bioengineering and biomedicine is immense.

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