Annette F. Dexter

Nanoparticles Mimicking Viral Surface Topography for Enhanced Cellular Delivery

Novel silica nanoparticles mimicking virus surface topography are prepared. It is demonstrated that increases in nanoscale surface roughness promote both binding of biomolecules and cellular uptake; thus, the cellular delivery efficiency is significantly increased (scale bars 20 μm).

The interfacial structure and Young's modulus of peptide films having switchable mechanical properties

We report the structure and Youngs modulus of switchable films formed by peptide self-assembly at the air–water interface. Peptide surfactant AM1 forms an interfacial film that can be switched, reversibly, from a high- to low-elasticity state, with rapid loss of emulsion and foam stability. Using neutron reflectometry, we find that the AM1 film comprises a thin (approx. 15 Å) layer of ordered peptide in both states, confirming that it is possible to drastically alter the mechanical properties of an interfacial ensemble without significantly altering its concentration or macromolecular organization. We also report the first experimentally determined Youngs modulus of a peptide film self-assembled at the air–water interface (E=80 MPa for AM1, switching to E<20 MPa). These findings suggest a fundamental link between E and the macroscopic stability of peptide-containing foam. Finally, we report studies of a designed peptide surfactant, Lac21E, which we find forms a stronger switchable film than AM1 (E=335 MPa switching to E<4 MPa). In contrast to AM1, Lac21E switching is caused by peptide dissociation from the interface (i.e. by self-disassembly). This research confirms that small changes in molecular design can lead to similar macroscopic behaviour via surprisingly different mechanisms.

Foaming properties of a peptide designed to form stimuli-responsive interfacial films

We have designed an amphipathic peptide, AM1, that can self-assemble at the air-water interface to form an interfacial ensemble capable of switching between a mechanically strong cohesive film state and a mobile detergent state in response to changes in the solution conditions. The mechanical properties of the AM1 ensemble in the cohesive film state are qualitatively equivalent to the protein β-LG, while in the mobile detergent state they are equivalent to the low molecular weight surfactant, SDS. In this work the foaming properties of AM1 are compared to those of β-LG and SDS at the same weight concentration and it is found that AM1 adsorbs rapidly to the interface, initially forming a dense foam like that formed by SDS and superior to β-LG. In addition, under solution conditions where interfacially adsorbed AM1 forms a cohesive film state the foam stability is high, comparable to β-LG. However when the interfacially adsorbed AM1 forms a foam under detergent-state conditions, the foam stability is poor. We have achieved control of foam stability through the design of a peptide that exhibits stimuli-responsive changes in the extent of intermolecular interactions between peptide molecules adsorbed at the air-water interface. These results illustrate the exciting potential of peptide surfactants to form a new class of stimuli-responsive foaming agents.

Tuneable Control of Interfacial Rheology and Emulsion Coalescence

Breaking point: Switchable peptide surfactants are used to demonstrate that the extent of cross-linking in an interfacial surfactant layer can control the rate of emulsion coalescence. Pictured is the rupture of an aqueous thin film where the peptide layer lacks sufficient strength to prevent hole formation, but nonetheless dramatically slows the rate of hole expansion.

Microbial Bio-Production of a Recombinant Stimuli-Responsive Biosurfactant

Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum‐derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single‐step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent‐, acid‐, and heat‐mediated precipitation of impurities were successfully applied as alternatives to post‐cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio‐produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam‐stabilizing and foam‐destabilizing states. Biotechnol. Bioeng. 2009;102: 176–187.

Interfacial and emulsifying properties of designed β-strand peptides.

The structural and surfactant properties of a series of amphipathic β-strand peptides have been studied as a function of pH. Each nine-residue peptide has a framework of hydrophobic proline and phenylalanine amino acid residues, alternating with acidic or basic amino acids to give a sequence closely related to known β-sheet formers. Surface activity, interfacial mechanical properties, electronic circular dichroism (ECD), droplet sizing and zeta potential measurements were used to gain an overview of the peptide behavior as the molecular charge varied from ±4 to 0 with pH. ECD data suggest that the peptides form polyproline-type helices in bulk aqueous solution when highly charged, but may fold to β-hairpins rather than β-sheets when uncharged. In the uncharged state, the peptides adsorb readily at a macroscopic fluid interface to form mechanically strong interfacial films, but tend to give large droplet sizes on emulsification, apparently due to flocculation at a low droplet zeta potential. In contrast, highly charged peptide states gave a low interfacial coverage, but retained good emulsifying activity as judged by droplet size. Best emulsification was generally seen for intermediate charged states of the peptides, possibly representing a compromise between droplet zeta potential and interfacial binding affinity. The emulsifying properties of β-strand peptides have not been previously reported. Understanding the interfacial properties of such peptides is important to their potential development as biosurfactants.

Bioproduction of highly charged designer peptide surfactants via a chemically cleavable coiled-coil heteroconcatemer

Designer peptides have recently attracted attention as self‐assembling fibrils, hydrogelators and green surfactants with the potential for sustainable bioproduction. Carboxylate‐rich peptides in particular have shown potential as salt‐resistant emulsifiers; however the expression of highly charged peptides of this kind remains a challenge. To achieve expression of a strongly anionic helical surfactant peptide, we paired the peptide with a cationic helical partner in a coiled‐coil miniprotein and optimized the polypeptide sequence for net charge, hydropathy and predicted protease resistance (via the Guruprasad instability index). Our design permitted expression of a soluble concatemer that accumulates to high levels (22% of total protein) in E. coli. The concatemer showed high stability to heat and proteases, allowing isolation by simple heat and pH precipitation steps that yield concatemer at 133 mg per gram of dry cell weight and >99% purity. Aspartate‐proline sites were included in the concatemer to allow cleavage with heat and acid to give monomeric peptides. We characterized the acid cleavage pathway of the concatemer by coupled liquid chromatography‐mass spectrometry and modeled the kinetic pathways involved. The outcome represents the first detailed kinetic characterization of protein cleavage at aspartate‐proline sites, and reveals unexpected cleavage preferences, such as favored cleavage at the C‐termini of peptide helices. Chemical denaturation of the concatemer showed an extremely high thermodynamic stability of 38.9 kcal mol−1, with cleavage decreasing the stability of the coiled coil to 32.8 kcal mol−1. We determined an interfacial pressure of 29 mN m−1 for both intact and cleaved concatemer at the air‐water interface, although adsorption was slightly more rapid for the cleaved peptides. The cleaved peptides could be used to prepare heat‐stable emulsions with droplet sizes in the nanometer range. Biotechnol. Bioeng. 2015;112: 242–251.

Mixed System of Eudragit S-100 with a Designed Amphipathic Peptide: Control of Interfacial Elasticity by Solution Composition

We report an interfacially active system based on an informational peptide surfactant mixed with an oppositely charged polyelectrolyte. The 21-residue cationic peptide, AM1, has previously been shown to respond reversibly to pH and metal ions at fluid interfaces, forming elastic films that can be rapidly switched to collapse foams or emulsions on demand. Here we report the reversible association of AM1 with the methacrylate-based anionic polymer Eudragit S-100. The strength of the association, in bulk aqueous solution, is modulated by added metal ions and by ionic strength. Addition of zinc ions to the peptide-polymer system promotes complex formation and phase separation, while addition of a chelating agent reverses the association. The addition of salt weakens peptide-polymer interactions in the presence or absence of zinc. At the air-water interface, Eudragit S-100 forms an elastic mixed film with AM1 in the absence of metal, under conditions where the peptide alone does not show interfacial elasticity. When zinc is present, the elasticity of the mixed film is increased, but the rate of interfacial adsorption slows due to formation of peptide-polymer complexes in bulk solution. An understanding of these interactions can be used to identify favorable foam-forming conditions in the mixed system.

Fabrication and characterization of hydrogels formed from designer coiled-coil fibril-forming peptides

Hydrogels are soft solids that represent attractive matrices for tissue engineering, wound healing and drug delivery. We previously reported an α-helical peptide, AFD19, that forms fibrils and hydrogels at pH 6, but precipitates under physiological conditions. We now show that a single targeted change in AFD19 yields peptide AFD36, which gels at physiological pH and in the presence of salt. Furthermore, we present a simple method for homogeneous sol–gel conversion through pH titration with sodium bicarbonate followed by loss of carbon dioxide. Chemical and thermal denaturation studies show AFD36 self-assembles to give stable α-helical structures, forming fibrils of 3.8–3.9 nm diameter at pH 4.0–7.0 as shown by small-angle X-ray scattering and atomic force microscopy. An AFD36 gel at 0.35% (w/v) showed an elastic modulus of 350 Pa. Mouse fibroblasts exhibited low cellular toxicity and spread morphologies when grown on the gel as a preliminary proof of principle towards cell culture studies. These peptide gels offer a molecularly simple, biodegradable alternative to polymer-based systems for biomedical applications.

Using nano-structured interfacial peptide films to create stimuli-responsive foams and emulsions

Proteins and peptides assemble nanostructured interfacial films when adsorbed at the fluid-fluid interface. We have measured the mechanical properties of these interfacial films with novel apparatus, the Cambridge interfacial tensiometer (CIT) and shown that the characteristic behaviour of the films can be modulated in response to changes in solution conditions. Changes in the mechanical properties of these interfacial films correspond to changes in the stability of both liquid droplets in emulsion systems and air bubbles in foam systems. We have developed a series of peptide surfactants (Pepfactants ) that are capable of stabilising foams and emulsions in a stimuli-responsive manner.