Apurba K. Das

Self-assembled peptide-based hydrogels as scaffolds for anchorage-dependent cells.

We report here the design of a biomimetic nanofibrous hydrogel as a 3D-scaffold for anchorage-dependent cells. The peptide-based bioactive hydrogel is formed through molecular self-assembly and the building blocks are a mixture of two aromatic short peptide derivatives: Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (arginine-glycine-aspartate) as the simplest self-assembling moieties reported so far for the construction of small-molecule-based bioactive hydrogels. This hydrogel provides a highly hydrated, stiff and nanofibrous hydrogel network that uniquely presents bioactive ligands at the fibre surface; therefore it mimics certain essential features of the extracellular matrix. The RGD sequence as part of the Fmoc-RGD building block plays a dual role of a structural component and a biological ligand. Spectroscopic and imaging analysis using CD, FTIR, fluorescence, TEM and AFM confirmed that FF and RGD peptide sequences self-assemble into beta-sheets interlocked by pi-pi stacking of the Fmoc groups. This generates the cylindrical nanofibres interwoven within the hydrogel with the presence of RGDs in tunable densities on the fibre surfaces. This rapid gelling material was observed to promote adhesion of encapsulated dermal fibroblasts through specific RGD-integrin binding, with subsequent cell spreading and proliferation; therefore it may offer an economical model scaffold to 3D-culture other anchorage-dependent cells for in-vitro tissue regeneration.

Enzyme-assisted self-assembly under thermodynamic control

The production of functional molecular architectures through self-assembly is commonplace in biology, but despite advances, it is still a major challenge to achieve similar complexity in the laboratory. Self-assembled structures that are reproducible and virtually defect free are of interest for applications in three-dimensional cell culture, templating, biosensing and supramolecular electronics. Here, we report the use of reversible enzyme-catalysed reactions to drive self-assembly. In this approach, the self-assembly of aromatic short peptide derivatives provides a driving force that enables a protease enzyme to produce building blocks in a reversible and spatially confined manner. We demonstrate that this system combines three features: (i) self-correction--fully reversible self-assembly under thermodynamic control; (ii) component-selection--the ability to amplify the most stable molecular self-assembly structures in dynamic combinatorial libraries; and (iii) spatiotemporal confinement of nucleation and structure growth. Enzyme-assisted self-assembly therefore provides control in bottom-up fabrication of nanomaterials that could ultimately lead to functional nanostructures with enhanced complexities and fewer defects.

Biocatalytic induction of supramolecular order

Supramolecular gels, which demonstrate tunable functionalities, have attracted much interest in a range of areas, including healthcare, environmental protection and energy-related technologies. Preparing these materials in a reliable manner is challenging, with an increased level of kinetic defects observed at higher self-assembly rates. Here, by combining biocatalysis and molecular self-assembly, we have shown the ability to more quickly access higher-ordered structures. By simply increasing enzyme concentration, supramolecular order expressed at molecular, nano- and micro-levels is dramatically enhanced, and, importantly, the gelator concentrations remain identical. Amphiphile molecules were prepared by attaching an aromatic moiety to a dipeptide backbone capped with a methyl ester. Their self-assembly was induced by an enzyme that hydrolysed the ester. Different enzyme concentrations altered the catalytic activity and size of the enzyme clusters, affecting their mobility. This allowed structurally diverse materials that represent local minima in the free energy landscape to be accessed based on a single gelator structure. Supramolecular gels show promise in diverse areas, including healthcare and energy technologies, owing to tunable properties that arise directly from the organization of their building blocks. Researchers have now been able to control this behaviour by combining enzymatic catalysis with molecular self-assembly. Although it seems counter-intuitive, gels that assembled faster showed fewer defects.

An investigation of the conductivity of peptide nanotube networks prepared by enzyme-triggered self-assembly

We demonstrate that nanotubular networks formed by enzyme-triggered self-assembly of Fmoc-L3 (9-fluorenylmethoxycarbonyl-tri-leucine) show significant charge transport. FT-IR, fluorescence spectroscopy and wide angle X-ray scattering (WAXS) data confirm formation of beta-sheets that are locked together viapi-stacking interactions. Molecular dynamics simulations confirmed the pi-pi stacking distance between fluorenyl groups to be 3.6-3.8 A. Impedance spectroscopy demonstrated that the nanotubular xerogel networks possess minimum sheet resistances of 0.1 MOmega/sq in air and 500 MOmega/sq in vacuum (pressure: 1.03 mbar) at room temperature, with the conductivity scaling linearly with the mass of peptide in the network. These materials may provide a platform to interface biological components with electronics.

Exploiting enzymatic (reversed) hydrolysis in directed self-assembly of peptide nanostructures

Enzyme-catalyzed reactions can be exploited to control molecular self-assembly under physiological conditions by converting nonassembling precursors into self-assembly building blocks. Two complementary approaches based on aromatic short-peptide derivatives that form molecular hydrogels are demonstrated. Firstly, it is shown that esterase-directed self assembly via hydrolysis of hydrophobic N-(fluorenyl-9-methoxycarbonyl) (Fmoc)-peptide methyl esters give rise to formation of transparent hydrogels composed of defined peptide nanotubes. The internal and external diameters of these tubes are highly tunable, depending on the amino acid composition and chain length of the building blocks. Secondly, protease-directed self-assembly of Fmoc-peptide esters is achieved via amide-bond formation (reversed hydrolysis) for combinations of Fmoc-threonine and leucine/phenylalanine methyl esters, producing fibrous hydrogels. Upon treatment with an esterase, these systems revert back to solution, thus providing a two-stage solution-gel-solution transition.

Sonication induced peptide-appended bolaamphiphile hydrogels for in situ generation and catalytic activity of Pt nanoparticles

The self-assembly of peptide based bolaamphiphiles is investigated upon sonication. We show the ability of peptide bolaamphiphiles 1 and 2 to form strong and rigid self-supporting hydrogels under physiological conditions upon sonication. The self-assembly of higher order supramolecular structures occurs through a synergic effect of hydrogen bonding and π-stacking interactions. Phenylalanine rich peptide bolaamphiphile 2 self-assembles into well-defined nanoribbons whereas tyrosine rich functional material 1 self-assembles into nanofibrillar structures that can be used as a template for in situ generation of platinum nanoparticles. Furthermore, hydrogenation reactions are catalyzed by in situ synthesized Pt nanoparticles such as p-phenylenediamine, which is prepared by the reaction of p-nitroaniline with Pt nanoparticles.

Enzyme-Activated Surfactants for Dispersion of Carbon Nanotubes†

N-Fluorenyl-9-methoxycarbonyl-protected amino acids are used as surfactants for carbon nanotubes and their interactions are modeled using quantum mechanical computations. These surfactants are then converted into enzymatically activated CNT surfactants that create homogeneous aqueous nanotube dispersions on-demand under constant and physiological conditions

Smart oligopeptide gels: in situ formation and stabilization of gold and silver nanoparticles within supramolecular organogel networks

Tripeptide with redox active chemical entities based smart organogels have been used for in situ formation and stabilization of gold and silver nanoparticles within the supramolecular gel networks and the gold nanoparticles are aligned in arrays along the gel nanofibers of peptide 1-toluene gels.

Lipase-catalyzed dissipative self-assembly of a thixotropic Peptide bolaamphiphile hydrogel for human umbilical cord stem-cell proliferation.

We report lipase-catalyzed inclusion of p-hydroxy benzylalcohol to peptide bolaamphiphiles. The lipase-catalyzed reactions of peptide bolaamphiphiles with p-hydroxy benzylalcohol generate dynamic combinatorial libraries (DCL) in aqueous medium that mimic the natural dissipative system. The peptide bolaamphiphile 1 (HO-WY-Suc-YW-OH) reacts with p-hydroxy benzylalcohol in the presence of lipase forming an activated diester building block. The activated diester building block self-assembles to produce nanofibrillar thixotropic hydrogel. The subsequent hydrolysis results in the dissipation of energy to form nonassembling bolaamphiphile 1 with collapsed nanofibers. The thixotropic DCL hydrogel matrix is used for 3D cell culture experiments for different periods of time, significantly supporting the cell survival and proliferation of human umbilical cord mesenchymal stem cells.

Photophysical study of a π-stacked β-sheet nanofibril forming peptide bolaamphiphile hydrogel

We describe the state of molecular self-assembly of a peptide based bolaamphiphile molecule using spectroscopic and microscopic techniques. The tryptophan and phenylalanine containing peptide bolaamphiphile forms a hydrogel upon sonication under physiological conditions. Sonication helps to reorient the peptide molecules by providing the required energy for the self-assembly process. The disassembly and self-assembly processes are influenced by various stimuli, including heating–cooling and shaking–rest methods. The extensive hydrogen bonding and π–π stacking interactions are responsible for the self-assembly process, which is confirmed by FT-IR, temperature dependent NMR and fluorescence spectroscopy studies. FT-IR and powder X-ray diffraction studies reveal that the gelator molecules self-assemble into an antiparallel β-sheet type structure. The TEM image of the hydrogel shows a well-defined amyloid-like nanofibrillar structure. The amyloid-like behaviour of the fibril forming peptide bolaamphiphile hydrogel is confirmed by ThT and Congo red binding studies. The effect of concentration, time and temperature on the self-assembly mechanism of the peptide bolaamphiphile hydrogel is investigated by time resolved fluorescence spectroscopy.