In recent years a great deal of research has focussed on understanding and exploiting self assembling peptides as they form fibrillar hydrogels for use in a variety of different applications, such as tissue engineering and drug delivery. A particular class of such peptide systems are ionic-complementary peptides, composed of alternating hydrophobic and hydrophilic amino acids. Their simple structure is generally seen to assemble into β sheet rich fibrils, and easy modification of the primary structure is possible to allow the inclusion of recognition motifs tailored for a specific use. This can be done simply via physical mixing. To maximise the potential of such systems it is important to understand the interactions that govern the self-assembly behaviour. Here a variety of different peptides have been studied to elucidate control of peptide conformation and fibril morphology. The ability to easily tune the mechanical strength of the hydrogel has been explored by mixing peptide systems. The peptide FEFEFKFK (FEKII) was seen to assemble into β sheet rich fibrils of ~3 nm in diameter. Control of pH and hence the charge state of the E and K side chains altered sample properties. Gelation at pH 2.8 occurred at a concentration between 20 30 mg ml 1. At pH 4, 5 and 10 where the peptide has a lower net charge gelation was lowered to ~10 mg ml 1. Mechanical properties varied with G' values of 20-1200 Pa as pH was altered. Stronger gels were formed with lower net peptide charge. Hierarchical fibre assembly was observed for positively charged peptides, with fibres forming from lateral association of fibrils. Negatively charged peptides at pH 10 showed no such hierarchical assembly, and lower fibril persistence length. This was related to the change in charge along the fibril structure. At pH 7, where the peptide has no net charge, precipitation occurred. This showed a net charge was required on the peptide to disperse fibrils and prevent aggregation. The work showed the importance of ionic-interactions in determining both network morphology and bulk properties, and also elucidated control of such behaviour. AEAEAKAK (AEKII) was shown to assemble into α helix fibres. Alanine (A) is less hydrophobic than F, and is a known helix former. The role of F and A in assembly was assessed by the design of peptides FEAEFKAK (FAIEKII) and FEFEAKAK (FAIIEKII). Mixing A with F disrupted the peptides' ability to form a β sheet network by lowering the driving force for assembly given by the F residues. Trace amounts of β sheet were observed at low concentration, but at a critical concentration β sheet content increased and gelation occurred. This was found to be pH dependent. FAIEKII formed β-sheet fibrils at a lower concentration than FAIIEKII. While FAIEKII was able to assemble into different fibril structures, FAIIEKII showed no specific aggregation. This not only highlighted the importance of Hydrophobicity as a key driving force to assembly but also how the grouping of these amino acids in the primary sequence can determine the overall assembly characteristics of the peptide. The peptides FEFEFKFKGGFEFEFKFK (FEKII18-1) and FEFEFKFKGGFKFKFEFE (FEKII18-2) were designed to co-assemble with FEKII. Individually both peptides were seen to assemble into β sheet fibrils. FEKII18-1 formed fibrils of 2.3 3.1 nm in size, a result of folding along the chain caused by intra molecular attractive ionic interactions. FEKII18-2 formed larger fibrils of 4.4 5.2 nm from a straightened peptide chain given by the change in charge distribution. When co assembled with FEKII mechanical properties were enhanced, with G' increasing from 40 Pa at 20 mg ml 1 to 2400 Pa, depending on the concentration of FEKII18-1/FEKII18-2 added to the system. This was a result of these peptides providing fibril connections acting as cross links. This work has detailed control over the assembly process via peptide conformation and fibril interactions and the effect this has on overall macroscopic sample properties. This is vital in determining the viability of such systems in various biomedical applications.

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Stimuli-responsive polymers and self-assembling peptides represent two classes of materials with interesting properties and great potential to be used as biomaterials. The conjugation of polymer with peptide offers a way to combine the controlled chemical, mechanical, and thermal properties of polymer with the functionality of designed bioactive group. Pure hybrid materials with the characteristics of individual components or systems containing hybrid materials became attractive for applications in drug delivery and tissue engineering. This work focused on systems where the thermo-responsive properties of a polymer were combined with the gelling properties of two different ionic-complementary peptides via conjugation. The prototypical thermo-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) was chosen due to its lower critical solution temperature (LCST) ~32°C being close to body temperature. Ionic-complementary oligo-peptides, containing the alternating hydrophobic/hydrophilic and charged/uncharged amino acids, phenylalanine (F), glutamic acid (E) and lysine (K), were selected as they are known to form β-sheet rich fibrillar networks at low concentrations. Two peptide sequences with different charge distribution were chosen: FEFEFKFK and FEFKFEFK which form self-supporting gels at ~17 and 10 mg ml-1 respectively. Polymer-peptide conjugates were used to confer self-assembling and thermo-responsive behaviour to the system.Thermo-responsive PNIPAAm-rich hydrogels were obtained by targeting different degrees of functionalisation of PNIPAAm with the self-assembling peptides. Two series of such systems were prepared by using either a thiol-modified FEFEFKFK or a thiol-modified FEFKFEFK peptide as the chain-transfer agent in the free radical polymerisation of NIPAAm. The resulting polymer/conjugate mixtures were studied by proton nuclear magnetic resonance (1H NMR). The polymer/conjugate ratios were calculated and showed that the conjugate fraction in the mixtures increased with increasing concentration of peptide used for the polymerisation. Static light scattering (SLS) and viscometry showed the aggregation of the polymer/conjugate mixtures presumably due to the presence of peptide. The values from gel permeation chromatography (GPC), which were mostly attributed to the unconjugated polymers, were higher than those obtained from 1H NMR and centrifugation for the conjugates. The polymer/conjugate mixtures formed self-supporting gels where the critical gelation concentration decreased with increasing conjugate content. Oscillatory rheology experiments confirmed gels had formed and revealed that their elastic modulus, G' varied from ~ 10 to 400 Pa depending on the sample. TEM and AFM studies proved the formation of β-sheet fibres of ~ 4.5 ± 1.5 nm in diameter. The PNIPAAm-rich hydrogels were also characterised by micro DSC to reveal their thermo-responsiveness and phase separation and showed the LCST at ~ 30°C. The results of the study showed that varying the peptide sequence did not have an effect on thermal, mechanical or morphological properties of the hydrogels. By exploiting the self-assembly of the ionic-complementary peptides, it was possible to create PNIPAAm-rich, thermo-responsive hydrogels with controllable properties.Further in the study pure PNIPAAm-FEFEFKFK conjugate was incorporated into the FEFEFKFK peptide matrix to create peptide-rich thermo-responsive composite gels. Two series of the composite gels were prepared by varying separately the peptide matrix and polymer-peptide conjugate concentration. Micro DSC measurements revealed an endothermic peak at ~ 30ºC characteristic of the LCST of PNIPAAm. Oscillatory rheology studies showed that the composite gels became stronger with increasing conjugate concentration (G' ~ 20 - 200 Pa). Network morphology was studied by SANS. Using contrast variation and contrast matching techniques it was possible to distinguish between the peptide fibres and the PNIPAAm chains. Below and above the LCST the scattering curves showed a q-1 behaviour which is typical of rod-like objects. TEM and AFM also proved the formation of fibres of ~4.0 ± 0.8 nm and ~4.5 ± 1 nm respectively. AFM studies showed that the fibres of the composite gels were decorated with polymer chains. The thermo-responsiveness and the gelation properties of these conjugate-based scaffolds have potential for use as drug delivery vehicles or tissue engineering scaffolds.

The present thesis entitled “Design, Synthesis and Characterization of Novel Nanomaterials” is divided into five chapters, staring with a general introduction. The remaining chapters focus on four different areas/projects that I have worked on. Chapter 1: Introduction to nanomaterials This chapter reviews the basic concepts of nanomaterials and their fabrication methods. Nanomaterials are defined as materials whose dimensions (at least one) are below 100 nm. One of the most exciting aspects of nanomaterials is that their properties may differ significantly from those of the corresponding bulk materials. Nanomaterials fabrication methods can be broadly classified according to whether the assembly follows either i) the bottom-up approach or ii) the top-down approach. These methods have been discussed with various examples including the self-assembly of proteins, peptides and small molecules. In the top-down approach synthetic procedures for Graphene Oxide and its application are discussed. All characterization techniques that are used for characterizing the nanomaterials are also described briefly. Chapter 2 Section A: Self-assembly of 1-Hydroxy benzotriazole (HOBT) in water The studies presented in Chapter 2 identifies HOBT as the smallest non-peptide building block that spontaneously self-assembles into hollow micro tubular structures upon evaporation of water. The tubes form under ambient conditions by rolling over of crystalline sheets of HOBT. The packing of HOBT in the tubes seem to be predominantly driven by intermolecular π-stacking interactions between the aromatic rings of HOBT. These structural and packing patterns are similar to those found in nanotubes formed by the self-assembly of peptides and other larger molecules. The cavities of these thermolabile microtubes act as molds for casting gold nanoparticles for the synthesis of gold microrods with monodisperse dimensions. The non-reacting inner surfaces of the cavities have been used to uniquely synthesize R6G-functionalized gold microrods. With these features, HOBT is an important novel non-peptide building block for accessing micro and nanometric materials for their applications in medicine, biology and molecular biotechnology. Section B: Controlling the orientation of self-assembly of HOBT microtubes The studies presented in this chapter address the self-assembly of HOBT into microtubular structures in different solvents of varying polarities (H2O and DCM:MeOH) to understand the role of solvent volatility and its direction on the orientation of the HOBT microtubes. HOBT self-assembles from DCM:MeOH mixtures in its bipolar canonical form and is coordinated with its water of hydration, similar to its crystals obtained from water. FTIR and TGA data shows that MeOH is also integrated with the microtubes. We observe for the first time that the orientation of microtubular self-assembly is controlled in the direction of evaporation of the solvent. We demonstrate further this feature by controlling the orientation of HOBT self-assembly in exclusively vertical direction through controlled vertical evaporation of the solvent mixture DCM:MeOH (9:1). Additionally, the unique transition between vertical and horizontal orientations for self-assembled HOBT microtubes is achieved by simple change of solvation between aqueous and organic solvents. These results reveal a dynamic relationship between the rate of evaporation of solvent and the rates of formation of different self-assembled morphologies. The rate of evaporation of the solvent primarily governs the rate of formation of the tubes, rather than their orientations in three dimensions. Chapter 3: Chemical origins of debris in Graphene Oxide (GO) This chapter is focused on the investigation of the carbonyl rich fragments arising from GO and provides an understanding of its formation. The fragments are expelled from GO due to an uncontrolled nucleophile driven reaction in aqueous medium leaving the holes on the sheet. These fragments are carbonyl rich small (5 ± 2 nm) nonaromatic molecules that form as by-products of oxidative chemical reactions that occur at the sp3 clusters on the basal surface of GO sheets when they are treated with nucleophilic bases under aqueous conditions. The structure and size of the debris, and hence that of the hole, depend on the size of the sp3 cluster on the sheet. These debris fall out of the GO sheet surface, leading to formation of nanometer sized holes. Formation of debris and hence the holes can be avoided by using anhydrous polar solvents. This work sheds new light on the fundamental structure of GO and the prevention of debris from it during redox reactions enabling better control over functionalization of the GO surface. Chapter 4: Measurement of mechanical properties of polypeptide fragment from Insulin like growth factor binding protein nanotubes by the Peak Force QNM method This chapter describes the discovery of Polypeptide fragment from an IGFBP-2. This fragment self-assembles spontaneously and reversibly into nanotubular structures under oxidizing conditions. These nanotubes were characterized by using Transmission electron microscopy. Notably as compared to the monomer, an increase in intrinsic fluorescence upon self-assembly. The thermal stability of these nanotubes is realized form the fluorescence studies. Peak Force Quantitative Nanomechanical Mapping method of AFM was used to measure the Young’s modulus of the nanotubes. These nanotubes were found to have Young’s modulus value of ~10 Gpa, which is comparable to those of bones presumably due to intermolecular disulphide bonds. These nanotubes will have potential applications in tissue engineering. Chapter 5: Probing the pathways of n→π* interaction in peptides This chapter deals with the theoretical study of n→π* interaction in designed peptidomimetics. The n→π* interaction involves the delocalization of the lone pair of the donor group into the antibonding orbital (π*) of a carbonyl group. However despite beeing extensively studied there exists a debate over the validation of these n→π* interaction which is reminiscent to Bürgi and Dunitz trajectory. This chapter present our findings that peptidomimetics containing the 5,6-dihydro-4H-1,3-oxazine (Oxa) and 5,6-dihydro-4H-1,3-thiazine (Thi) functional groups at the C-terminus of Pro selectively stabilizes the cis conformer by reverse n→πi-1* interaction. These systems have been used to study the n→πi1* interaction using Natural Bond Orbital (NBO) method. Our study reveals that the energetically most favorable trajectory of a nucleophile for a favorable n→π* interaction presumably to facilitate the overlap between the lonepair of the nucleophile and the antibonding orbital of the carbonyl group. The geometrical requirements for the optimum n→π* interaction depends on the relative orientations of the orbitals that are involved. This study has implications for more accurately identifying long distant n→π* interaction.