Peptide amphiphiles (PAs), peptides that self-assemble into hydrogels with a nanofibrous network, are interesting biomaterials due to their biocompatibility and biodegradability. Self-assembling peptide-based materials include a wide range of peptide motifs that form one-dimensional nanostructures in aqueous solution. Two different PAs are considered in this M.S. thesis work: lipidated peptides, and gas-releasing peptides (GRPs). These biomaterials have been developed to function as potential therapeutics that promote the growth of new blood vessels. The analyses conducted on the lipidated peptides, which were designed to include a peptide sequence that promotes angiogenesis, include cytotoxicity, viability, and tube formation assays. The GRPs were designed to release H2S, which is also capable of promoting angiogenesis. Several characteristic properties of the GRPs were analyzed, including morphology, mechanics, self-assembly, and gas release rates. Furthermore, cytotoxicity assays were conducted followed by the demonstration of gas uptake in endothelial cells. / Master of Science

Molecular self-assembly is the process of molecules able to associate into more ordered structures. Examples of self-assembling molecules is a class of ultrashort amphiphilic peptides with a distinct sequence motif, which consist of only three to seven amino acids. These peptides can self-assemble to form nanofibrous scaffolds, such as in form of hydrogels, organogels or aerogels, due to their amphiphilic structure which contains a dominant hydrophobic tail and a polar head group. Interestingly, these peptide scaffolds offer a remarkably similar fiber topography to that one found in collagen which is a dominant part of the extracellular matrix. The resemblance to collagen fibers brings a potential benefit in using these peptide scaffolds together with native human cells. Specifically, they can maintain high water content over 99 % weight per volume and are suitable for tissue engineering and regenerative medicine applications. Over the last decade, they have shown promising therapeutic potential in treating several diseases thanks to their high activity, target specificity, low toxicity, and minimal nonspecific and drug-drug interactions. This dissertation describes how to characterize and use ultrashort amphiphilic peptides for tissue engineering and biomedicine. The first chapter offers an overview of already reported self-assembling ultrashort peptides and their applications. As a proof-of-concept, ultrashort peptide scaffolds were used for osteogenic differentiation. Peptide nanoparticles were embedded into 5 peptide hydrogels with the goal to tune the stiffness of the peptide gels. Furthermore, the peptide scaffold was used for the generation of gold and silver nanoparticles after UV irradiation, which allowed the production of nanoparticles in the absence of any additional reducing agent. The mechanism of the generation of these nanoparticles was then investigated. The last chapter describes how tetrameric peptide solutions were utilized for 3D bioprinting applications. Compared to earlier reported self-assembling ultrashort peptide compounds, these tetrapeptides can form hydrogels at an extremely low concentration of 0.1% w/v in a relatively short time under physiological conditions. These promising findings suggest that the peptide solutions are promising bioinks for use in 3D bioprinting.

This dissertation describes the development of temperature and pH-responsive interfaces, where the emphasis is placed on tuning the responsivities within a physiological temperature range. This tuning is achieved through the utilization of polymeric building blocks, where each component is specifically synthesized to have a unique responsivity. The assembly of these components onto surfaces permits the fabrication of stimuli-responsive interfaces. In addition, this dissertation explores the use of a self-assembling peptide as a modular building block to modify the interface of hydrogel microparticles, resulting in the formation of a new biosynthetic construct. Hydrogels are three-dimensional, crosslinked polymer networks that swell in water. Over the years, hydrogels have been extensively explored as biomaterials due to their high water content, tunable mechanics, and chemical versatility. Two areas where hydrogels have received considerable interest are drug delivery and extracellular matrices. Unfortunately, developing structurally and functionally complex hydrogels from the top down is challenging because many parameters cannot be independently tuned in a bulk material. An alternative route would be to develop a library of building blocks, where each is tailored for a given function, and assemble these components into composite structures. The building block synthesized and utilized in this dissertation is a microgel. Microgels are a colloidal dispersion of hydrogel microparticles, ranging in size from 100 to 1000 nm in diameter. The microgels were prepared from environmentally responsive polymers, sensitive to both temperature and pH. Microgels have been used in the fabrication of polyelectrolyte layer-by-layer films, where the microgel serves as the polyanion and a linear polycation is used to “stitch” the particles together. In Chapters 3 and 4, stimuli-responsive interfaces are prepared from environmentally responsive microgel building blocks. In particular, Chapter 3 demonstrates tuning of the film response temperature by preparing several different microgels with differing ratios of two thermoresponsive polymers. Chapter 4 evaluates the influence of the pH environment on the thermoresponsivity of microgel films. While the pH environment was found to substantially affect some films, it is possible to prepare microgel films that behave independently of pH. The swelling/de-swelling of the films was also investigated by atomic force microscopy (AFM) as a function of both pH and temperature. It was determined that the AFM imaging parameters can drastically affect the measured film thicknesses (Appendix A) due to the soft, deformable nature of microgel films. The studies in these chapters illustrate the advantages of preparing composite structures from discrete components, where the functionality of the composite is dictated by the constituent particles. In Chapter 5, attention is placed on modifying the surface of microgel particles. Many of the traditional routes used to modify microgels involve the incorporation of co-monomers into the network or the addition of polymer shells. However, a new core/shell construct is presented, where a microgel core is coated with a self-assembling peptide shell. In this scenario, the peptide shell serves as a modular scaffold, where surface-localized functional groups can participate in reactions. Although there are still a number of questions remaining in regard to the assembly process and stability of the construct, initial experiments suggests that this is an interesting and promising structure to study. Finally, a discussion of future directions and possible experiments is provided in Chapter 6. Hopefully, this will serve as a guide for further exploration of the research presented herein. Microgels remain a rich class of materials to study and employ. While their synthesis is rather straightforward, their use often results in complex behavior and interesting phenomena. Understanding their behavior is a crucial step in realizing their full potential.

Self-assembling peptides have emerged as new nanobiomaterials and received considerable attention in the areas of nanoscience and biomedical engineering. In this category are ionic-complementary peptides, which contain a repeating charge distribution and alternating hydrophobic and hydrophilic residues in the amino acid sequence, leading to the unusual combination of amphiphilicity and ionic complementarity. Although their self-assembled nanostructures have been successfully applied as scaffoldings for tissue engineering, novel materials for regenerative medicine and nanocarriers for drug and gene/siRNA delivery, aspects of the assembly process remain unclear. Since many of these applications involve peptide-modified interfaces and surfaces, a better understanding and control of the peptide assembly on a surface are very crucial for future development of peptide-based applications in nano-biotechnology. This thesis contains two major parts: (i) fundamental study of the assembly of a model ionic-complementary peptide EAK16-II on surfaces and (ii) potential applications of such a peptide in surface modification and nanofabrication. In the fundamental study, EAK16-II assembly on negatively charged mica was first investigated via in-situ Atomic Force Microscopy (AFM). It was found that EAK16-II nanofiber growth on mica is surface-assisted and follows a nucleation and growth mechanism involving two steps: (i) adsorption of nanofibers and fiber clusters (from the bulk solution) on the surface to serve as the seeds and (ii) fiber elongation from the active ends of the seeds. Such a process can be controlled by adjusting the solution pH since it modulates the adsorption of the seeds and the growth rates. Unlike what is observed on mica, EAK16-II formed well-ordered nanofiber patterns with preferential orientations at angles of 60° or 120° to each other on hydrophobic highly ordered pyrolytic graphite (HOPG) surfaces, resembling the crystallographic structure of the graphite. Nanofiber formation on HOPG is also surface-assisted and adopts a nucleation and growth mechanism that can be affected by solution pH. The pH-dependent adsorption of peptides to HOPG is attributed to the resulting changes in peptide hydrophobicity. It was also found that EAK16-II assembly can be induced by the mechanical force of a tapping AFM tip. It occurs when the tip cuts the adsorbed EAK16-II nanofibers into segments that then serve as seeds for new nanofiber growth. This finding allows one to locally grow nanofibers at specific regions of the surface. The tip cutting has been combined with the effect that solution pH has on peptide assembly to develop a new AFM lithography method to fabricate local patterned peptide nanostructures on HOPG. To study the use of EAK16-II for surface modification applications, the wettability and stability of the peptide-modified surfaces were characterized. EAK16-II-modified mica becomes slightly hydrophobic as the water contact angle increases from <10° to 20.3 ± 2.9°. However, the hydrophobicity of the HOPG surface is significantly reduced, as reflected in a contact angle change from 71.2 ± 11.1° to 39.4 ± 4.3°. The EAK16-II-modified mica surface is stable in acidic solution, while the modified HOPG surface is stable in both acidic and alkaline solutions. The peptide-modified HOPG shows potential as a biocompatible electrode for (bio)molecular sensing. The ability of EAK16-II to form nanofibers on surfaces has also promoted research on peptide-based metallic nanowire fabrication. Our approach is to provide EAK16-II with metal ion binding ability by adding a GGH motif to the C-terminus. This new peptide EAK16(II)GGH has been found to form one-dimensional nanofibers while binding to Cu2+ ions. The dimensions of the nanofibers were significantly affected by the nature of the anions (SO42-, Cl- and NO3-) in the copper salt solution. This work demonstrates the potential usage of EAK16-II for nanowire fabrication.

Self-assembling peptides have emerged as new nanobiomaterials and received considerable attention in the areas of nanoscience and biomedical engineering. In this category are ionic-complementary peptides, which contain a repeating charge distribution and alternating hydrophobic and hydrophilic residues in the amino acid sequence, leading to the unusual combination of amphiphilicity and ionic complementarity. Although their self-assembled nanostructures have been successfully applied as scaffoldings for tissue engineering, novel materials for regenerative medicine and nanocarriers for drug and gene/siRNA delivery, aspects of the assembly process remain unclear. Since many of these applications involve peptide-modified interfaces and surfaces, a better understanding and control of the peptide assembly on a surface are very crucial for future development of peptide-based applications in nano-biotechnology. This thesis contains two major parts: (i) fundamental study of the assembly of a model ionic-complementary peptide EAK16-II on surfaces and (ii) potential applications of such a peptide in surface modification and nanofabrication. In the fundamental study, EAK16-II assembly on negatively charged mica was first investigated via in-situ Atomic Force Microscopy (AFM). It was found that EAK16-II nanofiber growth on mica is surface-assisted and follows a nucleation and growth mechanism involving two steps: (i) adsorption of nanofibers and fiber clusters (from the bulk solution) on the surface to serve as the seeds and (ii) fiber elongation from the active ends of the seeds. Such a process can be controlled by adjusting the solution pH since it modulates the adsorption of the seeds and the growth rates. Unlike what is observed on mica, EAK16-II formed well-ordered nanofiber patterns with preferential orientations at angles of 60° or 120° to each other on hydrophobic highly ordered pyrolytic graphite (HOPG) surfaces, resembling the crystallographic structure of the graphite. Nanofiber formation on HOPG is also surface-assisted and adopts a nucleation and growth mechanism that can be affected by solution pH. The pH-dependent adsorption of peptides to HOPG is attributed to the resulting changes in peptide hydrophobicity. It was also found that EAK16-II assembly can be induced by the mechanical force of a tapping AFM tip. It occurs when the tip cuts the adsorbed EAK16-II nanofibers into segments that then serve as seeds for new nanofiber growth. This finding allows one to locally grow nanofibers at specific regions of the surface. The tip cutting has been combined with the effect that solution pH has on peptide assembly to develop a new AFM lithography method to fabricate local patterned peptide nanostructures on HOPG. To study the use of EAK16-II for surface modification applications, the wettability and stability of the peptide-modified surfaces were characterized. EAK16-II-modified mica becomes slightly hydrophobic as the water contact angle increases from <10° to 20.3 ± 2.9°. However, the hydrophobicity of the HOPG surface is significantly reduced, as reflected in a contact angle change from 71.2 ± 11.1° to 39.4 ± 4.3°. The EAK16-II-modified mica surface is stable in acidic solution, while the modified HOPG surface is stable in both acidic and alkaline solutions. The peptide-modified HOPG shows potential as a biocompatible electrode for (bio)molecular sensing. The ability of EAK16-II to form nanofibers on surfaces has also promoted research on peptide-based metallic nanowire fabrication. Our approach is to provide EAK16-II with metal ion binding ability by adding a GGH motif to the C-terminus. This new peptide EAK16(II)GGH has been found to form one-dimensional nanofibers while binding to Cu2+ ions. The dimensions of the nanofibers were significantly affected by the nature of the anions (SO42-, Cl- and NO3-) in the copper salt solution. This work demonstrates the potential usage of EAK16-II for nanowire fabrication.

Hydrogels based on self-assembling peptides are believed to have potential for use in a wide range of biomedical and biodiagnostic applications. For many of these, control over various properties of the gels is essential for tuning the gels to fit certain constraints or requirements in terms physical properties such as diffusive properties and swelling. One important property to control for applications such as cell culture and drug delivery is its mechanical strength, and this study investigates three different strategies by which the individual peptide monomers can be modified in order to effect a change in the macromolecular self-assembled structure and therefore a bulk change in the mechanical stiffness. In chapter 4, two ionic-complementary octapeptides, FEFKFKFK and FEFQFKFK are described, with monomer charges of +2 and +1, respectively at physiological pH. FEFKFKFK was observed to form largely discrete fibrils, characteristic of similar systems, while FEFQFKFK formed fibril bundles – believed to be a limited form of an aggregation effect frequently seen in similar peptides with neutral charge. As a result of this structural change, FEFQFKFK was found to have values for the elastic and viscous moduli (which are often used to measure the ‘strength’ of a gel) between 5 and 10 times larger than those of FEFKFKFK at the same concentration. The same behaviour was seen in FEFKFKFK when the monomer charges were reversed by adjusting pH, suggesting that the monomer charge is indeed responsible for the bundling effect. In chapter 5, two branched peptides were designed and synthesized: KG17, with two arms consisting of self-assembling FKFEFKFK-motifs, and KG28 which had three such arms. Each branched peptide was doped into pure FKFEFKFK and the resulting gels investigated. While no obvious structural changes were observed for either dopant (save for a potential fibril parallelisation effect with KG17 observed in Small-Angle Neutron Scattering (SANS)), both were observed to increase the elastic and viscous moduli of the gels at overall peptide concentrations of 30 and 50 mg mL-1 (gels), but not at 10 mg mL-1 (viscous liquid). The most dramatic change was observed in the 50 mg mL-1 gels, suggesting that higher concentrations could enhance the effect of the dopants. In chapter 6, three thermo-responsive polymers (pTEGMA), of Degrees of polymerisation (DPs) 17, 47 and 142 were conjugated to CGFKFEFKFK and incorporated into a peptide hydrogel. Gels containing the non-conjugated versions of each polymer were also tested. While no changes in morphology were observed at the fibillar level, the polymer Lower Critical Solution Temperature (LCST) behaviour could be observed in SANS in all samples apart from the DP17 conjugate. However, in rheological tests gels doped with this conjugate appeared to show the strongest the elastic and viscous moduli. In general the conjugates appeared to increase the elastic and viscous moduli, particularly at temperatures above ca. 50°C. Rather than this being LCST behaviour, it was suggested that the polymers can act to enhance a natural thermo-response that was observed in the peptide, with the shortest polymer (DP17) experiencing the least steric hindrance and therefore having the strongest effect. It was postulated that this interaction could involve the screening of charge on the peptide fibril. Non-conjugated polymer appeared to have little effect on the mechanical properties, with elastic modulus values correlating strongly to the overall peptide concentration.

No description available.

An ideal drug delivery system should contain an appropriate therapeutic agent and biocompatible carrier. In this study, we investigated the ability of the all-complementary self-assembling peptide AC8 in stabilizing the anticancer compound and determined the in-vitro therapeutic efficacy of the peptide-mediated anticancer drug delivery. The all-complementary peptide AC8 was designed based on the amino acid pairing principle (AAP), which contains hydrogen bonding, electrostatic, and hydrophobic interaction amino acid pairs. AAP interactions make the peptide capable of self-assembling into β-sheet structure in solution in a concentration dependent manner. Peptide solution concentration is a key parameter in controlling the nanoscale assembling of the peptide. The critical assembly concentration (CAC) of the peptide was found ~ 0.01 mg/ml by several techniques. The all-complementary peptide AC8 was found to be able to stabilize neutral state of hydrophobic anticancer compound ellipticine in aqueous solution. The formation of peptide-ellipticine complex was monitored by fluorescence spectroscopy at different mass ratios of peptide-to-ellipticine. The anticancer activity of the complexes with neutral state of ellipticine was found to show great anticancer activity against two cancer cells lines, A-549 and MCF-7. This peptide-mediated anticancer delivery system showed the induction of apoptosis on cancer cells in vitro by flow Cytometry.

An ideal drug delivery system should contain an appropriate therapeutic agent and biocompatible carrier. In this study, we investigated the ability of the all-complementary self-assembling peptide AC8 in stabilizing the anticancer compound and determined the in-vitro therapeutic efficacy of the peptide-mediated anticancer drug delivery. The all-complementary peptide AC8 was designed based on the amino acid pairing principle (AAP), which contains hydrogen bonding, electrostatic, and hydrophobic interaction amino acid pairs. AAP interactions make the peptide capable of self-assembling into β-sheet structure in solution in a concentration dependent manner. Peptide solution concentration is a key parameter in controlling the nanoscale assembling of the peptide. The critical assembly concentration (CAC) of the peptide was found ~ 0.01 mg/ml by several techniques. The all-complementary peptide AC8 was found to be able to stabilize neutral state of hydrophobic anticancer compound ellipticine in aqueous solution. The formation of peptide-ellipticine complex was monitored by fluorescence spectroscopy at different mass ratios of peptide-to-ellipticine. The anticancer activity of the complexes with neutral state of ellipticine was found to show great anticancer activity against two cancer cells lines, A-549 and MCF-7. This peptide-mediated anticancer delivery system showed the induction of apoptosis on cancer cells in vitro by flow Cytometry.

No description available.