Peptides and peptide derivatives have shown great scope as biomaterials and for biomedicaltherapy application. It has been demonstrated that classes of these peptides can form fibrillar hydrogels making them a good candidate for ECM mimics. In particular, the ionic complementary peptides, composed of alternating hydrophobic and hydrophilic amino acidshave been reported as successful cell scaffolds. The simple structure of such ionic complementary peptides is generally seen to spontaneously self-assemble into β-sheet richfibrils in the presence of water. The highly aqueous environment, along with the inter meshing of fibres, results in an architecture akin to the natural ECM of the body, making peptide hydrogels highly suitable as cell culture scaffolds. The structure of such hydrogels, usually comprising 8-32 amino acids, has been widely reported as easily modifiable, thus, allowing for control of the final material properties. This study explores the potential use of a range of ionic-complementary peptides for the culture of primary bovine chondrocytes. Modifications and additions to peptide sequence, such as charge and amino acid substitution, were investigated. In all studies only 1 design parameter (sequence, charge etc.) was varied, to allow for better understanding of the effect of materials properties upon cell response. The encapsulation of primary bovine chondrocytes was undertaken, with the aim of providing a suitable cell scaffold capable of maintaining chondrocyte viability and function in vitro. Despite in vivo work being beyond the scope of this thesis, the properties of the hydrogel scaffold were designed with final aim of being suitable for use with matrix associated autologous chondrocyte implantation (MACI) in clinical therapy.

Peptides are emerging as promising therapeutics due to their inhibitory affinity towards protein-protein interactions (PPI). However, peptides have been limited mainly by their poor bio-stability and lack of cell permeability. Efforts to generate drug-like peptides have led to the development of macrocyclic peptides, which exhibit improved stability. Yet, most macrocyclic peptides still require the assistance of a cell penetrating peptide (CPP) for cellular entry. High throughput technologies have been exceptional tools for the discovery of peptides to interrupt PPIs. This work details the recent advancements we have made to improve our high throughput technique, mRNA display, to yield more therapeutically relevant peptides to inhibit PPIs. Our advancements are focused on cell permeability, protease stability, and secondary structure for enhanced affinity. Here we develop and optimize a cyclic CPP that can be included in future mRNA display libraries. We also tested the ability of our CPP to deliver an impermeable peptide cargo into cells. We rationally designed and tested linear and cyclic peptides to improve affinity to the BRCA1 protein. We used computational work to complement our experimental results for our CPPs and BRCA1 inhibitors. We examined peptides that arose from a library containing a mix of linear, monocyclic, and bicyclic peptides constructed using orthogonal cyclization chemistries. We rationally designed cyclic peptides and tested their affinity against Hsp70. We proposed a novel selection strategy to find optimal CPP motifs.

acase@tulane.edu / Development of antimicrobial peptides (AMPs) as next generation clinical antibiotics has been a pursuit of the scientific community for several decades. AMPs are attractive drug candidates because of their potent antibacterial activity and a low propensity for eliciting antibiotic resistant bacterial phenotypes. However, despite substantial efforts and myriad development approaches, AMPs have yet to make inroads in the clinic due to toxicity concerns and activity loss in vivo. We hypothesized that eukaryotic cytotoxicity and antibacterial activity loss are intricately related in that peptide-induced tissue or host cell damage corresponds to depletion of free peptide available to target bacterial cells. Using human red blood cells (RBCs) as a model eukaryotic cell, we demonstrate that a cross-section of AMPs lose appreciable antibacterial activity when preincubated with concentrated eukaryotic cells (1x109 red blood cells/mL) and that this behavior can be explained by plasma membrane binding. To approach this problem in a unique manner, we synthesized a combinatorial peptide library based on the potent AMP, ARVA, and screened the library for activity in the presence of concentrated RBCs. We isolated nine unique, but similar sequences from the screen. During the screening program, we discovered that RBC-peptide interactions lead to peptide degradation through the release of cytosolic proteases. We used this knowledge to design a consensus sequence based on the nine peptides isolated from the library screen and synthesized it using only D-isomer amino acids. The novel peptide displays excellent antimicrobial activity against several human pathogens in the presence and absence of concentrated RBCs, has reduced toxicity towards eukaryotic cells, and is not susceptible to cleavage by cellular proteases. We attempted to use this peptide, D-NOGCON, to combat P. aeruginosa in a mouse model of acute pneumonia, but were unable to ameliorate the negative outcomes associated with infection. We ultimately suggest alternative models of bacterial infection in which the peptide may be more effective and future approaches to further refining the sequence of D-NOGCON. / 1 / Charles Starr

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Mitochondria have evolved to play a vital role in both the life and death of a eukaryotic cell, through involvement in numerous cellular functions, such as the proficient production of energy from ATP biosynthesis and the regulation of programmed cell death. As a result, dysfunction in the biochemical processes housed within this organelle is implicated in diverse diseases, including cancer, diabetes, and neurodegenerative disorders. Advancing mitochondrial medicine by probing the subcellular biochemistry or targeting therapeutics into this organelle has motivated the development of effective mitochondrial delivery vectors. Thus, the rational design of novel mitochondrial-specific molecules, inspired by the success of cell-penetrating peptides, is described, whereby short synthetic peptides that retain the ability to traverse the plasma membrane, yet with mitochondrial-specificity were engineered. By modulating the overall physicochemical properties, through substitutions with both natural and synthetic amino acids, and monitoring the intracellular localization by confocal fluorescence microscopy, the requisite thresholds for achieving mitochondrial accumulation with a cationic peptide were elucidated. These systematic studies led to the development of a novel class of cationic yet lipophilic peptides, referred to as mitochondria-penetrating peptides (MPPs), which are readily cell permeable and preferentially localize into the mitochondria of living mammalian cells. The mechanisms of cellular uptake and mitochondrial matrix accumulation were investigated and the results from these studies suggest that MPPs utilize the negative membrane potential across these biological membranes to drive translocation. In addition, the effects of various chemical perturbations on the cellular and mitochondrial uptake, such as sequence, structure of the cation moiety, and chirality, were examined. The information obtained from these studies provided insight into the important features of these peptides and led to the design of an optimized molecule displaying pyridinium salt side chains. Moreover, MPPs were shown to be effective mitochondrial delivery vectors for diverse and bioactive small molecule cargo. In conclusion, the extensive biological and chemical characterization of MPPs revealed the importance of balancing the opposing characteristics of positive charge and lipophilicity to attain preferential sequestration into mitochondria, as well as provided evidence that these peptides will be suitable as mitochondrial delivery vectors.

Mitochondria have evolved to play a vital role in both the life and death of a eukaryotic cell, through involvement in numerous cellular functions, such as the proficient production of energy from ATP biosynthesis and the regulation of programmed cell death. As a result, dysfunction in the biochemical processes housed within this organelle is implicated in diverse diseases, including cancer, diabetes, and neurodegenerative disorders. Advancing mitochondrial medicine by probing the subcellular biochemistry or targeting therapeutics into this organelle has motivated the development of effective mitochondrial delivery vectors. Thus, the rational design of novel mitochondrial-specific molecules, inspired by the success of cell-penetrating peptides, is described, whereby short synthetic peptides that retain the ability to traverse the plasma membrane, yet with mitochondrial-specificity were engineered. By modulating the overall physicochemical properties, through substitutions with both natural and synthetic amino acids, and monitoring the intracellular localization by confocal fluorescence microscopy, the requisite thresholds for achieving mitochondrial accumulation with a cationic peptide were elucidated. These systematic studies led to the development of a novel class of cationic yet lipophilic peptides, referred to as mitochondria-penetrating peptides (MPPs), which are readily cell permeable and preferentially localize into the mitochondria of living mammalian cells. The mechanisms of cellular uptake and mitochondrial matrix accumulation were investigated and the results from these studies suggest that MPPs utilize the negative membrane potential across these biological membranes to drive translocation. In addition, the effects of various chemical perturbations on the cellular and mitochondrial uptake, such as sequence, structure of the cation moiety, and chirality, were examined. The information obtained from these studies provided insight into the important features of these peptides and led to the design of an optimized molecule displaying pyridinium salt side chains. Moreover, MPPs were shown to be effective mitochondrial delivery vectors for diverse and bioactive small molecule cargo. In conclusion, the extensive biological and chemical characterization of MPPs revealed the importance of balancing the opposing characteristics of positive charge and lipophilicity to attain preferential sequestration into mitochondria, as well as provided evidence that these peptides will be suitable as mitochondrial delivery vectors.

Self-assembling peptides have emerged as new nanobiomaterials and received considerable attention in the areas of nanoscience and biomedical engineering. One important type is the ionic-complementary peptide, which contains special patterns of positive and negative charge distribution. This thesis explores the application of this special type of peptides for the modification of electrode surfaces. The ionic-complementary peptide modified electrode was then further used to immobilize biologically active molecules, glucose oxidase in the present case, to construct a biosensor. There are two major parts in this thesis. In the first part, an ionic-complementary peptide, EFK16-II, was used to modify a highly ordered pyrolytic graphite (HOPG) electrode surface. The nanofibre structure of the self-assembling peptide on the electrode surface was characterized by atomic force microscopy (AFM). Attenuated total reflection fourier transform infrared sectroscopy (ATR-FTIR) spectra showed that upon addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EFK16-II molecules tend to be cross-linked among themselves. Cross-linking of the peptide diminishes the number of carboxyl groups available for immobilizing a sensing enzyme, i.e., glucose oxidase (GOx). A simple method based on pre-mixing the carbodiimide and GOx was developed; it inhibited peptide cross-linking and significantly improved enzyme immobilization. Biosensors constructed in this way showed increased overall signal intensity and a much higher sensitivity at 4.94mA M-1 cm-2, a six-fold increase compared to the previously-reported peptide-modified electrodes. In the second part, another ionic-complementary peptide, EAK16-II, was used to modify the HOPG electrode. AFM images showed that EAK16-II formed well-ordered nanofibre patterns on the electrode surface. The redox couple Fe(CN)63-/4- was used as a probe to detect the electrochemical properties of the EAK16-II modified electrode. The results showed that the electron transfer at the electrode surface does not change much before and after modification. GOx was immobilized onto the EAK16-II modified HOPG and showed a good response to the concentration change of glucose. Similar to the EFK16-II, inter- or intro-peptide cross-linking also occurs when the solution containing EDC and sulfo-NHS was injected onto EAK16-II modified electrode. The same method as in the first part was applied here to prevent peptide cross-linking. The sensitivity was improved from 0.53mA M-1cm-2 to 2.4mA M-1cm-2. A proposal for constructing a reagentless biosensor by immobilizing both enzyme and mediator onto the electrode was made. However, the results indicated that the mediator, ferrocene carboxylic acid (FCA), was not stable on the surface after being immobilized. A redox protein, cytochrome c (Cyt c), was also immobilized onto an EAK16-II modified electrode. Direct electron transfer (DET) between the redox center of Cyt c and the electrode was observed. However, cyclic voltammetry results indicated that the peptide did not help improve the DET of modified Cyt c. The results presented here demonstrate significant potential for ionic-complementary peptides for constructing electrochemical biosensors.

Self-assembling peptides have emerged as new nanobiomaterials and received considerable attention in the areas of nanoscience and biomedical engineering. One important type is the ionic-complementary peptide, which contains special patterns of positive and negative charge distribution. This thesis explores the application of this special type of peptides for the modification of electrode surfaces. The ionic-complementary peptide modified electrode was then further used to immobilize biologically active molecules, glucose oxidase in the present case, to construct a biosensor. There are two major parts in this thesis. In the first part, an ionic-complementary peptide, EFK16-II, was used to modify a highly ordered pyrolytic graphite (HOPG) electrode surface. The nanofibre structure of the self-assembling peptide on the electrode surface was characterized by atomic force microscopy (AFM). Attenuated total reflection fourier transform infrared sectroscopy (ATR-FTIR) spectra showed that upon addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EFK16-II molecules tend to be cross-linked among themselves. Cross-linking of the peptide diminishes the number of carboxyl groups available for immobilizing a sensing enzyme, i.e., glucose oxidase (GOx). A simple method based on pre-mixing the carbodiimide and GOx was developed; it inhibited peptide cross-linking and significantly improved enzyme immobilization. Biosensors constructed in this way showed increased overall signal intensity and a much higher sensitivity at 4.94mA M-1 cm-2, a six-fold increase compared to the previously-reported peptide-modified electrodes. In the second part, another ionic-complementary peptide, EAK16-II, was used to modify the HOPG electrode. AFM images showed that EAK16-II formed well-ordered nanofibre patterns on the electrode surface. The redox couple Fe(CN)63-/4- was used as a probe to detect the electrochemical properties of the EAK16-II modified electrode. The results showed that the electron transfer at the electrode surface does not change much before and after modification. GOx was immobilized onto the EAK16-II modified HOPG and showed a good response to the concentration change of glucose. Similar to the EFK16-II, inter- or intro-peptide cross-linking also occurs when the solution containing EDC and sulfo-NHS was injected onto EAK16-II modified electrode. The same method as in the first part was applied here to prevent peptide cross-linking. The sensitivity was improved from 0.53mA M-1cm-2 to 2.4mA M-1cm-2. A proposal for constructing a reagentless biosensor by immobilizing both enzyme and mediator onto the electrode was made. However, the results indicated that the mediator, ferrocene carboxylic acid (FCA), was not stable on the surface after being immobilized. A redox protein, cytochrome c (Cyt c), was also immobilized onto an EAK16-II modified electrode. Direct electron transfer (DET) between the redox center of Cyt c and the electrode was observed. However, cyclic voltammetry results indicated that the peptide did not help improve the DET of modified Cyt c. The results presented here demonstrate significant potential for ionic-complementary peptides for constructing electrochemical biosensors.

Piscine nodaviruses are members of genus Betanodavirus, which infects a large number of species of fish and causes massive mortality in larvae and juveniles. The disease causes great economic losses in aquaculture and sea-ranching. Virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were employed to screen the consensus sequences of paratopes by the phage display peptide technology. Using two biopanning methods to screen phages, their binding efficiency was examined by ELISA. The effects of treatment by different blocking buffers on the ELISA assays and the effects of the ELISA plates exposed to UV light were tested. The display phages with high binding efficiency were sequenced after the inserted fragment by PCR amplification. With NCBI BLAST, they were closely related to the claudin family and the consensus sequences were on the region of EL1 or EL2. Few non-claudin sequences demand further comparison with other kinds of receptors to ascertain the characters of these sequences.

Thesis (doctoral)--Christian-Albrechts-Universität zu Kiel, 2003.