The specific manner in which ions associate with a peptide surface is a subject of much research. The models currently proposed tend to rely either on computational results from overly simplified systems, or on observations of bulk solution behavior not applicable to peptide solvation. Herein we demonstrate a new platform for directly measuring specific ion interactions with peptides and use a pair of highly conserved model peptides to investigate specific mechanisms by which ions interact with a peptide surface. A system for investigation of charge selective ion-peptide interactions using a conical glass nanopore was designed. The glass nanopore was coated using layer-by-layer depositions of poly(diallyldimethylammonium) chloride and sodium poly(styrenesulfonate) to control the size and charge selectivity of the nanopore. The tip of this nanopore probe was encapsulated in a 5% agarose gel to prevent peptide fouling. This probe was then used to measure the partitioning of cations to or from the surface of two model peptides: nonpolar V5-120 and positively charged KV6-112 elastin-like polypeptide (ELP). Partitioning was measured by clamping the current through the pore at zero amps and measuring the resulting potential across the nanopore. This potential was used to determine the bulk concentration of electrolyte in a 1 mg/mL peptide in 0.1 M electrolyte solution. Measurements were made with a patch clamp using chloride salts with the cations potassium, lithium, cesium, ammonium, and guanidinium at both room temperature and in an ice bath to ensure that the peptides were in their unfolded state and thus that all possible binding sites were exposed to the solution. All salts were observed to partition to the peptide surface with much less affinity than water, resulting in an increase in the bulk electrolyte concentration with the exception of ammonium, which showed a greater affinity than water for the KV6-112 ELP in the ice bath measurements. These results demonstrate that cations do not favorably partition to nonpolar or cationic peptide surfaces.

Background: Despite the availability of a yearly vaccine and antivirals, the incidence of influenza infections remains high. The genome of the influenza virus can mutate rapidly, therefore novel influenza strains that may be resistant to the current vaccine or antivirals frequently enter the population. Because of the long production time necessary to produce a vaccine, new antivirals must be created to combat early stages of influenza outbreaks. The most effective antivirals will target a highly conserved and essential stage of virus replication. The influenza RNA-dependent RNA polymerase is a heterotrimeric complex composed of three subunits: PA, PB1, and PB2. The three components of the polymerase interact through well-defined domains and are essential for viral replication. Previously, influenza replication has been inhibited using a small synthetic peptide that mimics the interaction domain between PA and PB1 and inhibits the formation of the heterotrimeric complex. Problem and Hypothesis: Although the peptide could inhibit influenza replication, synthetic peptides are costly to produce and are not a viable option for large-scale production. This problem can often be overcome by attaching the peptide to a highly soluble carrier protein. We hypothesize that influenza replication can be inhibited by attaching a peptide, that mimics the binding domain between the PA and PB1 subunits, to a human elastin-like polypeptide (ELP) carrier protein. Methods and Results: The peptide and a nuclear localization sequence was genetically linked to a maltose binding protein (MBP) or ELP carrier protein. The MBP construct was purified by affinity chromatography using FPLC. A high yield of the ELP construct was obtained using inverse transition cycling, a method unique to ELPs because of their temperature-dependent solubility. The ELP construct was designed to be soluble at physiological temperature to limit cellular toxicity due to protein aggregation. The cytotoxicity of the ELP construct was assessed by monitoring the growth of A549 cells, a human lung epithelial cell line. The ELP construct did not have any adverse effects on A549 cell growth. Both constructs could localize to cell nuclei using their respective nuclear localization sequences and could also interact with the PA subunit, demonstrating their potential to inhibit influenza replication. Despite this, only the MBP construct was able to inhibit the replication of influenza. The MBP construct could inhibit the replication of both the H1N1 and H3N2 subtypes of influenza, indicating the recombinant protein had cross-strain activity. Conclusion: Linking a small peptide to carrier protein can result in high protein yields, however a carrier protein must be chosen that will maintain the peptides’ therapeutic activity. In this study, a small anti-influenza peptide inhibited influenza replication when attached to an MBP carrier protein, however was not able to inhibit influenza replication when attached to an ELP carrier protein. Although the peptide was ineffective when attached to this particular ELP carrier protein, different ELP proteins of various lengths and compositions may still be effective carrier proteins for an antiviral peptide. / Thesis / Master of Science (MSc) / The influenza virus causes seasonal outbreaks each year and can have life-threatening symptoms in the young and elderly. In addition, it can rapidly mutate through antigenic drift; therefore, a new vaccine is required each year. Pandemic influenza strains can enter the population when the virus undergoes genetic reassortment by antigenic shift. However, it can take a significant amount of time to formulate a vaccine against pandemic influenzas, which means antiviral drugs are often used as the first line of defense. New antivirals to treat influenza must be developed because resistance to the current influenza antivirals has steadily increased. In this work, we developed an antiviral peptide to disrupt a critical interaction in the influenza RNA-dependent RNA polymerase and inhibit virus replication. This peptide was previously conjugated to an E. coli MBP carrier protein, which would likely not be compatible in vivo. This thesis focused on attaching the antiviral peptide to an elastin-like polypeptide protein, which mimics human tropoelastin, and should be non-immunogenic in humans.

<p>The design and application of effective drug carriers is a fundamental concern in the delivery of therapeutics for the treatment of cancer and other vexing health problems. Traditionally utilized chemotherapeutics are limited in efficacy due to poor bioavailability as a result of their size and solubility as well as significant deleterious effects to healthy tissue through their inability to preferentially target pathological cells and tissues, especially in treatment of cancer. Thus, a major effort in the development of nanoscopic drug delivery vehicles for cancer treatment has focused on exploiting the inherent differences in tumor physiology and limiting the exposure of drugs to non-tumorous tissue, which is commonly achieved by encapsulation of chemotherapeutics within macromolecular or supramolecular carriers that incorporate targeting ligands and that enable controlled release. The overall aim of this work is to engineer a hybrid nanomaterial system comprised of protein and silica and to characterize its potential as an encapsulating drug carrier. The synthesis of silica, an attractive nanomaterial component because it is both biocompatible as well as structurally and chemically stable, within this system is catalyzed by self-assembled elastin-like polypeptide (ELP) micelles that incorporate of a class of biologically-inspired, silica-promoting peptides, silaffins. Furthermore, this methodology produces near-monodisperse, hybrid inorganic/micellar materials under mild reaction conditions such as temperature, pH and solvent. This work studies this material system along three avenues: 1) proof-of-concept silicification (i.e. the formation and deposition of silica upon organic materials) of ELP micellar templates, 2) encapsulation and pH-triggered release of small, hydrophobic chemotherapeutics, and 3) selective silicification of templates to potentiate retention of peptide targeting ability.</p> / Dissertation

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

<p>Traditional small molecule chemotherapeutics show limited effectiveness in the clinic as their poor pharmacokinetics lead to rapid clearance from circulation and their exposure to off-target tissues results in dose-limiting toxicity. The objective of this dissertation is to exploit a class of recombinant chimeric polypeptides (CPs) to actively target drugs to tumors as conjugation to macromolecular carriers has demonstrated improved efficacy by increasing plasma retention time, reducing uptake by healthy tissues, and enhancing tumor accumulation by exploiting the leaky vasculature and impaired lymphatic drainage characteristic of solid tumors. CPs consist of two principal components: (1) a thermally responsive elastin-like polypeptide (ELP) that displays a soluble-to-aggregate phase transition above a characteristic transition temperature (Tt); and (2) a cysteine-rich peptide fused to one end of the ELP to which small molecule therapeutics can be covalently attached (the conjugation domain). This work describes the development of CP drug-loaded nanoparticles that can be targeted to solid tumors by the external application of mild regional hyperthermia (39-43°C). </p><p>Highly repetitive ELP polymers were assembled by Plasmid Reconstruction Recursive Directional Ligation (PRe-RDL), in which two halves of a parent plasmid, each containing a copy of an oligomer, were ligated together to dimerize the oligomer and reconstitute the functional plasmid. Chimeric polypeptides were constructed by fusing the ELP sequence to a (CGG)8 conjugation domain, expressed in Escherichia coli, and loaded with small molecule hydrophobes through site specific attachment to the conjugation domain. Drug attachment induced the assembly of nanoparticles that retained the thermal responsiveness of the parent ELP in that they experienced a phase transition from soluble nanoparticles to an aggregated phase above their Tt. Importantly, the Tt of these nanoparticles was near-independent of the CP concentration and the structure of the conjugated molecule as long as it displayed an octanol-water distribution coefficient (LogD) > 1.5. </p><p>A series of CP nanoparticles with varying ratios of alanine and valine in the guest residue position was used to develop a quantitative model that described the CP transition temperature in terms of three variables - sequence, chain length, and concentration - and the model was used to identify CPs of varying molecular weights that displayed transition temperatures between 39°C and 43°C. A murine dorsal skin fold window chamber model using a human tumor xenograft was used to validate that only the thermoresponsive CP nanoparticles (and not the controls) exhibited a micelle-to-aggregate phase transition between 39-43°C in vivo. Furthermore, quantitative analysis of the biodistribution profile demonstrated that accumulation of these thermoresponsive CP nanoparticles was significantly enhanced by applying heat in a cyclical manner. It is hoped that this work will provide a helpful resource for the use of thermoresponsive CP nanoparticles in a variety of biomedical applications.</p> / Dissertation

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

abstract: Gold nanoparticles as potential diagnostic, therapeutic and sensing systems have a long history of use in medicine, and have expanded to a variety of applications. Gold nanoparticles are attractive in biological applications due to their unique optical, chemical and biological properties. Particularly, gold nanorods (GNRs) are increasingly used due to superior optical property in the near infrared (NIR) window. Light absorbed by the nanorod can be dissipated as heat efficiently or re-emitted by the particle. However, the limitations for clinical translation of gold nanorods include low yields, poor stability, depth-restricted imaging, and resistance of cancer cells to hyperthermia, are severe. A novel high-throughput synthesis method was employed to significantly increase in yields of solid and porous gold nanorods/wires. Stable functional nanoassemblies and nanomaterials were generated by interfacing gold nanorods with a variety of polymeric and polypeptide-based coatings, resulting in unique properties of polymer-gold nanorod assemblies and composites. Here the use of these modified gold nanorods in a variety of applications including optical sensors, cancer therapeutics, and nanobiomaterials were described. / Dissertation/Thesis / Ph.D. Chemical Engineering 2012

<div>Wounds are created in soft and hard tissue through surgery or disease. As the wound heals, the tissue is held in place using sutures or staples for soft tissue or plates, pins, or screws for hard tissues. These fixation methods inherently damage the surrounding healthy tissue. Surgical adhesives are a non-damaging alternative to these methods. In order to be effective, surgical adhesives must be biocompatible,</div><div>adhere strongly in a moist environment, and have mechanical properties similar to those of the native tissue.</div><div><br></div><div><div>To address the design criteria for surgical adhesives, we look to nature to find inspiration from compounds that provide these properties. Mussels use catechol-based</div><div>molecules to adhere to surfaces in wet and turbulent environments. Incorporating catechols into polymer systems can provide adhesion even in moist biological environments.</div><div>Mimics of elastomeric proteins from soft tissue can be used as backbones for soft and flexible adhesive systems. In particular, elastin-inspired proteins have a well-defined modular sequence that allows for a range of design choices. In this work, we explored the behavior of elastin- and mussel-inspired natural and synthetic polymers in biologically relevant environments.</div></div><div><br></div><div><div>First, the cytocompatibility of a catechol-containing poly(lactic acid) (cPLA) hard tissue adhesive was studied. The cPLA polymer was reacted with iron- or periodatebased</div><div>crosslinkers and compared to PLA. Fibroblasts grown directly on cPLA or cultured with leachate from cPLA had high viability but slower growth than cells on PLA. The periodate crosslinker was significantly cytotoxic, and cells grown on cPLA crosslinked with periodate had reduced metabolism and slowed growth. Cells grown on or in leachate from iron-crosslinked cPLA had similar viability, metabolism, and growth to cells on or in leachate from cPLA. The iron-crosslinked cPLA is a promising</div><div>cytocompatible adhesive for hard tissue applications.</div></div><div><br></div><div><div>Second, two elastin-like proteins (ELP) were developed that had pH-sensitive properties in solution and when crosslinked into hydrogels. Both ELPs had a large number of ionizable tyrosine and lysine residues, and one design also had a large number of ionizable histidine and aspartic acid residues. The stiffness of the hydrogels was maximized at pH values near the isoelectric point of the protein. The stoichometric ratio of crosslinker used affected hydrogel stiffness but did not significantly alter the pH-sensitivity of the gel. The crosslinked gel shrank when swelled at physiological pH. The pH-sensitive mechanical properties of hydrogels made from the two ELPs did not vary significantly. The tyrosine and lysine residues in one ELP were also</div><div>chemically blocked through acetylation to lower the isolectric point of the protein. The acetylated hydrogels had maximum stiffness at a pH near the isoelectric point of the acetylated ELP. The stiffness of both the native and acetylated gels were within the range of soft tissue. Through a combination of crosslinker ratio and chemical modification, the pH-responsive properties of the elastin-inspired hydrogels could be tuned.</div></div><div><br></div><div><div>Finally, adhesive proteins were created that were inspired by both elastin and mussels. An ELP was modified to include catechol groups (mELP). The ELP and mELP were optimized for adhesive use in a soft tissue system. A warm and humid environment was used to study the adhesion of these proteins on pig skin. Iron and (hydroxymethyl) phosphine crosslinkers increased the adhesive strength of both proteins, and periodate increased the adhesive strength of mELP. The adhesive strengths of the proteins were maximized when mELP was mixed with iron or when either protein were mixed with (hydroxymethyl)phosphine crosslinkers. These maximized adhesives were 12-17 times stronger than a commercially available sealant. In addition,</div><div>the iron and mELP adhesive formulation achieved high adhesive strengths even when cured for only ten minutes. This adhesive formula shows promise for adhesive</div><div>applications on soft tissue.</div></div>

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