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The world today needs novel solutions to address current challenges in areas spanning areas from sustainable manufacturing to healthcare, and biotechnology offers the potential to help address some of these issues. One tool that offers opportunities across multiple industries is the use of nonribosomal peptide synthases (NRPSs). These are modular

The world today needs novel solutions to address current challenges in areas spanning areas from sustainable manufacturing to healthcare, and biotechnology offers the potential to help address some of these issues. One tool that offers opportunities across multiple industries is the use of nonribosomal peptide synthases (NRPSs). These are modular biological factories with individualized subunits that function in concert to create novel peptides.One element at the heart of environmental health debates today is plastics. Biodegradable alternatives for petroleum-based plastics is a necessity. One NRPS, cyanophycin synthetase (CphA), can produce cyanophycin grana protein (CGP), a polymer composed of a poly-aspartic acid backbone with arginine side chains. The aspartic backbone has the potential to replace synthetic polyacrylate, although current production costs are prohibitive. In Chapter 2, a CphA variant from Tatumella morbirosei is characterized, that produces up to 3x more CGP than other known variants, and shows high iCGP specificity in both flask and bioreactor trials. Another CphA variant, this one from Acinetobacter baylyi, underwent rational protein design to create novel mutants. One, G217K, is 34% more productive than the wild type, while G163K produces a CGP with shorter chain lengths. The current structure refined from 4.4Å to 3.5Å. Another exciting application of NRPSs is in healthcare. They can be used to generate novel peptides such as complex antibiotics. A recently discovered iterative polyketide synthase (IPTK), dubbed AlnB, produces an antibiotic called allenomycin. One of the modular subunits, a dehydratase named AlnB_DH, was crystallized to 2.45Å. Several mutations were created in multiple active site residues to help understand the functional mechanism of AlnB_DH. A preliminary holoenzyme AlnB structure at 3.8Å was generated although the large disorganized regions demonstrated an incomplete structure. It was found that chain length is the primary factor in driving dehydratase action within AlnB_DH, which helps lend understanding to this module.
ContributorsSwain, Kyle (Author) / Nannenga, Brent (Thesis advisor) / Nielsen, David (Committee member) / Mills, Jeremy (Committee member) / Seo, Eileen (Committee member) / Acharya, Abhinav (Committee member) / Arizona State University (Publisher)
Created2022
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Description
Achieving effective drug concentrations within the central nervous system (CNS) remains one of the greatest challenges for the treatment of brain tumors. The presence of the blood-brain barrier and blood-spinal cord barrier severely restricts the blood-to-CNS entry of nearly all systemically administered therapeutics, often leading to the development of peripheral

Achieving effective drug concentrations within the central nervous system (CNS) remains one of the greatest challenges for the treatment of brain tumors. The presence of the blood-brain barrier and blood-spinal cord barrier severely restricts the blood-to-CNS entry of nearly all systemically administered therapeutics, often leading to the development of peripheral toxicities before a treatment benefit is observed. To circumvent systemic barriers, intrathecal (IT) injection of therapeutics directly into the cerebrospinal fluid (CSF) surrounding the brain and spinal cord has been used as an alternative administration route; however, its widespread translation to the clinic has been hindered by poor drug pharmacokinetics (PK), including rapid clearance, inadequate distribution, as well as toxicity. One strategy to overcome the limitations of free drug PK and improve drug efficacy is to encapsulate drug within nanoparticles (NP), which solubilize hydrophobic molecules for sustained release in physiological environments. In this thesis, we will develop NP delivery strategies for brain tumor therapy in two model systems: glioblastoma (GBM), the most common and deadly malignant primary brain tumor, and medulloblastoma, the most common pediatric brain tumor. In the first research chapter, we developed 120 nm poly(lactic acid-co-glycolic acid) NPs encapsulating the chemotherapy, camptothecin, for intravenous delivery to GBM. NP encapsulation of camptothecin was shown to reduce the drug’s toxicity and enable effective delivery to orthotopic GBM. To build off the success of intravenous NP, the second research chapter explored the utility of 100 nm PEGylated NPs for use with IT administration. Using in vivo imaging and ex vivo tissue slices, we found the NPs were rapidly transported by the convective forces of the CSF along the entire neuraxis and were retained for over 3 weeks. Based on their wide spread delivery and prolonged circulation, we examine the ability of the NPs to localize with tumor lesions in a leptomeningeal metastasis (LM) model of medulloblastoma. NPs administered to LM bearing mice were shown to penetrate into LM mets seeded within the meninges around the brain. These data show the potential to translate our success with intravenous NPs for GBM to improve IT chemotherapy delivery to LM.
ContributorsHouseholder, Kyle Thomas (Author) / Sirianni, Rachael W. (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Vernon, Brent (Committee member) / Caplan, Michael (Committee member) / Wechsler-Reya, Robert (Committee member) / Arizona State University (Publisher)
Created2018
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Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs

Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs generated using 1,4C-1,4Bis, a cationic polymer from our laboratory demonstrated significantly higher transgene expression and exhibited lower cytotoxicities when compared to similar assemblies generated using 25 kDa poly(ethylene imine) (PEI25k-GNRs), a current standard for polymer-mediated gene delivery. Additionally, sub-toxic concentrations of 1,4C-1,4Bis-GNR nanoassemblies were employed to deliver expression vectors that express shRNA ('shRNA plasmid') against firefly luciferase gene in order to knock down expression of the protein constitutively expressed in prostate cancer cells. The roles of poly(amino ether) chemistry and zeta-potential in determining transgene expression efficacies of PAE-GNR assemblies were investigated. The theranostic potential of 1,4C-1,4Bis-GNR nanoassemblies was demonstrated using live cell two-photon induced luminescence bioimaging. The PAE class of polymers was also investigated for the one pot synthesis of both gold and silver nanoparticles using a small library poly(amino ethers) derived from linear-like polyamines. Efficient nanoparticle synthesis dependent on concentration of polymers as well as polymer chemical composition is demonstrated. Additionally, the application of poly(amino ether)-gold nanoparticles for transgene delivery is demonstrated in 22Rv1 and MB49 cancer cell lines. Base polymer, 1,4C-1,4Bis and 1,4C-1,4Bis templated and modified gold nanoparticles were compared for transgene delivery efficacies. Differences in morphology and physiochemical properties were investigated as they relate to differences in transgene delivery efficacy. There were found to be minimal differences suggestion that 1,4C-1,4Bis efficacy is not lost following use for nanoparticle modification. These results indicate that poly(amino ether)-gold nanoassemblies are a promising theranostic platform for delivery of therapeutic payloads capable of simultaneous gene silencing and bioimaging.
ContributorsRamos, James (Author) / Rege, Kaushal (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Garcia, Antonio (Committee member) / Arizona State University (Publisher)
Created2014
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Description
Tissue approximation and repair have been performed with sutures and staples for centuries, but these means are inherently traumatic. Tissue repair using laser-responsive nanomaterials can lead to rapid tissue sealing and repair and is an attractive alternative to existing clinical methods. Laser tissue welding is a sutureless technique for sealing

Tissue approximation and repair have been performed with sutures and staples for centuries, but these means are inherently traumatic. Tissue repair using laser-responsive nanomaterials can lead to rapid tissue sealing and repair and is an attractive alternative to existing clinical methods. Laser tissue welding is a sutureless technique for sealing incised or wounded tissue, where chromophores convert laser light to heat to induce in tissue sealing. Introducing chromophores that absorb near-infrared light creates differential laser absorption and allows for laser wavelengths that minimizes tissue damage.

In this work, plasmonic nanocomposites have been synthesized and used in laser tissue welding for ruptured porcine intestine ex vivo and incised murine skin in vivo. These laser-responsive nanocomposites improved tissue strength and healing, respectively. Additionally, a spatiotemporal model has been developed for laser tissue welding of porcine and mouse cadaver intestine sections using near-infrared laser irradiation. This mathematical model can be employed to identify optimal conditions for minimizing healthy cell death while still achieving a strong seal of the ruptured tissue using laser welding. Finally, in a model of surgical site infection, laser-responsive nanomaterials were shown to be efficacious in inhibiting bacterial growth. By incorporating an anti-microbial functionality to laser-responsive nanocomposites, these materials will serve as a treatment modality in sealing tissue, healing tissue, and protecting tissue in surgery.
ContributorsUrie, Russell Ricks (Author) / Rege, Kaushal (Thesis advisor) / Acharya, Abhinav (Committee member) / DeNardo, Dale (Committee member) / Holloway, Julianne (Committee member) / Thomas, Marylaura (Committee member) / Arizona State University (Publisher)
Created2019
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Description
The list of applications of plasmonic nanoparticles in the fields of energy research, sensing, and diagnostics and therapeutics is continuously growing. Processes for the synthesis of the nanoparticles for such applications should incorporate provision to easily functionalize the particle formed and should ideally not use toxic reagents or create toxic

The list of applications of plasmonic nanoparticles in the fields of energy research, sensing, and diagnostics and therapeutics is continuously growing. Processes for the synthesis of the nanoparticles for such applications should incorporate provision to easily functionalize the particle formed and should ideally not use toxic reagents or create toxic by-products. The traditional methods of synthesizing nanoparticles generally are energy inefficient, requires stringent conditions such as high temperature, pressure or extreme pH and often produces toxic by-products. Although there exist a few solution-based methods to solve this problem, there is one avenue which has recently gained attention for nanoparticle synthesis: using biomolecules to facilitate nanomaterials synthesis. Using biomolecules for synthesis can provide a template to guide the nucleation process and helps to keep conditions biocompatible while also combining the step of functionalization of the nanoparticle with its synthesis through the biomolecule itself. The dissertation focuses on studying the bio-templated synthesis of two such noble metal nanoparticle which have biomedical applications: gold and platinum. In chapter 2, Gold Nanoparticles (GNP), with long-term stability, were synthesized using Maltose Binding Protein (MBP) as templating agent. The site of gold interaction on MBP was identified by X-ray crystallography. A novel gold binding peptide, AT1 (YPFGGSGGSGM), was designed based on the orientation of the residues in the gold binding site, identified through crystallography. This designed peptide was also shown to have stabilized and affected the growth rate of GNP formation, in similar manner to MBP. Further in chapter 3, a nanosensor was formulated using a variation of this GNP-MBP system, to detect and measure ionizing radiation dose for cancer radiation therapy. Upon exposure to therapeutic levels of ionizing radiation, the MBP‐based sensor system formed gold nanoparticles with a dose‐dependent color that could be used to predict the amount of delivered X‐ray dose. In chapter 4, a similar system of protein templated synthesis was introduced for platinum nanoparticle (PtNP). Here, GroEL, a large homo-tetradecamer chaperone from E.coli, was used as templating and stabilizing agent for reduction of K2PtCl4 ions to form PtNP. To understand how GroEL interacts with the PtNPs and thereby stabilizes them, single-particle cryo-electron microscopy technique was used to model the complex in solution. A 3.8-Å resolution 3D cryo-EM map of GroEL depicting the location of PtNP inside its central cylindrical cavity was obtained. Fitting a GroEL model to the map revealed Arginine-268 from two adjacent subunits of GroEL interacting with the PtNP surface. Finally in chapter 5, a solution to the potential issues of single particle data processing on protein nanoparticle complexes, specifically with 2D classification, was developed by creating masking algorithms.
ContributorsThaker, Amar Nilkamal (Author) / Nannenga, Brent L (Thesis advisor) / Acharya, Abhinav (Committee member) / Torres, Cesar (Committee member) / Mills, Jeremy (Committee member) / Rege, Kaushal (Committee member) / Arizona State University (Publisher)
Created2020