Doctor of Philosophy / Department of Biochemistry / John M. Tomich / Peptide-based delivery systems show great potential as safer drug delivery vehicles. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. We have designed and synthesized a set of 15 and 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated spheres with 50-150 nm diameters (confirmed by TEM and DLS). Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these structures are further stabilized by β-sheet hydrogen bonding and are stable at very low concentrations and even in the presence of SDS, urea and trypsin as confirmed by circular dichroism spectroscopy. Given sufficient time, they fuse together to form larger assemblies and trap compounds of different sizes within the enclosed space. They are prepared using a protocol that is similar to preparing lipid vesicles. We have shown that different concentrations of the fluorescent dye, 5(6)-Carboxyfluorescein can be encapsulated in these assemblies and delivered into human lens epithelial cells and MCF-7 cells grown on coverslips. Besides fluorescent dyes, we have delivered the plasmid (EGFP-N3, 4.7kb) into N/N 1003A lens epithelial cells and observed expression of EGFP (in the presence and absence of a selection media). In the case of large molecules like DNA, these assemblies act as nanoparticles and offer some protection to DNA against certain nucleases. Linear peptides that lacked a branching point and other branched peptides with their sequences randomized did not show any of the lipid-like properties exhibited by the branched peptides. The peptides can be chemically decorated with target specific sequences for use as DDS for targeted delivery.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18949号 / 工博第3991号 / 新制||工||1615(附属図書館) / 31900 / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 木村 俊作, 教授 瀧川 敏算, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Self-assembly is a fundamental process in the field of nanotechnology, where molecules organize into complex structures spontaneously or induced by environmental factors. Peptides, short chains of amino acids, can self-assemble into many types of nanostructures. The self-assembly of peptides is governed by noncovalent interactions, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, aromatic-aromatic interactions, and van der Waals forces. By varying the amino acid sequences and manipulating environmental parameters, these interactions can be modulated to obtain diverse supramolecular nanostructures, exhibiting a wide range of physical, chemical, and biological properties. Furthermore, the ability to control these properties opens up a world of possibilities in biomedical and biocatalytic applications. From drug delivery systems to enzyme mimics, as well as cancer treatments, the potential of these self-assembling peptides is vast and continues to be a vibrant area of research. Exploiting this potential, this dissertation delves into the design, synthesis, and investigation of self-assembling peptides for a range of applications. The introductory chapters of this document lay the groundwork, providing a comprehensive overview of self-assembly and its potential in biocatalytic and biomedical domains. The focus shifts in the later chapters to drug delivery applications, particularly in the delivery of hydrogen sulfide (H2S), and its implications in cardioprotection and cancer treatment. Finally, this document details an evaluation of self-assembled peptides in the context of biocatalysis using a combined experimental and computational approach. Chapter 3 discusses the design and synthesis of peptide-H2S donor conjugates (PHDCs) with an unusual adamantyl group. Several of PHDCs studied in this chapter self-assembled into novel nanocrescent structures observed under both conventional transmission microscopy (TEM) and cryogenic TEM (cryo-TEM). By varying the C-terminal amino acid with cationic, nonionic, or anionic amino acids, the PHDC morphologies remained unaffected, offering a robust peptide design for crescent-shaped supramolecular nanostructures. Chapter 4 discusses an extension of this project, introducing a cyclohexane in PHDCs instead of an adamantyl group. In this work, we designed and fabricated four constitutional isomeric PHDCs, which self-assembled into nanoribbons with different dimensions and large nanobelts. These morphologies exhibited varying cellular uptake and in vitro H2S release amounts, influencing their protective effects against oxidative stress induced by H2O2. With the knowledge of the impact of subtle changes in PHDC structures, Chapter 5 discusses our further design of three more PHDCs with the variation of side chain capping group, from an aromatic phenyl ring to a cyclohexane unit, to an aliphatic n-hexyl chain. In this chapter, we studied how changes in the hydrocarbon tail can influence the supramolecular nanostructures and their potential ability for colon cancer treatment. A final aspect of H2S delivery in Chapter 6 involves the creation of a stable PHDC with an extended H2S release profile. By integrating the H2S donor into a β-sheet forming peptide sequence with a Newkome-like poly(ethylene glycol) dendron, this PHDC self-assembles into spherical or fibril nanostructures with or without stirring. The H2S release was further studied by triggering release with various charged thiol molecules. Finally, another facet of this document focuses on three constitutional isomeric tetrapeptides containing a catalytic functional amino acid, His. Chapter 7 discusses these tetrapeptides, which self-assembled into nanocoils, nanotoroids, and nanoribbons based on the position of the His residue in the peptide sequence. Computational studies simulating the self-assembling process revealed the distribution of His residues and hydrophobic pockets, reminiscent of natural enzyme binding sites. A tight spatial distribution of His residues and hydrophobic pocket in nanocoils provided a picture for why this morphology exhibited the highest rate enhancement in catalyzing a model ester hydrolysis reaction. This study demonstrated how subtle molecular-level changes impact supramolecular nanostructures and catalytic efficiency. The final chapter details conclusions on all the research in this dissertation and discusses further directions of self-assembling peptides in the application of drug delivery and design of catalyst mimics. / Doctor of Philosophy / Self-assembly is a fascinating process in nanotechnology, where molecular building blocks come together to form complex structures. Peptides, which are short chains made up of amino acids, can play a crucial role in this process. They can organize themselves into various shapes due to different forces acting between their amino acid building blocks. By changing the arrangement of amino acids and adjusting the environment, scientists can create a wide range of nanoscale structures with unique properties from peptides. These self-assembling peptides have enormous potential in fields like medicine and catalysis. This dissertation describes how to design and make self-assembling peptides for various uses. Chapter 1 describes the general structure of the document, and Chapter 2 discusses the basics of self-assembly and how it can be applied in medicine and other areas. Chapters 3-6 focus on using self-assembling peptides to deliver hydrogen sulfide (H2S), a noxious gaseous molecule that is now recognized as a vital signaling molecule involved in various physiological processes. Several classes of peptide-H2S donor conjugates (PHDCs) are discussed in these chapters, including PHDCs that form nanoscale crescents, twisted ribbons, fibers, and other structures. These nanostructures show promise in protecting cells from harmful substances or can act as drugs in cancer treatment. We also investigate how different modifications affect their performance in biomedical applications. The final research chapter, Chapter 7, involves using self-assembling peptides as catalysts, molecules that speed up chemical reactions. By arranging the amino acids in different ways, peptides that form nanoscale coils, toroids, or ribbons-like structures were created. These different shapes influenced how well they catalyzed reactions. Computational modeling studies helped explain how small differences in molecular design led to big impacts on their catalytic abilities. The final chapter discloses conclusions on all the research in this dissertation and discusses the further directions of self-assembling peptides as medicines and catalysts.

<p>Multidrug resistance is a major issue in treatment and eradication of diseases. There are many mechanisms by which pathogens develop multi drug resistance. Here we focus on the ability of pathogens to evade drug treatment by establishing multi drug resistant reservoirs. In the case of HIV, the virus is able to evade drug treatment and forms both latent and active replicating reservoirs throughout the body. In the case of many bacterial pathogens, multidrug resistance reservoirs are established within mammalian cells, such as macrophages. Many classes of antibiotics are unable to penetrate mammalian cells, making intracellular bacteria difficult to clear</p><p>Previously our research group has developed a Trojan horse strategy to deliver antivirals to HIV cellular reservoirs. Ester based prodrug dimers of abacavir, a reverse transcriptase inhibitor, acted to both inhibit efflux transporters at the BBB and revert to the monomeric therapy in the reducing environments of the cell. Herein we present a new group of sterically hindered carbonate based disulfide linkers that shows improved payload delivery of abacavir and maintain the stability of prodrug molecules towards hydrolysis. We employed these linker molecules to synthesize prodrug dimers of the HIV latency reversal agent prostratin with the hope of targeting latent HIV reservoirs. Payload release studies as well as latency reversal experiments with a latently infected T-helper cell model confirmed that the prostratin carbonate homodimers (<b>ProS₂Me₂</b> and <b>ProS₂Me₄</b>) were able to revert to monomeric prostratin and reverse HIV latency. We next sought to synthesize a prostrain-protease inhibitor heterodimer. While our initial study of a prostratin-lopinavir heterodimer employing this linker strategy (<b>ProLpvS₂Me₂</b>) did not show significant HIV latency reversal activity, we hope to expand our heterodimer studies to achieve dual therapeutic molecules that can both reverse HIV latency and deliver antivirals to HIV reservoirs.</p><p>In order to combat intracellular bacteria our group has focused on development of a novel class of cell penetrating peptides with intrinsic broad spectrum antimicrobial activity that are based on a repeating amino acid triad which forms a cationic amphiphilic polyproline helix (CAPH) scaffold. The first member of this class, <b>P14LRR</b>, exhibited clearance of intracellular bacteria and concentration dependent co-localization within mammalian cells. In efforts to optimize antimicrobial activity we have expanded the CAPHs library by adjusting the chain length between the proline backbone and the guanadinium groups of the cationic amino acids. The first peptide from this expanded library, <b>P14GAP</b> showed much greater cell penetration and antimicrobial activity against a wide range of pathogenic bacteria. However, <b>P14GAP</b> also showed greater toxicity towards mammalian cells, increased hemolysis, and greater membrane binding with mammalian cells as compared to <b>P14LRR</b>. Here we describe the design and synthesis of <b>P14GAP-C1</b>, which contains a methylene between the proline backbone and the guanadinium group. This new analogue decreased the hemolysis activity as compared to <b>P14GAP</b>, although similar membrane binding with mammalian cells was observed. This improvement in hemolysis activity and a slight improvement in cell viability may allow us to use higher concentrations of peptide to treat multidrug resistant bacterial infections.</p><p> </p>