Author : Justin Stanley Plaut
Release : 2021
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Kind : eBook
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Book Synopsis Physicochemical Characterization of Bionanomaterials by : Justin Stanley Plaut

Download or read book Physicochemical Characterization of Bionanomaterials written by Justin Stanley Plaut. This book was released on 2021. Available in PDF, EPUB and Kindle. Book excerpt: In the first section of this dissertation, work involving the characterization of membrane-bound bioparticles is presented. Atomic force microscopy (AFM) is preeminent among techniques to characterize the morphological and nanomechanical properties of membrane-bound bioparticles, including extracellular vesicles, platelets, and liposomes. However, due to the sensitivity of the measurements and the fragility of the materials, there are significant challenges that must be overcome for the acquisition of accurate and high-quality data. A variety of commonly encountered issues, such as sample- or probe-related artifacts, are presented along methodological considerations to surpass such challenges. Extracellular vesicles are particularly difficult to characterize by AFM due to their diminutive size, on the order of tens to hundreds of nm, which results in a multitude of sample-specific artifacts. A study applying these methodological considerations to the quantitative, nanomechanical AFM characterization of matrix vesicles (MVs) is presented. MVs, a class of extracellular vesicles involved in physiological and pathological mineralization processes, are approximately 150 nm in diameter and are secreted from mineralization-competent cells, including chondrocytes, osteoblasts, and odontoblasts. These vesicles contain transporters and enzymatic payloads that enable sequestration of calcium cations and inorganic phosphate within the vesicle lumen to form calcium-phosphate minerals, which condense into semi-crystalline hydroxyapatite. AFM, with other physicochemical techniques, was used to measure the physical properties of MVs in both mineralizing and non-mineralizing conditions, which provided new insight into the structure and function of these membrane-bound bioparticles. In future work, MVs or synthetic MV could foreseeably be applied to the treatment of degenerative bone diseases and stronger linkages between pathological processes and MVs can be established. In the second section of this dissertation, work involving designed peptide biomaterials is presented. Oligopeptides, short polymers of amino acids, have a diverse set of naturally occurring chemical functionalities that can be harnessed to develop functional, shape-defined bionanomaterials. Solid-phase peptide synthesis (SPPS) enables the manufacture of peptides of arbitrary sequence and expands the natural chemical functionality through the inclusion of rare or synthetic amino acids, terminal capping, and other modifications. Three studies using N-terminal fluorenylmethyloxycarbonyl (Fmoc)-modified peptides are presented. Fmoc is an aromatic, polycyclic group that promotes peptide self-assembly through [pi]-[pi] stacking and contributes to the formation of hydrophobic pockets within which guest molecules, including diagnostic or therapeutic agents, can be loaded. Since Fmoc is a standard N-terminal protecting for SPPS, it presents as a no-cost modification for which inclusion reduces the complexity of the peptide synthesis. In each study, AFM was used to characterize the morphology of the peptide bionanomaterials. In the first study, a series of cell-penetrating triblock peptides, composed of a Fmoc-Fx hydrophobic block, a RADARADA amphiphilic block, and terminated with the HIV TAT 48-59 sequence, was developed. By varying the number of N-terminal phenylalanine residues, nanodrills of different morphologies and secondary structures were produced. An assembly model was developed with support by simulations in silico. The nanodrills were demonstrated to be effective vehicles for the enhanced intracellular delivery of hydrophobic molecules. In future work, the lessons learned from this study can help guide the design of simpler and more effective supramolecular delivery vehicles. In the second study, a series of triblock peptides, composed of an Fmoc-F assembly block, a permutated K,F,E amphiphilic block, and an R-G-D-amide cell-binding functional block, were developed through rational design and sequence permutation to identify a peptide suited for hydrogelation, P3. Since hydrogels produced from this peptide exhibit spontaneous gelation at 37oC, molecular-loading capability, and delayed drug release, P3 shows promise as an injectable hydrogel for drug or antigen delivery. Ongoing work will rigorously investigate the in situ hydrogelation and delivery functionality of these hydrogels. In the third study, a label-free protease sensor was developed by exchanging the functional block of P3 for the recognition site (AAN/G) for legumain, a cancer-associated protease, and a solubilizing unit (EEGSGEE). Upon cleavage of the peptide by legumain, the liberated Fmoc-FKFEAAN peptide self-assembles into [beta]-sheeted nanoplatelets that can be stained with thioflavin T for fluorescent signal amplification. The performance of the resulting label-free protease assay was then optimized in both buffer and human plasma. This assay can be adapted, in principle, to any protease of interest by changing the recognition sequence. Ultimately, this simple, inexpensive, and highly adaptable technology shows promise as a diagnostic assay for disease-associated protease biomarkers. In future work, more elaborate detection schemes, such as multiplexing of protease detection, or an adaptation to intracellular or cell-surface protease detection could be implemented. Ultimately, the distinct applications of the peptides in these studies serve as an illustration of the diverse structural and functional properties achievable with designed peptide bionanomaterials.