Graduation Year

2013

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Physics

Major Professor

Donald T. Haynie

Keywords

aqueous feedstocks, biomaterials, crosslinking, electrospinning, self-organization, stability

Abstract

This dissertation has aimed to fabricate polypeptide based biomaterial and characterize physical properties. Electrospinning is used as a tool for the sample fabrication. Project focused on determining the feasibility of electrospinning of certain synthetic polypeptides and certain elastin-like peptides from aqueous feedstocks and to characterize physical properties of polymer aqueous solution, cast film and spun fibers and fiber mats. The research involves peptide design, polymer electrospinning, fibers crosslinking, determining the extent of crosslinking, fibers protease degradation study, fibers stability and self-organization analysis, structure and composition determination by various spectroscopy and microscopy techniques and characterization of mechanical properties of individual suspended fibers.

Fiber mats of a synthetic cationic polypeptide poly(L-ornithine) (PLO) and an anionic co-polypeptide of L-glutamic acid and L-tyrosine (PLEY) of defined composition have been produced by electrospinning. Fibers were obtained from polymer aqueous solution at concentrations of 20-45% (w/v) in PLO and at concentrations of 20-60% (w/v) in PLEY. Applied voltage and spinneret-collector distance were also found to influence polymer spinnability and fibers morphology. Oriented fibers were obtained by parallel electrodes geometry. Fiber diameter and morphology was analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM).

PLO fibers exposed on glutaraldehyde (GTA) vapor rendered fiber mats water-insoluble. A common chemical reagent, carbodiimide was used to crosslink PLEY fibers. Fiber solubility in aqueous solution varied as a function of crosslinking time and crosslinker concentration. Crosslink density has been quantified by a visible-wavelength dye-based method. Degradation of crosslinked fibers by different proteases has been demonstrated.

Investigation of crosslinked PLEY fibers has provided insight into the mechanisms of stability at different pH values. Variations in fiber morphology, elemental composition and stability have been studied by microscopy and energy-dispersive X-ray spectroscopy (EDX), following the treatment of samples at different pH values in the 2-12 range. Fiber stability has been interpreted with reference to the pH dependence of the UV absorbance and fluorescence of PLEY chains in solution. The data show that fiber stability is crucially dependent on the extent of side chain ionization, even after crosslinking.

Self-organization kinetics of electrospun PLO and PLEY fibers during solvent annealing has been studied. After being crosslinked in situ, fibers were annealed in water at 22 °C. Analysis by Fourier transform infrared spectroscopy (FTIR) has revealed that annealing involved fiber restructuring with an overall time constant of 29 min for PLO and 63 min for PLEY, and that changes in the distribution of polymer conformations occurred during the first 13 min of annealing. There was a substantial decrease in the amount of Na+ bound to PLEY fibers during annealing. Kinetic modeling has indicated that two parallel pathways better account for the annealing trajectory than a single pathway with multiple transition states.

Bacteria have been engineered to make novel 250-mer elastin-like polypeptides (ELPs). Each was predicted to have an absolute net charge of less than 0.05 electron charges per amino acid residue in aqueous solution at neutral pH. Polymer structure in solution has been assessed by Circular dichroism spectroscopy (CD) and dynamic light scattering (DLS). Suitability for materials manufacture has been tested by electrospinning.

Here, we have also tested the hypothesis that blending polypeptides of radically different amino acid composition will enable the realization of novel and potentially advantageous material properties. Aqueous polymer feedstock solutions consisted of pure ELP or ELP blended with a synthetic polypeptide, PLEY, which is highly ionized at neutral pH and spinnable. Morphology analysis of blended fibers by SEM has revealed the formation of a surprising variety of structures that are not seen in fibers of ELP or PLEY alone, for example, hollow beads. Analysis of blended fibers by fluorescence microscopy showed that there was little or no phase separation, despite the large difference in electrical properties between ELP and the synthetic polymers.

Structure and composition of PLO, PLEY, ELPs and blends and electrospun fibers made of these polymers have been determined and compared. CD and FTIR have been utilized to obtain structural information on these polymers in aqueous solution, cast films and fibers. Fiber composition has been analyzed by EDX. Protein adsorption has been analyzed by quantitative fluorescence microscopy. The polymers adopted random coil-like conformations in aqueous feedstocks at neutral pH and in dehydrated cast films and fibers on glass, and the fibers comprised numerous counterions, according to spectral analysis. Adsorption of model proteins and serum proteins onto hydrated and crosslinked fibers depended on the electrical charge of the proteins and the fibers. The surface charge density of the fibers will be comparable to, but less than, the charge density on the outer leaflet of the plasma membrane of usual eukaryotic cells.

Mechanical properties of a series of as-spun and crosslinked PLO and PLEY nanofibers with various diameters have been analyzed by using the pure bending mode and AFM technique. Aligned nanofibers were deposited on top of a microsized groove etched on a glass substrate. AFM tip was used as a probe, which could apply a measurable deflection and force onto the suspended nanofiber at a force calibration mode, so that the Young's modulus of a single nanofiber can be calculated based on the basic beam bending theories. The Young's moduli of the studied peptide nanofibers increased significantly with decreased fiber diameters. This study has also demonstrated that crosslinked electrospun PLO and PLEY fibers have a higher Young's modulus compared with their as-spun counterparts.

Taken together, the results will advance the rational design of polypeptides for peptide-based materials, especially materials prepared by electrospinning. It is believed that this research will increase basic knowledge of polymer electrospinning and advance the development of electrospun materials, especially in medicine and biotechnology. The study has yielded two advances on previous work in the area: avoidance of an animal source of peptides and avoidance of inorganic solvent. The present results thus advance the growing field of peptide-based materials. Non-woven electrospun fiber mats made of polypeptides are increasingly considered attractive for basic research and technology development in biotechnology, medicine and other areas.

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