MMP-2 Sensitive Self-assembled Peptide Nanoscaffolds for Neural Tissue Engineering

• Author / Creator

  Koss, Kyle M

• Neurotrophic factor peptide analogues are capable of promoting healing by neurogenesis, enhancing neuronal survival and attenuating glial activation. In vivo peptide delivery is difficult due to susceptibility to proteases, but they are considered ideal therapeutics due to specificity, potency, and size (i.e. diffusion). Self-assembling peptide (RADA)4 is a novel class of peptide that, upon injection, assembles into a 3D nanoscaffold, capable of storing water and molecules. Synthesizing peptide therapeutics onto this nanoscaffold may afford protection from proteases, whilst utilizing resident enzymes (i.e. Matrix Metalloproteinase 2 (MMP-2)) for localized, ‘on-demand’ release. In this thesis, we study the self-assembly of engineered peptides into enzymatically sensitive nanoscaffolds for proteolytic induced release kinetics, gial activity, and PC-12 neurite-outgrowth. Specifically, peptides with high ((RADA)4-GG-GPQG+IASQ (CP1)) and low ((RADA)4-GG-GPQG+PAGQ (CP2)) MMP-2 sensitivity were investigated ('+' = scissile bond). These peptides were shown to self-assemble, measured incrementally by morphology, over 24 hours and CP1 and CP2 favored bundles of nanofibers when compared to (RADA)4 morphology. Fractal dimensions in these nanostructures were significant after 2 hours, suggesting diffusion limitations of fractal nanofibers. Methionine-Valine-Guanine (MVG; DP1), a brain-derived neurotrophic factor secretion stimulant, and Aspartic acid-Guanine-Guanine-Leucine (DGGL; DP2), a cilliary neurotrophic factor analogue, were tethered to CP1 and CP2, by automated peptide synthesis, and mixed with (RADA)4-IKVAV as a means of promoting cell adherence. Nanoscaffold self-assembly, enzyme-induced release of DP1 and DP2, glial activation and resulting PC-12 acetylcholine-esterase signaling and neurite outgrowth were evaluated. Release kinetics were shown to be related to the high (CP1) and low (CP2) activity cut sites and by their concentrations. Peptide release could be controlled between 0 and 100%, iii over 32 days, depending on nanoscaffold design. Nanoscaffold seeded microglia were visually ramified and MTT levels were found to be highest in pure (RADA)4 nanoscaffolds; suggesting increased adhesion and survival. Ramified microglia, as well as negligible astrocyte scarring and axonal cell death were also observed upon an intra-cerebral injection into the brains of 2-day post-natal rat pups. MMP-2 released peptide-drugs promoted differentiation in PC-12 cells; observed neurite extension and increased acetylcholine esterase signaling for nanoscaffolds composed of 10% v/v (RADA)4-IKVAV, 10% v/v (RADA)4-CP1/CP2-MVG/DGGL, and 80% (RADA)4. Finally, optimal seeding capacity was observed upon addition of 10% v/v (RADA)4-IKVAV to the nanoscaffolds. (RADA)4 nanoscaffolds were observed to self-assemble, exhibit microglia compatibility without affecting their response to lipopolysaccaride stimulation, and able to promote neural differentiation for PC-12 cells upon release of DP1 or DP2. Thus a biomimetic, fully synthetic self-assembling nanoscaffold has been demonstrated for a tuneable drug release system with good microglia and intra-cerebral biocompatibility capable of inducing neural differentiation by peptide cleavage.

• Subjects / Keywords

  • Neural Tissue Engineering
  • Drug Delivery
  • Self-assembling peptides
  • Cell Scaffold

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R38C9RM0Q

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.