Characterization of the stability and bio-functionality of tethered proteins on bioengineered scaffolds: implications for stem cell biology and tissue repair.
Various engineering applications have been utilized to deliver molecules and compounds in both innate and biological settings. In the context of biological applications, the timely delivery of molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superiority of long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared with conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway, and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial-derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation, and neurite morphogenesis. Enzyme-linked immunosorbent assays (ELISA) revealed that GDNF, covalently tethered onto polycaprolactone (PCL) electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signaling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilization of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days, further validating prolonged functionality of the protein, well beyond the minutes to hours timeframe observed for soluble proteins under the same culture conditions. This study provides important evidence of the stability and functionality kinetics of tethered molecules.
From the Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, Australia, 3010 and.
Ting-Yi
TY
Wang
the Research School of Engineering, The Australian National University, Canberra, Australia, 0200.
Kiara A F
KA
Bruggeman
From the Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, Australia, 3010 and.
Rebecca K
RK
Sheean
From the Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, Australia, 3010 and.
Bradley J
BJ
Turner
the Research School of Engineering, The Australian National University, Canberra, Australia, 0200 david.nisbet@anu.edu.au.
David R
DR
Nisbet
From the Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, Australia, 3010 and cparish@unimelb.edu.au.
Clare L
CL
Parish
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015151:Immunoblotting; D051379:Mice; D008855:Microscopy, Electron, Scanning; D057139:Nanofibers / Q000737:chemistry* / Q000648:ultrastructure; D058953:Neural Stem Cells / Q000187:drug effects / Q000378:metabolism; D011091:Polyesters / Q000737:chemistry*; D011247:Pregnancy; D055550:Protein Stability; D057905:Stem Cell Research; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry*; D014945:Wound Healing
D051100:Glial Cell Line-Derived Neurotrophic Factor; D055767:Immobilized Proteins; D011091:Polyesters; C016240:polycaprolactone
Biomaterials; Neurons; Neurotrophic Factor; Polycaprolactone; Protein Degradation; Protein Engineering; Protein Stability; Protein Tethering