Title:
Controlled release of GLP-1 from affinity-based protein microspheres and quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by macrophages
Controlled release of GLP-1 from affinity-based protein microspheres and quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by macrophages
Author(s)
Padmore, Trudy J.
Advisor(s)
Champion, Julie A.
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Abstract
Peptide drugs are highly specific and efficacious; interest in them remains high despite the challenges of rapid clearance and degradation. To overcome these limitations peptide delivery systems must prolong release while protecting biological activity. Glucagon-like peptide 1 (GLP-1) stimulates insulin secretion from pancreatic β-cells and is used for treatment of type 2 diabetes. However, GLP-1 undergoes rapid deactivation by proteolytic degradation and its size leads to a short, 2-minute half-life. An affinity-mediated delivery system can prolong half-life, while modification of its amino acid sequence can confer protease resistance. Here we describe a strategy to prolong the release of active Glucagon-like peptide 1 (GLP-1) using Src homology 3 (SH3) domain protein microparticles that bind GLP-1 through affinity-mediated interactions. GLP-1 modified with SH3-binding peptides of weak and strong affinities (GLP-1-SBPs) facilitate tunable release from SH3 microparticles. Release rates of GLP-1 from SH3 microspheres were strongly dependent on the SH3 binding peptide affinity, with the weaker binding GLP-1-SBP13 releasing 40% of its total cargo and the stronger binding GLP-1-SBP2 releasing 20% over a 7-day period. Released GLP-1 stimulated significant increases in Beta cell number, while insulin secretion stimulation was not significant in comparison to basal untreated level. Mathematical models qualitatively replicated affinity-dependent release trends. Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by macrophages. Asbestos fibers induce chronic lung inflammation and have been associated (WHO estimate) with 105 lung cancer deaths per year worldwide. In the lung, immune cells (macrophages) attempt to engulf inhaled foreign materials (like asbestos), secreting inflammatory molecules. The inability of macrophages to effectively remove asbestos leads to chronic inflammation and disease. Although the health effects of asbestos have been extensively investigated, this study examines the role of fiber length on phagocytosis and molecular inflammatory responses so as to characterize fiber toxicity at the cellular level. A major challenge is obtaining fibers of the same diameter, d, but differing in length, L. Glass fibers, with d ~1micron, were used as a model for asbestos. Samples with different measured length distributions were prepared: aggressive crushing for short fibers populations and successive sedimentation for long fibers populations. The interactions of MH-S murine alveolar macrophages with the fibers were analyzed by time-lapse video microscopy, to qualitatively describe attempted phagocytosis in real time, and by flow cytometry, to quantitatively measure attachment and internalization of fibers. Short fibers were observed to bind to macrophages, being internalized with little effort. Conversely, macrophages literally wrestled with long fibers over many hours, usually with unsuccessful internalization. Short fibers were twice as likely to be internalized as the long fibers. Persistent macrophage activation, measured by secretion of pro-inflammatory biomolecules, is a critical indicator of fiber toxicity and a pathological hallmark. The production of the pro-inflammatory biomolecules tumor necrosis factor alpha (TNF-a), interleukin 1 alpha (IL-1a) and cyclooxygenase-2 (COX-2), was quantified by ELISA after exposure of the macrophages to glass fibers of varying lengths. TNF-a was secreted in a dose-dependent manner for both short and long fibers. However, exposure to long fibers resulted in greater secretion of the cytokine compared to that of equivalently dosed short fibers. These results suggest that the persistence of long fibers due to incomplete internalization plays a critical role in persistent macrophage activation. Parameters derived from fiber length distributions along with cytokine dose-response curves were used to develop mathematical models that propose critical fiber lengths. These lengths were 20 micron and 27 micron respectively for TNF-a and IL-1a respectively. Critical lengths facilitate a deeper understanding of the role that single physic-chemical parameter of length plays in molecular events and disease outcomes.
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Date Issued
2016-11-30
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Text
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Dissertation