Synthesis and characterization of carboxy-silk-galactosamine conjugates for tissue engineering applications
Montero, Maria.
2020
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Thesis (M.S.)--Tufts
University, 2020.
Submitted to the Dept. of Biomedical Engineering.
Advisor: David Kaplan.
Committee: Mark Cronin-Golomb, and Joseph Brown.
Keywords: Biomedical engineering, Chemistry, and Biology.
Galactose modified polymers have a range of applications in tissue engineering. Synthetic and natural polymers have, specifically, ... read morebeen modified with galactose moieties for enhancement of hepatocyte culture, development of galectin assays, and targeted drug delivery. First, we review two emerging fields with potential applications for carboxy-silk conjugated with galactosamine, including targeted galectin and liver tissue engineering. Galectins are a family of β-galactoside binding proteins expressed by various cell types and their implication in the progression of different diseases makes them potential targets for therapy. Likewise, the lectin family transmembrane asialoglycoprotein receptor has been a key target for liver tissue engineering. This receptor is crucial for the removal of target glycoproteins form circulation and vital for the metabolism of galactose. Here, we introduce a new method to modify carboxy silk with galactosamine utilizing common carbodiimide conjugation techniques. The extraction time of silk fibroin was varied to capture a large range of initial silk molecular weights, including 1 minute, 30 minutes, and 120 minutes. Serine residues on regenerated silk fibroin are carboxylation modification targets. Carboxy-serine, glutamic acid, and aspartic acid are galactosamine conjugation targets during carbodiimide coupling. Additionally, lysine residues are potential targets for glycation with galactosamine. We use analytical techniques to characterize the carboxy-silk galactosamine conjugates and correlate galactosamine substitution to initial molecular weight of silk fibroin. Presence of galactosamine in silk conjugates is confirmed via proton nuclear magnetic resonance. Notably, the carboxylation intermediate step cleaves silk fibroin to approximately the same molecular weight, 66 kDa, regardless of the initial silk fibroin molecular weight. Protein hydrophobicity is affected by the carboxylation process, yielding a more hydrophilic polymer. Although the carboxylation process affects protein molecular weight and hydrophobicity, it does not impact the β-sheet formation capacity glycine-alanine-glycine-alanine-glycine-serine repeats. Increase in galactosamine conjugation was correlated to decreasing molecular weight of initial silk fibroin as determined by liquid chromatography-mass spectrometry. The hydroxyl groups on galactosamine make it highly hydrophilic, however, it does not affect the hydrophilicity of silk conjugates as compared to its carboxy-silk counterpart. Furthermore, the presence of galactosamine on silk-conjugates does not significantly affect the β-sheet formation capacity of silk fibroin. Finally, we determined the capacity of the carboxy-silk-galactosamine conjugates to form two and three-dimensional scaffolds for potential use in biomedical engineering applications. Films and lyophilized sponges were developed from 1- and 30-minute extracted silk galactosamine conjugates and with no demonstrated mass loss following a 24-hour water incubation period. Self-sustaining, enzymatically crosslinked hydrogels were only successfully developed with 4% and 8% weight by volume 30-minute extracted carboxy-silk galactosamine conjugates. The 30-minute extracted carboxy-silk hydrogel intermediate had a decreased compressive modulus compared to its native silk fibroin counterpart, from 10.7 kPa to 3.2 kPa of 4% weight/volume hydrogels. The compressive modulus of 4% and 8% hydrogels further decreased following galactosamine conjugation, 3.2 and 4.6 kPa to 2.3 and 2.5 kPa for carboxy-silk and silk-galactosamine conjugates, respectively. Thus, we synthesized, characterized, and fabricated carboxy-silk-galactosamine conjugates for potential use in tissue engineering.read less - ID:
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