(599a) Synthesis of Degradable Nanotubes by Tubulin Template Polymerization

Introduction: The cytoskeleton, a bone-like structure floating within the cytoplasm, is a scaffold that provides the cell with structure and shape. One of the filamentous components of the cytoskeleton are microtubules with diameter of 25 nm and several micrometers in length. Microtubules are polymers of alpha and beta tubulin dimers. Tubulin, which can polymerize from both ends in vitro, consists of chemically functional surfaces with well-defined patterns of amino acid side chains that provide a wide variety of active sites for modification. The size and surface properties of microtubules have been exploited for template-directed nucleation of palladium and gold nanoparticles. The objective of this work was to use tubulin as a template for the synthesize of degradable polymeric nanotubes/nanofibers as components of scaffolds for tissue engineering applications. Methods: The purified tubulin protein was purchased from Cytoskeleton. The microtubules were assembled in vitro in a buffer solution of 20 mM PIPES (pH 6.8), 80 mM NaNO3, 0.5 mM Mg(NO3)2, and 1 mM EGTA by adding 0.25 M GTP and, after 30 min, 10 mM taxol at 37C. The assembled microtubules were chemically fixed in 0.5% glutaraldehyde. The microtubule formation and fixation was confirmed by TEM. Tubulin ATRP-tubulin initiator was synthesized by adding 2-bromo-2-methylpropionyl bromide into the microtubule suspension. After reaction, the mixture was dialyzed against distilled water to remove non-tubulin-macroinitiator components. Results: unsaturated (lactide-co-ethylene oxide-co-fumarate) degradable macromer (PLEOF; 10/90 LA/EG) in water was added to 200 ìL of tubulin macroinitiator under N2/Ar atmosphere. Oxygen-free catalyst solution (6.4 mg of CuBr and 14.0 mg of 2,2'-bipyridine in 1.00 mL of degassed water) was added to initiate polymerization. The reaction was stopped after 2 h by dilution with water. The ATRP initiator was chemically attached to the tubulin surface and polymerization of degradable PLOEF macromer was initiated by activation of ATRP initiator. The polymerization of the PLEOF macromer was successfully initiated from the microtules surface. Polymerization resulted in a decrease in the aspect ratio of the tubulin. Some fragmentation of the tubulin was observed due to the acidic nature of the ATRP initiator attached to the microtubules.