Materials composed of theDrosophilaHox protein Ultrabithorax are biocompatible and nonimmunogenic

Abstract

Although the in vivo function of the Drosophila melanogaster Hox protein Ultrabithorax (Ubx) is to regulate transcription, in vitro Ubx hierarchically self-assembles to form nanoscale to macroscale materials. The morphology, mechanical properties, and functionality (via protein chimeras) of Ubx materials are all easily engineered. Ubx materials are also compatible with cells in culture. These properties make Ubx attractive as a potential tissue engineering scaffold, but to be used as such they must be biocompatible and nonimmunogenic. In this study, we assess whether Ubx materials are suitable for in vivo applications. When implanted into mice, Ubx fibers attracted few immune cells to the implant area. Sera from mice implanted with Ubx contain little to no antibodies capable of recognizing Ubx. Furthermore, Ubx fibers cultured with macrophages in vitro did not lyse or activate the macrophages, as measured by TNF-α and NO secretion. Finally, Ubx fibers do not cause hemolysis when incubated with human red blood cells. The minimal effects observed are comparable with those induced by biomaterials used successfully in vivo. We conclude Ubx materials are biocompatible and nonimmunogenic.

Identification of Multiple Dityrosine Bonds in Materials Composed of the Drosophila Protein Ultrabithorax

“…Indeed, the sequence conservation of both the N‐terminus and the homeodomain in Hox proteins suggests these regions may interact. Furthermore, the N‐terminus has a large impact on the DNA‐binding affinity of the homeodomain . Finally, the ANCHOR algorithm identifies the N‐terminus as a region likely to engage in protein interactions.…”

“…Production of Ubx Materials : Protocols were used as established in the Bondos lab for expression, purification, and assembly of Ubx and Ubx fusion proteins into materials . In brief, the ubx gene, cloned into pET‐19b vector, was transformed into Rosetta (DE3) pLysS cells (Novagen).…”

“…Fibrils associate laterally to generate macroscopic films, which are the building blocks for various macroscale Ubx architectures such as fibers, sheets, and bundles . Ubx materials have many useful properties, including cytocompatibility, biocompatibility, and nonimmunogenicity . Ubx materials can be functionalized (i) with full‐length proteins via gene fusion, (ii) with DNA by sequence‐specific recognition, and (iii) with nanoparticles by noncovalent surface interactions .…”

The Effect of Protein Fusions on the Production and Mechanical Properties of Protein‐Based Materials

“…Ubx materials are biocompatible, strong, and remarkably extensible. Furthermore, proteins fused to Ubx retain their activity once incorporated into materials . The proteins fused to Ubx were selected to test a range of sizes, structures, stabilities, solubilities, and charges.…”

Functionalization of Ultrabithorax Materials with Vascular Endothelial Growth Factor Enhances Angiogenic Activity

“…Cell suspensions were incubated with Ubx fibers with diameters ranging from 7 to 10 μm, wrapped 2−4 times around inoculation loops to allow cells to attach to fibers and cultured as previously described. 53,54 For time course experiments, cells cultured on Ubx were transferred to new wells containing M199 without serum for 0 to 4 h. While ECs seeded on VEGF-Ubx were untreated, 40 ng/mL of soluble VEGF in 1 mL of media was added to ECs seeded on Ubx alone to compare cell responses to immobilized versus soluble VEGF. In all immunofluorescence experiments, 500 μL of a freshly made 10% paraformaldehyde solution in PBS (16 mM Na 2 HPO 4 , 2.6 mM KCl, 1.2 mM K 2 HPO 4 , 68 mM NaCl) was added to the existing culture media yielding a final concentration of 4%, and 53,54 were seeded onto gelatin-coated wells and allowed to reach confluency.…”