Hemoglobin-Conjugated Gelatin Microsphere as a Smart Oxygen Releasing Biomaterial

In this study, a novel micrometric biomaterial acting as a cyclic oxygen releasing system is designed. Human hemoglobin (Hb) is conjugated to the surface of gelatin microspheres (GM) to produce gelatin hemoglobin oxygen depot (G-HbOD). G-HbOD is obtained by means of two different conjugation strategies. The degree of conjugation of GM surfaces in terms of free amino groups by using HPLC is first evaluated. By following the strategy A (G-HbOD_A), Hb is conjugated to GM by means of the formation of a polyurethane linker. In the strategy B (G-HbOD_B) the conjugation occurs via amide bound formation. Physical and morphological differences between G-HbOD_A and G-HbOD_B are investigated by means of Fourier Transform Infrared Spectroscopy (FTIR), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Differences in oxygen uptake/release kinetics are found depending on the conjugation strategy and it is proved that G-HbOD works under repeated cycles in microfluidic chip. Moreover, G-HbOD is also able to work as oxygen depot in the early stages of 3D cell cultures.