Low modulus biomimetic microgel particles with high loading of hemoglobin.

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.