Porosity and semipermeability of hemoglobin-loaded polymeric nanoparticles as potential blood substitutes

Porosity and semipermeability, allowing life-sustaining small molecules to penetrate, but hemoglobin (Hb) and other enzymes to cut off, predominantly affect the functionalities of the Hb-loaded polymeric nanoparticles (HbPNPs) as blood substitutes. In this article, HbPNPs formulated in the size range of 110-122 nm were prepared by a modified double-emulsion method with poly(lactic acid) (PLA)-based polymers. The influences of the main preparation conditions, including solvent composition, stirring speed, Hb concentration and polymer matrix, on the porosity were investigated in details. To evaluate the porosity of HbPNPs, a novel nondestructive testing method based on molecular weight cut-off (MWCO) was developed, and an effusion approach was applied to investigate the pore size in the particle shells with poly(ethylene glycol)s (PEGs) of different molecular weights (PEG200, PEG400, PEG600) as probes. Moreover, in vitro diffusion behaviors of ascorbic acid and reduced glutathione from HbPNPs fabricated with various polymer matrices were studied. The MWCO of HbPNPs by changing solvent composition, stirring speed, Hb concentration, and polymer composition varied from 200 to 600, especially the PEGylation of the polymer, which exhibited obvious influence on the MWCO of HbPNPs. Ascorbic acid with molecular weight 176.1 could diffuse into PEGylated nanoparticles with mPEG content of 5-30 wt % freely, while reduced glutathione with molecular weight 307.3 could not penetrate when mPEG content reached 30 wt %. These results suggest that the HbPNPs optimized with MWCO between 400 and 600 can facilitate the transport of all those life-sustaining small molecules.