Progressive Saturation Improves the Encapsulation of Functional Proteins in Nanoscale Polymer Vesicles

To develop a technique that maximizes the encapsulation of functional proteins within neutrally charged, fully PEGylated and nanoscale polymer vesicles (i.e., polymersomes). Three conventional vesicle formation methods were utilized for encapsulation of myoglobin (Mb) in polymersomes of varying size, PEG length, and membrane thickness. Mb concentrations were monitored by UV-Vis spectroscopy, inductively coupled plasma optical emission spectroscopy (ICP-OES) and by the bicinchoninic acid (BCA) assay. Suspensions were subject to protease treatment to differentiate the amounts of surface-associated vs. encapsulated Mb. Polymersome sizes and morphologies were monitored by dynamic light scattering (DLS) and by cryogenic transmission electron microscopy (cryo-TEM), respectively. Binding and release of oxygen were measured using a Hemeox analyzer. Using the established "thin-film rehydration" and "direct hydration" methods, Mb was found to be largely surface-associated with negligible aqueous encapsulation within polymersome suspensions. Through iterative optimization, a novel "progressive saturation" technique was developed that greatly increased the final concentrations of Mb (from < 0.5 to > 2.0 mg/mL in solution), the final weight ratio of Mb-to-polymer that could be reproducibly obtained (from < 1 to > 4 w/w% Mb/polymer), as well as the overall efficiency of Mb encapsulation (from < 5 to > 90%). Stable vesicle morphologies were verified by cryo-TEM; the suspensions also displayed no signs of aggregate formation for > 2 weeks as assessed by DLS. "Progressive saturation" was further utilized for the encapsulation of a variety of other proteins, ranging in size from 17 to 450 kDa. Compared to established vesicle formation methods, "progressive saturation" increases the quantities of functional proteins that may be encapsulated in nanoscale polymersomes.