Compositions and methods for improved encapsulation of functional proteins in polymeric vesicles
Inventors
Ghoroghchian, P. Peter • Yewle, Jivan Namdeo
Assignees
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Publication Number
US-10987409-B2
Publication Date
2021-04-27
Expiration Date
Abstract
Methods of preparing polymersome-encapsulated functional protein suspensions may include thermally blending an amount of a block copolymer with an amount of a low molecular weight polyethylene glycol (PEG) for at least 30 minutes, mixing and cooling a resulting PEG/polymer formulation to room temperature, adding an aliquot of a solution of the functional protein to a sample containing the PEG/polymer formulation, and performing at least three dilution steps in which polymersomes that are generated are progressively saturated with the functional protein. The aliquot of the solution of the functional protein added may have a to the PEG/polymer sample of around 0.5:1 to 1.5:1 by volume, and the thermal blending may be performed at 90-100° C.
Core Innovation
The invention relates to an improved method for preparing a suspension of polymersome-encapsulated functional protein to address poor protein encapsulation observed with conventional thin-film rehydration and direct hydration. The method uses a thermal blending approach in which a quantity of a block copolymer is blended with a quantity of a low molecular weight polyethylene glycol (PEG) at 90–100 °C for at least 30 minutes, followed by mixing and cooling the PEG/polymer formulation to room temperature.
A functional protein aliquot is then added to a sample containing the PEG/polymer formulation at a defined volume ratio, followed by at least three additional dilution steps. In each additional dilution step, an additional amount of the functional protein solution is added, the resulting dispersion is mixed, and the dispersion is sonicated for at least 30 minutes. This serial dilution and sonication sequence is used to progressively saturate the polymersomes with the functional protein.
After the at least three dilution steps, surface-associated protein is removed from polymersomes by proteolysis using a pronase treatment, followed by dialysis. The resulting polymersome-encapsulated functional protein is described as showing increased myoglobin loading/encapsulation efficiency, preserved oxygen-binding functionality, and stability against aggregation over extended storage, with generalizability to proteins in a specified molecular weight range and using different PEO-b-PBD variants and multiple functional protein examples.
Claims Coverage
The independent claim provides a complete workflow for preparing a suspension of polymersome-encapsulated functional protein, defined by inventive features comprising thermal blending of a block copolymer with low molecular weight PEG, progressive protein saturation via at least three dilution steps with sonication, and removal of surface-associated protein via proteolysis and dialysis. Dependent claims further refine blend temperature and specific ratios/step structure and specify example functional proteins and related proteolysis/dilution structures.
Thermally blended PEG/block copolymer polymersome medium
Thermally blending a quantity of a block copolymer with a quantity of a low molecular weight polyethylene glycol (PEG) for at least 30 minutes at 90–100 °C, followed by mixing and cooling a resulting PEG/polymer formulation to room temperature.
Progressively saturating polymersomes using serial dilution with sonication
Adding an aliquot of a solution of the functional protein to a sample containing the PEG/polymer formulation at a defined volume ratio; subsequently performing at least three additional dilution steps so that polymersomes that are generated are progressively saturated with the functional protein, wherein each additional dilution step comprises adding an additional amount of the solution of the functional protein, mixing a resulting dispersion of the functional protein in the PEG/polymer formulation, and sonicating the resulting dispersion for at least 30 minutes.
Proteolysis and dialysis to remove surface-associated protein
Removing surface-associated protein from polymersomes in the suspension of the polymersome-encapsulated functional protein, after the at least three dilution steps, using proteolysis and dialysis.
Overall, the claim coverage centers on forming a PEG/block copolymer polymersome medium by thermal blending, increasing functional protein encapsulation by progressive saturation through at least three serial dilution steps with sonication, and removing surface-associated protein via pronase proteolysis followed by dialysis.
Stated Advantages
Increases myoglobin loading/encapsulation efficiency.
Preserves oxygen-binding functionality.
Improves stability against aggregation over extended storage.
Documented Applications
Polymersome-encapsulated functional proteins including myoglobin (metmyoglobin/oxymyoglobin) and other proteins spanning an indicated molecular weight range, demonstrating generalizability to multiple functional proteins such as hemoglobin, BSA, IgG, catalase, fibrinogen, and apoferritin.
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