Compositions and methods for improved encapsulation of functional proteins in polymeric vesicles

1. A method of preparing a suspension of a polymersome-encapsulated functional protein, comprising:

a) thermally blending a quantity of a block copolymer with a quantity of a low molecular weight polyethylene glycol (PEG) for at least 30 minutes, wherein the thermal blending is performed at 90-100° C., wherein the low molecular weight PEG has a molecular weight no greater than that of PEG500;

b) mixing and cooling a resulting PEG/polymer formulation to room temperature;

c) adding an aliquot of a solution of the functional protein to a sample containing the PEG/polymer formulation, wherein a ratio of the added aliquot to the PEG/polymer formulation is between 0.5:1 and 1.5:1 by volume;

d) subsequently performing at least three additional dilution steps such that polymersomes that are generated are progressively saturated with the functional protein, wherein each additional dilution step comprises:

adding to the sample 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; and

e) removing surface-associated protein from polymersomes in the suspension of the polymersome-encapsulated functional protein, after the at least three dilution steps using proteolysis, wherein using proteolysis comprises:

treating the PEG/polymer/protein sample with a 0.4 wt % pronase solution for at least 18 hours at room temperature; and

allowing dialysis of the mixed PEG/polymer/protein sample at 4° C. for at least twelve hours.

2. The method of claim 1 , wherein performing the at least three additional dilution steps comprises performing a first, a second, and a third dilution step in a serial fashion, wherein:

adding the additional amount of the solution in the second step comprises adding a second amount of the solution of the functional protein such that a ratio of the second amount to the PEG/polymer formulation is 2:1 by volume; and

adding the additional amount of the solution in the third step comprises adding a third amount of the solution of the functional protein such that a ratio of the third amount to the PEG/polymer formulation is 5:1 by volume.

3. The method of claim 1 , wherein performing the at least three dilution steps further comprises performing a fourth dilution step, wherein adding the additional amount of the solution in the fourth step comprises adding a fourth amount of the solution of the functional protein such that a ratio of the fourth amount to the PEG/polymer formulation is 5:1 by volume.

4. The method of claim 1 , wherein the solution of the functional protein comprises a 150 mg/mL solution of oxymyoglobin in phosphate buffered saline (PBS).

5. The method of claim 4 , further comprising preparing the solution of the functional protein by:

combining a solution of 150 mg/mL metmyoglobin (metMb) in phosphate buffered saline with sufficient amount of 1 wt % sodium dithionite (Na 2 S 2 O 4 ) to reduce to the metMb to oxymyoglobin (oxyMb).

6. The method of claim 1 , wherein the block copolymer comprises an amphiphilic diblock copolymer.

7. The method of claim 6 , wherein the amphiphilic diblock copolymer comprises poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD).

8. The method of claim 1 , wherein thermally blending the quantity of the block copolymer with the quantity of the low molecular weight PEG for at least 30 minutes comprises thermally blending 5-15 mg of poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD) with 5-15 mg of PEG500 for at least one hour.

9. The method of claim 1 , wherein: thermally blending the amphiphilic diblock copolymer with the low molecular weight PEG for at least 30 minutes comprises

thermally blending 10 mg of poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD) with 10 mg of PEG500 for one hour; and

adding the aliquot of the solution of the functional protein comprises adding 10 μL of an oxymyoglobin solution to the sample of the PEG/polymer formulation.

10. The method of claim 1 , wherein the thermal blending is performed at 95° C.