Compositions and Methods for Improved Encapsulation of Functional Proteins in Polymeric Vesicles

RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62/187,942, filed on July 2, 2015, and U.S. Non-Provisional Patent Application Serial No. 15/198,836, filed on June 30, 2016, both of which are hereby incorporated by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] Work on this invention was supported by funds from the National Institute of Health (Study ID# 1R43CA159527-01A1 and Study ID# 1R43AI096605-01). The United States Government therefore has certain rights in this invention.

BACKGROUND

[0003] Natural and synthetic proteins offer an incomparable array of unique biological functions that may be exploited for human therapeutic applications. Their clinical utility, however, is often limited by biochemical instability, poor pharmacologic properties, and potential to induce adverse immunogenicity. Incorporation of biomolecules, such as proteins, in long-circulating vehicles with attached polyethylene glycol (PEG) polymer chains (i.e., PEGylated vehicles), such as nanoparticles, may mitigate such issues. However, the stable encapsulation of large quantities of functional proteins in PEGylated vehicles has proven to be challenging. Conventional encapsulation techniques, which were originally developed for small-molecule drug delivery, require the input of high energies and/or the use of organic solvents for particle formation, and are therefore unsuitable for use with biologically complex and more labile macromolecules.

[0004] In particular, examples of conventional encapsulation techniques may include thin-film rehydration, direct-hydration, and electro-formation, which may be used to encapsulate small molecules and proteins with unique biological function into polymersomes generated from poly(ethylene oxide)-block-poly(butadiene) (PEO-b- PBD). For example, methylene blue (mBlue; Mw = 319.85 g/mol) may be used as a model small molecule, and myoglobin (Mb; Mw =17,600 Da) may be used a model protein with unique biological function (i.e., oxygen storage). The efficiencies of encapsulating methylene blue and myoglobin into PEO-b-PBD using the thin-film rehydration and direct hydration techniques have been compared. In particular, quantification of the maximum encapsulation of fully functional myoglobin was based on a number of characteristics, using these established techniques. For example, the concentration and the reduction-oxidation reaction ("redox") state of iron in the heme group of myoglobin were respectively measured using inductively coupled plasma optical emission spectroscopy (ICP-OES) and UV-Vis absorption spectroscopy (also referred to as spectrophotometry). The morphologies and stabilities of polymersome- encapsulated myoglobin (PEM) were respectively verified by cryogenic transmission electron microscopy (cryo-TEM) and by dynamic light scattering (DLS). Equilibrium oxygen binding and release at various partial pressures of oxygen were measured using a Hemeox analyzer. While the thin-film rehydration and direct hydration techniques allowed for successful methylene blue encapsulation, encapsulation of myoglobin was uniformly poor. Therefore, improved methods for generating PEM will be beneficial for human therapeutic applications.

SUMMARY

[0005] Various embodiments include methods of preparing polymersome- encapsulated functional protein suspensions by 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. In some embodiments, a ratio of the added aliquot to the PEG/polymer sample is around 0.5: 1 to 1.5: 1 by volume. In some embodiments, the thermal blending is performed at 90-100 °C. In some embodiments, each dilution step includes 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.

[0006] In some embodiments, performing the at least three dilution steps includes performing a first, a second, and a third dilution step in a serial fashion. In some embodiments, adding the additional amount of the solution in the first step includes adding a first amount of the solution of the functional protein such that a ratio of the first amount to the PEG/polymer formulation is around 1: 1 by volume. In some embodiments, adding the additional amount of the solution in the second step includes 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 around 2: 1 by volume. In some embodiments, adding the additional amount of the solution in the third step includes 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 around 5: 1 by volume.. Embodiment methods may also include performing a fourth dilution step in which adding the additional amount of the solution in the fourth step includes 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 around 5: 1 by volume. Embodiment methods may further also include removing surface-associated protein from polymersomes in the suspension of the polymersome-encapsulated functional protein using proteolysis after the at least three dilution steps. In some embodiments, using proteolysis includes treating the mixed PEG/polymer/protein sample with a 0.4 wt% pronase solution for at least 18 hours at room temperature, and allowing dialysis of the

PEG/polymer/protein sample at 4°C for at least twelve hours.

[0007] In some embodiments, the solution of the functional protein may be a 150 mg/mL solution of oxymyoglobin (oxyMb) in phosphate buffered saline (PBS).

Embodiment methods may also include 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 oxyMb. In some embodiments, the block copolymer may be an amphiphilic diblock copolymer. In some embodiments, the amphiphilic diblock copolymer may be poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD). In some embodiments, thermally blending an amount of the amphiphilic diblock copolymer with an amount of the low molecular weight PEG for at least 30 minutes includes thermally blending 5-15 mg of poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD) with 5-15 mg of 500 kDa PEG (PEG500) for at least one hour. In some embodiments, thermally blending an amount of the amphiphilic diblock copolymer with an amount of the low molecular weight PEG for at least 50 minutes includes thermally blending 10 mg of poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD) with 10 mg of 500 kDa PEG (PEG500) for one hour. In some embodiments, thermally blending the amount of the amphiphilic diblock copolymer with the amount of the low molecular weight PEG for at least 30 minutes may include thermally blending 10 mg of poly(ethylene oxide)-block-poly(butadiene) (PEO-b- PBD) with 10 mg of 500 kDa PEG (PEG500) for one hour. In some embodiments, adding the aliquot of the solution of the functional protein may include adding 10 μΕ of a solution of oxyMb to the sample containing the PEG/polymer formulation. In some embodiments, the thermal blending may be performed at around 95 °C.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate exemplary embodiments, and together with the descriptions of various embodiments, serve to explain the features herein.

[0009] FIG. 1 is a table of results from encapsulating a variety of proteins using existing techniques.

[0010] FIG. 2 is a table of properties for two poly(ethylene oxide)-block- poly(butadiene) (i.e., PEO-b-PBD) diblock copolymers and their polymersome formulations used for small molecule and protein encapsulation.

[0011] FIGs. 3 A and 3B are graphs showing results from optimization of various steps in the direct hydration protocol to improve encapsulation of myoglobin in polymersomes of a particular PEO-b-PBD formulation.

[0012] FIGs. 3C and 3D are graphs showing the rate of myoglobin oxidation and the loss of surface-associated myoglobin from proteolysis as functions of time.

[0013] FIG. 3E is a graph showing final weight percentage compared to encapsulation efficiency of myoglobin using various ratios of myoglobin to phosphate buffered saline solution.

[0014] FIG. 4 is a table showing encapsulation results from using the progressive saturation protocol and OB29 polymersomes to encapsulate a range of proteins according to the various embodiments.

[0015] FIG. 5A is a schematic representation of components of polymersomes prepared according to the various embodiments.

[0016] FIG. 5B is schematic representation of the existing thin-film rehydration protocol for forming polymersome-encapsulated myoglobin.

[0017] FIG. 5C is a schematic representation of the existing direct hydration protocol for forming polymersome-encapsulated myoglobin.

[0018] FIGs. 6A and 6B are graphs showing results from encapsulation of methylene blue into polymersomes formed from a particular PEO-b-PBD formulation using existing protocols.

[0019] FIGs. 6C and 6D are graphs showing results from encapsulation of myoglobin into polymersomes formed from a particular PEO-b-PBD formulation using existing protocols.

[0020] FIG. 7A is a schematic representation of the progressive saturation protocol for forming polymersome-encapsulated myoglobin in various embodiments.

[0021] FIGs. 7B and 7C are graphs illustrating results from encapsulation of myoglobin into polymersomes formed from a particular PEO-b-PBD formulation using the progressive saturation protocol of FIG. 7A. [0022] FIG. 7D is a table summarizing the results shown in FIGs. 7B and 7C.

[0023] FIG. 7E is a table showing the partial pressure of oxygen required to achieve 50% saturation (P ₅₀ ) obtained from O ₂ equilibrium curves for of free myoglobin and for polymersome-encapsulated myoglobin, prior to proteolysis and after pronase treatment, that was prepared by progressive saturation.

[0024] FIG. 8A is a graph showing results from encapsulation of myoglobin into polymersomes formed from two particular PEO-b-PBD formulations using the progressive saturation technique of the various embodiments.

[0025] FIGs. 8B and 8C are cryo-TEM images of vesicles in polymersome- encapsulated myoglobin suspensions formed from each of the particular PEO-b-PBD formulations using the progressive saturation technique of the various embodiments.

[0026] FIG. 8D is a graph showing the average hydrodynamic diameters of a polymersome-encapsulated myoglobin suspension formed from a particular PEO-b- PBD formulation using the progressive saturation technique of the various

embodiments.

[0027] FIG. 8E is a graph showing the oxygen equilibrium curves for free

oxymyoglobin and oxygenated polymersome-encapsulated myoglobin suspension formed from a particular PEO-b-PBD formulation using the progressive saturation technique of the various embodiments.

DETAILED DESCRIPTION

[0028] The various embodiments will be described in detail with reference to the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. References made to particular examples and implementations are for illustrative purposes, and are not intended to limit the scope of the claims.

[0029] The various embodiments include improved methods for generating PEM, which may include identifying the key parameters of the thin-film rehydration and direct hydration protocols that prevent efficient uptake and/or compromise protein function, and performing iterative optimization of such key parameters. As a result, the various embodiments provide a generalizable technique that enables the encapsulation of increased quantities of functional proteins within neutrally charged and fully PEGylated polymer vesicles. That is, the various embodiments include a new "progressive saturation" method for encapsulating myoglobin in polymersomes.

[0030] Nanoparticle vehicles may overcome many challenges associated with the delivery of functional proteins to enable the clinical development of diverse macromolecular pharmaceuticals. Nanoparticle vehicles may include, for example, liposomes (i.e., self-assembled vesicles of natural phospholipids) and polymersomes (i.e., self-assembled polymer vesicles of block copolymers), as well as micelles, perfluorocarbon emulsions, and others. To that end, polymersomes, have

advantageous properties as compared to conventional liposomes, as liposomes typically have high membrane permeability and low stability in vivo. There have been, however, few comparative studies to establish and validated a single, scalable and generalized strategy for encapsulating large amounts of protein in neutrally charged and/or PEGylated polymersomes. By optimizing and combining different steps from various liposome-based encapsulation methods, the various embodiments provide a new progressive saturation technique that allows improved encapsulation of functional proteins in nanoscale polymer vesicles. The various embodiments demonstrate a tradeoff between the degree of polymersome loading (i.e., weight percentage of protein-to-polymer) and the encapsulation efficiency of protein (with respect to the initial quantity that was employed for polymersome formation) that may be achieved. Moreover, in the various embodiments, a proteolysis step accurately quantifies the amounts of both encapsulated protein (i.e., the desired outcome) as well as surface associated (i.e., non-specifically bound) product that may be obtained in polymersome suspensions formed by the progressive saturation protocol. While there are some reports of large amounts of protein loading within polymersomes at high efficiencies using existing liposome encapsulation techniques, such reports do not involve differentiating between encapsulated and surface-associated protein.

Therefore, the progressive saturation technique in the various embodiments may provide a more robust, scalable, and generalizable strategy for encapsulation of proteins in fully PEGylated and neutrally charged polymersomes in quantities and at efficiencies that may enable further translational development.

[0031] Selective and potent modulation of protein function in mammalian cells is the principal activity of most molecular therapeutics, where the vast majority of available agents are organic small molecules (less than or equal to 800 Da in size). Recent studies suggest, however, that only a small percentage of the human proteome is susceptible to small molecule-based therapy. Moreover, the functional diversity of proteins that are successfully targeted by small molecules remains very low. That is, around 40% of all prescription drugs target a single class of proteins, namely the G- protein coupled receptors. However, use of small-molecule therapy is limited since small drug molecules are intrinsically unable to cope with the extended contact surfaces found at many biologically important interfaces.

[0032] Biomacromolecules such as proteins have recently shown significant clinical utility, in large part due to their ability to overcome these significant limitations associated with traditional small molecule therapies. When compared to the interaction of a small-molecule with its biological target, macromolecular therapeutics have higher folding energies (typically around 7-20 kcal/mol) that allow for the adoption of larger and more precise three-dimensional configurations, which are often required for efficient binding and/or control of complex biological function. As such, macromolecules may achieve superior binding selectivity and more potent on-target activity. Currently, a small number of macromolecular therapies in use, including the approximately 200 protein drugs available worldwide, have demonstrated a high potential as new leads in drug development. Nevertheless, several barriers have hindered the ready development of macromolecules as human therapeutics, including: (i) the difficulty and/or expense of commercial scale production, (ii) biochemical instability that occurs in pathophysiologic environments or with prolonged storage, (iii) short circulatory half-lives and large steric hindrance that prevent effective tissue penetration, and (iv) risks associated with their potential to promote severe adverse effects, such as the induction of anti-idiotypic antibodies and/or immune complex formation. To overcome some of these limitations, most pharmaceutical compounds either employ biocompatible polymers (e.g., polyethylene glycol (PEG) or hyaluronic acid) or liposomes (i.e., lipid based vesicles) for protein complexation and in vivo delivery.

[0033] Synthetic nanoparticles may exhibit superior properties to enhance drug delivery when compared to more conventional formulations. In particular, among the classes of nanoparticles, polymersomes (i.e., self-assembled polymer vesicles comprised of amphiphilic block copolymers) may provide a beneficial nanoscale delivery platform. While lipid-based vesicles (i.e., liposomes) have been extensively utilized in biomedical research, there are material limitations inherent to these phospholipid-based drug delivery vehicles, including compromised suspension stability, premature drug release, and limited product shelf-life. In contrast, polymersomes are formed from higher molecular weight amphiphilic block

copolymers that impart a broad and tunable range of carrier properties. For example, polymersomes enable: (i) facile and stable loading of diverse therapeutic payloads through non-covalent interactions, (ii) mechanical stability that is 5 to 50 times greater than that of liposomes or micellar structures constructed from similar molecular weight copolymers, (iii) economic and large scale production that removes the need for costly post-manufacturing purification, and (iv) diversity in biochemical properties, which are imparted by their construction from a variety of copolymer compositions. Such properties may include fully PEGylated surfaces and tunable in vivo circulation times, site-specific targeting, environmental responsiveness, and complete biodegradation.

[0034] The incorporation of proteins into nanoparticles may enhance their

pharmacologic performance and improve their on-target activity. Methods that have been developed for encapsulating proteins into nanoparticles have utilized

electrostatic interactions to incorporate a handful of highly anionic proteins, or chemical or genetic modification of the original protein for efficient and reproducible nanoparticle formation. Examples of such method include thin-film rehydration (i.e., rehydration of dried polymer), which results in low yields of polymersome- encapsulated protein. Another example method is direct hydration, the use of which is generally limited to small-scale preparations (e.g., less than one mL). Another example method is electro-formation, which provides useful results for only a limited number of proteins (i.e., highly charged proteins). Another example method is hollow-fiber extrusion, which involves extrusion of preformed vesicles in the presence of protein solution. While the hollow-fiber extrusion technique has been used for large-scale preparations of liposome-encapsulated protein, elevated temperatures and pressures are required for polymersome formation, which has limited its widespread applicability.

[0035] Existing techniques require the input of thermal, electric, ultrasonic, or mechanical energy for particle formation, or alternatively the use of organic cosolvents, which may damage the structure and/or function of the protein, making encapsulation more challenging and limited in utility. Therefore, in various encapsulation techniques, a need exists for a generalized method that enables the incorporation of large quantities of native protein in neutrally charged and/or

PEGylated nanoparticles.

[0036] While adoption of various liposome encapsulation techniques has enabled facile incorporation of small molecules within polymersomes, these methods cannot directly be applied for scalable encapsulation of the functional proteins. Often, there is a trade-off in the maximum concentration of the aqueous protein that may be encapsulated (i.e., mg protein/mL solution), the final loading ratio of protein-to- polymer that comprises the polymersome structure (i.e., w/w% protein/polymer), and/or the protein encapsulation efficiency (i.e., the percentage of the initial protein suspension that is retained). Further, the value of each of these parameters is highly dependent on the nature of the protein, the exact block copolymer formulation, and the encapsulation method that is utilized. For example, the table in FIG 1 shows existing results from encapsulation of various proteins into polymersomes. The various embodiments provide an alternative, optimized and reproducible method to efficiently encapsulate increased quantities of functional proteins in polymersomes. The newly developed "progressive saturation" technique of the various embodiments is readily scalable, highly reproducible, and generalizable for producing increased quantities of polymersome-encapsulated protein that may enable new and diverse biomedical applications.

[0037] In various embodiments, PEO-b-PBD copolymers are used to form

polymersomes that possess fully PEGylated surfaces. Such surfaces, being uncharged and nondegradable; provide an ideal system for ensuring vesicle integrity and minimizing unwanted protein interactions or modifications. Two different molecular weight PEO-b-PBD polymers, "OB 18" diblock copolymer and "OB29" diblock copolymer, are employed to determine the generalizability of the results as they pertain to polymersomes of different minimal sizes, PEG lengths, and membrane core thicknesses. FIG. 2 provides a table showing a comparison of various properties of OB 18 and OB29. Methylene blue (mBlue; Mw = 319.85 g/mol), which is highly stable in aqueous suspension and has a strong near-infrared absorbance enabling ready spectrophotometric detection, is used as a model small molecule to establish various baseline parameters for encapsulation using existing methods. Such baseline parameters include aqueous suspension concentration, final weight percentage, and encapsulation efficiency. Myoglobin (Mb; Mw =17,600 Da), which has a size and thermal stability (i.e., denaturation above 60 °C) comparable to other small proteins with therapeutic potential, was used as a model protein. Myoglobin also has a strong ultraviolet (UV) absorbance that enables ready identification of its functional status, as determined by the redox state of its iron-containing heme group. Myoglobin has additionally been employed in other studies, enabling comparisons of results to other encapsulations using existing techniques, as discussed above with respect to FIG. 1. Methylene blue is easily encapsulated in PEO-b-PBD polymersomes formed by thin- film rehydration at elevated temperatures, yielding final weight ratios of mBlue-to- polymer of 4.1 and 5.0 w/w% when formed at 40 and 60 °C, respectively. However, similar conditions only led to myoglobin degradation.

[0038] When vesicles are formed by thin-film rehydration, as the film of dry copolymer is hydrated, lamellae (aka sponge-like structures) are first formed as the hydrophilic blocks in the film swell. Further swelling leads to transformation into hexagonally packed vesicles and finally into fully dispersed polymersomes. When thin-film rehydration is attempted in solutions of soluble small molecules (or proteins), these water-soluble species adsorb to the surfaces of the budding lamellae, which subsequently adopt a spontaneous (or preferred) curvature. During formation, these membranes preferentially bend away from the aqueous compartment that contains the higher concentration of adsorbing species, thereby excluding the water- soluble agents from vesicle encapsulation. Ultimately, the input of energy can overcome this spontaneous surface tension in order to promote vesicle encapsulation. The amount of energy that is required scales with the size of the adsorbed molecule and the membrane thickness of the vesicle. Thus, while it is easy to disrupt liposomes and enable effective small molecule and protein loading by thin-film rehydration by the input of thermal (and/or sonic) energy, such input is only only enables effective encapsulation of small molecules into polymersome suspensions. In the direct hydration method, which was developed as a hybrid of solvent dispersion and homopolymer addition, the hydrophilic polymer PEG500 dimethyl ether (DME) is used to disrupt the interactions of hydrophobic chains in the forming polymer lamellae . With subsequent additions of aqueous solution, self-assembly of vesicles from budding lamellae that have dispersed protein is promoted and results in improvements in aqueous encapsulation; encapsulation efficiencies as high as 37% have been observed. Using direct hydration at 23 °C, polymersome-encapsulated myoglobin suspensions may have encapsulation efficiencies greater than 10%, with the encapsulated myoglobin species exhibiting good suspension properties and the characteristic absorption spectra of intact protein. The final loading of myoglobin in these polymersome-encapsulated suspensions, however, was found to be only around 0.3 w/w% Mb/polymer. Upon addition of a protease solution to induce proteolysis of all surface associated (i.e., non-specifically bound) protein, the final Mb composition of PEM suspensions was found to be even lower— that is, less than 0.1 w/w%

Mb/polymer. For translational therapeutic applications, the loading of therapeutic proteins within the aqueous cavities of polymersome vehicles is ultimately the metric that must be maximized in order to minimize the amount of associated carrier that is introduced to a subject. Therefore, such encapsulation using standard direct hydration is insufficient.

[0039] In various embodiments a progressive saturation protocol provides for efficient generation of PEM suspensions. The generalizability of progressive saturation for protein encapsulation is further established by utilizing a variety of different proteins, ranging from 17-450 kDa, yielding nanoscale polymersomes in quantities that may enable preclinical investigations of many novel therapeutic compositions. In particular, a difference between the progressive saturation method and direct hydration may involve adding five subsequent volumes of the functional protein solution to dilute the PEG/polymer mixture in lieu of additional dilutions with phosphate buffered saline (PBS).

[0040] Specifically, the progressive saturation method of the various embodiments involves heating 10 mg of polymer and 10 mg of PEG at around 95 °C for around 1 h. The sample mixture may be centrifuged and cooled to room temperature. A

metmyoglobin (metMb) solution (e.g., 150 mg/mL in PBS) may be reduced to oxymyoglobin (oxyMb) with sodium dithionite (Na ₂ S ₂ O ₄ ) (e.g., 1 wt%). From the resulting oxyMb solution, a portion (i.e., aliquot) may be added to the sample mixture at a ratio of 1 : 1 by volume, and mixed thoroughly followed by sonication at room temperature for around 30 min. In particular, the aliquot may be 10 μΕ of the oxyMb solution. The sample mixture may be further diluted with a number of dilution steps. Specifically, each dilution step may involve addition of a volume of the oxyMb solution (e.g., 150 mg/mL in PBS), followed by thorough mixing and sonication at room temperature for around 30 minutes. The volumes of oxyMb solution used in the dilution steps may be amounts in which ratios of the oxyMb solution to the original sample mixture are 1: 1, 2: 1, 5: 1, and 5: 1 by volume 10 μΕ, followed by 20, 50 and 100 μΕ. After the dilution steps, the resulting sample may be sonicated for an additional 30 min at room temperature, followed by dialysis for at least 30 h at around 4 °C, employing a 1000 kDa molecular weight cutoff membrane. Surface associated myoglobin may be removed by proteolysis via treatment with 0.4 wt % pronase solution, followed by dialysis for at least 12h at around 4 °C (e.g., molecular weight cutoff of 1000 kDa). In various embodiments, myoglobin encapsulation of the resulting polymersome suspension may be measured before and after proteolysis. Specifically, concentration of myoglobin may be measured using inductively coupled plasma optical emission spectroscopy (ICP-OES), while redox states of iron in the heme group of myoglobin may be quantified using UV-Vis absorption spectroscopy.

[0041] These progressive saturation steps provided favorable results for encapsulating myoglobin in OB 18 polymersomes, as shown in FIGs. 3A-3E. For example, FIG. 3 A shows the final weight percentage of Mb-to-polymer (i.e., w/w% Mb/polymer) in polymersome-encapsulated myoglobin obtained when no sonication was used (as in conventional techniques) and by using sonication, according to the progressive saturation protocol (i.e., for around 30 min at room temperature after each dilution step). A shown in FIG. 3A, a final weight percentage of Mb-to-polymer of around 6 (i.e., w/w% Mb/polymer) may be an achieved result from polymersome-encapsulated myoglobin created by a protocol that includes such sonication. Therefore, sonicating the sample for around 30 min at room temperature after each dilution step may increase encapsulation efficiency by more than 30 times based on the final weight percentage resulting from polymersome-encapsulated myoglobin generated via direct hydration.

[0042] FIG. 3B shows the final weight percentage of Mb-to-polymer (i.e., w/w% Mb/polymer) in polymersome-encapsulated myoglobin obtained using a

metmyoglobin solution (as in conventional techniques) and by using oxyMb as in the progressive saturation technique. FIG. 3C shows the rate of myoglobin oxidation (expressed as a percentage of metMb formed over time) as a function of myoglobin exposure to different solution conditions. FIG. 3D shows the amount of surface- associated Mb removed (% Mb loss) as a function of proteolysis time for various oxyMb volumes. FIG. 3E shows the final weight percentage of Mb-to-polymer (i.e., w/w% Mb/polymer) compared to encapsulation efficiency (% Mb EE) in

polymersome-encapsulated myoglobin generated using various ratios for the volumes of oxyMb solution to PBS used in the dilution steps. In particular, the samples in FIG. 3E were proteolyzed for 18 h to remove surface-associated myoglobin, and quantified using UV-Vis absorption spectroscopy.

[0043] Therefore, the final Mb-to-polymer weight ratios that were obtained in generating polymersome-encapsulated myoglobin using progressive saturation according to various embodiments (i.e., 4-6 w/w% Mb/polymer) may be significantly improved compared to polymersome-encapsulated myoglobin generated using the direct hydration protocol (i.e., 0.1-0.3 w/w% Mb/polymer). Without wishing to be bound to a particular theory, the loading capacity achieved using progressive saturation steps may be due to incomplete polymersome formation during the initial dilution step, and further encapsulation being accomplished with each subsequent addition of protein solution.

[0044] Developing the progressive saturation protocol included optimizing and combining various steps from multiple liposome formation methods. Factors influencing the final concentrations of myoglobin, the relative loading levels that could be achieved within the OB 18 polymersome carrier (i.e., w/w%

protein/polymer), and the efficiency of myoglobin encapsulation were systematically evaluated. Factors such as the molecular weight of the polymer, the oxidation state and concentration of the protein, the pH and nature of the buffered solution, the exact polymer hydration conditions (i.e., time, temperature, and blending technique), the number and duration of sonication steps, and the addition or avoidance of freeze-thaw cycles all had effects on the concentration and the fidelity of the final polymersome- encapsulated protein product.

[0045] Further, compared to polymersome-encapsulated myoglobin created using existing techniques, polymersome-encapsulated myoglobin created by progressive saturation also exhibits an increase in the final concentrations of Mb. For example, the final concentration of Mb in polymersome-encapsulated myoglobin generated via direct hydration is less than around 0.5 mg/ML in solution, while that of

polymersome-encapsulated myoglobin generated via progressive saturation in the various embodiments may be greater than around 2.0 mg/mL in solution. [0046] Using cryo-TEM to verify vesicle morphologies, suspensions of polymersome- encapsulated myoglobin developed using progressive saturation showed no signs of aggregate formation when maintained at 4 °C, 23 °C, and 37 °C for longer than one month. The progressive saturation technique may be further utilized for the successful encapsulation of a variety of other proteins ranging in size from 17 to 450 kDa, within PEO-b-PBD polymersomes.

[0047] Without wishing to be bound to a particular theory, there may be a direct tradeoff between Mb encapsulation efficiency and the final weight ratios of Mb-to- polymer that could be achieved based on the concentration of free Mb that was used for each addition step. Aqueous encapsulation of protein is preferred to surface- associated protein in order to assure that the final product meets the objectives for utilizing a polymersome delivery vehicle— that is, to improve biochemical stability, to increase circulatory half-life, to minimize adverse side effects, and to achieve controlled release of the associated protein. The various embodiment techniques may be employed using different proteins that vary over a large range of molecular weights and sizes, including those associated with therapeutically relevant antibodies and enzymes. For example, the progressive saturation technique may be utilized to encapsulate myoglobin in a PEO-b-PBD-based polymersome system comprised of the OB29 diblock copolymer. In various embodiments, the progressive saturation technique may be utilized to encapsulate any of a number of other proteins, including, but not limited to, antibodies (e.g., immunoglobulin G (IgG) and functional enzymes (e.g., catalase).

[0048] As described above with respect to FIG. 2, when compared to the OB 18 diblock copolymer, the OB29 diblock copolymer has a smaller molecular weight and generates polymersomes with a shorter PEG brush border (1.3 vs. 3.9 kDa), thinner bilayer membrane (9.6 nm vs. 14.8 nm), and smaller average hydrodynamic diameter (130 vs. 200 nm). In various embodiments, using progressive saturation to

encapsulate myoglobin in OB29 polymersomes provides similar results to those from OB 18 polymersomes. In various embodiments, progressive saturation technique may be applied using any PEG-based polymersome-forming block copolymer, including any amphiphilic polymer comprised of PEG and a hydrophobic block that is a biodegradable polymer (e.g., a biodegradable polyester, poly(amide), poly(peptide), poly(nucleic acid), etc.). Examples of biodegradable polyesters that may form the hydrophobic block include, but are not limited to, poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(caprolactone), poly(methyl caprolactone), poly(hydroxybutyrate), poly(hydroxyvalerate), poly(hdyroxyhexanoate),

poly(hydroxyoxtanoate), and poly(trimethylene carbonate).

[0049] The generalizability of the progressive saturation technique is further demonstrated by analogous results from encapsulation of several larger proteins using OB29 polymersomes. FIG. 4 shows encapsulation results from using the progressive saturation protocol and OB29 polymersomes to encapsulate myoglobin, hemoglobin (Hb) (64 kDa), bovine serum albumin (BSA) (66 kDa), IgG (150 kDa), catalase (250 kDa), fibrinogen (340 kDa), and apoferritin (450 kDa).

[0050] The invention is intended to be illustrated but not limited by the following examples.

EXPERIMENTAL

[0051] Comparative and quantitative studies were performed in order to establish a generalizable method for producing scalable quantities of neutrally-charge and fully PEGylated polymersomes that encapsulate functional protein. Differences in small molecule and protein encapsulation were examined by employing polymersome formulations comprised of OB 18 and OB29 diblock copolymers. As described above with respect to FIG. 2, these two PEO-b-PBD polymers and the polymersomes formed therefrom differ with respect to molecular weight and, ultimately, vesicle membrane thicknesses. Methylene blue (mBlue; Mw = 319.85 g/mol) was used as a model small molecule and myoglobin (Mb; Mw =17,600 Da) as a model protein with unique biological function (i.e., oxygen storage and release). FIG. 5A shows a representation of polymersomes made of amphiphilic diblock copolymers, as well as water-insoluble agents and water-soluble species that may be encapsulated in or attached to

polymersomes. For example, conventional vesicle formation techniques that were employed to incorporate water-soluble agents within PEO-b-PBD polymersomes included thin- film rehydration and direct hydration, protocols for which are shown in FIGs. 5B and 5C, respectively. The encapsulations of methylene blue and myoglobin in PEO-b-PBD polymersomes generated by each of the thin-film rehydration and direct hydration methods were compared. In order to quantify the encapsulation of fully functional protein capable of oxygen binding, the iron concentration in

polymersome-encapsulated myoglobin was measured by ICP-OES, and the redox states of iron in the heme group of myoglobin measured by UV-Vis absorption spectroscopy.

[0052] Compared to PEM created using existing techniques, PEM created by progressive saturation exhibit an increase in the final concentrations of Mb (e.g., from less than around 0.5 mg/mL in solution to greater than around 2.0 mg/mL in solution), and an increase in the final weight ratio of Mb to polymer that could be reproducible obtained (from less than 1 w/w% Mb/polymer to greater than around 3-4 w/w% Mb/polymer). Further, PEM created by progressive saturation show an increase in the overall efficiency of protein encapsulation (from less than around 5% to greater than around 90%) in the PEM suspensions. Using cryo-TEM to verify vesicle morphologies, suspensions of PEM developed using progressive saturation display no signs of aggregate formation for longer than one month at 4 °C, 23 °C, and 37 °C.

[0053] Materials

[0054] PEO(3900)-b-PBD(6500) (OB18) and PEO(1300)-b-PBD(2500) (OB29) were purchased from Polymer Source (Dorval, Quebec, Canada). Horse skeletal muscle Mb, bovine blood hemoglobin (Hb), bovine serum albumin (BSA), catalase (C), fibrinogen (F), sodium hydrosulfite, poly(ethylene glycol) dimethyl ether (PEG; Mn = -500), protease from Streptomyces griseus ("pronase"), and dichloromethane (DCM) were purchased from Sigma-Aldrich (St. Louis, USA). Horse spleen apoferritin (aFr) was purchased from Alfa Aesar (Ward Hill, USA). Immunoglobulin G (IgG) was purchased from LEE Biosolutions (St. Louis, USA). Dialysis tubing and vials were purchased from Spectrum Laboratories (Rancho Dominguez, USA). Sodium chloride, potassium chloride, sodium phosphate dibasic, potassium phosphate monobasic, mBlue, and Triton X-100 were purchased from Fisher Scientific (Suwanee, USA). All chemicals were of reagent grade unless otherwise stated.

[0055] The particle sizes were measured using Delsa™Nano, a dynamic light scattering (DLS) instrument (Beckman Coulter, Indianapolis, USA). Mb and mBlue concentrations were determined by absorption spectroscopy using a Genesys™ 10S UV-Vis spectrophotometer (Thermo Scientific, Suwanee, USA). The concentrations of all proteins in polymersome suspension were further measured using a Micro BCA Protein Assay Kit, utilizing UV-Vis spectrophotometry and by following the manufacturer's protocols (Pierce Biotechnology, Inc; Rockford, IL, USA). Iron concentrations in polymersome-encapsulated Mb suspensions were determined using a Vista-PRO CCD ICP-OES (Varian, USA). Oxygen equilibrium binding was studied using a Hemox™- Analyzer (TCS Scientific Corp, New Hope, PA, USA). Electro- formation was performed using Gene Pulser (Bio-Rad, Hercultes, CA, USA).

[0056] Methods

[0057] Thin-film rehydration method

[0058] 10 mg of OB 18 polymer was dissolved in 200 μΐ, of DCM. The polymer solution was deposited on Teflon wafers (15mm 15mm) that were subsequently dried for 30 min at room temperature (RT). The films were further kept under vacuum overnight at RT to ensure DCM evaporation. For methylene blue encapsulation, polymer films were then hydrated with methylene blue solution (21 mg/mL) in phosphate buffered saline (PBS; 10 mM, pH 7.4) for 24-48 h at 23, 40 or 60 °C. The samples were sonicated for 30 min at room temperature, followed by (xlO) fireeze- thaw cycles using liquid nitrogen. The samples were dialyzed (MW cutoff = 100 kDa) for 30 h at RT. For myoglobin encapsulation, polymer films were hydrated with myoglobin solution (150 mg/mL) in PBS (10 mM, pH 7.4) for 60 h at 23, 40, and 60 °C. The samples were then sonicated for 30 min at RT followed by dialysis (MW cutoff = 1000 kDa) for 30 h at 4 °C.

[0059] Direct hydration method [0060] 10 mg of OB 18 and 10 mg of PEG were heated in a 1.5 mL centrifuge tube for 20 min at 95 °C. The samples were mixed and cooled to room temperature, followed by the addition of 10 μΐ. of methylene blue solution (21 mg/mL) or myoglobin solution (150 mg/mL) in PBS (10 mM, pH 7.4). The samples were then diluted with 20, 70, and 900 μΐ. of PBS and well mixed after each addition/dilution (via vortexing). The samples were then dialyzed for 30 h at room temperature or at 4 °C (molecular weight cutoff of 1000 kDa) to remove unencapsulated methylene blue or myoglobin, respectively.

[0061] Quantification of mBlue/Mb

[0062] The amounts of methylene blue or myoglobin that were encapsulated in purified polymersome suspensions were determined by measuring solution absorbance at 665 nm (mBlue) or at 410 nm (Mb), using a UV-Vis spectrophotometer. Calibration curves for methylene blue and myoglobin were developed using serial dilutions of known concentrations. To measure the iron content in polymersome-encapsulated myoglobin suspensions (as a corroboration of myoglobin concentration in that sample), 5-10% (v/v) of Triton X-100 was added, the mixture was digested by heating in aqua regia for 3 h at 98 °C, and was subsequently diluted with deionized water. ICP-OES was performed on experimental samples and their iron content was determined in comparison to this standard calibration curve. The concentrations of myoglobin (as calculated by UV-Vis absorbance spectroscopy) were compared to those obtained via ICP-OES or via the Micro BCA Assay (secondary UV-Vis method) for each suspension. Loading of aqueous encapsulants in the polymersomes was quantified and expressed as the final weight percentages of encapsulant-to-polymer that comprised the vesicles in suspension (e.g., w/w% Mb/polymer).

[0063] Quantification of metMb

[0064] The amount of metmyoglobin (metMb, i.e., oxidized Mb with a Fe(III)-heme group) in polymersome suspensions was quantified using a modified UV-Vis absorption protocol that was previously established for the measurement of

cyanomethemoglobin levels. In brief, the absorbance of myoglobin was measured at 630 nm (L^) against a blank reference (deionized water). One drop of KCN solution (1 part 10% KCN and 1 part 50 mM phosphate, pH 7.6) was added and mixed with the treated myoglobin samples. This reaction step was necessary to convert metMb to cyanometmyoglobin (cyanoMb), which does not absorb at 630 nm. After 2 min, the absorbance was measured at 630 nm (L ₂ ) against the deionized water, which served as the blank reference. The concentration of metMb was determined using Equation 1:

[metMb] (mM)=^ xD _! (Eq. 1),

1 ^X E where E = 3.7 (cm mM) ^"1 and is the extinction coefficient of metMb at 630 nm, and Di is the dilution factor in this experiment (cuvette length = 1 cm).

[0065] To determine the concentration of myoglobin, one drop of 20% K ₃ (Fe(CN) ₆ ) was added and mixed with the treated myoglobin sample. The solution was allowed to react for 2 min and an additional drop of 10% KCN was added and mixed. The absorbance of the sample was then measured at 540 nm (L ). The concentration of total Mb was determined using Equation 2:

[total Mb] (mM) = ^ X D ₂ (Eq. 2),

1 X E where E = 11.3 (cm mM) ^"1 and is the extinction coefficient for cyanometMb at 540 nm; D ₂ is the dilution factor (cuvette length = 1 cm).

[0066] The percentage of metMb in the original solution was determined using Equation 3 :

[metMb] (%)= x 100 (Eq. 3).

^{L J J} [metMb] + [total Mb]

[0067] Structural characterization of polymersomes

[0068] Polymersome suspensions were diluted in PBS solution and their

hydrodynamic diameters were measured by DLS using a standard 1.5 mL semi-micro Plastibrand polystyrene cuvette (VWR, Atlanta, USA). The morphologies of blank polymersomes and polymersome-encapsulated myoglobin were visualized by cryo- TEM (JEOL 2100F, USA). In brief, polymersome samples were suspended in a microperf orated grid, rapidly vitrified using liquid ethane (-183 °C), and loaded onto a cryogenic sample holder for cryo-TEM imaging at 200 kV.

[0069] Encapsulation of mBlue and Mb in polymersome suspensions using

conventional methods

[0070] To establish a baseline for comparisons of small molecule and protein encapsulation in polymersome suspensions, the final concentrations, weight percentages (i.e., weight of encapsulated agent compared to the weight of the polymer that comprises the nanoparticle), and efficiencies of encapsulation for methylene blue were determined with OB 18 polymersomes formed by the thin-film rehydration technique. FIG. 6A shows the weight percentage results of the final polymersome composition for encapsulation of methylene blue at 40 °C (i.e., 4.1 w/w%

mBlue/polymer) and 60 °C. (i.e., 5.0 w/w% mBlue/polymer). When thin-film rehydration was attempted at room temperature (i.e., 23 °C), the encapsulation of methylene blue was found to be negligible (results not shown), possibly due to the observation that the polymer films did not swell after 48-72 h of hydration. PEO-b- PBD-based polymersomes require the input of energy for vesicle formation, which is usually supported by using elevated temperatures (e.g., greater than 45 °C).

[0071] To improve the efficiency of encapsulation at lower temperatures, which would be necessary when employing labile proteins, encapsulation of mBlue was also studied by the direct hydration method. FIG. 6B shows the final weight percentage of mBlue-to-polymer in polymersome suspensions created using direct hydration at 23 °C (i.e., 1.2 w/w% mBlue/polymer).

[0072] Next, polymersome-encapsulated myoglobin suspensions formed at 23 °C by thin-film rehydration were initially found to be comprised of around 2.7 w/w%

Mb/polymer. After the addition of a proteolysis step to any remove surface- associated Mb (i.e., free protein that was nonspecifically bound), the final composition of the polymersomes was found to be only 0.5 w/w% Mb/polymer, indicating that very small amounts of protein were being encapsulated within polymersomes. FIG. 6C shows the final weight percentage of Mb-to-polymer in polymersome suspensions created using thin-film rehydration at 23 °C both before and after the added proteolysis step.

[0073] In order to improve the concentrations and the final weight percentages of myoglobin in polymersome-encapsulated myoglobin suspensions, polymersome generation at higher temperatures was again attempted utilizing thin-film rehydration at 40 and 60 °C. Such tests, however, only resulted in protein denaturation and aggregation. In contrast, polymersome-encapsulated myoglobin suspensions prepared by direct hydration at 23 °C displayed good colloidal properties and the characteristic absorption spectra of intact myoglobin, yet the final loading ratio of Mb-to-polymer in these polymersome-encapsulated myoglobin suspensions was low. FIG. 6D shows the final weight percentage of Mb-to-polymer in polymersome suspensions created using direct hydration at 23 °C both before proteolysis (i.e., showing 0.3 w/w%

Mb/polymer) and after proteolysis (i.e., 0.1 w/w% Mb/polymer).

Modifications to Conventional Processes

[0074] Features of both the direct hydration and thin-film rehydration techniques were iteratively evaluated in experimental conditions in order to improve polymersome- encapsulation of functional protein.

[0075] Effects of sonication

[0076] Following the direct hydration protocol, upon addition of OB 18 polymer and PEG, the sample was mixed, cooled to RT, and 10 μΕ of Mb solution (150 mg/mL) in PBS (10 mM, pH 7.4) was added. The sample was then further diluted with 10, 20, 50, and 100 μΕ of Mb solution, followed by mixing and sonication for either: A) 0 min or B) 30 min after each additional dilution step. All samples were then dialyzed for 30 h at 4 °C (molecular weight cutoff of 1000 kDa). The final Mb concentrations, weight percentages of Mb-to-polymer, and the efficiencies for Mb encapsulation in the resultant polymersome suspensions were measured by UV-Vis absorption

spectroscopy, ICP-OES and compared. [0077] In attempting encapsulation of Mb in OB 18 polymersomes, and by employing the direct hydration protocol for vesicle formation, the weight ratios of Mb-to-polymer that were reproducibly obtained in the final PEM suspensions were found to again be very low (e.g., around 0.2 w/w% Mb/polymer). The encapsulation efficiency, however, could be increased by more than 30 times if the samples were sonicated for 30 min at room temperature after each dilution step (i.e., sonicating after introducing additional volumes of aqueous solution to dilute the concentration of polymer in suspension). As discussed above with respect to FIG. 3 A, the relative amount of Mb in PEM suspension could be increased to around 6.0 w/w% Mb/polymer, supporting the addition of this sonication step to the original direct hydration protocol.

[0078] Effects of blending technique (dissolving polymer in organic solvent vs.

addition of heat)

[0079] The effects of utilizing an organic solvent were compared to adding heat to blend OB 18 with a PEG500 homopolymer to improve polymer dissolution during the first step of the direct hydration protocol. These strategies were compared with respect to the final yield of polymersome formation and, ultimately, to the

concentrations and efficiencies of protein encapsulation that could be obtained by each method. If the two polymers were first mixed by dissolution in DCM (followed by polymersome formation after organic solvent evaporation), the final weight ratio of Mb-to-polymer in PEM suspensions was around 2 w/w% Mb/polymer. In comparison, initial heating of dry OB 18 and PEG500 to 95 °C for 1 h improved mixing and promoted more efficient polymersome generation, yielding a significantly higher final weight ratio of Mb-to-polymer in the final PEM suspensions (i.e., around 5 w/w% Mb/polymer), corresponding to a greater amount of encapsulated protein.

[0080] Following the direct hydration protocol, 10 mg of OB 18 and 10 mg of PEG were either blended by heating at 95 °C for 1 h, or mixed by dissolution in DCM (50 μΕ) followed by drying under vacuum at room temperature overnight. Further encapsulation was done using the same protocol with the addition of 30 min of sonication after each dilution step. Mb concentrations in the final suspensions were determined by UV-Vis absorption spectroscopy and ICP-OES and compared. [0081] Effects of Mb oxidation state (i.e., utilizing oxyMb vs. metMb for polymersome-encapsulation)

[0082] Myoglobin encapsulation was found to be further augmented when the starting Mb stock solution was first reduced with sodium dithionite to convert all

metmyoglobin (i.e., metMb) to the oxmyoglobin (i.e., oxyMb) form. OxyMb contains a central heme group with iron in the ferrous state (i.e., Fe(II)), which improves the solubility of the protein when compared it its metMb form that contains Fe(III). This oxyMb solution was further desalted via dialysis prior to its utilization in all of the subsequent dilution steps in the direct hydration protocol, which was found to be necessary to increase the loading of Mb in PEM suspensions (i.e., the final weight ratio of Mb-to-polymer). As discussed above with respect to FIG. 3B, when oxyMb was used in the initial protocol step, PEM suspensions comprised of 6 w/w%

Mb/polymer were formed, which was a statistically significant improvement over the 4 w/w% Mb/polymer obtained when metMb was utilized.

[0083] The direct hydration protocol was modified to expose the initial mixture of polymer and PEG to 1 h (instead of 20 min) of heating at 95 °C. The effect of the iron oxidation state of the heme group of Mb on the efficiency of polymersome- encapsulation was studied by using oxyMb (i.e., Fe(II)Mb) vs. metMb for each dilution step. MetMb solution was prepared by dissolving lyophilized Mb in PBS; the same solution was treated with 1 wt% Na ₂ S ₂ O ₄ to obtain the reduced Mb form

(oxyMb). Mb encapsulation in polymersomes was measured by UV-Vis absorption spectroscopy and ICP-OES, and compared.

[0084] Effects of sonication and temperature on Mb oxidation

[0085] More than 40% of the oxyMb that was used in the initial step for polymersome- encapsulation was found to be reoxidized to metMb within 2 h at 50 °C. In contrast, only around 15% metMb was generated from the initial oxyMb solution if lower temperatures were employed for polymersome formation (e.g., heating for 2 h at 40 °C). The rate of Mb oxidation at 50 °C was also significantly higher than that at 40 °C, regardless of the addition of sonication or the power that was utilized, as discussed above with respect to FIG. 3C. As such, it was determined that sonication had no effect on Mb oxidation and it was thus preferentially employed to both promote polymer mixing and to provide interfacial energy to augment polymersome formation.

[0086] The initial Mb solution (at 150 mg/mL) was reduced with Na ₂ S ₂ O ₄ and subjected to various conditions, including heating at 40 °C (with or without

sonication) or at 50 °C for 2-5 h. Mb oxidation was determined by measuring the percentages of metMb in the total polymersome-encapsulated Mb suspensions, using the cyanomethemoglobin method.

[0087] Effects of proteolysis

[0088] Upon formation, PEM suspensions were treated with 0.4% pronase solution for up to 18 h at room temperature in order to examine the duration of time required for the complete digestion of any surface-associated (i.e., non-specifically bound) Mb. It was observed that all surface-associated Mb was digested in 2 h and that neither increasing pronase exposure time nor concentration further augmented Mb loss, thus indicating that only encapsulated Mb was retained, as discussed above with respect to FIG. 3D.

[0089] Mb was encapsulated in OB 18 polymersomes using different initial solution concentrations of protein (i.e., 50, 75, and 150 mg/mL) followed by dialysis for at least 30 h at 4 °C (molecular weight cutoff of 1000 kDa). The samples were

subsequently treated with 0.4 wt% pronase solution for 18 h at room temperature and again dialyzed overnight at 4 °C. Mb encapsulation in polymersomes (before and after proteolysis) was measured by UV-Vis absorption spectroscopy and ICP-OES, and compared.

[0090] Improvement of Mb encapsulation efficiency (i.e., %Mb EE)

[0091] Five sets of experiments were done with various Mb-to-PBS volume ratios (i.e., "Mb:PBS") in order to establish the optimal Mb concentration to use in each subsequent dilution step in our modification of the original "direct hydration" protocol. Notably, when the Mb:PBS increased, the final w/w% Mb/polymer in the PEM suspensions also increased; but, the Mb encapsulation efficiency (i.e., %Mb EE) decreased as a result. In other words, the final Mb-to-polymer mass ratio was maximized when all dilutions steps were conducted using a maximally concentrated Mb solution (i.e., Mb:PBS = 190:0 and 150 mg/mL oxyMb). As discussed above with respect to FIG. 3E, the %Mb EE was largest when the Mb:PBS was minimal (i.e., 10: 180). As the amount of protein in the final polymersome suspension is ultimately the metric that must be optimized for therapeutic administration (in order to minimize the amount of associated carrier polymer that is introduced to a subject), it was determined that a pure Mb solution (150 mg oxyMb/mL) would be used for each dilution step in the ultimate encapsulation protocol, maximizing the final w/w% Mb/polymer in PEM suspensions.

[0092] Following the basic direct hydration protocol, 10 mg polymer and 10 mg of PEG were initially heated in 1.5 mL microcentrifuge tubes for 1 h at 95 °C and subsequently cooled to RT. The mixtures were then diluted by adding 10, 10, 20, 50, and 100 μΕ of diluents. Two different solutions were used and compared for each of the 5 dilution steps: PBS and/or Mb suspensions (i.e., 150 mg/mL Mb in PBS). The final (v/v) ratio of Mb to PBS (i.e., "Mb:PBS") used as diluents in steps 1, 2, 3, 4, and 5 were 10: 180, 20: 170, 40: 150, 90: 100, and 190:0, respectively. The samples were then proteolyzed using 0.4 wt% pronase and again dialyzed overnight at 4 °C

(molecular weight cutoff of 1000 kDa). Mb encapsulation was measured using UV- Vis absorption spectroscopy. The Mb encapsulation efficiencies were calculated using Equation 4:

Mb Encapsulation Efficiency= [ 1 - ^ ¹ ^ ² ] ^χ 100 (Eq. 4),

Where vi = Initial volume of the unencapsulated Mb (mL), Ci = Initial concentration of unencapsulated Mb (mg/mL), v ₂ = volume of polymersome-encapsulated Mb obtained after dialysis and proteolysis (mL), and c ₂ = concentration of encapsulated Mb obtained after dialysis and proteolysis (mg/mL).

[0093] Using progressive saturation to generate polymersome-encapsulated protein suspensions. [0094] By incorporating each of the steps in the various embodiments, a progressive saturation technique was established, represented in FIG. 7A, which vastly improved upon the results of the original direct hydration protocol discussed above with respect to FIG. 5C. Using the progressive saturation protocol, the final content of Mb in OB18-based PEM suspensions was found to be 6.1 and 3.2 w/w% Mb/polymer before and after proteolysis, respectively. Quantification of the iron content (numbers of intact heme groups) in each of the polymersome suspensions by ICP-OES

corroborated UV-Vis measurements of protein concentration. As shown in FIG. 7B, the final loading ratios of Mb in the polymersomes were found to be 7.9 and 5.1 w/w% Mb/polymer before and after proteolysis, respectively. As shown in FIG. 7C, the percentage of metMb (with respect to the total Mb content in these suspensions) was determined by UV-Vis absorbance spectroscopy and found to be around 8% and 6% in non-proteolyzed (PEM-SE) and proteolyzed (PEM-E) samples, respectively. FIG. 7D is a table of the measured properties (i.e., results) for the OB18-based PEM suspensions, as discussed with respect to FIGs. 7B and 7C.

[0095] Stability of polymersome-encapsulated protein suspensions

[0096] OB18-encapsulated Mb suspensions were prepared using the optimized progressive saturation technique. The samples were stored at 4, 23, and 37 °C for 3 weeks. At predetermined time points, the samples were diluted with PBS and the mean particle size and distributions were determined by DLS.

[0097] Equilibrium binding of oxygen in polymersome-encapsulated Mb suspensions

[0098] The equilibrium binding and dissociation curves for oxygen in suspensions of free and polymersome-encapsulated Mb were obtained at 37 °C using a Hemox™- Analyzer. Samples were allowed to saturate to a pO ₂ of 147 mmHg (using compressed air) and then deoxygenated (using a compressed nitrogen stream). The absorbance of oxygenated and deoxygenated free and polymersome-encapsulated Mb suspensions was recorded as a function of pO ₂ via dual wavelength spectroscopy. Oxygen equilibrium curves were fit to a four-parameter (A ₀ , A _∞ , P ₅₀ , n) Hill model (Equation 5). In this model, A ₀ and A _∞ represent the absorbance at 0 mmHg and at 147 mmHg, respectively. The pO ₂ represents the partial pressure of oxygen; and, P ₅₀ represents the partial pressure of O ₂ where the sample is 50% saturated with oxygen. Lastly, n represents the cooperativity coefficient for the sample.

Y _ Abs-Ao _ —2^— _(Eq . ₅₎ .

A _∞ - Ao ρθ!?+ Ρί} ₀ '

[0099] FIG. 7E shows the P ₅₀ values (in mmHg) obtained for a free myoglobin (Mb) sample, a polymersome-encapsulated myoglobin sample prior to proteolysis (PEM- SE) that was prepared using the progressive saturation technique, and a polymersome- encapsulated myoglobin sample after pronase treatment (PEM-E) that was prepared using the progressive saturation technique.

[0100] Characterization of the final PEM suspensions

[0101] The size distributions of the final OB 18- and OB29-based PEM suspensions were measured by both DLS and cryo-TEM. FIG. 8A provides the average

hydrodynamic diameter of particles in OB18-based and OB29-based PEM

suspensions prepared using progressive saturation, as assessed by DLS. Cryo-TEM images of vesicles in OB18-based and OB29-based PEM suspensions are shown in FIGs. 8B and 8C, respectively. These results confirmed a mean particle diameter of approximately 200 nm for OB 18 polymersomes, and 130 nm for OB29

polymersomes. The stability of the OB18-based PEM suspensions were further examined over three weeks and at various temperatures, with the polymersomes, demonstrating no aggregation based on the consistent particle numbers and stable size distributions in suspension. FIG. 8D shows the average hydrodynamic diameters of particles, as determined by DLS, in OB18-based PEM suspensions that were prepared by progressive saturation at various temperatures (i.e., 4 °C, 23 °C, and 37 °C) as a function of time. Finally, the functional status of encapsulated Mb in the PEM suspensions (i.e., retention of Mb's ability to bind and release oxygen) was verified by dual wavelength spectroscopy. FIG 8E shows oxygen equilibrium curves for free oxyMb and oxygenated OB18-based PEM suspensions. Error bars denote standard deviation of the mean, n > 3 experimental replicates per condition. The oxygen equilibrium curves, P ₅₀ (i.e., the partial pressure of O ₂ where the Mb is 50% saturated with oxygen) of PEM were very similar to those of free Mb in solution.

[0102] Polymersome-encapsulation using block copolymers and proteins of varying molecular weight

[0103] The generalizability of the progressive saturation technique was tested using proteins of various sizes: i.e., Mb (17 kDa), hemoglobin (Hb; 64 kDa), bovine serum albumin (BSA; 66 kDa), immunoglobulin G (IgG: 150 kDa), catalase (250 kDa), fibrinogen (340 kDa), and apoferritin (450 kDa); each protein was dissolved in PBS (10 mM, pH 7.4) at its maximal solubility, corresponding to final suspension concentrations of 150, 150, 40, 20, 50, 50, and 25 mg/mL, respectively. The progressive saturation protocol was followed to encapsulate these proteins in OB29 polymersomes. Free proteins were separated by dialysis for at least 30 h at 4 °C (molecular weight cutoff of 1000 kDa). Surface associated protein was removed by proteolysis via treatment with 0.4 wt% pronase solution followed by overnight dialysis at 4 °C (molecular weight cutoff of 1000 kDa). Protein encapsulation (before and after proteolysis) in polymersome suspensions was quantified via the micro-BCA assay, utilizing UV-Vis spectrophotometry and by following the manufacturer's protocols (Pierce Biotechnology, Inc; Rockford, IL, USA). The final concentrations of protein were divided by those of polymer and expressed as the final weight ratios of protein-to-polymer that comprised the polymersomes in suspension (e.g., w/w% Mb/polymer).

[0104] Statistical analysis

[0105] Data are presented as the mean ± the standard deviation of the mean (SD). A minimum of three experimental replicates was used for each condition. One-way analysis of variance (ANOVA) was conducted using GraphPad software (San Diego, USA). A p value of < 0.05 was considered statistically significant.