Biomacromolecules 2005, 6, 2172-2181
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Polymersome Encapsulated Hemoglobin: A Novel Type of Oxygen Carrier Dian R. Arifin and Andre F. Palmer* Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556 Received February 28, 2005; Revised Manuscript Received April 28, 2005
Bovine hemoglobin (Hb) was encapsulated inside polymer vesicles (polymersomes) to form polymersome encapsulated Hb (PEH) dispersions. PEH particles are 100% surface PEGylated with longer PEG chains and possess thicker hydrophobic membranes as compared to conventional liposomes. Polymersomes were self-assembled from poly(butadiene)-poly(ethylene glycol) (PBD-PEO) amphiphilic diblock copolymers with PBD-PEO molecular weights of 22-12.6, 5-2.3, 2.5-1.3, and 1.8-0.9 kDa. The first two diblock copolymers possessed linear hydrophobic PBD blocks, while the later possessed branched PBD blocks. PEH dispersions were extruded through 100 and 200 nm pore radii membranes. The size distribution, Hb encapsulation efficiency, P50, cooperativity coefficient, and methemoglobin (metHb) level of PEH dispersions were consistent with values required for efficient oxygen delivery in the systemic circulation. The influence of different molecular weight diblock copolymers on the physical properties of PEH dispersions was analyzed. PBDPEO copolymers with molecular weights of 22-12.6 and 2.5-1.3 kDa completely dissolved in aqueous solution to form polymersomes, while the other two copolymers formed a mixture of solid copolymer precipitates and polymersomes. PEHs self-assembled from 22-12.6 and 2.5-1.3 kDa PBD-PEO copolymers possessed Hb loading capacities greater than PEG-LEHs, PEGylated actin-containing LEHs, and nonmodified LEHs, although their sizes were smaller and their hydrophobic membranes were thicker. The Hb loading capacities of these polymersomes were also higher than lipogel encapsulated hemoglobin particles and nanoscale hydrogel encapsulated hemoglobin particles. PEH dispersions exhibited average radii larger than 50 nm and exhibited oxygen affinities comparable to human erythrocytes. Polymersomes did not induce Hb oxidation. The interaction between Hb and the membrane of 2.5-1.3 kDa PBD-PEO polymersomes improved the monodispersity of these particular PEH dispersions. These results suggest that PEHs could serve as efficient oxygen therapeutics. 1. Introduction 1.1. Liposome Encapsulated Hemoglobin. Liposome encapsulated hemoglobin (LEH) dispersions constitute one major class of cellular hemoglobin-based oxygen carriers (HBOCs).1-5 The circulation half-life of nonmodified LEHs is very short (∼4-12 h6,7). Moreover, LEH particles are prone to aggregate and fuse together after several days of storage.8 The most promising strategy to improve the circulation half-life and stability of LEH particles relies on surface modification with poly(ethylene glycol) (PEG).3,9 PEG is a biologically inert polymer, which has been extensively used in drug delivery systems and is declared to be safe for in-vivo use by the Food and Drug Administration.3,9,10 It was proposed that PEG conjugation creates a steric hydrophilic barrier surrounding each LEH particle, protecting PEG-LEHs from opsonizing plasma proteins, and thus increasing their intravascular persistence.9,11 The steric barrier created by PEG conjugation also prevents PEG-LEH aggregation and fusion and thus stabilizes PEG-LEH dispersions.12-14 The colloidal state of LEH dispersions grafted * To whom correspondence should be addressed.
with 0.3 mol % of 5000 Da PEG (the concentration of lipid was ∼57 mg/mL) was shown to be stable for 1 year.8,15 In addition, the methemoglobin (metHb) level of PEG-LEH dispersions can be suppressed by coencapsulation of reductants or catalases.8,16,17 It was also observed that PEG surface modification also improved the rheology, hemodynamic properties, and biocompatibility of LEH dispersions.14,15,18 However, the shielding effect imparted by PEGylated lipids is limited by the maximum amount of PEGylated lipids that can be incorporated into the liposome bilayer before phase separation occurs and separate PEG-lipid micelles are formed.19 The most optimum PEG surface coverage on liposomes was reported to be 10 mol % of 5 kDa PEG.20 These PEG-LEH dispersions exhibited a circulation halflife of ∼48 h in rabbits.20 The maximum surface concentration of PEG that can be conjugated onto liposome bilayers appears to decrease with increasing PEG molecular weight, due to the increasing hydrophilicity of the PEG-lipid.19,21 Therefore, there is a physical limit when optimizing the steric shielding and biocompatibility of PEG-LEH dispersions.9 A recent study on complement (C) activation showed that while short PEG chain (5000 Da or less) grafting prolongs liposome intravascular circulation, it may not effectively prevent C
10.1021/bm0501454 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/04/2005
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activation in-vivo.22 C activation is associated with anaphylactic reactions and activation of other proteolytic plasma cascades.22 1.2. Polymersome Encapsulated Hemoglobin. It is possible to prepare vesicles that are 100% PEGylated, with larger PEG molecular weights as compared to conventional liposomes. Amphiphilic diblock copolymers with hydrophilic volume or mass fractions ranging from 0.20 to 0.40 have been shown to form polymer vesicles, also called polymersomes, in pure water or aqueous solutions.23-25 Polymersomes, which are composed solely of diblock copolymers, overcome the disproportionalities of PEG-lipids by increasing both the hydrophobic and the hydrophilic masses of the blocks.21,24-26 As a result of increasing the hydrophobic portion of the copolymer, vesicles with thicker membranes can be prepared. Polymersomes with PEG chains longer than 20 kDa can be readily prepared by varying the hydrophilic blocks of the copolymers. Moreover, the diameter of polymersomes can be easily engineered from 50 nm to 50 µm, depending on the vesicle preparation method.21,25 While the membrane thickness of liposomes and other natural membranes are universally 3-4 nm, the hydrophobic core thickness of polymersomes can be engineered to exceed 4 nm, by simply varying the copolymer hydrophobic block molecular weight.19,21,24,25,27 The thicker membrane of polymersomes imparts better stability and mechanical strength as well as influences the permeability of polymersomes to small molecules.19,23,28 Giant polymersomes with membrane thickness ∼8 nm were shown to be almost an order of magnitude mechanically resilient, and at least 10 times less permeable to water than liposomes.25,28 This reduces problems associated with osmotic pressure gradients encountered with liposomes, which compromises their mechanical strength.29 It was observed that the membrane stability of polymersomes increased with increasing membrane thickness, while the surface elasticity was scale-independent.24,30 Polymersomes were stable in saline solutions for several months, and for at least 5 days in blood plasma under wellmixed quasi-physiological conditions.21,25,26 Because polymersomes are 100% PEGylated with longer PEG chains (20 kDa or more) as compared to PEG-LEHs, the colloidal state of PEH dispersions (even at copolymer concentrations as high as 57 mg/mL) can be expected to be stable for a storage period of at least 1 year, similar to PEG-LEH dispersions.8 Polymersomes exhibited no in-surface thermal transitions (lamellar surface transition and membrane area expansion with increasing temperature) up to 60 °C. In contrast, common phospholipids exhibited thermal transitions at ∼2325 °C.6,25 If the surface transition is at, or near, the vesicle preparation or storage temperature, leakage of the vesicle contents will occur. However, temperatures higher than 60 °C should be avoided because PEG chains are more susceptible to hydrolysis in this temperature range.21 The hydrophilic PEG brushes of polymersomes can be synthesized to be much longer than those typically conjugated to the surface of liposomes,21,25 thus imparting superior camouflage ability, a high level of biocompatibility, and preventing the polymeric surface from interacting with blood proteins. In-vitro studies demonstrated that the dense PEG
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brush of polymersomes prevented phagocytic ligand deposition and/or phagocyte adhesion.25 PEG chains present on the densely populated polymersome surface automatically adopt a mushroom-brush intermediate conformation and/or a brush conformation due to steric repulsion between neighboring PEG chains.22,31 It was observed that PEG brushes adopting these conformations were more effective in preventing phagocytosis and suppressing the activation of the human C system.22 Longer PEG chains (20 kDa or more), which were found to be more effective in suppressing C activation, can be easily synthesized for subsequent self-assembly into polymersomes.22,32 Incubation of cell cultures with polymersomes showed no adverse effects or vesicle-cell adhesion after 5 days under turbid conditions,21 and after 48 h under stagnant conditions.25 A mixture of polymersomes with whole blood also showed no signs of adhesion to red blood cells (RBCs) for a few hours without sustained mixing. In addition, infusion of 5 mg of polymersomes into rats (300-400 g body weight) did not induce toxic effects over 8 weeks.21 Although complement activation occurs with particles >200 nm in diameter, because polymersomes are 100% PEGylated with longer PEG brushes (20 kDa or more) as compared to conventional liposomes, complement activation mediated by polymersomes with diameters ∼200 nm is expected to be significantly attenuated. The circulation half-life and in-vivo stability of polymersomes are expected to surpass those of liposomes. The circulation half-lives of 100 nm-diameter polymersomes with PEG brushes ranging from 1200 to 3680 Da and hydrophobic core thickness ) 9.6-14.8 nm were shown to be 10-20 h longer as compared to PEG-liposomes with similar sizes.21 The circulatory half-life of polymersomes appears to depend mainly on the molecular weight of the PEG brush, and not on the hydrophobic core thickness, due to the obfuscation of the hydrophobic core by the dense PEG brush. Therefore, there is flexibility in the choice of hydrophobic core chemistry.21 The in-vivo clearance mechanism of polymersomes appears to be similar to that of PEG-liposomes, that is, through opsonizing plasma protein deposition onto the membrane, which mediates clearance by the phagocytes of the liver and spleen.21,25 Considering the aforementioned advantages of polymersomes as compared to PEG-liposomes, the potential of polymersomes as a unique in-vivo delivery system has started to gain attention. In this study, we will demonstrate that polymersomes could be used as a promising new vehicle for the storage and transport of hemoglobin (Hb) in the context of a cellular HBOC. To our knowledge, this is the first study that explores the possibility of employing polymersomes for this application. We will show that Hb can be readily encapsulated inside polymersomes, thus creating polymersome encapsulated Hb (PEH) dispersions. The potential of PEHs to transport oxygen will be investigated by evaluating the oxygen affinity, encapsulation efficiency, and metHb level of various PEH dispersions. Moreover, diblock copolymers with four different molecular weights will be used to produce PEHs. Two copolymers consist of linear hydrophobic blocks, while the other two consist of
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Figure 1. Chemical structure of PBD-PEO diblock copolymer with linear (A) or branched PBD blocks (B). The block inside the parentheses is hydrophobic, while the block inside the square bracket is hydrophilic. Table 1. Number-Averaged Molecular Weight (Mn), PEG Length, Hydrophilic Mass Faction (fhydrophilic), and Hydrophobic Membrane Thickness (d) of PBD-PEO Diblock Copolymers Used in This Study diblock copolymer
Mn (kDa)
PEG length (kDa)
f hydrophilic
d (nm)
22-12.6 kDa (L1) 5-2.3 kDa (L2) 2.5-1.3 kDa (B1) 1.8-0.9 kDa (B2)
34.6 7.3 3.8 2.7
12.6 2.3 1.3 0.9
0.36 0.32 0.34 0.33
∼35 12-13 9-10 8-9
branched hydrophobic blocks. The influence of different molecular weight diblock copolymers on the physical properties of PEH dispersions will also be investigated. 2. Materials and Methods 2.1. Materials. In this study, bioinert poly(ethylene oxide) (PEO) and polybutadiene (PBD) were chosen as the hydrophilic and hydrophobic blocks, respectively, in the diblock copolymer chemical structure.21,24 A material is considered to be bioinert if it is nonreactive toward blood plasma proteins or with living tissues. Bioinert PEO is the structural equivalent of PEG.25 PBD is known to be bioinert as it is used as the hydrophobic block of triblock copolymers, which are employed to modify the surface of biomaterials such as Dacron to prevent surface-induced thrombosis.33 A study performed by Photos et al.21 supported this assertion. Infusion of empty polymersomes composed of PBD-PEO copolymer into rats showed no toxic effects over an 8-week period.21 Bovine aortic endothelial cells and vascular smooth muscle cells incubated with 5 mg/mL of empty PBD-PEO polymersomes showed 88.4% (endothelial cells) and 91.1% (muscle cells) cell survival after 5 days incubation.21 Poly(butadiene(1,4 addition)-b-ethylene oxide) with a linear PBD block and poly(butadiene(1,2 addition)-b-ethylene oxide) with a branched PBD block were purchased from Polymer Source (Dorval, QC, Canada). The structural properties of PBD-PEO copolymers used in this study are summarized in Table 1. The hydrophobic membrane thickness (d) of polymersomes was taken or extrapolated from the literature.21,24 PBD blocks of the diblock copolymers L1 and L2 are linear, while PBD blocks of the diblock copolymers B1 and B2 are branched. The chemical structures of PBD-PEO copolymers with linear or branched PBD blocks are shown in Figure 1. These copolymers are nearly monodisperse based on the measured ratios of the weightaveraged to number-averaged molecular weights (Mw/Mn < 1.1).
For convenience, PEHs composed of 22-12.6 kDa PBDPEO copolymers will be referred to as L1 PEHs, PEHs composed of 2.5-1.3 kDa PBD-PEO copolymers will be referred to as B1 PEHs, PEHs composed of 5.0-2.3 kDa PBD-PEO copolymers will be referred to as L2 PEHs, and PEHs composed of 1.8-0.9 kDa PBD-PEO copolymers will be referred to as B2 PEHs. Likewise, their corresponding controls (empty polymersomes) will be referred to as L1 controls, B1 controls, L2 controls, and B2 controls, respectively. 2.2. Extraction and Purification of Hb from Bovine Erythrocytes. Bovine Hb was used as a convenient and compatible replacement to human Hb for the preparation of PEH dispersions.34 Bovine erythrocytes were extracted, centrifuged, and collected in 3.8% sodium citrate by Animal Technologies, Inc. (Tyler, TX). Bovine Hb was then extracted from erythrocytes according to a previously described procedure in the literature.29,35-38 2.3. Assay of Hb Solution. The Hb concentration of the purified Hb stock solution was quantified as previously described.29 The Hb solution was subjected to SDS-PAGE analysis to determine its level of purity. Hb solutions that were 99.9% pure were used to prepare PEH dispersions. Freshly purified Hb was diluted with phosphate buffered saline (PBS, at pH 7.3) to 300 mg/mL. Five mM of N-acetylL-cysteine (NAC) was added to suppress Hb oxidation.39 Hb stock solutions with metHb levels less than 5% were used for subsequent experiments. 2.4. PEH Preparation. PBS at pH 7.3, which mimics the salinity and pH of blood plasma, was used as the PEH extrusion buffer. PEHs were prepared by first forming a thin film containing 1 mg of diblock copolymer onto the walls of a 50 mL round-bottom glass flask using a Buchi R-205 rotary evaporator (Buchi Analytical, Inc., New Castle, DE) from a chloroform stock solution containing 1 mg/mL of diblock copolymer. The thin film of diblock copolymer was subsequently rehydrated with 300 mg/mL of Hb stock solution containing 5 mM of NAC.39,40 Polydisperse polymersomes were formed in this initial step. To regulate the size of the initially formed polymersomes, the dispersion was extruded 20 times through 100 and 200 nm pore radii polycarbonate membranes (Avanti Polar Lipids, Inc., Birmingham, AL; and Avestin, Inc., Ottawa, Canada).29,41-46 Empty polymersomes were also prepared to serve as controls using the same preparation procedure, however, without Hb present. In this case, the hydrating Hb solution was replaced with PBS. PEH and control dispersions were used immediately after preparation. 2.5. Size Distribution and Hb Encapsulation Efficiency. The size distribution and Hb encapsulation efficiency of PEH dispersions were measured using an Eclipse asymmetric flow field-flow fractionator (AFFFF) coupled in series to a 18angle Dawn EOS multi-angle static light scattering photometer (MASLS) equipped with a linearly polarized 30 mW gallium-arsenide laser operating at 690 nm, and an Optilab DSP differential interferometric refractometer (DIR) (Wyatt Technology Corp., Santa Barbara, CA). Light scattering spectra were analyzed using the ASTRA software (Wyatt Technology Corp.). A detailed theoretical background and
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experimental protocol for using AFFFF-MASLS-DIR to determine vesicle size distribution and Hb encapsulation efficiency can be found in the literature.29 The mobile phase for all experiments was PBS at physiological pH ) 7.3 and 37 °C, filtered through 0.2 µm filters. This setup is capable of measuring particles with molecular weights ranging from ∼500 to 108 Da, and mean square radii ranging from 10 to 500 nm. In a previous publication,29 we developed a novel technique for measuring Hb encapsulation efficiency. Specifically, we used the DIR to measure the change in refractive index of eluting unencapsulated Hb, which was separated from the encapsulated Hb suspension (PEH). This was used to calculate the Hb encapsulation efficiency of PEH dispersions.29 However, Hb-loaded vesicles and particles in the literature46-48 were made using different phospholipid or polymer concentrations and compositions as compared to our formulations. Therefore, we calculated the weight ratio of entrapped Hb to copolymers (R) comprising the membrane, such that the Hb loading capacities of PEHs measured in this work can be compared to the Hb loading capacities reported in the literature.46-48 R was calculated by dividing the entrapped Hb concentration in mg/mL with the copolymer concentration in mg/mL. The estimated entrapped Hb concentration was calculated by multiplying the Hb encapsulation efficiency with the initial Hb concentration (300 mg/ mL). Measurements were done in triplicate (starting from the copolymer film formation step) to obtain mean ( SD values. 2.6. Oxygen Binding Properties and MetHb Level. PEH dispersions were dialyzed overnight using 100 000 Da molecular weight cutoff dialysis bags (Spectrum Laboratories, Inc., Fort Lauderdale, FL) in PBS at 2-3 °C to separate unencapsulated Hb from PEH particles (1:1000 v/v dialyzed sample: PBS). The oxygen binding curves of dialyzed PEH dispersions were measured using a Hemox-Analyzer from TCS Scientific Corp. (New Hope, PA) at physiological temperature (37 °C). The P50 and cooperativity coefficient (n) of PEH dispersions was regressed from the measured oxygen binding curves by fitting the oxygen binding curves to the Adair equation. To measure the encapsulated metHb level, dialyzed PEHs were first lysed using 15 mM Triton-X49 (Sigma-Aldrich, St. Louis, MO) in PBS. The metHb concentration encapsulated inside PEH particles was assayed using a UV-visible spectrophotometer (Varian, Inc., Palo Alto, CA).29 3. Results and Discussion 3.1. Size Distribution and Average Radius of PEH Dispersions. 3.1.1. Formation of Polymersomes in Competition with Other Self-Assembled Structures in Solution. L2 (5-2.3 kDa) and B2 (1.8-0.9 kDa) diblock copolymers were observed to have poor solubility in aqueous solution, while L1 (22-12.6 kDa) and B1 (2.5-1.3 kDa) diblock copolymers completely dissolved in PBS or Hb solutions. During the rehydration step in either Hb or PBS solutions, L2 and B2 diblock copolymers formed solid copolymer precipitates, micelles, and polymersomes. Even with vigorous sonication, the copolymer precipitates did not
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completely dissolve in aqueous solution. In agreement with the literature,27,50 polymersome formation was favored upon an increase in the total molecular weight of the copolymers. The morphology of self-assembled copolymer aggregates in solution is governed by a balance between contributions to the free energy of aggregation, which include: (1) corechain stretching, (2) interfacial energy, and (3) intercoronal chain interactions.27 Thus, the morphology of self-assembled structures can be controlled by many factors which influence one or more of the three free-energy contributions.27 The Gibbs free energy becomes more negative with increasing total copolymer molecular weight.50 Thus, the morphology shifts from micelles/open bilayer structures to vesicles with increasing total copolymer molecular weight to decrease the interfacial tension between the hydrophobic portion of the copolymer and the aqueous solution.50 Because the bending modulus of polymers increases with increasing molecular weight, the formation of vesicles should be easier in longchained copolymers as compared to short-chained copolymers.27 However, other factors also determine the morphology of copolymer aggregates, such as the length of the hydrophilic block27 and the hydrophilic/hydrophobic block ratio.51 The aggregate structure changes from spherical to cylindrical micelles to vesicles to micrometer-size compound micelles with decreasing length of the hydrophilic block27 and hydrophilic/hydrophobic block ratio.51 3.1.2. Size Distribution of Polymersome Dispersions Composed of 22-12.6 kDa (L1) and 2.5-1.3 kDa (B1) PBD-PEO Copolymers. Because L1 and B1 copolymers completely dissolved in PBS or Hb solutions, we will discuss these copolymers first. L1 and B1 soluble copolymers could have also self-assembled in aqueous solution to form micelles in addition to polymersomes. However, the presence of micelles was not detected in the AFFFF-MASLS-DIR chromatograms. Therefore, we deduced that most or all of the L1 and B1 diblock copolymers self-assembled into polymersomes. The differential size distributions of control and PEH dispersions are shown in Figures 2 and 3, respectively. L1 control dispersions were more monodisperse than B1 control dispersions, regardless of the extruder membrane pore size utilized, which was expected. Because the molecular weight of the PEO block of L1 (12.6 kDa) was significantly greater as compared to that of B1 (1.3 kDa), we expected the colloidal state of L1 to be better than B1 due to the superior steric shielding afforded by the higher molecular weight PEO block in L1. However, B1 PEH dispersions were more monodisperse than L1 PEH dispersions, regardless of the extruder membrane pore size utilized. The size distribution widths of L1 PEH and L1 control dispersions were almost similar (differed by ∼10 nm), but the size distribution of B1 PEH dispersions was significantly more monodisperse than B1 control dispersions. Therefore, it appeared that Hb encapsulation inside B1 polymersomes drove the formation of monodisperse B1 PEH dispersions. In the case of LEH dispersions, it was previously observed that encapsulated Hb molecules initially formed complexes with the lipid bilayer, which was subsequently followed by
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Figure 2. Differential size distributions of polymersome control dispersions extruded through 200 (solid lines) and 100 nm (dotted lines) pore radii membranes. Panel A: polymersomes composed of 22-12.6 kDa PBD-PEO copolymers. Panel B: polymersomes composed of 5-2.3 kDa PBD-PEO. Panel C: polymersomes composed of 2.5-1.3 kDa PBD-PEO. Panel D: polymersomes composed of 1.8-0.9 kDa PBD-PEO.
Figure 3. Differential size distributions of PEH dispersions extruded through 200 (solid lines) and 100 nm (dotted lines) pore radii membranes. Panel A: polymersomes composed of 22-12.6 kDa PBDPEO copolymers. Panel B: polymersomes composed of 5-2.3 kDa PBD-PEO. Panel C: polymersomes composed of 2.5-1.3 kDa PBDPEO. Panel D: polymersomes composed of 1.8-0.9 kDa PBD-PEO.
intercalation of Hb molecules into the hydrophobic region of lipid bilayers.52 Hb-lipid complex formation and intercalation were primarily attributed to the hydrophobic interaction between Hb and the lipid membrane. To date, little is known regarding protein adsorption onto pure polymeric bilayers. However, Pata and Dan predicted that incorporation of transmembrane proteins into pure polymeric bilayers is
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possible.53 In other words, it is theoretically possible for Hb molecules to intercalate into the hydrophobic core of polymersomes. It appeared that the intercalation of Hb molecules into the hydrophobic membranes of polymersomes composed of branched hydrophobic blocks (B1) promoted the formation of monodisperse vesicle dispersions. Past studies12,54,55 reported that vesicle dispersions stabilized by the spontaneous curvature mechanism (indicated by a vesicle bending constant . kT where k is Boltzmann’s constant and T is 37 °C) exhibited narrower size distributions as compared to those stabilized by the thermal undulation mechanism (bending constant ≈ kT or the thermal energy). In liposomes, it was observed that incorporation of wedge-shaped PEG-lipids into bilayers composed of tubular-shaped phospholipids induced lateral expansion of the bilayers, which rigidified the bilayers and shifted the vesicle stabilization mechanism to spontaneous curvature.13,56 We postulate that Hb intercalation into the hydrophobic core of B1 polymersomes also induced membrane lateral expansion, which increased the rigidity of the membranes and consequently shifted the vesicle stabilization mechanism to spontaneous curvature. Therefore, monodisperse B1 PEH dispersions were formed. However, it is not clear why this phenomenon occurred in B1 PEH dispersions, but not in L1 PEH dispersions. Nevertheless, in designing PEH dispersions for use as a HBOC, it is very important to obtain a monodisperse dispersion, because the circulation half-life of these vesicles is highly dependent on their size.57 Hence, encapsulation of Hb into polymersomes with branched PBD blocks (B1), which appeared to improve their monodispersity, contributed favorably to the design of PEH as a HBOC. 3.1.3. Average Radii of Polymersome Dispersions Composed of 22-12.6 kDa (L1) and 2.5-1.3 kDa (B1) PBD-PEO Copolymers. Our experimental results indicate that the average radii of B1 dispersions were larger than L1 dispersions by 20-30 nm for PEH dispersions and by 90150 nm for control dispersions, regardless of the extruder membrane pore sizes utilized. Because the bending modulus of copolymers increases with increasing molecular weight,27,58 the formation of large vesicles which have less curvature as compared to small vesicles is favored by copolymers with lower molecular weight (in this case, B1 copolymer). Vesicles with radii larger than 50 nm are desired to prevent their filtration by the capillaries, and the kidney tubules.3 Here, we demonstrated that PEH particles with radii larger than 50 nm could be readily produced. The average radii of L1 PEH particles were larger than those of L1 control particles, whereas the average radii of B1 PEH particles were smaller than those of B1 control particles. We first postulated that Hb encapsulated in the aqueous core of polymersomes resulted in the osmotic pressure inside polymersomes being larger than the osmotic pressure of the exterior environment (PBS buffer). Therefore, the osmotic pressure gradient promoted diffusion of water into polymersomes, and caused L1 PEHs to swell. Osmosis of water into and out of the aqueous core of vesicles was previously encountered with LEHs, which accounted for their limited colloidal stability.29
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Polymersome Encapsulated Hemoglobin Table 2. The P50, Cooperativity Coefficient (n), MetHb Level, Encapsulation Efficiency (E%), and Weight Ratio of Estimated Entrapped Hb to Copolymer (R) of PEH Dispersions
R (mg Hb/ mg copolymer)
pore radius (nm)
P50 (Torr)
n
200 100
22.2 29.7
2.4 2.5
22-12.6 kDa (L1)
