Bioconjugate Chem. 2008, 19, 1025–1032
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Biocompatible and Biodegradable Polymersome Encapsulated Hemoglobin: A Potential Oxygen Carrier Shahid Rameez, Houssam Alosta, and Andre F. Palmer* Department of Chemical and Biomolecular Engineering, The Ohio State University 231A Koffolt Laboratories, 140 West 19th Avenue, Columbus, Ohio 43210. Received December 14, 2007; Revised Manuscript Received March 18, 2008
This work describes the development of polymersome-encapsulated hemoglobin (PEH) self-assembled from biodegradable and biocompatible amphiphilic diblock copolymers composed of poly(ethylene oxide) (PEO), poly(caprolactone) (PCL), and poly(lactide) (PLA). In the amphiphilic diblock, PEO functions as the hydrophilic block, while either PCL or PLA can function as the hydrophobic block. PEO, PCL, and PLA are biocompatible polymers, while the last two polymers are biodegradable. PEH dispersions were prepared by extrusion through 100 nm pore radii polycarbonate membranes. In this work, the encapsulation efficiency of human and bovine hemoglobin (hHb and bHb) in polymersomes was adjusted by varying the initial concentration of Hb. This approach yielded Hb loading capacities that were comparable to values in the literature that supported the successful resuscitation of hamsters experiencing hemorrhagic shock (1). Moreover, the Hb loading capacities of PEHs in this study can also be tailored simply by controlling the diblock copolymer concentration. In this study, typical Hb/diblock copolymer weight ratios ranged 1.2-1.5, with initial Hb concentrations less than 100 mg/mL. The size distribution, Hb encapsulation efficiency, oxygen affinity (P50), cooperativity coefficient (n), and methemoglobin (metHb) level of these novel PEH dispersions were consistent with values required for efficient oxygen delivery in the systemic circulation (2-5). Taken together, our results demonstrate the development of novel PEH dispersions that are both biocompatible and biodegradable. These novel dispersions show very good promise as therapeutic oxygen carriers.
INTRODUCTION Liposome-encapsulated hemoglobin (LEHb) comprises one subset of cellular Hb-based O2 carriers (HBOCs). The architecture of these particles consists of a purified and concentrated Hb solution that is encapsulated inside the aqueous core of the liposome. Lipids are for the most part biocompatible. This property has motivated the development of liposomes as drug and protein carriers (3, 5, 6). Liposomes lack controlled release mechanisms and have several other pharmacokinetic limitations (e.g., limited half-life in the circulation) which minimize the effective delivery of therapeutic molecules (7). In order to mimic the carbohydrate coat on cell membranes and thus prolong the circulatory half-life of liposomes, biocompatible poly(ethylene glycol) (PEG) was attached to a small fraction (5-10 mol %) of the lipid headgroups on LEHb particles. This resulted in increased circulatory half-life of LEHb particles, due to decreased recognition by the reticuloendothelial system (RES). However, the beneficial effects gained by the prolonged circulation time of PEGylated LEHb is attenuated by significant particle accumulation in the liver and spleen. Splenic sequestration of LEHb particles saturates the RES, thereby limiting the effectiveness of the body’s natural defense system against foreign bodies . To counteract these effects, we propose to use vesicles composed of amphiphilic diblock copolymers, instead of lipids, that have the ability to degrade once sequestered in the spleen. Biocompatible diblock copolymers that consist of a PEO block plus a degradable block, such as PEO-poly(lactic acid) (PEO-PLA) or PEO-poly(caprolactone) (PEO-PCL), are promising candidates for the preparation of polymer vesicles that have the native ability to evade the RES and degrade once sequestered in the spleen or liver (8-10). * Corresponding author. E-mail:
[email protected].
Self-assembled polymer vesicles composed of diblock copolymer amphiphiles where one block is hydrophilic and the other block is hydrophobic are called polymersomes (4). These vesicles are synthetic in nature. The amphiphilic diblocks are similar in structure to lipids, but possess much larger molecular weights (4). In aqueous solution, the diblocks form an arrangement where the hydrophobic blocks tend to associate with each other to minimize direct exposure to water, whereas the more hydrophilic blocks face inner and outer hydrating solutions and thereby form the two interfaces of a typical bilayer membrane (11). Polymersomes are composed of synthetic polymers of molecular weights more typical of polypeptides (order of 10 kDa), whereas lipids possess molecular weights less than 1 kDa (4). Lipid membranes appear conducive to various cellular processes (endocytosis, cell division, receptor clustering, etc.) because of the flexibility afforded by the low molecular weight lipids, which made them an attractive candidate for an oxygen carrier over the last few decades. Polymersomes can be prepared from diblock copolymers by the same techniques used to prepare liposomes such as rehydration, sonication, and extrusion, which can generate unilamellar micrometer-sized vesicles as well as monodisperse submicrometer-sized vesicles (12). Diblock copolymers consisting of poly(ethylene glycol-blactide) (PEG-PLA) or poly(ethylene glycol-b-caprolactone) (PEG-PCL) have been used to prepare controlled release polymer vesicles (13). One study (13) demonstrated the encapsulation of anticancer agents in polymersomes and their ability to release drug to cancer cells. Another study demonstrated degradable polymersomes being used in the growth arrest and shrinkage of rapidly growing tumors (14). In addition, polymersome-encapsulated paclitaxel has exhibited slow, steady release of the anticancer drug paclitaxel in human breast cancer cells. Paclitaxel-polymersomes have exhibited desirable restrained release profile and long-term stability.The formulations
10.1021/bc700465v CCC: $40.75 2008 American Chemical Society Published on Web 04/29/2008
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Table 1. Number-Averaged Molecular Weight (Mn), PEG Length, Hydrophilic Mass Faction (fhydrophilic), and Hydrophobic Membrane Thickness (d) of the Biodegradable and Biocompatible Diblock Copolymers Used in This Study diblock copolymer
Mn (kDa)
PEG length (kDa)
fhydrophilic
d (nm)
PEO-b-PCL-1 PEO-b-PCL-2 PEO-b-PLA-1 PEO-b-PLA-2
1.65 15 10 2.45
0.55 5 5 0.55
0.33 0.33 0.33 0.25
∼8 ∼18 ∼17 ∼8
also showed comparable activity to the drug alone in inhibiting the growth of MCF-7 human breast cancer cells (15). Polymersomes made from either PEG-PLA or PEG-PCL form stable vesicles (16). Dynamic light scattering provides an accurate measurement of the vesicle size distribution. Periodic measurements over a period of time has shown that PEO-PBD polymersomes are stable whereas vesicles made with degradable copolymer PEO-PLA disassemble over a period of days at room temperature (16). By changing the copolymer composition, especially the molecular weight and type of hydrophobic block, the release of encapsulated material from polymersomes can be adjusted accordingly (17). Biodegradable polymersomes also have the potential to be targeted to specific sites in the body, as has been shown by the specific interaction of antihuman serum albumin conjugated polymersomes with a human serum albumin coated sensor surface (17). In this work, we prepare biodegradable and biocompatible polymersomes encapsulating Hb and show that they are promising candidates for hemoglobin-based oxygen carriers (HBOCs). To our knowledge, this is the first study that explores the possibility of employing biodegradable and biocompatible polymersomes to be used as HBOCs.
Figure 2. Comparison of Hb encapsulation efficiency in PEH dispersions extruded through 200 nm diameter polycarbonate membranes using different sources of Hb.
EXPERIMENTAL PROCEDURES Materials. Four biodegradable and biocompatible diblock copolymers composed of poly(ethylene oxide-b--caprolactone) and poly(ethylene oxide-b-lactide) (both DL and L forms) were used in this study. Two of these diblocks were composed of poly(ethylene oxide-b-caprolactone), and two were composed of poly(ethylene oxide-b-lactide). The diblock copolymers were purchased from Polymer Source Inc. (Dorval QC, Canada). For convenience, PEH composed of poly(ethylene oxide-b--caprolactone) and poly(ethylene oxide-b-lactic acid) will be referred to as PEO-b-PCL-1, PEO-b-PCL-2, PEO-b-PLA-1, and PEOb-PLA-2 (Table 1). Likewise, their corresponding controls (i.e., empty polymersomes) will be referred to as PEO-b-PCL-1 (control), PEO-b-PCL 2 (control), PEO-b-PLA-1 (control), and PEO-b-PLA-2 (control) (Table 1).
Figure 1. Chemical structure of (A) poly(ethylene oxide-b--caprolactone) and (B) poly(ethylene oxide-b-lactide). The block inside the parentheses is hydrophilic (PEO), while the block inside the square bracket is hydrophobic (PCL or PLA).
Figure 3. Comparison of bHb encapsulation efficiency in PEH dispersions extruded through 200 nm diameter polycarbonate membranes determined via the centrifugation and dialysis methods.
The chemical structures of PEO-b-PCL and PEO-b-PLA are shown in Figure 1, and the structural properties of the four diblock copolymers are summarized in Table 1. Extraction and Purification of bHb and hHb from RBCs. Sterile bovine red blood cells (bRBCs) were purchased from QUAD 5 (Ryegate, MT), while human red blood cells (hRBCs) were obtained from the American Red Cross (Columbus, OH). Both bRBCs and hRBCs were initially prepared in the same manner for filtration. RBCs were harvested by centrifugation for 15 min at 4500 rpm and 4 °C. Collected cells were washed three times with a 0.9% (w/v) saline solution. The washed cells were then lysed on ice with 15 mOsM phosphate buffer solution (pH 7.2) for 1 h. The lysate was passed through a glass wool filter three times, then filtered through a paper filter to remove the majority of cell debris. The filtrate was then passed through a four stage hollow fiber setup ranging from 0.2 µm to 50 kDa in molecular weight cutoff (MWCO). The Hb retentate from the 50 kDa MWCO filter was used for the experiments in this study. This method was used to both purify and concentrate the Hb used in this study. Assay of Hb Solution. SDS-PAGE was used to determine the purity of fractionated Hb. The purity of Hb bands in each lane of the SDS-PAGE gel was assessed from the relative band
Biodegradable and Biocompatible PEH
Figure 4. Effect of diblock copolymer concentration on bHb encapsulation efficiency in PEH dispersions extruded through 200 nm diameter polycarbonate membranes.
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PEH Preparation. 50 mg of diblock copolymer dried into a thin film was mixed with 1 mL of purified Hb in PBS at pH 7.3 as the extrusion buffer, in order to mimic the salinity and pH of blood plasma. The mixture was hydrated for at least 1 h. To increase the efficiency of Hb entrapment in higher molecular weight copolymers, the copolymer/Hb mixture was subjected to 3 to 5 freeze/thaw cycles by alternatively placing the vials in a dry ice bath and warm water bath. After full hydration, the sample was extruded 20 times through 200 nm diameter polycarbonate membranes (Avanti Polar Lipids Alabaster, AL). Empty polymersomes were prepared in the same manner and used in the study as controls. Instead of hydrating with Hb solution, PBS was used as the hydration buffer. Both PEH and controls were used in the study immediately after preparation. Dialysis and centrifugation methods were used to separate unencapsulated Hb from PEH particles. In the dialysis method, extruded PEH samples were dialyzed overnight using 300 kDa MWCO dialysis bags (Spectrum Laboratories Inc. Fort Lauderdale, FL) in PBS at 2-3 °C at a 1:1000 v/v dialyzed sample/ PBS ratio in order to remove unencapsulated Hb. In the centrifugation method, extruded PEH samples were centrifuged at 14 000 rpm for 60 min. The supernatant containing unencapsulated Hb was discarded, while the PEH pellet was resuspended in PBS. This process was repeated 3 times until a clear supernatant was obtained. PEH Size Distribution. An Eclipse asymmetric flow field flow fractionator (AFFFF) (Wyatt Technology Corp., Santa Barbara, CA) coupled in series to an 18-angle Dawn Heleos multiangle static light scattering (MASLS) photometer (Wyatt Technology Corp., Santa Barbara, CA) was used to measure the size distribution of PEHs and controls. The MASLS photometer was equipped with a 50 mW GaAs linearly polarized laser operating at a laser wavelength of 658 nm. ASTRA software (Wyatt Technology Corp., Santa Barbara, CA) was used to analyze the light scattering spectra and calculate the particle size distribution of the particle dispersions. Hb Encapsulation Efficiency and Methemoglobin Level. In order to measure the Hb encapsulation efficiency and encapsulated methemoglobin (metHb) level, centrifuged PEHs were first lysed using 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS. The encapsulation efficiency was calculated using the following equation:
[
Hb Encapsulating Efficiency (%) ) 1 -
Figure 5. Oxygen equilibrium curves of PEH dispersions extruded through 200 nm diameter polycarbonate membranes comprising (A) bHb and (B) hHb. Here, Y is the fraction of Hb saturated with oxygen and pO2 is the partial pressure of oxygen.
intensities using the Quantity One 1-D analysis software (BioRad Laboratories, Hercules CA). bHb with 97% purity and hHb with 96% purity were used to prepare PEHs. The freshly prepared Hb was buffer-exchanged with phosphate buffered saline (PBS) (pH 7.3). The metHb levels of the Hb solutions used in this study were always less than 3%.
]
V1C1 - V2C2 × 100 V1C1 (1)
where V1 ) Initial volume of unencapsulated Hb and PEH particles (mL); C1 ) Initial concentration of Hb (mg/mL); V2 ) Volume of PEH particles obtained after centrifugation (mL); C2 ) Concentration of encapsulated Hb obtained after lysing PEHs (mg/mL). The metHb levels of Hb encapsulated inside PEH particles were assayed via UV-vis spectroscopy (Thermo Electron Corp. Madison, USA) using the cyanomethemoglobin method detailed elsewhere (2-5, 7, 18). The absorbance of Hb extracted from lysed PEH dispersions was measured at 630 nm (L1) against a blank reference (deionized water). Three drops of a 1:1 potassium cyanide solution made from equal parts of 10% KCN and 50 mM phosphate buffer were added to the lysed PEH samples. This converted methemoglobin to cyanomethemoglobin, which does not absorb at 630 nm. After 2 min, the
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Figure 6. Oxygen affinity (P50) of PEHs extruded through 200 nm diameter polycarbonate membranes.
Figure 7. Cooperativity coefficient of PEHs extruded through 200 nm diameter polycarbonate membranes.
absorbance was read at 630 nm (L2) against a blank reference. The concentration of metHb was determined as follows: L1 - L2 D1 (2) [MetHb]mM ) E -1 where E ) 3.7 (cm × mM) is the extinction coefficient of metHb at 630 nm. The metHb level (%) is then expressed as follows: [MetHb]mM × 100 (3) [MetHb](%) ) [MetHb]mM + [tHb]mM To determine the concentration of Hb, 3 drops of 20% potassium ferricyanide K3(Fe(CN)6) was added to 3 mL samples diluted appropriately (D2). The solution was allowed to react for 2 min before 3 drops of 10% KCN was added. The absorbance was then measured at 540 nm (L3). The concentration of Hb was determined as follows: L3 (4) [tHb]mM ) D2 E
where E ) 11.0 (cm × mM)-1 is the extinction coefficient for metHb at 540 nm. Oxygen Equilibrium Binding Properties. The oxygen equilibrium binding curves of PEH dispersions were measured using a Hemox-Analyzer (TCS Scientific Corp. New Hope, PA) at physiological temperature (37 °C) (4, 7, 18) Briefly, 1 mL of PEH dispersion was added to a mixture containing 4 mL Hemox buffer, 20 µL of Additive-A, and 10 µL of antifoaming agent (TCS Scientific Corporation). The PEH dispersion was allowed to saturate to a pO2 of 145 ( 2 mmHg using compressed air. The PEH dispersion was given extra time to saturate since oxygen takes a longer time to diffuse across the PEH membrane. After that, the gas stream was switched to pure N2 to deoxygenate the PEH dispersion. The absorbances of oxy- and deoxyHb in solution were recorded as a function of pO2 via dual wavelength spectroscopy. A six-parameter (A0, A∞, A1, A2, A3, A4) Adair model (5) and a four-parameter (A0, A∞, P50, n) Hill model (6) were used to fit the oxygen equilibrium curves of PEH dispersions in order to regress the Adair parameters, as
Biodegradable and Biocompatible PEH
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Table 2. Mean Diameter, PEG Length, P50, Cooperativity Coefficient (n), Encapsulated MetHb Level and Membrane Thickness (d) of LEHs, PEG-LEHs, LEAcHbs, Polymerized Hemoglobin, and PEHs mean diameter (nm)
PEG length (kDa)
P50 (mmHg)
230 250
0.55 2
33.2 25.1
272 281
2 2
28.5 27.5
200 200
12.6 2.3
22.2 28.1
238 260
none none
31.0 22.4
n
d (nm)
metHb level (%)
ref
PEG-LEH 3-4 3-4
