Biomacromolecules 2005, 6, 414-424
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Photopolymerization of Bovine Hemoglobin Entrapped Nanoscale Hydrogel Particles within Liposomal Reactors for Use as an Artificial Blood Substitute Jaqunda N. Patton and Andre F. Palmer* Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556 Received September 10, 2004; Revised Manuscript Received October 30, 2004
Lipogel particles encapsulating bovine hemoglobin (BHb) were synthesized via photopolymerization of poly(N-isopropylacrylamide) (pNIPA) and poly(acrylamide) (pAAm) monomers within liposomal reactors. Nanoscale hydrogel particles (NHPs) encapsulating bovine hemoglobin, which represent a hybrid between acellular and cellular hemoglobin based oxygen carriers, were formed upon solubilization of the lipid bilayer of lipogel particles encapsulating BHb. Lipogels and NHPs encapsulating BHb constitute a new class of blood substitute that prevents both dissociation of hemoglobin (Hb) and in vivo exposure of acellular Hb, while allowing oxygen transport through the polymer matrix. pNIPA and pAAm particles encapsulating BHb displayed oxygen affinities ranging from 9.9 ( 1.9 to 14.4 ( 0.1 mmHg for lipogels, methemoglobin levels ranging from 9.3 ( 3.7% to 26.0 ( 5.0% for lipogels and NHPs, and encapsulation efficiencies ranging from 34.2 ( 3.4% to 97.4 ( 15.8% for lipogels and NHPs. Interestingly, the methemoglobin level of pNIPA particles was reduced 61% by coencapsulating the reducing agent, N-acetylcysteine. Fractionation and light scattering results showed that lipogels and NHPs were spherical and exhibited narrow size distributions. The colloidal osmotic pressure of pNIPA and pAAm lipogels ranged from 3.71 ( 0.02 to 206.87 ( 0.42 mmHg, depending on UV-irradiation time, type of buffer, and polymer composition. These results demonstrate that hemoglobin can be encapsulated within hydrogel based particles for use as an artificial blood substitute. 1. Introduction There is an urgent need for oxygen carriers due to the impending shortage of donor blood and the risks associated with the use of transfused blood including the transmittance of infectious diseases and immunosuppressive effects.1 Hemoglobin based blood substitutes that have been developed to date have been classified as either acellular (stroma free hemoglobin, intramolecularly cross-linked hemoglobin, conjugated hemoglobin, and polymerized hemoglobin) or cellular (hemoglobin encapsulated in liposomes and polymer capsules) based oxygen carriers.2 Hydrogel particles, which have been used for drug delivery, drug targeting, and enzyme immobilization, have not been examined as artificial blood substitutes.3 In this work, hydrogel based oxygen carriers are introduced as a novel confinement system for hemoglobin for use as an artificial blood substitute. Acellular hemoglobin based blood substitutes are designed to prevent dissociation of tetrameric hemoglobin into Rβ dimers, which are toxic to cells in the body, prevent rapid extravasation, and increase colloidal osmotic activity.4 Cellular hemoglobin based blood substitutes, on the other hand, are designed to mimic red blood cells (RBCs) by encapsulating hemoglobin inside the aqueous core of a membrane shell to avoid exposing the body’s tissues to free hemoglobin.5 Liposome encapsulated hemoglobin (LEHb) particles greater * To whom correspondence should be addressed. E-mail:
[email protected].
than 200 nm in diameter were found to be subject to rapid removal by the reticuloendothelial system (RES) due to a complement mediated phenomena.6,7 To avoid rapid removal by the RES and remain large enough to encapsulate sufficient hemoglobin for oxygen transport, researchers turned to PEGylation of LEHbs.8 Animal studies have shown that though the circulation half-life is improved by PEGylation (up to 65 h in rabbits), the hemoglobin content remains insufficient to adequately transport oxygen.6,9 In addition, a significant amount of hemoglobin succumbed to conversion to methemoglobin over a 24 h period.10 Researchers have since incorporated enzymes and substrates in LEHbs to reduce methemoglobin formation. However, challenges to the development of LEHb include the need to increase hemoglobin encapsulation efficiency and prevent detrimental interactions between the hemoglobin molecule and cells, tissues, organs, and phospholipids in the lipid vesicles, which reportedly catalyze oxidation of hemoglobin to methemoglobin.9 Only animal studies of LEHbs have been conducted to date, though it is expected that human clinical studies will soon follow. Polymer capsules are the only other hemoglobin confinement system investigated to date for use as an artificial blood substitute. Hemoglobin encapsulated in polymer capsules retained 2,3-diphosphoglycerate and exhibited an oxygen dissociation curve similar to that of RBCs.12,13 Upon encapsulating hemoglobin, the protein maintained over 50%
10.1021/bm049432i CCC: $30.25 © 2005 American Chemical Society Published on Web 12/14/2004
Photopolymerization of NHPs
of its oxygen carrying capacity, but ultimately denatured.14 Other attempts at forming hemoglobin microcapsules were also made but continued to have the limitation of rapid hemoglobin release.15-17 Most recently (2003), researchers investigating hemoglobin encapsulated microcapsules determined the optimum conditions for preparing microcapsules containing hemoglobin with a mean size of 10 µm, a P50 of 26.00 mmHg, a Hill coefficient of 2.4, and an encapsulation efficiency of 93%.15 These particles were far too large, however, to be used as oxygen carriers. Chang and Wu have recently synthesized even smaller biodegradable nanocapsules (70-400 nm) encapsulating bovine hemoglobin. Chang’s group has also prepared hemoglobin nanocapsules that show reduction in methemoglobin levels via incorporation of a methemoglobin reduction system.5,13,18-20 Although use of a biodegradable capsule removes the issue of accumulation of lipid in the RES, which arises from the use of LEHb, there is still the problem of free hemoglobin being released into the blood stream upon degradation of the membrane. Previous studies focused on the use of biodegradable polymers with the intent that the polymer would safely deteriorate in the body to avoid the accumulation of components in vital organs. However, the issue of the release of acellular hemoglobin, which is toxic, is not addressed. By using nonbiodegradable polymers that are biocompatible, hydrogel based oxygen carriers will not result in the release of acellular hemoglobin and will avoid adverse immunogenic and antigenic effects due to the in vivo presence of the polymer components. Instead, it is hypothesized that these particles will ultimately succumb to extravasation through the RES similarly to outdated RBCs. Furthermore, the solid, spherical shape of these capsules is not the most efficient RBC substitute since it is not a pliable membrane, and remains subject to rapid removal by the RES. Even rigid RBCs formed during storage have shown a reduced ability to pass through microcirculatory beds, which can result in decreased microvascular flow, local hypoxia, and potential multiple organ failure.21 In this work hemoglobin is encapsulated inside nanoscale hydrogel particles (NHPs) for use as a potential oxygen carrier. A hydrogel is a 3-dimensional network of hydrophilic polymers that swell and absorb aqueous solutions.22 Hydrogels offer several advantages compared to the above confinement systems used to encapsulate hemoglobin. Hydrogel particles are mechanically more stable than liposomes, have a greater loading capacity, and have swelling and shrinking properties that can be controlled more so than liposomal systems.23,24 The permeability of the gel matrix can also be controlled by tuning the size of the pores in the gel, which can be controlled experimentally by varying the amount of UV-irradiation time, concentration of cross-linker, and the polymer composition used to synthesize the particle.11,26 Furthermore, the flexible, rubbery texture of hydrogels should be less susceptible to recognition and subsequent removal by the RES than rigid polymer capsules.22 In light of this, hydrogels can be used as an immobilizing support for hemoglobin to design blood substitutes that are potentially free of immunogenic and antigenic effects.
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This study considers not only the use of NHPs but also the use of NHPs enclosed in a lipid bilayer (lipogels) as a hemoglobin confinement system. Few studies have been conducted to examine preparation of hydrogel-liposome complexes such as lipogels, and most of these have been at the microscale.23,27-28 Encapsulating hydrogel particles within liposomes has been found to increase the mechanical strength of liposomal systems.23,29 Further mechanical stability can be obtained by covalently attaching the hydrogel core to the lipid bilayer.30 Hence, lipogels encapsulating bovine hemoglobin can be engineered with controlled permeability to oxygen while exhibiting the properties of both hydrogels and liposomes. Oxygen uptake and release by lipogels and NHPs will be governed in part by intracellular oxygen diffusion through the hydrogel matrix since there is little or no resistance to oxygen transport across the lipid bilayer in the case of lipogels and since there is no distinct cellular barrier around NHPs. Engineering the permeability of the NHP should allow control of the pore size, and therefore the resistance of the hydrogel matrix, to oxygen transport. Swelling is expected to effect increased oxygen delivery due to an increased gel pore size. Likewise, shrinking will result in reduced oxygen delivery due to a decreased gel pore size. This phenomena was tested for hydrogels by Guzma´n et al.11, and it was shown that restrictions in the diffusion path due to the polymer matrix caused a 1/3 reduction in the diffusion coefficient of oxygen compared to its value in water.11 This work examines the preparation and characterization of lipogels and NHPs for use as a potential artificial blood substitute and presents the first analysis of this new class of hydrogel based oxygen carrier. Bovine hemoglobin was cross-linked within the polymer matrix of unanchored poly(acrylamide) and poly(N-isopropylacrylamide) NHP that were trapped within a liposomal reactor. To assess the feasibility of using hydrogel based oxygen carriers as an artificial blood substitute, the size distribution, oxygen affinity, percentage of methemoglobin (metlevel), hemoglobin encapsulation efficiency, and colloidal osmotic pressure (COP) of lipogels and NHPs were examined. 2. Experimental Section 2.1. Materials. L-R-Phosphatidylcholine derived from egg yolk and dissolved in chloroform (EPC, transition temperature Tm ) - 10 °C, MW 760 g/mol) was obtained from Avanti Polar Lipids (Alabaster, AL). Acrylamide (AAm), N-isopropylacrylamide (NIPA), 2,2-diethoxyacetophenone (DEAP), N,N-methylenebisacrylamide (MBA), and nonionic detergent Triton X-100 were purchased from Aldrich (Milwaukee, WI). Phosphate salts, Trizma HCl, Trizma Base, and NaCl, which were used to make the various buffers, were obtained from Sigma-Aldrich (St. Louis, MO). All chemicals were used as received without additional purification. The water used in all experiments was purified with a Milli-Q (Millipore) water system to a resistivity of 18.1 M Ω cm. 2.2. Hemoglobin Preparation. Tetrameric bovine hemoglobin was isolated from bovine RBCs as follows. First,
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bovine RBCs in 3.8% sodium citrate (Animal Technologies, Inc., Tyler, TX) were washed 3 times with 3 volumes of isotonic saline solution at 4 °C. Then the RBCs were lysed with 2 volumes of hypotonic, 15 mOsM phosphate buffer (PB), pH 7.4, which was maintained at 4 °C. Cell debris was removed via ultrafiltration using 0.05µm hollow fiber cartridges (Spectrum Laboratories, Inc., Rancho Dominguez, CA) in a chromatography refrigerator (Isotemp Plus Refrigerator, Fisher Scientific, Pittsburgh, PA) maintained at 4 °C. Bovine hemoglobin was then reconcentrated using 50 000 g/mol MWCO hollow fiber cartridges, also at 4 °C. The concentration of hemoglobin and methemoglobin in the stock solution of extracted hemoglobin was determined using an adaptation of the cyanomethemoglobin method.31,32 A 1 mL sample was placed in a UV-vis cuvette with a dilution D1 that yielded an absorbance 75% and was consistently >50%. The average hemoglobin encapsulation efficiency ranged from 57% to 97%, and the error due to interference of lipid, micelles, and the NHPs in solution is quantified by the standard deviation of a minimum of 3 independent measurements for each experiment. Table 6 reveals a 61% decrease (99.9% confidence) in methemoglobin upon addition of an equimolar amount of NAC and hemoglobin, which has been shown to reduce the rate of methemoglobin formation in hemoglobin vesicles.48 The encapsulation efficiency is also seen to decrease, as expected, with equimolar encapsulation of NAC and hemoglobin. NAC was observed to have no significant effect on the metlevel of pAAm particles. It should be noted that the metlevel of pAAm NHPs is significantly less than that of pNIPA exposed to the same amount of UV irradiation. It was observed that when the hydrating solution was prepared with pNIPA, cross-linker, and photoinitiator, the metlevel of the bovine hemoglobin stock solution increased from 2% in the hydrating solution. However, the pAAm hydrating solution prepared with bovine hemoglobin exhibited a reduced metlevel of 0.6% compared to 1% methemo-
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globin in the stock bovine hemoglobin. Therefore, pAAm may have acted as a reducing agent for methemoglobin. This explains the lower metlevel of pAAm particles compared to pNIPA, and why NAC did not reduce the metlevel since it was reduced by pAAm. Over time, hydrating solutions made with both monomers were found to increase metlevels. From this, it can be inferred that the photoinitiator and/or crosslinker oxidizes bovine hemoglobin within the liposomal reactor, and polymerization of pAAm reduces its ability to reduce the oxidized bovine hemoglobin. Another source of hemoglobin oxidation arises due to the lipid used to form the liposomal reactor, EPC, which has been confirmed by several researchers to rapidly oxidize hemoglobin.49,50 For this reason, a control experiment was conducted by encapsulating bovine hemoglobin into EPC liposomes. The control experiment showed that the hydrating solution metlevel increased significantly from 1.8 ( 0.2% to 14.9 ( 1.8% after dialysis and did not show a significant increase after UV irradiation. These results are consistent with the conclusion that EPC oxidized hemoglobin to methemoglobin. It also reveals that UV irradiation up to 8 min did not compromise the integrity of the hemoglobin as it was polymerized within the hydrogel. It is important to maintain metlevels as low as possible in order to increase the efficacy of cellular based oxygen carriers. Though, metHb levels
