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Langmuir 2006, 22, 2212-2221
Physical Properties of Hemoglobin-Poly(acrylamide) Hydrogel-Based Oxygen Carriers: Effect of Reaction pH Jaqunda N. Patton and Andre F. Palmer* Department of Chemical and Biomolecular Engineering, UniVersity of Notre Dame, Notre Dame, Indiana 46556 ReceiVed October 26, 2005. In Final Form: December 13, 2005 This work examines the physical properties of bovine hemoglobin (BHb) chemically cross-linked to a pH responsive polymer (poly(acrylamide)) with the goal of taking advantage of the polymer’s pH sensitivity to generate low-P50 oxygen carriers for application in physiological conditions characterized by deviations from normal pH. BHbhydrogel-based oxygen carriers encapsulating 10-16 g/dL BHb displayed P50s < 10 mmHg when encapsulated inside of liposomes (i.e. Hb-LGs) and 95% using SDS-PAGE.2 Synthesis of Polyacrylamide Lipogels and Nanoscale Hydrogel Particles. Hb-pAAm nanoscale hydrogel-based oxygen carriers were prepared via UV-induced polymerization within liposomal reactors using the procedure described by Patton and Palmer.1,2 The referenced work entailed cross-linking 150 mg/mL of BHb with the hydrogel-forming species, whereas in this study 300 mg/mL of BHb was cross-linked with the hydrogel-forming species. NAC was coencapsulated with Hb and AAm in the liposomal reactors to prevent or limit the formation of metHb. Oxygen carriers were synthesized in PBS over a range of physiologically significant pH (pH 7.0, 7.4, and 7.8). The liposome solution (EPC concentration 26.3 mM) was formed via agitation of the rehydrated lipid film followed by manual extrusion through 600, 400, and 200 nm pore diameter polycarbonate membranes (Avanti Polar Lipids, Inc., Birmingham, AL). Details of the extrusion procedure were previously reported.1,8,10,49-52 The lipid bilayer surrounding Hb-LGs was solubilized with 15 mM Triton X-100 in PBS at the same pH as the Hb-LG solution (i.e. 2:1 dilution with 30 mM Triton X-100) to yield a solution of detergent micelles, mixed micelles composed of detergent and phospholipids molecules, and Hb-NHPs. The concentration of Hb in the final NHP solution was 500 nm). The small peak in the particle size distribution 3 days after synthesis ∼10 nm in diameter reflects the presence of a small fraction of micelles remaining after diafiltration. This peak dominated the light scattering intensity before diafiltration with an intensity of 100 au, which verifies removal of the majority of micelles. In Figure 4, the peak corresponding to individual particles has a greater light scattering intensity (100 au) compared to aggregates (80 au), 3 days after synthesis. However, 5 days later, the intensity of the peak corresponding to particles decreased dramatically to 500 nm). In general, particles aggregate as a consequence of an imbalance between attractive van der Waals forces and repulsive electrostatic forces.59 Therefore, a dispersion of oxygen carriers will be colloidally stable if the surfaces of the particles have a charge density or potential that is sufficient for the particles to repel one another. The increase in intensity and size of the aggregate peak is an indicator of the physical instability of Hb-NHPs.61 The aforementioned change in size and aggregation of the dispersions can be explained by considering the ζ potential of the particles. Oxygen carriers must be larger in size compared to Hb tetramers, which are ∼6 nm in diameter, since acellular tetrameric Hb can extravasate through blood vessels into surrounding tissue, thus sequestering NO, which leads to vasoconstriction and systemic hypertension.5,9,11,12,14,15 As mentioned earlier, acellular tetrameric Hb is cytotoxic; so, it is important to prevent direct contact between tetrameric Hb and tissues.5,9 In addition, oxygen carriers that are 20-50 nm in diameter can readily diffuse through endothelial fenestra, and particles larger than 5 µm can be rapidly removed by the RES.5,8 It should also be noted that there is evidence that particles greater than ∼200 nm in the circulation can induce pulmonary hypertension.5,9,40 BHb cross-linked to pAAm formed hydrogel-based oxygen carriers within the size range suitable for use as an oxygen carrier. Although the size of Hb-NHPs was observed to be larger than 200 nm due to particle aggregation, this phenomena can be eliminated via surface modification of the particles with poly(ethylene glycol).5 Furthermore, our novel technique for synthesis of hemoglobinhydrogel-based oxygen carriers within a liposomal reactor permits synthesis over a range of sizes that is determined by the pore diameter of the polycarbonate membrane used during liposome extrusion, which may be as small as 25 nm.1,8 The size of Hb-LGs was not affected by changes in reaction pH as a consequence of the impermeability of the lipid bilayer to hydrophilic ions. However, Hb-NHPs increased in size with increasing reaction pH. ζ Potential. Measurements of ζ potential, which determine whether colloids in a dispersion will aggregate, yield information about the colloidal stability of particle dispersions.59-62 The larger the magnitude of the ζ potential, the more stable the dispersion. Full electrostatic stabilization of stored colloidal solutions is realized with ζ potentials of at least |30| mV and optimally >|60| mV.62 Limited flocculation occurs between |5| and |15| mV, where flocculation refers to a state of slow particle aggregation.62 Particle dispersions injected into the blood stream will be under the influence of anions, cations, and nonionic molecules that will alter the particles’ ζ potential. For this reason, it is desirable to design oxygen carriers with ζ potentials similar to that of RBCs to prevent aggregation of blood colloids and to prevent adverse interactions with blood components. Agglomeration of RBCs occurs at approximately -15 mV in blood, and maximum dispersion occurs at -100 mV.62 (59) Ricˇka, J.; Tanaka, T. Macromolecules 1984, 17, 2916-2921. New, R. R. C. In Liposomes: A Practical Approach; IRL Press: New York, 1997; pp 100103. (60) Heurtault, B.; Saulnier, P.; Pech, B.; Proust, J.-E.; Benoit, J.-P. Biomaterials 2003, 24, 4283-4300. (61) Riddick, T. M. Control of Colloid Stability through Zeta Potential: With a Closing Chapter on its Relationship to CardioVascular Disease; Livingston Publishing Co.: Wynnewood, PA, 1968. (62) Pelton, R. H.; Pelton, H. M.; Morphesis, A.; Rowell, R. L. Langmuir 1989, 5, 816-818.
Hb-Poly(acrylamide) Hydrogel-Based Oxygen Carriers
Control experiments were conducted to examine the stability of empty lipogel and NHP particles. The ζ potential of empty particles (lipogels and NHPs) shown in Figure 5 reveals that lipogels were most stable at pH 7.0 at both 25 and 37 °C. The magnitude of the ζ potential of lipogels decreased at pH 7.4 and then increased only slightly as the pH increased to 7.8. Since van der Waals attractive forces are relatively insensitive to changes in pH and electrolyte concentration, the change in ζ potential is primarily due to changes in electrostatic repulsive forces of the lipogel or lipid bilayer. The electrostatic repulsion of the particle will depend on the charge of the particle, which in turn will change depending on how close the pH is to the isoelectric point (pI) of pAAm and EPC, where the pI is the pH at which there is an equal number of negative and positive charges.56 There is no significant change in the magnitude of the ζ potential of lipogels between a pH of 7.4 and 7.8. The ζ potential of lipogel controls reveals that the particles fall within the range of limited flocculation at pH 7.4 and 7.8. The ζ potential of solubilized lipogels, which contains micelles, mixed micelles (composed of phospholipid and nonionic detergent molecules), and NHPs was determined from intensity-weighted averages of the particle velocity. For this reason, the electrophoretic mobility of the solubilized lipogel dispersion has a smaller electrophoretic mobility compared to the NHP system with micelles removed. Nevertheless, both systems exhibited ζ potentials that were similar in magnitude and showed negligible change over the pH range examined. In a buffer with high ionic strength such as PBS, the electrophoretic mobility and, therefore, the electrostatic stabilization of the system are reduced, which tips the balance toward attractive van der Waals interactions. However, PBS was used in this study to examine the ζ potential of hydrogel-based oxygen carriers, since it has an ion concentration similar to that of plasma. The ζ potential of temperaturesensitive pNIPA hydrogels was also found to be independent of environmental changes in high ionic strength solutions because of reduction in electrostatic interactions.2,63 These results lead to the conclusion that Hb-NHPs do not exhibit colloidal stability in PBS. However, the stability of these particles in plasma may be achieved by using a polymer composition that has a greater ζ potential or by covalently conjugating poly(ethylene glycol) (PEG), which has been used to increase the colloidal stability of polymer capsules and liposomes, to the surface of these particles.5,27 In general, proteins are made up of numerous amino acids that are bound by peptide linkages. Consequently, it is likely that some of BHb’s ionizable groups remain exposed to aqueous solvent molecules when mixed with AAm and NAC in the liposomal reactor. During the cross-linking process, the ionizable groups of BHb interact with NAC and AAm. These interactions between the various groups contribute to the change in ζ potential of Hb-LGs and Hb-NHPs compared to empty lipogels and NHPs. At pH 7.0, the magnitude of the ζ potential of Hb-LGs (Figure 6) decreased upon cross-linking with Hb at 25 and 37 °C, compared to empty particles (Figure 5). The magnitude of the ζ potential of Hb-LGs increased with an increase in pH from 7.0 to 7.4 and was unchanged between pHs 7.4 and 7.8 at 25 °C. At 37 °C, the magnitude of the ζ potential of Hb-LGs is observed to fluctuate. It first increases between pHs 7.0 and 7.4 and then decreases between pHs 7.4 and 7.8. The ζ potential of RBCs exhibits similar behavior, first decreasing between pHs 4 and 9 and then increasing for pHs > 9.61 (63) Whittam, R. Transport and Diffusion in Red Blood Cells; The Williams & Wilkins Co.: Baltimore, MD, 1964.
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The ζ potential of solubilized Hb-LGs shown in Figure 6 was larger in magnitude compared to the control dispersions shown in Figure 5. The pI of BHb is 6.8, with carboxyl and amide groups existing as NH3+ and COO- near the pI.56 When the pH is > 7.0, BHb becomes negatively charged, and the amide and carboxyl groups exist as NH2 and COO-.56 It can be inferred that the oxygen carrier then becomes more electrostatically stabilized upon cross-linking pAAm and BHb via ionization of BHb. Likewise, the pI of the lipid bilayer and the hydrogel matrix will affect the charge and electrostatic stabilization of the oxygen carriers. The slight decrease in the magnitude of the ζ potential above pH 7.4 may also be due to the presence of the Hb-pAAm cross-linked hydrogel core, which becomes increasingly ionized with increased pH. As counterions diffuse into the Hb-pAAm cross-linked hydrogel matrix, the charge of the particles is neutralized, thus resulting in a decrease in the magnitude of the ζ potential (i.e. reduction in colloidal stability).43 Figure 6 also shows that the magnitude of the ζ potential of Hb-NHPs decreased as the pH was increased. As the pH increased, the particles also increased in size as discussed previously. With an increase in size, the charge density of Hb-NHPs was reduced, which corresponds to a reduction in magnitude of the ζ potential (i.e. reduced colloidal stability). Again, these results are consistent with DLS results, since at 25 °C Hb-NHPs were smaller in size. Small particles possess a higher charge density and are thus stabilized primarily by electrostatic interactions. The ζ potential of stored human RBCs, LEHbs, and polymer capsules was found to be -16.8, -21, and -73 mV, respectively.13,58,64 Polymer capsules and LEHbs have been stabilized via conjugation of PEG to the particle surface.5,27,65 The magnitude of the ζ potential of Hb-LGs and Hb-NHPs were shown to be within the range of limited flocculation. The ζ potentials were within the values of agglomeration for RBCs. For this reason, in the future it will be necessary to PEGylate Hb-LGs and Hb-NHPs to achieve colloidal stability of these suspensions. Hb-LG-based oxygen carriers may be PEGylated using the same protocols used to PEGylate LEHb, since a lipid bilayer surrounds the NHP. NHPs, on the other hand, may be PEGylated after solubilization of the lipid bilayer and removal of the micelles from solution. PEGylation of NHPs may be achieved after evaporation of the solvent followed by resuspension in a solution of reactive PEG molecules. Oxygen Affinity. Figures 7 and 8 reveal that both Hb-LGs and Hb-NHPs showed similar oxygen binding behavior with changes in reaction pH. The only factors that will influence the oxygen affinity of hemoglobin-based oxygen carriers are those that will hinder or enhance the ability of the protein to change structural conformations, such as allosteric affectors (e.g. chloride ions and protons) and hydrogel polymer chain flexibility. The size and ζ potential of the hydrogel-based oxygen carriers will not directly affect oxygen affinity. The P50 of Hb cross-linked to pAAm hydrogel-based oxygen carriers (7 mmHg 1). The effect of reaction pH on the P50 and Hill coefficient (n) of Hb-LGs and Hb-NHPs is more readily seen in Figure 9, which depicts a bar graph of the oxygen affinity on the primary (64) McCarthy, M. R.; Vandegriff, K. D. Biophys. Chem. 2001, 92, 103-117. (65) Fronticelli, C.; Bucci, E.; Orth, C. J. Biol. Chem.1984, 259, 1984110844.
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Patton and Palmer
axis and symbols plotting the Hill coefficient, n, on the secondary axis for Hb-LGs and Hb-NHPs as a function of pH. The oxygen carriers exhibited increased oxygen affinity (i.e. reduced P50) at pH 7.0, compared to the oxygen affinity at a normal physiological pH of 7.4. This means that oxygen was bound and released at lower oxygen tensions at pH 7.0. The P50s were approximately the same for both Hb-LGs and Hb-NHPs at pH 7.8 (Figure 9). Physiological conditions characterized by reduced oxygen tensions due to decreased pH will benefit from enhanced oxygen delivery at low oxygen tensions, which is provided by high oxygen affinity oxygen carriers. The behavior of the P50 of Hb-pAAm hydrogel-based oxygen carriers presented above is counterintuitive to that predicted by the Bohr effect for acellular Hb when the protein is incubated and the dissociation curve measured over a range of pH. Typically, Hb experiences an increase in P50 with decreasing pH (i.e. the Bohr effect) due to structural bias of the T state for protons.42 Since the T state, which has an increased P50 (i.e. decreased oxygen affinity), dominates in the presence of increasing concentrations of H+, decreasing the pH yields an increased P50.56 The effect of reaction pH on the oxygen affinity of HbLGs and Hb-NHPs can be attributed to structural changes in Hb due to cross-linking Hb with pAAm at different pHs. The degree of cross-linking and hence the number of resultant covalent chemical bonds between BHb and pAAm will depend on the ionization of BHb, pAAm, and EPC, which will become more negative with increased pH above their respective pIs. It is likely that structural changes in the oxygen carrier occurred as a consequence of increased flexibility of the polymer chains with increased preparation pH. The reaction pH should only affect the initial oxygen affinity and cooperativity of the oxygen carrier, and the typical Bohr effect should occur in vivo with changes in blood composition due to the structural bias of the T-state for protons. The Hill coefficient of Hb-NHPs increased with increasing pH, which reflects an increased ability of the protein to shift conformations upon binding each successive oxygen molecule due to the increased flexibility of the hydrogel matrix. In general, the Hill coefficient of the oxygen carriers is less than that of acellular BHb (n ) 2.5) due to chemical cross-linking BHb to the hydrogel matrix, which limit conformational shifts between the T and R states.1 The high cooperativity of cross-linked hemoglobin has been reported previously for intramolecularly cross-linked human hemoglobin, which had a Hill coefficient of 2.4, and polymerized bovine hemoglobin, which exhibited Hill coefficients ranging from 1.0 to 2.0.15,65 Structural changes in BHb were also previously shown to affect the pH sensitivity of the oxygen affinity for intramolecularly cross-linked BHb yielding a reduction in the Bohr effect.40,42,66 Likewise, the increase in P50 with an increase in pH from 7.0 to 7.4, which was observed for Hb-pAAm hydrogel-based oxygen carriers, indicates possible stabilization of the R state upon synthesis of the oxygen carrier. The oxygen affinity of Hb-LGs appear to be more sensitive than Hb-NHPs to reaction pH, as evidenced in the difference between the P50 of Hb-LGs and Hb-NHPs at pH 7.0 and 7.4 (Figure 9). This may be due to accessibility of the oxygen carriers to the allosteric effector of BHb, chloride ions (Cl-). The difference between the P50 of Hb-NHPs and Hb-LGs decreased as the pH increased from 7.0 to 7.8, becoming equal at pH 7.8. The charge on BHb at pH 7.0 is very small, since it is very near to the pI of BHb (pI ) 6.8). As the pH increases above the pI, the charge of the protein increases and it becomes more negatively
charged. Charge equilibrium requirements then limit the influx of chloride ions into the hydrogel matrix. Chloride ions are known to elicit an increase in the P50 of acellular BHb.66 The lipid bilayer of Hb-LGs prevents access of chloride ions from the bulk solution to BHb cross-linked to the hydrogel matrix. Therefore, as the charge of BHb cross-linked within Hb-NHPs becomes more negative (i.e. as the pH increases), P50 of HbNHPs converges to that of Hb-LGs. The P50 of cellular-based oxygen carriers is typically designed to mimic that of human RBCs. LEHbs have been synthesized with P50s ranging from 19 to 35 mmHg.8 However, Hb-LGs and Hb-NHPs were designed to function as high oxygen affinity oxygen carriers with P50s ranging from 5 to 15 mmHg to target oxygen delivery to tissues with low oxygen tensions. Hb-NHPs and Hb-LGs were found to yield an increase in oxygen tension as the pH decreased from normal physiological pH of 7.4 to 7.0. Therefore, Hb-pAAm hydrogel-based oxygen carriers synthesized at pHs less than 7.4 are expected to deliver more oxygen at lower oxygen tensions as the pH is reduced below normal physiological pH. Hemoglobin Encapsulation Efficiency. It is desirable to encapsulate large amounts of Hb to optimize the ability of oxygen carriers to transport oxygen with minimal dosage. Table 1 shows that increasing the pH resulted in an increase in Hb encapsulation efficiency, which ranged from 35% (10.5 g/dL BHb) to 53% (15.9 g/dL BHb). LEHbs exhibited Hb encapsulation efficiencies of 14.8% in PBS.8 These results show that Hb-LGs and HbNHPs can achieve Hb encapsulation efficiencies much greater than that of LEHbs. A major drawback to the use of LEHbs is low Hb encapsulation efficiencies, which is reflected in the high encapsulation ratio (i.e. ratio of the mass of phospholipid to the mass of encapsulated Hb) of LEHbs, which ranges from 0.62 to 3.85.8,13 The ratio of the mass of phospholipid to the mass of Hb cross-linked within hydrogel-based oxygen carriers ranges from 0.1 to 0.3.1,2 Hence, hydrogel-based oxygen carriers exhibit a more superior encapsulation ratio compared to LEHbs. MetHb Level. It has been suggested that the percentage of metHb in oxygen carriers needs to be maintained below 10% to yield oxygen carriers that can deliver oxygen effectively.67 Hb is more readily oxidized to metHb at pHs < 7.0, which explains why the lowest level of metHb was observed at high pHs in Figure 10. A major factor contributing to the high levels of metHb of hydrogel-based oxygen carriers is the chemical nature of the lipid used to form the liposomal reactor. In particular, EPC has been shown to enhance Hb oxidation.8,26-27,67 This lipid was used in this study, since it was readily available. However, the high level of metHb may be reduced by using a combination of lipids such as DMPC, cholesterol, and DMPG, which have been successfully used to prepare LEHbs with levels of metHb < 3%.8 The presence of the reducing agent, NAC, does not appear to reduce metHb over time. Previous experiments in the absence of NAC revealed that NAC had no effect on the size distribution, shape, or oxygen affinity of hemoglobin-pAAm hydrogel-based oxygen carriers.1 These results also suggested that the high reactivity of AAm with hydroxyl, amino, and sulfhydryl groups may limit the use of NAC as a metHb reducing agent during formulation of hemoglobin-poly(acrylamide) hydrogel-based oxygen carriers.1 Therefore, the reducing capabilities of the metHb reductant may have been exhausted during synthesis. It is noteworthy that when hydrogel-based oxygen carriers were synthesized at a pH of 7.8, the metHb level of the solution was
(66) Linberg, R.; Conover, C. D.; Shum, K. L.; Shorr, R. G. L. Artif. Cells Blood Substitutes 2003, 26, 133-148.
(67) Takeoka, S.; Sakai, H.; Mano, Y.; Seino, Y.; Nishide, H.; Tsuchida, E. Bioconjugate Chem. 1997, 8, 539-544.
Hb-Poly(acrylamide) Hydrogel-Based Oxygen Carriers
less compared to that at lower pH. To avoid this, the reductant should be loaded into the gel after synthesis.
Conclusions Hydrogel-based oxygen carriers were synthesized with the pH-sensitive polymer pAAm to develop an oxygen carrier that will target oxygen delivery to tissues with low oxygen tensions elicited by decreased physiological pH. The size of Hb-LGs was insensitive to changes in pH, and Hb-NHPs responded to pH in accordance with gel swelling theory on the basis of ion migration into the gel matrix. Hb-LG suspensions were shown to be physically stable, not succumbing to particle aggregation after over 1 month of storage. The ζ potential of Hb-LGs and Hb-NHPs remained within the range of limited flocculation, even under the influence of a high ionic strength and physiologically similar salt environment afforded by PBS. However, it will be necessary to engineer the polymer composition of the hydrogel polymer matrix to increase the colloidal stability of the oxygen carriers and thus make them suitable for in vivo applications. The oxygen affinity of Hb-LGs and Hb-NHPs increased when synthesized at pH 7.4 and 7.0. This is a useful property for delivering oxygen to tissues with low oxygen tensions and that are anoxic due to conditions characterized by reduced pH. The oxygen carriers exhibited high Hb encapsulation efficiencies, which increased with reaction pH. Hb encapsulation efficiency may be controlled by altering the reaction pH, and may be more important than achieving a higher oxygen affinity if arterial oxygen tensions are normal. However, if arterial oxygen
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tensions are low, high oxygen affinity oxygen carriers are desired, since they will deliver oxygen more efficiently at low oxygen tensions. Levels of metHb of oxygen carriers were >10%; however, levels of metHb may be further reduced by using a lipid combination that does not oxidize Hb and by loading the oxygen carriers with metHb reducing agents as opposed to synthesizing the oxygen carrier in the presence of the metHb reducing agent. It is expected that synthesized hydrogel-based oxygen carriers will maintain the ability of the hydrogel matrix to swell and shrink in response to changes in pH due to changes in blood composition, offset from the properties that the oxygen carriers exhibit upon formulation at a specific reaction pH. The ζ potential of the dispersions is also expected to respond to changes in blood composition, since salt concentration and solution pH are known to influence the ζ potential of colloids, including RBCs and liposomes.61 In conclusion, this work describes the preparation and characterization of oxygen carriers synthesized at discrete pHs to yield oxygen carriers with high oxygen affinities. Acknowledgment. We acknowledge the help of Dennis Birdsell from the Center for Environmental Science and Technology at the University of Notre Dame for use of the center’s Brookhaven ZetaPlus ζ potential and particle size analyzer. This work was supported, in part, by Grant BES-0196432 from the National Science Foundation (Arlington, VA). LA052883J
