Hemoglobin-Based Oxygen Carrier Microparticles: Synthesis

Article pubs.acs.org/Biomac

Hemoglobin-Based Oxygen Carrier Microparticles: Synthesis, Properties, and In Vitro and In Vivo Investigations Yu Xiong,*,† Axel Steffen,† Kristin Andreas,‡ Susanne Müller,§ Nadine Sternberg,† Radostina Georgieva,†,∥ and Hans Baü mler*,† †

Institute of Transfusion Medicine and Berlin-Brandenburg Center for Regenerative Therapies, §Department of Experimental Neurology and Center for Stroke Research Berlin, and ‡Tissue Engineering Laboratory and Berlin-Brandenburg Center for Regenerative Therapies, Department of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany ∥ Department of Medical Physics, Biophysics and Radiology, Medical Faculty, Trakia University, ul. “Armeiska” 11, 6000 Stara Zagora, Bulgaria S Supporting Information *

ABSTRACT: Bovine hemoglobin microparticles (Hb-MPs) as suitable oxygen carriers are fabricated easily by three key steps: coprecipitation of Hb and CaCO3 to make Hb-CaCO3-microparticles (Hb-CaCO3-MPs), cross-linking by glutaraldehyde (GA) to polymerize the Hb and dissolution of CaCO3 template to obtain pure Hb-MPs. The Hb entrapment efficiency ranged from 8 to 50% corresponding to a hemoglobin quantity per Hb-MP of at least one-third of that in one erythrocyte. The Hb-MPs are spherical, with an average diameter of 3.2 μm and high oxygen affinity. The methemoglobin level was increased after preparation, but can be reduced to less than 7% with ascorbic acid. Phagocytosis assays showed low immunogenicity of Hb-MPs if the particles were cross-linked with low concentration of GA and treated with sodium borohydride. Magnetiteloaded Hb-MPs circulated up to 4 days after intravenous application.

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INTRODUCTION

of vasoconstriction and can lead to suitable blood substitutes.10,11 To produce larger HBOCs, Hb can be encapsulated in nanoor microparticles/capsules with different methods such as interfacial polycondensation,12 double emulsion-solvent diffusion/evaporation,13,14 encapsulation of Hb in liposome/vesicles (HbV),15 or nano artificial RBCs.16 Hollow multilayer microcapsules fabricated by layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes onto colloidal particles followed by core removal are also intensively investigated because of their broad applications, particularly in biorelated fields such as controlled drug delivery, biosensors and bioreactors.17 The use of porous inorganic CaCO3 microparticles (CaCO3−MPs) as template for fabrication of those multilayer microcapsules has advances due to the biocompatibility and high adsorption capacity of the CaCO3−MPs and the mild fabrication conditions.18,19 Moreover, the coprecipitation of biomacromolecules with CaCO3 to form biomacromolecules-CaCO3 hybrid-microparticles has become more interesting since biomacromolecules can be captured by CaCO3−MPs with the coprecipitation method more effectively than by simple adsorption onto preformed CaCO3−MPs.20

The use of human blood for blood transfusions has limitations such as a limited shelf life of blood products, risk of disease transmission, immunomodulation, risk of hemolytic transfusion reactions, and logistical constraints.1,2 Unmodified stroma-free hemoglobin is not suitable as a blood substitute as it produces severe problems: short circulation time in vivo, nephrotoxicity, high affinity to nitric oxide (NO), and related vasoconstriction and hypertension.3,4 Diverse modifications of hemoglobin (Hb) based on intra- and intermolecular cross-linking or encapsulation have been tested to find a suitable oxygen carrier. Hemoglobin-based oxygen carriers (HBOCs) have been investigated intensively during the last 30 years with the aim to develop a universal blood substitute. Early generation HBOCs such as diaspirin intramolecular cross-linked Hb (DCLHb), weakly polymerized Hb like Hemopure and PolyHeme have prolonged intravascular retention time but caused substantial hypertension.1,2,5−7 Sakai et al. and Cabrales et al. demonstrated that the extent of the vasoconstriction and hypertension decreases with increasing size of the HBOC.8,9 Furthermore, HBOC with high pO2 at 50% saturation of Hb (p50) can release excessive amounts of oxygen into the systemic circulation and also induces vasoconstriction.10 Therefore, polymerization of Hb leading to larger HBOC with low p50 (high oxygen affinity) provides a better solution for prevention © 2012 American Chemical Society

Received: July 13, 2012 Revised: August 29, 2012 Published: September 17, 2012 3292

dx.doi.org/10.1021/bm301085x | Biomacromolecules 2012, 13, 3292−3300

Biomacromolecules

Article

Scheme 1. Fabrication Scheme of Hemoglobin Microparticles

temperature for 1 h under oxygen-free conditions, followed by three washing steps with Ampuwa. The CaCO3 template was removed by treatment of Hb-CaCO3-MPs with EDTA solution (0.2 M, pH 7.4, 20 mL). Finally, the resulting Hb-MPs were centrifuged (1000 g, 2 min), washed three times with Ampuwa, and resuspended in Ampuwa for further use. Magnetite-loaded Hb-MPs were fabricated by adding magnetite nanoparticles (100:1 volume ratio) to the Na2CO3 previous to coprecipitation. The iron amount in Hb-MPs and magnetite loaded Hb-MPs was measured using inductively coupled plasma optical emission spectrometry (ICP-OES; JY 38 Plus, Jobin-Yvon ISA, France). Concentration of Hb and Methemoglobin (MetHb). Concentration of Hb stock solution and MetHb-content were determined by the standard cyanomethemoglobin method25 using a UV−visspectrophotometer (Hitachi U2800, Hitachi High-Technologies Corporation). The light scattering of the particles was compensated applying the absorption value at 597 nm (isosbestic point for MetHb and cyanomethemoglobin). Hb Entrapment Efficiency (EE) of CaCO3-MPs. Hb amount in Hb-CaCO3-MPs was determined as the difference between the total Hb amount applied (Hbt) and the Hb amount determined in the supernatant (Hbf) after coprecipitation and after each washing step. The EE % was calculated according to the following equation EE % = (Hbt - Hbf) × 100%/Hbt. The measurements were performed with a microplate reader (PowerWave 340, BioTek Instruments GmbH) at 415 nm. Zeta-Potential. Zeta-potential was measured in 0.25 mM NaCl solution using a Zetasizer nano ZS instrument (Malvern Instruments Ltd., U.K.). Confocal Laser Scanning Microscopy (CLSM). CLSM images were taken with a confocal microscope LSM 510 Meta (Carl Zeiss MicroImaging GmbH, Jena Germany) equipped with a 100× oilimmersion objective (numerical aperture 1.3) applying excitation wavelength 488 nm and a long pass emission filter 505 nm. Scanning Electron Microscopy (SEM). For SEM analysis, samples were prepared by applying a drop of the particle suspension to a glass slide and then drying overnight. After that, the samples were sputtered with gold. Measurements were conducted using a Gemini Leo 1550 instrument at an operation voltage of 3 keV. Oxygen Dissociation Curve (ODC). ODC measurements were performed as described by Zhang et al., with modifications.26 The partial oxygen pressure (pO2) was detected by an oxygen electrode (GMH 3630, Greisinger electronic GmbH, Germany). Briefly, Hb or Hb-MPs were suspended in phosphate buffer (pH 7.4, 0.1 M Cl1−) and were held at 37 °C. The absorption (A) at 576, 580, and 588 nm was measured at pO2 values between 0 mmHg (Adeoxy) and 160 mmHg (Aoxy). The absorption at 584 nm (isosbestic point for oxygenated Hb and deoxygenated Hb) was used to exclude evaporation and light scattering. The value of oxygen saturation (Y) at a given pO2 was calculated according to the following equation: Y = (A − Adeoxy)/(Aoxy − Adeoxy). Oxygenation and deoxygenation were operated by gas with air or argon, a complete deoxygenation of Hb and Hb-MPs was achieved by addition of SDT. Immune Response to Hb-MPs. The phagocytic activity of granulocytes and monocytes after addition of Hb-MPs was measured in vitro in human whole blood using a Phagotest kit (Glycotope-

These hybrid-microparticles provide a suitable platform for both multilayer microcapsules fabricated by LbL-technology17,20,21 and biopolymer microparticles. In this article, we present bovine hemoglobin microparticles (Hb-MPs) as oxygen carriers with a low p50 fabricated by a novel simple technique recently developed by Bäumler and Georgieva.22,23 The Hb-MPs were easily fabricated after three key steps: coprecipitation of Hb and CaCO3 to make HbCaCO3-microparticles (Hb-CaCO3-MPs), cross-linking by glutaraldehyde (GA) to polymerize the Hb and dissolution of CaCO3-template to obtain pure Hb-MPs. The morphological, physicochemical and immunological properties of the Hb-MPs were characterized. The oxygen-carrying functionality of the Hb-MPs was also evaluated. Furthermore, magnetite-loaded Hb-MPs were tested in vivo in Wistar rats concerning their circulation in the bloodstream and distribution in the body applying magnetic resonance imaging (MRI).

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MATERIALS AND METHODS

Materials. Hemoglobin (Hb) was extracted from bovine red blood cells by hypotonic hemolysis.24 Briefly, fresh bovine whole blood (obtained from Schlachtbetrieb GmbH Perleberg, anticoagulated with EDTA) was centrifuged at 2500 g for 10 min at 4 °C, the packed red blood cells (RBCs) were washed three times with ice cold PBS, 5 volume of ice cold Ampuwa (aqua ad iniectabilia; Fresenius Kabi Deutschland GmbH) was added to 1 volume of washed RBCs. The solution was stirred at 4 °C overnight and then centrifuged at 10000 g for 1 h at 4 °C. The supernatant was filtered through a 0.1 μm polyethersulfone filter (Sartorius AG, Germany) and stored as stock solution at −80 °C until use. The purity of Hb was controlled by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE, Supporting Information, Figure S1). Glutaraldehyde (GA), calcium chloride (CaCl2), sodium carbonate (Na2CO3), and phosphate buffered saline (PBS), pH 7.4, were purchased from Sigma-Aldrich; ethylenediaminetetraacetic acid (EDTA) and sodium dithionite (SDT) were purchased from Fluka; sodium hydroxide (NaOH) was purchased from Carl Roth; Ampuwa and sterile 0.9% NaCl solution were purchased from Fresenius Kabi Deutschland GmbH. Citrate-coated magnetite particles (diameter = 10 nm), 1% (w/w) suspension in water, were purchased from Magnetic fluids, Berlin, Germany. Preparation of Hb-MPs. Hb-MPs were fabricated using a novel technique as previously described by Bäumler and Georgieva (Scheme 1).22 Briefly, 5 mL and equal concentration of solutions of Na2CO3 and CaCl2 containing Hb were rapidly mixed in a 100 mL beaker under vigorous stirring (Bibby Scientific CB161 Magnetic Stirrer, level 3) for 30 s at room temperature to produce the Hb-CaCO3-MPs.20 To calculate the Hb entrapment efficiency of CaCO3−MPs, the concentrations of Na2CO3 and CaCl2 solutions were 0.15, 0.25, and 0.5 M, the concentrations of Hb in CaCl2 solution were 2.5, 5.0, 7.5, 10.0, and 15.0 mg mL−1. To characterize the Hb-MPs, 0.25 M Na2CO3, and CaCl2 containing 15.0 mg mL−1 Hb were used. The obtained Hb-CaCO3-MPs were separated by centrifugation (300 g, 1 min) and washed three times with Ampuwa. The particles were then suspended in GA solution (0.01−1.0%, 20 mL) and incubated at room 3293


Biomacromolecules

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Figure 1. (a) Hb entrapment efficiencies of CaCO3-MPs and (b) Hb content of Hb-CaCO3-MPs (absolute amount of the entrapped Hb in MPs, percentage by weight) by different CaCl2/Na2CO3 concentrations and Hb initial concentrations. Biotechnology GmbH, Heidelberg, Germany). The phagocytosing cell fractions were quantified by flow cytometry (FACS-Canto II, Becton and Dickinson, Franklin Lakes, NJ, U.S.A.). Circulation and Biodistribution. In vivo magnetic resonance imaging (MRI) was performed using a 7 T rodent scanner (Pharmascan 70/16, Bruker BioSpin, Ettlingen, Germany) and a 1H-RF volume resonator with an inner diameter of 72 mm. Wistar rats (3 months old, approximately weight 400 g) were placed on a heated circulating water blanket to ensure constant body temperature of 37 °C. Anesthesia was induced with 4% and maintained with 3−2% isoflurane (Forene, Abbot, Wiesbaden, Germany), delivered in 0.5 L min−1 of 100% O2 via a facemask under constant ventilation monitoring (Small Animal Monitoring and Gating System, SA Instruments, Stony Brook, New York, U.S.A.). T2*-weighted imaging was performed using a 2D-gradient-echo sequence (FLASH) with TR/TE = 619.7/7.2 ms, flip angle 30°, and 2 averages for 25 slices with a slice thickness of 1.0 mm, a FOV of 7.5 × 7.5 cm, and a matrix size of 128 × 128. MRI data evaluation was carried out with Analyze 5.0 software (AnalyzeDirect, Inc., Lenexa, U.S.A.). A region of interest (ROI) was assigned to each liver and the muscle of the foreleg, resulting in 3D object maps for each single rat. Normalized mean intensities of the liver were calculated referring to the intensity of the limb muscle by dividing signal-to-noise ratio (SNR) values of the liver by the SNR values of the muscle. Prior to the injection of the particles, 1 mL animal blood was taken. Than Hb-MPs and magnetite-loaded Hb-MPs with a particle number 1 × 109 suspended in 1 mL of sterile 0.9% NaCl-2% HSA were slowly injected (over 30 min) intravenously into the tail vein of a control and

a test animal, respectively. The animals were scanned before the injection, every 10 min in the first hour after injection and 4 days later. The animal experiments were approved by the local animal research authorities.

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RESULTS Hb Entrapment Efficiency (EE) of CaCO3-MPs. The entrapment efficiency of Hb in the CaCO3-MPs was studied using different concentration of the starting materials Hb, CaCl2 and Na2CO3. The Hb entrapment efficiency of the CaCO3-MPs rose with increasing concentration of CaCl2 and Na2CO3 solutions, but decreased with increasing initial Hb concentration. In our study, the Hb entrapment efficiency ranged from 8 to 50% of the initial amount of Hb (Figure 1a). The Hb content in MPs (absolute amount of the entrapped Hb in Hb-CaCO3-hybrid particles) in percentage by weight is shown in Figure 1b. At constant CaCl2/Na2CO3 concentration, the Hb content of MPs increased with increasing hemoglobin concentration. At constant Hb concentration, the Hb content of MPs was significantly higher for lower CaCl2/Na2CO3 concentration (0.15 and 0.25 M) compared with 0.5 M CaCl2/Na2CO3. Hb content of MPs of 8% was achieved with 15 mg mL−1 Hb and 0.25 M CaCl2 and Na2CO3. Only very little Hb has been found in supernatants of the washing steps after the cross-linking of Hb and dissolution of 3294


Biomacromolecules

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Table 1. Properties of Hb-MPs phagocytic activity of Hb-MPs, cross-linked with 1% GA 0.1% GA 0.01% GA 0.01% GAa a

size [μm]

MetHb level [%]

± ± ± ±

32.8 ± 5.9 25.3 ± 5.4 18.6 ± 3.8 15.0 ± 5.1 6.9 ± 2.0b

3.3 3.2 3.2 3.2

0.7 0.7 0.8 0.7

zeta potential [mV] −36.3 −34.3 −22.7 −25.2

± ± ± ±

0.8 6.4 2.9 3.8

granulocytes [%]

monocytes [%]

p50 [mmHg]

22.5 ± 5.8 17.9 ± 7.7 6.8 ± 2.9 4.6 ± 1.5

33.7 ± 10.8 24.0 ± 17.0 8.9 ± 6.0 4.9 ± 2.1

7.0 ± 2.0 7.0 ± 1.9

Particles quenched with glycine and reduced with NaBH4. bParticles after a postreduction with ascorbic acid.

Figure 2. Light transmission images (upper panel) and confocal micrographs of Hb-MPs (middle panel) cross-linked with different concentrations of GA, (a) 1.0%, (b) 0.1%, (c) 0.01%, and (d) 0.01%, and additionally quenched with glycine and reduced with NaBH4. Lower panel shows fluorescence profiles of selected Hb-MPs marked in middle panel.

Figure 3. SEM image of (a) CaCO3 microparticles and (b) Hb particles cross-linked with 0.01% GA, quenched with glycine and reduced with NaBH4.

CaCO3 (less than 3% of entrapped Hb). After dissolution of CaCO3 the pure Hb-MPs were obtained (Hb content 100%). MetHb Concentration of the Hb-MPs. During the fabrication of Hb-MPs in the presence of oxygen a part of the hemoglobin converts into MetHb where the iron in the heme group is in the ferric state. MetHb cannot bind and release oxygen. Therefore, a high amount of metHb should be avoided. The level of MetHb was 4.0% in Hb stock solution and increased to more than 30.0% in Hb-MPs. The MetHb level decreased with decreasing GA concentration used in the

cross-linking step (Table 1). Treatment of the particles with sodium borohydride (NaBH4) reduced the MetHb level down to 15.0% and a postreduction with ascorbic acid at pH 7.4 in oxygen free condition resulted in a further decrease to less than 7.0%. Morphology and Particle Size. Hb-MPs produced by our method are spherical and exhibit a particle size distribution between 2.5 and 4.5 μm with an average diameter of 3.2 μm (Table 1, particle size has been calculated with LSM Image Browser Release 4.2 by measurement of 100 particles per 3295


Biomacromolecules

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Figure 4. Functionality of Hb-MPs. (a) Absorption spectra of Hb and Hb-MPs. (b) Oxygen dissociation curves of Hb and Hb-MPs.

Figure 5. MRI of Wistar rats after application of 1 × 109 Hb-MPs: (a) control animal injected with Hb-MPs, 0; (b) test animal injected with magnetite loaded Hb-MPs; (c) normalized mean intensities in the liver (signal-to-noise ratio (SNR) of the liver divided by the SNR of the limb muscles) as a function of the time after injection; (d) regions of interests (ROI) for calculation of the normalized mean intensity in the liver.

Scanning electron micrographs of CaCO3−MPs without Hb and Hb-MPs cross-linked with 0.01% GA, quenched with glycine, and reduced with NaBH4 are shown in Figure 3. The porous structure and roughness of CaCO3 particles can be easily recognized. Hb-MPs are shrunken as a result of removing of CaCO3 template and drying upon sample preparation for SEM measurements. Due to the fact that Hb is uniformly distributed in the whole volume of the particles and cross-

sample). We applied different GA concentrations in order to find optimal cross-linking conditions. The particle size was not influenced by the GA concentration. A strong autofluorescence of the particles was observed due to the GA cross-linking (Figure 2a−c). Quenching by glycine after cross-linking and reduction with NaBH4 during the dissolution step considerably reduced the autofluorescence (Figure 2d). 3296


Biomacromolecules

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linked the Hb-MPs do not collapse during the drying procedure and remain their spherical form. The surface of the Hb-MPs is smoother in comparison to the surface of CaCO3 particles and the Hb-MPs looked like softballs. Zeta-Potential. The Hb-MPs show a negative zetapotential, which was dependent on the cross-linker concentration. The higher the concentration of GA, the more amino groups of Hb reacted with GA, correlating with the negative zeta-potential of the particles. It was slightly increased after quenching with glycine, which can be attributed to the attachment of new carboxyl groups to the particle surface (Table 1). Functionality of Hb-MPs. The ability of oxygenation and deoxygenation of the Hb-MPs was demonstrated by UV−vis spectroscopy (Figure 4a). After preparation the Hb-MP exhibited three maxima peaks at 414, 542, and 576 nm, which are the characteristic absorption peaks of oxygenated Hb (OxyHb). Addition of sodium dithionite to the suspension of Hb-MPs resulted in a red shift of the 414 nm peak (Soret peak) to 432 nm, appearance of a peak at 556 nm, and disappearance of the peaks at 542 and 576 nm. Such spectral changes are characteristic for the deoxygenation of Hb and confirm the ability of Hb-MPs to bind and release oxygen similarly like free Hb. Figure 4b shows the oxygen dissociation curve (ODC) of Hb-MPs cross-linked with 0.01% GA, quenched with glycine and reduced with sodium borohydride. The p50 value measured for free Hb was 26.5 mmHg in accordance with literature values.26,27 The p50 value of 7 mmHg measured for the Hb-MPs shows that the particles exhibit high oxygen affinity. Phagocytosis Assay. The Hb-MPs were incubated with heparinized whole blood for 30 min at 37 °C and the phagocytic activity of granulocytes and monocytes were analyzed. The percentage of granulocytes and monocytes phagocytozing Hb-MPs decreased with decreasing concentration of GA from more than 30% for cross-linking with 1% GA to less than 10% if the particles were cross-linked with 0.01% GA. In the case where 0.01% GA cross-linked Hb-MPs were additionally quenched with glycine and reduced with NaBH4, the phagocytosis rate even reached values below 5% (Table 1). Circulation and MRI Study. The Hb-MPs were further tested in vivo in six Wistar rats concerning their circulation in the bloodstream and distribution in the body. Two of them were investigated applying magnetic resonance imaging (MRI). All animals showed normal behavior after injection of Hb-MPs. For the MRI study, Hb-MPs and magnetite-loaded Hb-MPs with an iron amount of 0.35 and 13.8 mg, respectively, were applied. A particle number of 1 × 109 was slowly injected intravenously into the tail vein of a control and a test Wistar rat. Figure 5b demonstrates the strong reduction of the normalized mean intensity observed in the liver of the test rat after injection of magnetite labeled Hb-MPs. In contrast, the normalized intensity in the liver of the control animal remains constant (Figure 5a). The normalized mean intensity measured in the liver of the test animal four days later was lower again, which indicates that a part of the particles still circulated during this period of time (Figure 5b,c). Using their autofluorescence, the circulating Hb-MPs were also confirmed qualitatively in blood samples taken at different times after injection by flow cytometry.

Article

DISCUSSION

Porous CaCO3-MPs are able to encapsulate protein effectively.18 If the protein is simultaneously coprecipitated with CaCO3, the protein capture by CaCO3-MPs will be more effective.20 To find the optimal concentrations of the reactants in terms of low material consumption, high amount of particles and high Hb-entrapment in MPs, and different salt and Hb concentrations have been tested. In our study, the Hb entrapment efficiency ranged from 8 to 50% of the initial amount of Hb. At constant CaCl2/Na2CO3 concentration, the entrapment efficiency decreased with increasing Hb concentration as a result of saturation effect of CaCO3-MPs because the capture capacity of CaCO3-MPs is limited. At constant Hb concentration, the entrapment efficiency increased with increasing concentration of CaCl2 and Na2CO3 solutions, because more particles are formed and more Hb is captured by CaCO3-MPs. Hb-content of 8% (weight content) in CaCO3-MPs was achieved with 15 mg mL−1 Hb and 0.25 M CaCl2 and Na2CO3. We used this concentration of salt and Hb for particle characterization and further investigations. By taking a density of 1.6 g mL−1 of the CaCO3-MPs19 and the average diameter of 3.2 μm, one can calculate 2.20 pg Hb (2.05 × 107 Hb tetramers) per single particle and a content of 11.5 pg Hb per 90 fL (12.8 g dL−1) CaCO3-MPs. It is known that the average amount of Hb in one human erythrocyte (RBC) is approximately 30 pg per 90 fL,28 hence, the Hb content in the CaCO3-MPs was roughly 30−35% of the Hb content of the RBCs. After cross-linking, dissolution and washing about 3% of captured Hb was lost, that means approximately 2 × 107 Hb tetramers were still in one particle. Hb-MPs can also be prepared with the method named protein activation and spontaneous self-assembly (PASS).29 PASS is based on the dithiothreitol (DTT) triggered dissociation of internal disulfide bridges and their rearrangement as intermolecular cross-linkers of protein molecules finally forming protein particles. However, in the case of Hb, the application of DTT leads to an excessive methemoglobin (MetHb) formation and impairs the oxygen carrier function of Hb-MPs. For this reason, we applied the commonly used crosslinker GA. The Hb polymerization with GA in a free solution results in an inhomogeneous product with undefined morphology and a broad size distribution. After a 2 h incubation of Hb with GA in a molar ratio of 1:60, only 15− 500 Hb tetramers are polymerized.30 In contrast, the Hb-MPs produced by the method described here are spherical, with well-defined morphology. After 1 h incubation with GA (0.01%, Hb-GA molar ratio 1:68), approximately 2.00 × 107 Hb tetramers were cross-linked in one microparticle. This is clearly more effective than polymerization of Hb in a free solution. In other words, the consumption of the toxic GA is strongly reduced. Recently hemoglobin-based capsules for use as blood substitutes fabricated by covalent layer-by-layer assembly are reported by the Liś group.31 By this method, spherical MnCO3 microparticles were used as templates for hollow capsule assembly. Hb and dialdehyde heparin (DHP) were alternately adsorbed; DHP was used as one of the wall components and a cross-linker. The MnCO3 templates were then dissolved after treatment with EDTA to obtain the Hb hollow microcapsules.31 However, this covalent layer-by-layer assembly requires an extremely long cross-linking time. Each Hb/DHP layer assembly requires 12 h for cross-linking with DHP,31 so to 3297


Biomacromolecules

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Table 2. Comparison of Parameters of Different HBOCs vs Hb-MPs DCLHb1 Hb source Hb content, g/dL p50, mmHg Hill coefficient MetHb viscosity, cp avg weight or size

PolyHeme1

hemopure1

human 10 32

human 10 20−22

bovine 12−14 40

Hemoglobin-Based Oxygen Carrier Microparticles: Synthesis

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