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Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Novel Hemoglobin Particles—Promising New-Generation Hemoglobin-Based Oxygen Carriers *Hans Bäumler, *Yu Xiong, †Zhi Zhao Liu, †Andreas Patzak, and *‡Radostina Georgieva *Institute of Transfusion Medicine and Berlin-Brandenburg Center for Regenerative Therapies; †Institute of Vegetative Physiology, Charité-Universitätsmedizin Berlin, Berlin, Germany; and ‡Department of Medical Physics, Biophysics and Image Diagnostics, Medical Faculty, Trakia University, Stara Zagora, Bulgaria

Abstract: During the last 30 years, artificial oxygen carriers have been investigated intensively with the aim to develop universal blood substitutes. Favorably, hemoglobin-based oxygen carriers (HBOCs) are expected to meet the sophisticated requirements. However, the HBOCs tested until now show serious side effects, which resulted in failure of clinical trials and Food and Drug Administration disapproval. The main problem consists in vasoconstriction triggered by nitric oxide (NO) scavenging or/and oxygen oversupply in the pre-capillary arterioles. HBOCs with a size between 100 nm and 1 µm and high oxygen affinity are needed. Here we present a highly effec-

tive and simple fabrication procedure, which can provide hemoglobin particles (HbPs) with a narrow size distribution of around 700 nm, nearly uniform morphology, high oxygen affinity, and low immunogenicity. Isolated mouse glomeruli are successfully perfused with concentrated HbP suspensions without any observable vasoconstriction of the afferent arterioles. The results suggest no oxygen oversupply and limited NO scavenging by these particles, featuring them as a highly promising blood substitute. Key Words: Artificial oxygen carriers—Hemoglobin—Submicron particles—Oxygen affinity.

Approximately every 2 s someone in the United States needs a blood transfusion (1). In 2008 alone, 20 million units of packed red blood cells (RBCs) were transfused in Germany and in the United States (2,3). Transfusion of blood and blood products still comprise several risks, for example, mismatched transfusions, transfusion-related acute lung injury, immunomodulation, hemolytic transfusion reactions, bacterial or viral infections, and logistical constraints (4). The World Health Organization reported in 2011 that blood donations are still not routinely tested for transmissible infections in 39 countries, and that in developing countries 47% of the donations are tested in laboratories without quality assurance (5). Hence,

artificial oxygen carriers without the abovementioned risks are urgently needed. Due to its short circulation time and nephrotoxicity, stroma-free hemoglobin (Hb) cannot be used as a blood substitute (6). Diverse Hb modifications such as intra- and intermolecular cross-linking, conjugation with macromolecules, or encapsulation (4) have been intensively studied to overcome these problems. However, serious side effects occurred during application of most of these products in vivo, particularly hypertension caused by vasoconstriction (4,7). The mechanisms of vasoconstriction caused by hemoglobin-based oxygen carriers (HBOCs) are currently still under discussion. There are two main hypotheses: (i) oxygen oversupply caused by HBOCs with low oxygen affinity; and (ii) nitric oxide (NO) scavenging by small HBOCs (8,9). NO is an important vasodilator that is produced by the endothelial cells and is released into the smooth muscle tissue of the blood vessel (10). Stroma-free Hb and nano-sized HBOCs can pass through the gaps between the endothelial cells, bind the NO, and cause vasoconstriction (11,12). Thus, the synthesis of

doi:10.1111/aor.12331 Received November 2013; revised February 2014. Address correspondence and reprint requests to Dr. Radostina Georgieva, Institute of Transfusion Medicine, CharitéUniversitätsmedizin Berlin, Charitéplatz 1, Berlin 10117, Germany. E-mail: [email protected] Presented in part at the 4th International Symposium on Artificial Oxygen Carriers, held September 28, 2013 in Yokohama, Japan. Artificial Organs 2014, 38(8):708–714

PROMISING NEW-GENERATION HBOCS HBOCs larger than 100 nm to avoid HBOC extravasation through the gaps may be crucial to solve the problem of NO scavenging in the vessels. Taking into account that particles in the size range 1–3 µm can be strongly phagocytosed (13,14) and particles larger than 5 µm can block the microcirculation at higher concentrations, the preferable size of HBOCs should be between 100 nm and 1 µm. Additionally, the new-generation HBOCs should have a high oxygen affinity to prevent premature oxygen release in the pre-capillary arterioles, oxygen oversupply, and vasoconstriction caused by an autoregulatory mechanism (9). Here we present unique hemoglobin particles (HbPs) synthesized by our recently introduced procedure (15–17) as promising HBOCs matching the above-mentioned requirements for blood substitutes: particle size below 1 µm and high oxygen affinity to avoid vasoconstriction of small blood vessels. MATERIALS AND METHODS Materials Bovine serum albumin (BSA), FITC-BSA, TRITC-BSA, glutaraldehyde (GA), calcium chloride (CaCl2), manganese chloride tetrahydrate, sodium carbonate (Na2CO3), phosphate-buffered saline pH 7.4, glycine, and sodium borohydride were purchased from Sigma-Aldrich (Munich, Germany); ethyldiaminetetraacetic acid (EDTA) and sodium dithionite (SDT) were purchased from Fluka (Buchs, Switzerland); sodium hydroxide was purchased from Carl Roth (Karlsruhe, Germany); Ampuwa and sterile 0.9% NaCl solution were purchased from Fresenius Kabi Deutschland GmbH (Bad Homburg, Germany). Human serum albumin (HSA) solution 20% was purchased from Grifols Deutschland GmbH, Frankfurt am Main, Germany. Preparation and characterization of hemoglobin particles (HbPs) Hb was obtained from bovine RBCs by hypotonic hemolysis as described earlier (18) and stored as stock solution at −80°C until use. HbPs and albumin particles (APs) were fabricated in a modified and improved manner based on a novel technique as previously described (15–17). The co-precipitation, cross-linking, and dissolution steps were carried out under oxygen-free conditions. When needed, FITCBSA or TRITC-BSA (ratio = 1:100) was added to Hb during the coprecipitation step to produce fluorescently labelled HbPs. Concentrations of Hb and MetHb in stock solutions were determined by the cyano-methemoglobin method (19) using a

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UV-VIS-spectrophotometer (Hitachi U2800, Hitachi High-Technologies Corporation, Krefeld, Germany). The light scattering of the particles was compensated by the absorption value at 597 nm (isosbestic point for MetHb and cyanomethemoglobin). Hb entrapment efficiency (EE) 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 ability of HbPs to bind and deliver oxygen was investigated by spectral analysis in the range from 380 to 700 nm. The measurements were performed with a microplate reader (PowerWave 340, BioTek Instruments GmbH). Deoxygenation of HbPs was achieved by addition of 1.5 mg/mL SDT to the particle suspension. The hydrodynamic diameter and the zeta-potential of the HbPs were measured by dynamic light scattering using a Zetasizer nano ZS instrument (Malvern Instruments Ltd., Malvern, UK) in 0.9% NaCl solution. The particle size was also measured from scanning electron microscopy (SEM) images (Gemini Leo 1550, Oberkochen, Germany) using ImageJ 1.44p software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD, USA). For SEM analysis, samples were dried overnight on a glass slide, sputtered with gold and measured at an operation voltage of 3 keV. Confocal laser scanning microscopy (CLSM) images were taken with LSM 510 Meta (Carl Zeiss MicroImaging GmbH, Jena, Germany) microscope with a 100× oil-immersion objective. The behavior of HbPs in human blood was investigated in whole blood samples and platelet-rich plasma (PRP). Typically 500 µL of whole blood or PRP was gently mixed with 100 µL of 20% (v/v) HbP suspension or sterile physiological saline (control). Whole blood samples were incubated in a water bath at 37°C for 45 min. PRP samples were incubated at the standard storage conditions of platelet concentrates (on a platelet agitator at 25°C) for 45 min. The platelet number was detected before and after incubation using a hematology analyzer ABX Micros 60 (HORIBA Europe GmbH, Berlin, Germany). The number of activated platelets was measured using PE- or FITC-labeled mouse anti-human CD41a or 62p by flow cytometry and CLSM. The samples were also microscopically evaluated for platelet or particle aggregation as well as for interaction with RBCs using the CLSM. The phagocytic activity of leukocytes after addition of FITC-labeled HbPs was measured in human whole blood using a Phagotest kit (GlycotopeBiotechnology GmbH, Heidelberg, Germany) and was quantified by flow cytometry (FACS-Canto II, Becton & Dickinson, Franklin Lakes, NJ, USA). Artif Organs, Vol. 38, No. 8, 2014

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Microperfusion of arterioles of lung and glomeruli The experiments were performed in accordance with the regulations of the Office for Health and Social Matters of Berlin, Germany. Isolation and microperfusion of afferent arterioles were performed from male C57BL/6 adult mice (FEM, CharitéBerlin, Germany) as described previously (20) using a set of perfusion pipettes (illustrated in Fig. 3a) hand made from glass tubes (Vestavia Scientific, Vestavia Hills, AL, USA) and assembled on an electronically controlled micro-manipulator. Dulbecco’s modified Eagle medium (GIBCO, Darmstadt, Germany) was used for dissection and as bath solution (0.1% albumin). To provide a physiological flow in the afferent arterioles, the pressure in the pressure head was set to 100 mm Hg (flow of 50 nL/min). All perfusion experiments were performed within 120 min after sacrificing the mice. After a 10 min of stabilization period at 37°C, the perfusate was exchanged with BSA (30 mg/mL), Hb (30 mg/mL), APs, and HbPs (each 10% by volume corresponding to a protein content of 30 mg/mL) for 11 min. Finally, angiotensin II (Sigma, Hamburg, Germany) (10−12 to 10−6 mol/L) was applied at 2-min intervals for each concentration to provoke vasoconstriction. The perfused arterioles were continuously imaged and recorded in digital movies using a microscope camera. Digital pictures were serially acquired from the digital movie for each experiment, and were used to measure arteriolar luminal diameter and the distribution of fluorescence in the vessels. The data are presented as absolute diameters (µm) or relative values, that is, percentage (%) of the initial diameter. Circulation and biodistribution In vivo circulation tests were performed with 3-month-old Wistar rats and TRITC-BSA labeled HbPs (animal experiments approved by the local animal research authorities). Anesthesia was induced with 4% and maintained with 2–3 % isoflurane (Forene, Abbot, Wiesbaden, Germany), delivered in 0.5 L/min of 100% O2 via a face mask under constant ventilation monitoring (Small Animal Monitoring & Gating System, SA Instruments, Stony Brook, NY,

FIG. 1. Scheme of particle preparation via the coprecipitation, cross-linking, dissolution technique. Two inorganic solutions, for instance Na2CO3 and CaCl2, containing one or more biopolymers are mixed, resulting in CaCO3 particles with entrapped biopolymers. After cross-linking the biopolymers, CaCO3 is dissolved and the pure biopolymer particles remain in the solution (modified from Xiong et al. [16]).

USA). Approximately 20% of the animal’s blood was replaced by a suspension of HbPs in 0.9% NaCl2 % HSA (volume concentration 15%). The HbPs were detected and counted in blood samples taken after 0, 4, 24, and 96 h by flow cytometry. RESULTS AND DISCUSSION The coprecipitation–cross-linking–dissolution (CCD) procedure for fabrication of biopolymer particles is schematically represented in Fig. 1. Purified bovine Hb was coprecipitated with either CaCO3 or MnCO3. In the case of using MnCO3, human serum albumin was added immediately after coprecipitation to avoid aggregation. After cross-linking, the inorganic compounds were completely removed by dissolution using EDTA, followed by several washing steps as confirmed by inductively coupled plasma optical emission spectrometry. Interestingly, the size, morphology, and Hb entrapment efficiency of the resulting HbPs were significantly different for both inorganic templates. Table 1 summarizes characteristic values for HbPs obtained by coprecipitation with CaCO3 or MnCO3, respectively. HbPs obtained with

TABLE 1. Characteristic size, zeta-potential, entrapment efficiency, oxygen affinity, and immunogenicity of HbPs templated on CaCO3 or MnCO3 HbPs templated on CaCO3 MnCO3

Size (µm)

Zeta-potential (mV)

Hb in HbPs related to Hb in RBCs (%)

3.2 ± 0.7 0.7 ± 0.1

−11.1 ± 0.9 −2.1 ± 0.8

35 80

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p50 (mm Hg) 7.0 6.0

Activity of blood phagocytes after 30-min incubation (%) 0°C

37°C

2.4 ± 1.6 0.1 ± 0.1

4.6 ± 1.6 3.9 ± 3.3

PROMISING NEW-GENERATION HBOCS CaCO3 are spherical microparticles with a diameter between 2.5 and 4.5 µm. In contrast, HbPs produced by coprecipitation with MnCO3 emerge peanutshaped, with longest and shortest diameter of 800 and 600 nm, respectively. Measured by dynamic light scattering, the average size appears around 710 nm. The Hb EE of the HbPs was calculated as a percentage of the Hb content in a single human RBC (30 pg (21)). Using CaCO3 for HbPs fabrication, the EE depended on the applied concentrations of CaCl2 and Na2CO3 solutions and on the initial Hb concentration. Under optimal conditions, a content of 11.5 pg Hb per 90 fL HbPs was calculated, which corresponds to 30–35% of the Hb content of an RBC (16). However, in MnCO3 the Hb EE was more than two times 23.7 pg per 90 fL particles, corresponding to 80% of the Hb content of an RBC (15). The ability of oxygenation and deoxygenation of the HbPs was demonstrated by UV-VIS spectroscopy (Fig. 2). Oxygenated HbPs exhibited three maxima peaks at 414, 542, and 576 nm which are the characteristic absorption peaks of oxygenated Hb (OxyHb). After deoxygenation of the HbPs, a red shift of the 414 nm peak (Soret peak) to 432 nm occurred. The peaks at 542 and 576 nm disappeared, and a peak at 556 nm was detected instead. The spectral behavior is characteristic for the oxygenation/ deoxygenation of Hb and confirms the ability of HbPs to bind and release oxygen. The p50 value of 6 to 7 mm Hg measured for the HbPs is three to four times lower than that of free Hb (26.5 mm Hg), thus a high oxygen affinity is achieved. This is one very important requirement for the new-generation HBOCs. 1 0,9 OxyHb

0,8

DeoxyHb

Absorption

0,7

Oxygenated HbPs

0,6

Deoxygenated HbPs

0,5 0,4 0,3 0,2 0,1 0 380

430

480

530

580

630

680

Wavelength [nm] FIG. 2. Absorption spectra of OxyHb, deoxygenated (DeoxyHb), oxygenated and deoxygenated HbPs.

Hb

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Another challenging issue for the new HBOCs is to avoid vasoconstriction due to NO scavenging. The HbPs that were synthesized using the CCD technique were large enough to avoid penetration through the endothelial gaps. Moreover, using MnCO3 for the coprecipitation, submicron particles with a narrow size distribution around 700 nm are obtained, hitting the optimal size range between 100 nm and 1 µm. We examined these particles in terms of vasoconstrictive effects measuring the diameters of afferent arterioles of isolated glomeruli from mice as an in vitro model. The microvessels in the kidney just preceding the glomerular capillary network, the afferent arterioles, play a crucial role in the regulation of renal blood flow and are very sensitive to changes in NO bioavailability (22). The glomerular filtration rate depends strongly on the resistance of these vessels. Angiotensin II (Ang II) plays an important role in the control of arteriole diameter constricting afferent arterioles significantly in a dose-dependent manner (23,24). The vasoreactivity of the arteriole to Ang II also increases with decreasing NO bioavailability (23,24). Isolated afferent arterioles attached to their glomeruli were perfused with particle suspensions (Fig. 3a–c) and protein solutions for 11 min. The luminal diameters of the vessels in steady state (after 5-min perfusion) were chosen as initial diameters. After perfusion, the afferent arterioles were treated with Ang II in progressive doses to check their vasoreactivity. We measured the influence of pure Hb solution on vessel tone and its vasoreactivity to Ang II. Perfusion with BSA served as a control. The arteriolar tone was not significantly affected during 11-min perfusion with either BSA or Hb solutions. However, the response of the afferent arterioles to Ang II was significantly increased by Hb. At a concentration of 10-10 M Ang II, the luminal diameter of arterioles perfused with Hb decreased to 6.3 ± 2.5% of its initial size, whereas under the same conditions the control vessels perfused with BSA revealed 71.0 ± 7.7% of their initial diameter measured immediately before Ang II addition (Fig. 3b). This observation is in agreement with results from other studies (8,25,26) showing stroma-free Hb induces strong vasoconstriction. The influence of HbPs on arteriolar tone and its vasoreactivity to Ang II was then investigated and compared with the influence of APs produced by the same procedure as a control. Similar behavior of the vessels perfused with HbPs and APs was observed. During perfusion, a slight diameter reduction to approximately 90% of the initial size was observed Artif Organs, Vol. 38, No. 8, 2014

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FIG. 3. Microscopic images of microperfused afferent arterioles of glomeruli (a–c, mice) and lung (d, Wistar rat). (a) Two holding pipettes held the free end of an arteriole (left) and an attached glomerulus (right), respectively. An inner pipette inside of the left holding pipette was advanced into the lumen of the arteriole. The arteriole is perfused with particles. (b) Reaction of an afferent arteriole perfused with Hb solution after addition of 10-10 M Ang II; the arteriole is occluded. (c) Reaction of afferent arteriole after addition of 10-10 M Ang II after perfusion with HbPs; the diameter of the vessel is only slightly changed. (d) Fluorescence image of a perfused lung arteriole with a suspension of fluorescently labeled HbPs particles (particle concentration 25%) showing uniform distribution of the fluorescence inside the vessels.

for vessels perfused with HbPs as well as with APs. This was probably caused by reduced outcoming flow from the pipette into the arteriole due to particle sedimentation inside the pipette. Neither the tone of afferent arterioles nor their reactivity to Ang II was affected by HbPs in comparison with the APs (85.2 ± 18.9% and 78.2 ± 10.0% of the initial diameters for HbPs and APs, respectively at 10-10 M Ang II). Thus, the construction of HbPs diminished the NO scavenging effect of free Hb. As a second in vitro model, we perfused isolated lungs of rats with highly concentrated suspensions of fluorescently labeled HbPs and followed their distribution inside the lung arterioles by fluorescence microscopy. As can be seen in Fig. 3d, the fluorescence is uniformly distributed inside the vessels, suggesting that no occlusion by the HbPs occurs. Additionally, no significant changes of the HbPs concentration could be detected after 4-h perfusion as measured by flow cytometry. The behavior of HbPs in human blood was investigated in vitro with respect to the interaction of the particles with the blood cells. No aggregation of HbPs and no interaction between RBC and HbPs

were seen under the microscope (Fig. 4a). It can also be seen that the physiological ability of RBC to form rouleaux is not impaired by the presence of HbPs. Incubation of HbPs in platelet rich plasma for 45 min at 37°C did not change the platelet number in comparison with the control sample incubated with sterile physiological saline. Only 0.2% activated platelets were found, which is comparable with the control sample (15). The response of phagocytosing leukocytes was investigated using a standard phagocytosis kit for heparinized whole blood. The test was performed for 30 min at 37°C, and indicated a low phagocytosis rate of 3–5% of the total blood phagocytes (Table 1). In vivo tests were performed with Wistar rats and TRITC-BSA labeled HbPs (Fig. 4b) to follow their biodistribution and circulation. Approximately 20% of the estimated total blood volume of the animals was replaced by a suspension of HbPs in 0.9% NaCl-2% HSA with a volume concentration of approximately 15%. The HbPs were counted in blood samples taken immediately after injection and then after 4, 24, and 96 h by flow cytometry. Within 4 h, the number of detected HbPs in the blood

FIG. 4. In vitro and in vivo behavior of HbPs in blood. (a) FITC-BSA-labeled HbPs in whole human blood after incubation and shear stress in vitro. (b) TRITC-BSA-labeled HbPs. (c) TRITC-BSA-labeled HbPs in a blood sample of a Wistar rat taken 30 h after exchange of approximately 20% of the estimated blood volume with a suspension of HbPs.

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PROMISING NEW-GENERATION HBOCS decreased by 50%. However, particles were still detectable by flow cytometry and observable by CLSM after 30 h (Fig. 4c) and even 4 days after injection (16). Here, the HbPs were prepared on CaCO3 without any surface modification, but they were always suspended in physiological NaCl solution supplemented with 2% HSA. In this their surface is always “modified” by HSA that is known to adsorb easily on charged and noncharged surfaces. The long circulation time, together with the low phagocytosis activity, presumes the low immunogenicity of the particles. In further studies, we certainly intend to study deeply the immunological response and the production of antibodies to our novel HbPs. However, glutaraldehyde polymerized bovine Hb has been shown to provoke only a “low antigenicity” in clinical studies, although it differs from human Hb by as much as 17 amino acids in the R chains and 24 amino acids in the beta chains (8). Previous studies on Hemopure (also based on bovine Hb; former: Biopure Corp., Cambridge, MA, USA; now: OPK Biotech LLC, Cambridge, MA, USA) have shown that its administration elicits IgG antibodies in humans that react with bovine and human Hb, but do not appear to facilitate complementmediated hemolysis in vitro. IgE antibodies have not been detected in the serum samples of volunteers supporting the conclusion that the risk for Type 1 (IgE antibody-mediated) hypersensitivity reactions after repetitive administration is negligible (27). CONCLUSION In conclusion, we presented here hemoglobin particles as an enormously promising new type of hemoglobin-based oxygen carriers. The fabrication is based on a simple technique, which exploits the hemoglobin capture ability of insoluble inorganic salts like CaCO3 and MnCO3. The fabrication method can provide particles with a narrow size distribution in the submicron range and nearly uniform morphology. The HbPs have a high oxygen affinity, which is necessary to prevent a premature release of oxygen in the precapillary arterioles. Microperfusion experiments showed that concentrated HbP suspensions can easily pass through the arterioles of kidney and lung. Remarkably, afferent glomerular arterioles perfused with HbPs behaved similar to the control group perfused with albumin particles concerning arteriolar tone and reactivity to angiotensin II. In contrast, stroma-free Hb solutions significantly enhanced the vasoreactivity to Ang II. These findings, together with the positive results of preliminary in vivo experiments, are promising prerequisites of

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the new type HbPs as a candidate for a novel blood substitute. Acknowledgments: We cordially acknowledge Prof. A.R. Pries, Institute of Vegetative Physiology, Charité-Universitätsmedizin Berlin, for the support of this work with the lung perfusion experiments. We also acknowledge the financial support of the European Community (EFRE-ProFIT 10139827 and IRSES-612673 DINaMIT). REFERENCES 1. American Red Cross. Blood Facts and Statistics. Available at: http://www.redcrossblood.org/learn-about-blood/blood -facts-and-statistics. Accessed October 10, 2013. 2. Funk M, Günay S, Lohmann A, Witzenhausen C, Henseler O. Paul-Ehrlich-Institut Haemovigilance Report 1997-2008 Assessment of reports of serious adverse transfusion reactions. 2008. p. 1–26. 3. Whitaker BI, Schlumpf K, Schulman J, Green J. Report of the US Department of Health and Human Services. The 2009 National Blood Collection and Utilization Survey Report. Washington: US Department of Health and Human Services, Office of the Assistant Secretary for Health, 2011. 4. Jahr JS, Sadighi A, Doherty L, Li A, Kim HW. Hemoglobinbased oxygen carriers: history, limits, brief summary of the state of the art, including clinical trials. In: Mozzarelli A, Bettati S, eds. Chemistry and Biochemistry of Oxygen Therapeutics: From Transfusion to Artificial Blood. Chichester, UK: John Wiley & Sons, Ltd., 2011;301–16. 5. World Health Organization. Blood safety: Key global fact and figures in 2011. 2011; Available at: http://www.who.int/ bloodsafety/global_database/GDBS_Summary_Report_2011 .pdf. Accessed October 10, 2013. 6. Chang TMS. Blood substitutes based on nanobiotechnology. Trends Biotechnol 2006;24:372–7. 7. Chen J-Y, Scerbo M, Kramer G. A review of blood substitutes: examining the history, clinical trial results, and ethics of hemoglobin-based oxygen carriers. Clin (São Paulo) 2009;64: 803–13. 8. Riess JG. Oxygen carriers (“blood substitutes”) raison d’être, chemistry, and some physiology blut ist ein ganz besondrer saft. Chem Rev 2001;101:2797–920. 9. Winslow RM. Current status of blood substitute research: towards a new paradigm. J Intern Med 2003;253:508–17. 10. Ignarro LJ. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci 1987;84:9265–9. 11. Cabrales P, Sun G, Zhou Y, et al. Effects of the molecular mass of tense-state polymerized bovine hemoglobin on blood pressure and vasoconstriction. J Appl Physiol 2009;107:1548–58. 12. Sakai H, Hara H, Yuasa M, et al. Molecular dimensions of Hb-based O2 carriers determine constriction of resistance arteries and hypertension. Am J Physiol Circ Physiol 2000; 279:H908–15. 13. Champion JA, Walker A, Mitragotri S. Role of particle size in phagocytosis of polymeric microspheres. Pharm Res 2008; 25:1815–21. 14. Rudt S, Müller RH. In vitro phagocytosis assay of nano- and microparticles by chemiluminescence. III. Uptake of differently sized surface-modified particles, and its correlation to particle properties and in vivo distribution. Eur J Pharm Sci 1993;1:31–9. 15. Xiong Y, Liu ZZ, Georgieva R, et al. Nonvasoconstrictive hemoglobin particles as oxygen carriers. ACS Nano 2013;7: 7454–61. Artif Organs, Vol. 38, No. 8, 2014

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