Review pubs.acs.org/Biomac
Synthetic Approaches to RBC Mimicry and Oxygen Carrier Systems Christa L. Modery-Pawlowski, Lewis L. Tian, Victor Pan, and Anirban Sen Gupta* Department of Biomedical Engineering, Case Western Reserve University, Cleveland Ohio 44106, United States
Reproduced with permission from Doshi et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 21495−9 and Merkel et al. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 586−91. Copyright 2009, 2011 National Academy of Sciences of the United States of America.
ABSTRACT: Whole blood or red blood cell (RBC) transfusions are highly significant, clinically, for blood replacement therapies in traumatic injuries, presurgical conditions, and anemias. However, natural RBC-based products suffer from limited shelf life due to pathological contamination and also present risks of refractoriness, graft-versus-host disease, immunosuppression, and acute lung injury. These issues can be only partially resolved by pathogen reduction technologies, serological blood testing, leukoreduction, and specialized storage; hence, they severely affect the efficacy and safety of the blood products. Consequently, there is a significant interest in synthetic RBC analogues that can mimic its oxygen-transport properties while allowing convenient manufacture, reproducibility, long shelf life, and reduced biological risks. To this end, the current Review provides a comprehensive description and discussion of the various research approaches and current state-of-the-art in synthetically mimicking RBC’s oxygen-carrying biochemical properties, as well as the biophysical parameters (shape, size and mechanical modulus) that influence RBCs’ hemodynamic transport properties in blood flow.
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INTRODUCTION
and reduced biological risks.14 Also, synthetic blood substitutes can reduce the need for blood typing, making all products available to any patient. To this end, a large volume of research has been carried out in the area of synthetic RBCs, platelet substitutes, and synthetic plasma expanders.5,15−24 In a recent review, we have described the various research approaches in platelet substitutes.25 Analogous to that, here we present a comprehensive review and analysis of the current state-of-theart products and research approaches in RBC mimicry and substitute oxygen carriers. We discuss the pros and cons of these approaches, emphasizing the biological and physicomechanical parameters of design. Synthetic Approaches in Mimicking the Biological Function of RBCs. The primary biological function of natural RBCs (Figure 1A) is to transport hemoglobin-bound oxygen and carbon dioxide to and from tissues, respectively. Hemoglobin is a tetramer of two α- and two β-polypeptide chains, each bound to an iron-containing heme group capable of binding one oxygen molecule (Figures 1B,C). The binding of oxygen by the protein is positively cooperative.26 As a result, small change in oxygen partial pressure (pO2) results in a large change in the amount of oxygen bound or released by hemoglobin due to the equilibrium principle (Figure 1D). The
Whole blood transfusions or isolated red blood cell (RBC) transfusions are clinically significant for blood replacement therapies in traumatic injuries (e.g., in accidents and battlefield wounds) and in presurgical settings (e.g., transplants), as well as for treatment of chronic and acute anemias.1,2 However, only about 40% of the U.S. population is eligible to donate blood, which is not sufficient for the high demand.3 In addition, natural blood-based products suffer from limited shelf life due to risks of pathological contamination. For example, RBC suspensions can be stored only for about 20−40 days .4,5 One reason for this limited storage is degeneration of cell membrane integrity, leading to the release of hemoglobin and 2,3-DPG, leaving the RBCs ineffective.6 Furthermore, these products present risks of refractoriness, graft-versus-host disease, transfusion-associated immunosuppression, and acute lung injury.7−10 These risks can be only partially reduced by current pathogen reduction technologies (e.g., psoralen-based or riboflavin-based UV irradiation techniques), extensive serological testing of donor blood, leukoreduction, and specialized storage protocols, but these result in high cost.11−13 Altogether, these issues severely affect the availability, efficacy, and safety of these blood components. Consequently, there is a significant clinical interest in synthetic analogues of blood components that can mimic and amplify the corresponding biological functions, while simultaneously providing advantages of convenient manufacture, consistent quality, long storage life, © 2013 American Chemical Society
Received: January 15, 2013 Revised: February 28, 2013 Published: March 4, 2013 939
dx.doi.org/10.1021/bm400074t | Biomacromolecules 2013, 14, 939−948
Biomacromolecules
Review
such as a reduced need for compatibility testing (minimum antigenicity), the ability to sterilize by ultrafiltration or low heat, and the ability to transport oxygen in plasma more efficiently compared to RBCs because of the lack of interference by a cell membrane.34 However, administration of unmodified cell-free hemoglobin was found to have unacceptably short circulation residence times because of tetramer dissociation and binding to plasma haptoglobin followed by reticulo-endothelial clearance and elimination by the spleen, liver, and kidneys.17,35−38 Additionally, cell-free hemoglobin can precipitate in the loop of Henle causing severe renal toxicity.39 Natural RBCs regulate their oxygen affinity through the action of 2,3-diphosphoglycerate (2,3-DPG), a molecule present inside the cells. The lack of 2,3-DPG in cell-free hemoglobin results in unfavorably high oxygen affinity, which affects oxygen release when needed. In addition, oxygen molecules in cell-free formulations come in direct contact with the vessel wall, yielding abnormally high oxygen delivery to surrounding tissues.40 Furthermore, cell-free hemoglobin results in reduced cardiac output from vasoconstriction by inhibiting endothelial cell relaxing factors.15,41 For example, cell-free hemoglobin can bind nitric oxide (NO), thereby causing vasoconstriction and leading to hypertension.42 Finally, cell-free hemoglobin can change the colloidal osmotic pressure of the blood, altering the blood volume.34 To resolve the plasma dissociation problem, research has been carried out to create chemical cross-links between the α and β chains, for example, with bis-(3,5-dibromosalicyl) fumarate.15 HemAssist (Baxter Healthcare Corporation, U.S.A.), a diaspirin-cross-linked hemoglobin, showed an increase in circulation time up to 12 h compared to 300 mmHg) to be effective physiologically.74 Also, PFC emulsions have other side-effects like mild thrombocytopenia and complement system activation.75 Resolving these issues and refinement of this technology is necessary to establish its clinical viability. The Fe2+ porphyrins have shown promising oxygen carrying capabilities, but little translational research has been carried out on this design for a synthetic RBC substitute. The foremost issue of these porphyrins is their irreversible oxidation in aqueous media, ultimately inactivating their oxygen carrying capabilities. The current research in the field is still in the chemical formulation stages, and its application to synthetic RBCs will be dependent on establishing the reproducibility of the formulations, as well as the biological safety of their in vivo performance. Another challenge in the design of synthetic RBC analogues and their clinical translation is their suboptimal pharmacokinetic profile and pro-inflammatory risks. These issues may be associated with the shape, size mechanical modulus parameters and surface charge of the synthetic designs. Recent reports from several groups have demonstrated the importance of these physicomechanical parameters toward influencing the biodistribution and function of blood cells.97,98 Future Perspectives. Semisynthetic and synthetic oxygen carriers continue to be a highly sought after technology in the clinic, to minimize the issues associated with allogeneic blood transfusions. Despite significant research in this area regarding RBC-inspired particle mimicry and Hb or PFC-based oxygen transport systems, an ideal oxygen carrier with sufficient clinical safety and efficacy is yet to be achieved. Future refinement of these approaches would require (i) significant advancement in recombinant technologies for producing Hb in bacteria costeffectively on a large scale, (ii) modification of PFC-based and Fe2+ porphyrin-based systems to ensure reproducibility of the formulations and their in vivo safety, and (iii) possible integration of particle technologies that allow mimicry of RBCs’ physicomechanical parameters with the biochemical functionalities of oxygen carrier molecules on a single synthetic platform. This merger of biophysical and biochemical parameters can lead to a superior design and optimum performance of synthetic oxygen carrier systems.
weight, and covalently secured the Hb to the particles via ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry.90 These Hb-loaded particles proved to be robust by retaining their size, shape, deformability, and loaded Hb when subjected to high shear in microfluidic devices. Li et al. have reported on liposome-encapsulated actinhemoglobin (LEAcHb) synthetic RBCs with similar shape properties as natural human RBCs, using a polymerized actin core.91 Although these particles were much smaller (∼136.8 nm) than RBCs, the biconcave shape along with the mechanical support of the membrane improved the half-life from ∼8 h to >72 h. Mimicking Surface Charge of RBCs. It has been well established that for both negatively and positively charged particles, the extent of phagocytosis increases with increasing absolute zeta potential values.92 A correlation between surface charge and opsonization has also been demonstrated in vitro, with neutrally charged particles showing much less opsonization than charged particles.93 However, the surface charge of natural RBCs in the body is negative. It is suggested that the negative surface charge of RBCs inhibits their aggregation by creating an electrostatic repulsion force over a distance of 20 nm.94 Therefore, there has been some interest in determining whether mimicking surface charge is important for design of synthetic RBC substitutes. To this end, Xu et al. have investigated varying the surface charge of hemoglobinencapsulating PEG−PLA−PEG nanoparticles (
