Artificial Cells, Blood Substitutes, and Biotechnology, 34: 551566, 2006 Copyright Q Informa Healthcare ISSN: 1073-1199 print/1532-4184

online DOI: 10.1080/10731190600973808

Evolution of Artificial Cells Using Nanobiotechnology of Hemoglobin Based RBC Blood Substitute as an Example
Thomas Ming Swi Chang
Faculty of Medicine, McGill University, Montreal, Quebec, Canada

Abstract: The original artificial red blood cells have evolved into oxygen carriers in the form of polyhemoglobin and conjugated hemoglobin. Clinical conditions requiring only oxygen carriers are responding well to these types of oxygen carriers without the need for a complete artificial red blood cell. For those conditions requiring more than just oxygen carriers, new generations of polyhemoglobin containing antioxidant enzymes are being developed. Though a complete artificial red blood cell comparable to red blood cell is still a dream, development in lipid membrane artificial red blood cells and biodegradable polymeric nano artificial red blood cells are steps towards this possibility. The many years of neglect on basic research in the area of blood substitutes have resulted in the lack of important basic knowledge needed for the rapid development of blood substitutes suitable for clinical use. This is further hampered by the mistaken conception that blood substitute is a single entity. We need to look at blood substitutes as consisting of progressively more complicated entities, e.g. oxygen carriers, oxygen carriers with antioxidant activity, and complete red blood cell substitutes. Each of these entities is not applicable to all clinical conditions, but is suitable for specific applications.
Keywords: Antioxidants; Artificial cells; Blood substitutes; Catalase; Hemoglobin; Nanobiotechnology; Nanoencapsulation; Oxygen radicals; Polyethylene glycol; Polyhemoglobin; Polylactide; Superoxide dismutase This author is the principle investigator of the following ongoing grant supports that he gratefully acknowledges: term grant of the Canadian Institutes of Health Research and the Research Group (dequipe) award on Blood Substitutes in Transfusion Medicine from the Hemovigillance and Transfusion Medicine Program of the Quebec Ministry of Health. Address correspondence to Thomas Ming Swi Chang, Faculty of Medicine, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6. E-mail: artcell.med@mcgill.ca 551

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INTRODUCTION The first artificial cells prepared in 1957 are in the form of artificial red blood cells [1,2]. They are of cellular dimensions, containing hemoglobin and red blood cell enzymes. Unlike red blood cells, the enclosing membrane is made of ultrathin artificial membrane. At that time there was no interest in blood substitutes; as a result, the principle of artificial cells has evolved into many other areas of application for the replacement of cell and organ functions [24]. The earliest routine clinical use of artificial cells is in the form of artificial cells containing adsorbents for hemoperfusion [5]. This has long been a routine method of treating accidental or suicidal poisoning, especially where dialysis is not readily available [5,6]. Other areas include artificial cells containing enzymes for enzyme therapy, islets for diabetes, hepatocytes for liver failure, delivery systems for medication and hormone and other areas [2,4,711]. Recent interest in biotechnology, molecular biology, nanotechnology, and regenerative medicine has stimulated even more extensive evolution and development of artificial cells into numerous other areas (Fig. 1) [9]. The evolution of artificial cells is a very large and complex area. As a result, only the evolution of artificial red blood cells into nanobiotechnology for hemoglobin based blood substitutes [12] will be used here as an example.

Figure 1. Present status of artificial cells: large variations possible in contents, membrane materials and dimensions.

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ARTIFICIAL RED BLOOD CELLS The first artificial red blood cell was prepared in 1957 consisting of hemoglobin and red blood cell enzymes enclosed in an ultrathin spherical artificial membrane of cellular dimensions [1] (Fig. 2). This solves many of the problems related to red blood cells (Fig. 1). However, after infusion they are rapidly removed from the circulation. It was found that the removal of sialic acid from red blood cell membrane resulted in the rapid removal of the rbc on reinfusion [3,4]. As a result, studies were carried out to modify the membrane properties [24]. We found that negative charge surface or polysaccharide linked surface significantly increased the circulation time, though this was still not enough. The next step was to prepare smaller nanobiotechnologybased artificial red blood cells as follows. Nanobiotechnology is the assembling of biological molecules into nanodimension structures (nano dimension in size, in membrane thickness or in diameter of tubular structures).

POLYHEMOGLOBIN In 1964, Chang used bifunctional agents, diacids, to crosslink hemoglobin molecules into polyhemoglobin membranes or very small 1 micron or submicron polyhemoglobin [2]. The reaction is as follows: Cl-CO-CH2 8 -CO-Cl HB-NH2 -HB-NH-CO-CH2 8 -CO-NH-HBSebacyl Chloride Hemoglobin Polyhemoglobin

In 1971, he used another bifunctional agent, glutaraldehyde, to crosslink hemoglobin into soluble polyhemoglobin [13]. The same study also shows that this stabilizes and prolongs the enzymatic activities of catalase crosslinked into the polyhemoglobin. The reaction is as follows: H-CO-CH2 3 -CO-H HB-NH2 -HB-NH-CO-CH2 3 -CO-NH-HBGlutaraldehyde Hemoglobin Polyhemoglobin

Glutaraldehyde crosslinked polyhemoglobin has evolved into the presentday polyhemoglobin blood substitutes. Two promising polyHb products have been developed independently by two groups, based on the above basic principle of glutaraldehyde crosslinked polyHb. One is pyridoxalated glutaraldehyde human polyHb [14,15]. This was developed for animal testing followed by clinical trials. As will be discussed later, it is important to mention that this preparation contains less than 1% tetrameric Hb. This product has been shown in

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Figure 2. Evolution of artificial red blood cell into nanobiotechnology of rbc substitutes.

Phase III clinical trials to successfully compensate for extensive blood loss in trauma surgery by maintaining the Hb level, with no reported side effects [15]. This group has infused up to 20 liters of polyHb into

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individual trauma surgery patients. They have published their clinical trials on 171 patients using polyHb in trauma surgery. In the U.S., this product has been approved for compassionate uses in patients. They have just completed a Phase III trial to study the pre-hospital use of this polyhemoglobin at the site of injury and in the ambulance. Since human red blood cells from outdated blood are in short supply, a glutaraldehyde-crosslinked bovine polyHb with less than 4% tetrameric Hb, has been developed and tested in animals and patients [16,17]. This bovine polyHb has been approved for routine clinical use in patients in South Africa, a region with higher incidence of human immunodeficiency virus [18]. In North America, this polyHb has been approved for compassionate uses in patients and for routine uses in veterinary medicine. Another way to solve the problem of short supply of outdated human red blood cells is the extraction of Hb from red blood cells of placentas that are discarded after birth. This is being used for the preparation of glutaraldehyde human polyHb [19].

CONJUGATED HEMOGLOBIN In 1964, Chang showed that when diamine is added to hemoglobin, diacids can conjugate hemoglobin to polymer to form conjugated hemoglobin [24]. Wong in 1968 extended this basic principle to form hemoglobindextan conjugates [20]. Davis group in 1970 [21] and Iwashita et al. in 1980 extended this further to form conjugate hemoglobin using polyethylene glycol. Winslows group [22,23] has prepared another conjugated hemoglobin consisting of hemoglobin conjugated with maleimidepolyethylene glycol. This will be described in more detail in another paper in this proceeding. This [23] and another PEG-Hb of Lius group in China [19] is in ongoing clinical trials.

INTRAMOLECULARLY CROSSLINKED TETRAMERIC HEMOGLOBIN In 1968, Bunn and Jandl crosslinked hemoglobin intramolecularly to form crosslinked tetrameric hemoglobin. In 1979, Walder et al. extended this into diaspirin crosslinked tetrameric hemoglobin that has been developed and tested in clinical trials [23]. Vasoactivity is one of the effects observed with the infusion of this blood substitute. Burhop and Estep [25] also reported small subendocardial lesions in animal studies.

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RECOMBINANT HEMOGLOBIN Human hemoglobin from red blood cells needed for preparing blood substitutes is not in plentiful supply. In 1990, Hoffman et al. were able to produce recombinant human Hb using genetically engineered Escherichia coli. In 1998, Doherty et al. prepared a new recombinant human Hb with no vasoactivity by mutagenesis of the distal heme pocket in order to change the reactivity of Hb for nitric oxide. Other ongoing approaches for recombinant Hb are being actively carried out [26,27]. The short supply of human red blood cells as source for human Hb is such that recombinant human Hb can be an important source of human Hb for use in the preparation of different types of Hb based blood substitutes.

WHY SOME TYPES OF BLOOD SUBSTITUTES INCREASE VASOACTIVITY AND OTHERS DO NOT Vasopressor effect has been observed in some of the hemoglobin based blood substitutes [23,24,2830]. We are gaining more basic knowledge of the reasons why some of the modified Hb solutions cause vasoconstriction and why there are variations between different types of modified Hb. The most commonly accepted theory is as follows. The intercellular junctions of the endothelial lining of vascular wall allow single tetrameric Hb to enter into the interstitial space. There, Hb acts as a sink in binding and removing nitric oxide needed for maintaining the normal tone of smooth muscles. This results in the constriction of the blood vessels and other smooth muscles, especially those of the esophagus and the GI tract. The evidence for this is as follows. The use of polyHb with <1% tetrameric Hb did not show vasopressor effects, even when large volumes of 10 units were infused [14,15]. With polyHb containing less than 4% tetrameric Hb, there were only slight vasopressor effects when very large volumes were used [16,17]. In the case of 100% intramolecularly crosslinked or recombinant tetrameric Hb, even smaller volume would cause vasopressor effects and increased smooth muscle contractions [24,25]. With another type of polyHb containing more than 30% tetrameric Hb, significant vasoactivity and increased smooth muscle contractions could be observed when using larger volumes. Further support comes from the observation that recombinant Hb no longer increases vasoactivity when modified to prevent it from binding nitric oxide [31]. Furthermore, Buccis group showed that a non-extravasating hemoglobin polymer did not have pressor effects [32]. Winslow and his collaborators showed that a high molecular radius maleimide-PEG conjugated Hb that does not cross the intercellular junction also does not have vasopressor

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Figure 3. One of the theories of vasopressor effects.

effects [33]. Thus, there is strong support for the hypothesis of the removal of nitric oxide caused by tetrameric Hb crossing the intercellular junction resulting in vasoconstriction. There is another school of thought that proposes that, unlike Hb inside red blood cells, modified Hb in solution releases oxygen prematurely at high concentration in the arterioles, resulting in vasoconstriction [23,33]. This is because the flow of red blood cells is laminar whereas modified Hb, being a solution, is in close contact with the vascular wall. Furthermore, for modified Hb in solution there is no diffusion barrier from a red blood cell membrane. It is likely that both hypotheses play some roles in vasoactivity but perhaps to different degrees and under different circumstances. Nevertheless, opponents to the above two hypotheses argue that one cannot compare different types of modified Hbs since there are major differences in the method of preparation, the chemistry involved, and the oxygen affinity. We therefore use the same glutaraldehyde method to prepare PolyHb, each containing different percentage of tetrameric Hb [34]. These were characterized to ensure that they all have the same oxygen affinity. Thus the preparations are prepared from the same chemical method and have the same oxygen affinity. Our analysis [34] shows that. There is no vasoactivity if the preparation contains less than 1% molecular hemoglobin. There is only minimal vasoactivity for preparation containing 4% molecular hemoglobin. Higher % of molecular hemoglobin results in

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significant increases in vasopressor effects. However, what is even more important is our finding that high percentage (78%) of tetrameric Hb causes marked changes in ECG after infusion [34]. Even the preparation with 38% of tetrameric Hb causes minor elevation of the ST segment; those with 16% of tetrameric Hb cause minimal elevation of the ST segment. Those with low percentage did not show any significant changes. The ECG changes could be related to vasoactivity and thus decrease in oxygen supply to the myocardium. This seems to support Burhop and Esteps [25] suggestion that nitric oxide removal could be the reason for the observation of small subendocardial lesions in some primates and swine after being infused with crosslinked tetrameric modified Hb. All the findings discussed above seem to show that the presence of significant amounts of tetrameric Hb is an important cause for vasoactivity. Thus, it is now generally accepted that it would be better to use polyHb or conjugated Hb of sufficient dimensions that contain little or no molecular tetrameric Hb. Thus Buccis group prepared large zerolink bovine polyHb of 25,000 kDa and showed that these did not have vasopressor effects [32]. Other groups crosslink recombinant Hb into recombinant polyHb [26]. As far as the second hypothesis [33] of the effects of premature release of high concentrations of oxygen is concerned, it will be useful to carry out studies using modified Hb with different P50 prepared using the same method. If this is confirmed, then instead of aiming for high P50 we may have to change our concept and to prepare modified Hb with a lower P50 to prevent the release of high concentrations of oxygen at the arterioles [23]. The total result will then be to prepare polyhemoglobin or conjugated Hb with low P50 and containing minimal tetrameric Hb molecules.

NEW GENERATIONS OF RED BLOOD CELL SUBSTITUTES The original artificial red blood cells [1,2] have evolved into oxygen carriers in the form of polyhemoglobin and conjugated hemoglobin. These are useful in clinical conditions requiring only oxygen carriers. For those conditions requiring more than just oxygen carriers, new generations of polyhemoglobin containing antioxidant enzymes are being developed. Development in lipid membrane artificial red blood cells and biodegradable polymeric nano artificial red blood cells are steps towards the possibility of complete artificial red blood cell substitutes. However, it will be expected that the new generations of blood substitutes will be more costly and more complicated to produce. Thus for those conditions that only need oxygen carriers, one may still want to use the simpler and less costly firstgeneration hemoglobin based rbc blood substitutes.

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PolyHb Crosslinked with Antioxidant Enzymes Even back in 1971, Chang reported the use of glutaraldehyde to crosslink hemoglobin with catalase to form a polyhemoglobin-catalase complex (Fig. 3) [13]. With the more recent need for antioxidant enzyme in blood substitutes, we have extended this to prepare a new generation of polyHb based on crosslinking polyHb with superoxide dismutase and catalase (PolyHb-SOD-CAT) [35]. This can transport oxygen and at the same time remove oxygen radicals so as to lessen the effects of ischemiareperfusion injuries. The intestine is one of the organs that is most likely to be injured by ischemia-reperfusion in sustained severe hemorrhagic shock. We found that PolyHb-SOD-CAT, unlike PolyHb, did not cause a significant increase in oxygen radicals when it was used to reperfuse rat intestine after 90 minutes of ischemia [36]. Another example is in the obstruction of arteries due to thrombosis or other causes that can result in stroke, myocardial infarction and other conditions. PolyHb is in solution and therefore more likely to be able to perfuse partially obstructed vessels compared to red blood cells. However, if arterial obstruction and lack of oxygen is prolonged, reperfusion with an oxygen carrier alone may also result in ischemia reperfusion injuries. Thus we found that in a rat stroke model, after more than 60 minutes of ischemia, reperfusion with polyHb resulted in significant increase in the breakdown of the bloodbrain barrier and an increase in brain oedema [37]. On the other hand, polyHb-SOD-CAT can supply oxygen without causing these adverse changes [37]. Hsia has reported another approach based on the use polynitroxylated cross-linked Hb [38].

PolyHb Crosslinked with Tyrosinase Malignant tumors are well vascularized but have abnormal microcirculation, resulting in under-perfusion by red blood cells and therefore lower tissue oxygen tension. Attempts have been made to decrease the blood supply even further by the use of agents that inhibit angiogenesis to prevent the growth of blood vessels to the tumor, but on the other hand, radiation therapy and chemotherapy work better with better tissue oxygen tension. As polyhemoglobin and conjugated hemoglobin are in solution, they can perfuse the abnormal microcirculation of tumors more effectively than red blood cells. Thus conjugated hemoglobin has been studied for supplying more oxygen for radiation therapy [21]. PolyHb has been shown to decrease the rate of growth and increase the lifespan in a rat model of gliosarcoma brain tumor when used as an adjunct for

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Figure 4. Polyhemoglobin crosslinked with enzymes to form polyhemoglobin enzymes like polyhemoglobin-superoxide dismutase-catalase (PolyHb-SOD-CAT).

Figure 5. Biodegradable polyethylene glycol-polylactide membrane nanodimension artificial red blood cells.

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chemotherapy [17]. We have recently crosslinked tyrosinase with Hb to form a soluble polyHb-tyrosinase complex [39]. This has the dual function of supplying the oxygen needed for optimal chemotherapy or radiation therapy and also lowering the systemic levels of tyrosine needed for the growth of melanoma. We have shown that intravenous injections of polyhemglobin-tyrosinase can delay the growth of the melanoma without causing adverse effects or changes in the growth of the treated animals [39].

Artificial Red Blood Cells The longterm dream is to prepare artificial red blood cells having the same properties as biological cells. Our earlier work shows that artificial red blood cells containing hemoglobin and rbc enzymes has an oxygendissociation curve comparable to that of red blood cells [1] and rbc enzymes such as carbonic anhydrase [2] and catalase [40] in these artificial red blood cells continue to function. For example, artificial cells containing Hb and catalase have been used to replace the antioxidant functions in mice with an inborn defect in red-blood-cell catalase [40]. The major problem was a short circulation time resulting from rapid uptake by the reticuloendothelial system [3,4]. As discussed earlier, one way of solving this is to keep decreasing the size by using bifunctional crosslinkers to form polyhemoglobin or conjugated hemoglobin. This approach has been evolved and extended by many groups into the present soluble polyhemoglobin and conjugated hemoglobin in ongoing clinical trials. Another extension is to retain the original principle of the membrane enclosed type of artificial red blood cells [1,2]. The results obtained by a number of groups are as follows:

Hb Lipid Vesicles In 1980, Djordjevich and Miller prepared small lipid-membrane artificial red blood cells approximately 0.2 mm in diameter and showed that these remain in the circulation for a longer period. Extensive research on bilayer-lipid membrane artificial red blood cells has been carried out by many groups and in particular by Rudolphs group [41,42] and Tsuchidas group [43,44]. Polyethylene(PEG)-lipid vesicles are especially effective in increasing the circulation time [42]. PEG-lipid Hb lipid vesicles have been developed and scale up for preclinical animal studies with promising results [43,44]. The potential shortage of Hb has led this group to study the incorporation of synthetic heme into lipid vesicles to avoid

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the need for using Hb [42,44]. They have also linked synthetic heme to recombinant albumin to form an oxygen carrier [43]. More details will be reported in another paper in this proceeding.

Biodegradable-Copolymer Membrane Nano Artificial Red Blood Cells Containing Hb and Red Blood Cell Enzymes Polylactide is degraded in the body into water and carbon dioxide and the rate of degradation can be adjusted by variations in molecular weight and use of ultrathin membrane. As a result, the rate of degradation can be adjusted to prevent accumulation in the reticuloendothelial system. Polylactic acid encapsulated Hb and enzymes have been prepared in the micron range [7]. More recently, we have prepared polylactide(PLA) Hb nanocapsules in the nanodimension of 80180 nanometer containing Hb and all the rbc enzyme system [45,46]. Superoxide dismutase catalase and metHb reductase can be included with the Hb. These artificial red blood cells containing the metHb reductase system can enzymatically convert metHb to Hb since nanocapsules are permeable to glucose and other small hydrophilic molecules. Reducing agents can also diffuse into the nanocapsules to convert metHb to Hb as has been demonstrated by in vitro studies [47]. In order to increase the circulation time, we have synthesized a number of new polyethylene glycol(PEG)-polylactide copolymers for the membrane of the nano artificial red blood cells [47]. One of these, Nanocap-17-1.5-15 K-XL, when used for the membrane of nanocapsules, can increase the circulation time of the nanodimension artificial red blood cells to nearly double that of polyhemogolobin [47]. This result can be extrapolated to a half time of about 41.5 hours in human. The PEG-PLA Hb nanocapsules are likely to have even higher circulation time in humans compared to PolyHb. This is because the reticulo-endothelial systems in rats are much more efficient than in humans in removing particulates like nanocapsules as compared to PolyHb solution. This is a significant and important increase that would allow longer function after infusion and thus decrease the need for donor blood.

IS THERE A FUTURE FOR BLOOD SUBSTITUTES? In all regions of the world, one of the important and urgent uses of blood substitutes is in an accident, civil unrest or war, where severe traumatic injuries require transfusion on the spot or in the ambulance. Here, donor

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blood cannot be used until after the patients have been admitted to the hospital and the delay could be fatal. In this regard, the ongoing Phase III clinical trial by Gould et al. using polyhemoglobin for onthespot and ambulance use is particularly important. Blood substitute is urgently needed in regions of the world where there is a severe shortage of donor blood because of cultural or religious belief that make it less likely for people to donate blood. There are also regions with higher incidence of infective agents like HIV and thus higher potential for the presence of infective agents in donated blood or insufficient infectionfree donor blood. Here, the use of blood substitutes to avoid the need for donor blood would be important and urgent. In regions where there is now minimal potential of infective agents in donor blood because of the availability of screening methods, blood substitutes still have a number of advantages over donor blood. What is even more important is that we should not forget the numerous patients infected by contaminated donor blood as the result of the outbreaks of HIV and hepatitis C that continued until proper screening tests were developed much later. Would anyone want to take the responsibility to assure the public that there will not be outbreaks of other infective agents and recurrence of contaminated donor blood infecting large numbers of the population? For example, not too long ago, the World Health Organization, government agencies and granting agencies were placing extensive resources and efforts to prepare for a possible avian flu epidemic. However, none seem to pay much attention to blood substitutes despite the fact that it will again be disastrous if blood substitute is not available. Even if screening tests are available, there will not be enough donors if a large proportion of the population is carrying the infective agent. This being the case, should anyone insist that blood substitutes must have all the functions of red blood cells before they can be used clinically? This is like saying that hemodialysis should not be used to maintain tens of thousands of renal failure patients alive until hemodialysis is identical to the biological kidneys! Or that hemoperfusion should not be continued to be used to save the many patients with suicidal or accidental overdose of medication or chemicals? As someone who has worked on artificial cells for 50 years, I can predict that red blood cell substitutes that have exactly the same functions of their biological counterpart are still a distant dream. On the other hand, it has evolved into blood substitutes like polyhemoglobin and conjugated hemoglobin that are useful as oxygen carriers. As such, these oxygen carriers have important roles for those clinical applications where only oxygen carriers are required. For other clinical applications that need more than just oxygen carriers, new generations of blood substitutes are being developed.

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34. Yu, B.L., Liu, Z.C., Chang, T.M.S. (2006). PolyHb with different percentage of tetrameric Hb and effects on vasoactivity and electrocardiogram. Artificial Cells, Blood Substitutes & Biotechnology 34: 159174. 35. DAgnillo, F., Chang, T.M.S. (1998). PolyHb-superoxide dismutase. Catalase as a blood substitute with antioxidant properties. Nature Biotechnology 16(7): 667671. 36. Chang, T.M.S., DAgnillo, F., Razack, S. (1998). A second generation Hb based blood substitute with antioxidant activities, in Blood Substitutes: Principles, Methods, Products and Clinical Trials, T.M.S. Chang, Ed., Karger: Basel, Vol. 2, pp. 178186. 37. Powanda, D., Chang, T.M.S. (2002). Cross-linked polyHb-superoxide dismutase-catalase supplies oxygen without causing blood brain barrier disruption or brain edema in a rat model of transient global brain ischemia-reperfusion. Artificial Cells, Blood Substitutes & Immob. Biotechnology 30: 2542. 38. Buehler, P.W., Mehendale, S., Wang, H., Xie, J., Ma, L., Trimble, C.E., Hsia, C.J., Gulati, A. (2000). Resuscitative effects of polynitroxylated alpha-alpha-cross-linked Hb following severe hemorrhage in the rat. Free Radic Biol Med. 29(8): 764774. 39. Yu, B.L., Chang, T.M.S. (2004). In vitro and in vivo effects of polyHb tyrosinase on murine B16F10 melanoma. Melanoma Res. 14: 197202. 40. Chang, T.M.S., Poznansky, M.J. (1968). Semipermeable microcapsules containing catalase for enzyme replacement in acatalsaemic mice. Nature 218(5138): 242245. 41. Rudolph, A.S., Rabinovici, R., Feuerstein, G.Z. (1997). Red Blood Cell Substitutes: Basic Principles and Clinical Applications, Marcel Dekker, Inc.: New York. 42. Philips, W.T., Klepper, R.W., Awasthi, V.D., Rudolph, A.S., Cliff, R., Kwasiborski, V.V., Goins, B.A. (1999). Polyethylene glyco-modified liposomeencapsulated Hb: a long circulating red cell substitute. J. Pharm. Exp. Therapeutics 288: 665670. 43. Tsuchida, E., Komatsu, T., Yanagimoto, T., Sakai, H. (2002). Preservation stability and in vivo administration of albumin-heme hydrid solution as an entirely synthetic oxygen-carrier. Polymer Adv. Technol. 13: 845850. 44. Kobayashi, K., Tsuchida, E., Horinouchi, H. (2005). Artificial Oxygen Carriers, Springer Verlag: New York. 45. Yu, W.P., Chang, T.M.S. (1996). Submicron polymer membrane Hb nanocapsules as potential blood substitutes: preparation and characterization. Artificial Cells, Blood Substitutes & Immobilization Biotechnology 24: 169184. 46. Chang, T.M.S., Yu, W.P. (1998). Nanoencapsulation of Hb and rbc enzymes based on nanotechnology and biodegradable polymer, in Blood Substitutes: Principles, Methods, Products and Clinical Trials, T.M.S. Chang, Ed., Karger: Basel, Vol. 2, pp. 216231. 47. Chang, T.M.S., Powanda, D., Yu, W.P. (2003). Ultrathin polyethyleneglycol-polylactide copolymer membrane nanocapsules containing polymerized Hb and enzymes as nano-dimension red blood cell substitutes. Artificial Cells, Blood Substitutes & Biotechnology 31(3): 231248.