FUTURE DEVELOPMENTS IN MODIFIED HEMOGLOBIN AS RED BLOOD CELL SUBSTITUTES
Thomas Ming Swi Chang,O.C.,M.D.,Ph.D.,F.R.C.P.(C) Director, Artificial Cells & Organs Research Centre. Professor of Physiology, Medicine & Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, Quebec, Canada, H3G 1Y6.
Introduction click
Modified Hemoglobin click (See also:Future Prospectives for Artificial Blood" from Trends in Biotechnology, February 1999, 17:61-67)
First Generation Modified Hemoglobin click
Second Generation Modified Hemoglobin click (see especially 2 recent papers in Nature Biotechnology volume 16, 1998: Chang's group 667-671 and Lemon's group 672-676 )
Third Generation Modified Hemoglobin click
Others including Fluorochemicals click
References click
Fluids for volume replacement have been in routine clinical use for many years. When fluid replacement alone is not enough, red blood cell or whole blood is the only next step at present. The present effort in the development of blood substitute is to produce a substitute for red blood cells. At one time there was some discussion that red blood cell substitutes had to be exactly the same as blood in efficacy and safety. Are we correct in making this assumption? Most of us who have been involved in the research and development of red blood cell substitutes look at the first generation of blood substitutes as an intermediate step between volume replacement and red blood cells. In other words, the first generation blood substitute is not prepared to have the same efficacy and safety as red blood cells because it is something in-between. Furthermore, there are also different types of first generation blood substitutes such that each may be best used for some specific applications. In this regard, we can learn much from the development of fluids used in volume replacement.
The first volume replacement fluid did not start with the same efficacy and safety as plasma. For nearly a hundred years we have saline which does not have even the same electrolyte composition as plasma. It is by far less effective or safe when compared to plasma. It was then improved to Ringer's lactate. Then it was further improved by adding colloid to Ringer's lactate for colloid osmotic pressure. However, it is very important to note that despite this type of increasing improvement, we do not discard saline or Ringer's lactate. Saline is still being used for volume replacement in dialysis and in other situations. Furthermore, one does not add colloids to Ringer's lactate all the time. Indeed, Ringer's lactate is used by itself most of the time and colloid is only needed in some special situations.
In the case of blood substitutes, we now have a number of first generation blood substitutes that are already in phase one, two and three clinical trials with some nearly ready for routine clinical use in patients. This promising result should propel us to further develop second and third generation products. However, as for fluid replacement systems, it does not mean that once these are developed, we have to discard the first generation products. Unless the condition demands it, there is no need to use a more expensive and more complicated second and third generation blood substitute, if the first generation blood substitute can be used. So it is a matter of what specific situation you want to use it for.
(Figure 1. Two major groups of modified hemoglobin). Hemoglobin in red blood cells is in tetrameric form. In addition, there is 2,3-DPG which allows the hemoglobin to release oxygen as required therefore having a good P50. Outside the red blood cell the hemoglobin breaks down into dimers and 2,3-DPG is not available for binding in the plasma. Accordingly, hemoglobin gets excreted very quickly and it does not release oxygen well. Modified hemoglobin is an attempt to solve these problems.
There are two main classes of modified hemoglobin. (1) Molecular modifications based on crosslinked polyhemoglobin, conjugated hemoglobin and tetrameric hemoglobin of the crosslinked or recombinant types. (2) Encapsulated hemoglobin based on lipid vesicles and the more recent use of nanotechnology for preparing biodegradable polymeric nanocapsules.
FIRST GENERATION BLOOD SUBSTITUTE
The first generation blood substitute is based on molecular modification. The first approach is to crosslink hemoglobin with a cross-linking reagent. The first crosslinker used is a diacid (Chang, 1964, 1965, 1966, 1972) and the next one is glutaraldehyde(Chang,1971, Payne,1973).There is also intramolecular cross-linking first using bis(N-maleimidomethyl)ether (Bunn & Jandl, 1968). There is also conjugated hemoglobin (Chang, 1964, 1965, 1966, 1972), especially the soluble type(Wong,1988, Iwashita, 1992, Shorr,1996).
Improvements in cross-linking reagents and modifying agents have been ongoing (Benesch et al 1975,Walden et al 1979, Estep et al 1992, Kluger, 1996, Bucci et al, 1996, Hsia et al, 1996). There is also recombinant human hemoglobin with possible intramolecular modifications (Shoemaker et al 1994, Fattor & Mathew, 1996, Olson,1996, Caspirin, 1996).
Potential concerns related to HIV in donor blood have stimulated a tremendous amount of research and development into blood substitutes in the last 10 years. A large number of dedicated scientists and developers have been involved in research into crosslink hemoglobin as the first generation blood substitutes. Examples include groups of Abuchowski, Agishi, Bakker, Benesch, Bonhard, Bund & Jandl, Biro, Bucci, Caspirin, Chang, DeVenuto, Estep, Faivre, Feola, Fratantoni, Gould, Greenburg, Hedlund,Hess, Hori, Hsia, Iwashita, Jesch, Jones, Keipert, Klugger, Magnin, Manning, McKenzie, Messmer, Moss, Muldoon, Nose, Olson, Pristoupil, Pluira, Segal, Sekiguchi, Sideman, Shoemaker, Shorr, Valeri, Walder, Winslow, Wong and many other groups. Each group has made important contributions to allow the first generation modified hemoglobin approach to be taken to the present stage.
The stage we are now in is very exciting. The present status of clinical trials using first generation blood substitutes has already been discussed in another article in this website. The first generation blood substitute now in clinical trial includes different types of blood substitutes. Each of these will most likely be best for certain specific applications. This progress has encouraged many to look forward to the second and third generation blood substitutes.
EXAMPLES OF FUTURE GENERATIONS OF BLOOD SUBSTITUTES
1. NITRIC OXIDE:
Blood vessels can be very schematically represented by the smooth muscle outside, endothelial cells surrounding the inside of the vessel, and finally the circulating blood inside the vessel. A number of mechanisms control the release of nitric oxide from the endothelial cells. Nitric oxide plays an important role in controlling the vascular tone. Lowering nitric oxide results in vasoconstriction. Increasing nitric oxide results in v asodilatation. Nitric oxide also acts on the nerve plexus and other sites in the body.
(Figure 2. Tetrameric hemoglobin removes nitric oxide). The intercellular junctions of the endothelial cell layer allow tetrameric hemoglobin to cross from the circulating blood. Since hemoglobin binds nitric oxide, these tetrameric hemoglobin on leaving the circulation act as a sink in removing nitric oxide resulting in vasoconstriction. There may be other effects including those on the nerve plexus. Blood substitutes prepared from polyhemoglobin, encapsulated hemoglobin and conjugated hemoglobin normally contains a small amount of tetrameric hemoglobin. This tetrameric hemoglobin can also cross the intercellular junction of the endothelial cells. One common way to avoid this is to remove as much tetrameric hemoglobin as possible from the preparation. Another way is to add pharmaceutical agents to counteract the effects. Another group is using recombinant technology to change one amino acid to block the nitric oxide adsorption site of the resulting recombinant hemoglobin. By doing this, the vasoconstriction effects have been markedly minimized (see recent paper in Nature Biotechnology 16:672-676,1998 by Lemon's group).
What happens if one uses polyhemoglobin, conjugated hemoglobin or encapsulated hemoglobin containing no tetrameric hemoglobin? Theoretically, there will not be a sink of tetrameric hemoglobin to remove nitric oxide and all the effects associated with this should be eliminated. However, nitric oxide can also enter the blood stream from the other side of the endothelial cells. How do hemoglobin blood substitutes behave towards this source of nitric oxide? Can one expect any effects when a large amount of blood substitute is infused? In the same way, how does hemoglobin in red blood cells behave towards this source of nitric oxide?
2. S-NITROSOTHIOLS
The present concept is that the major role of hemoglobin in red blood cells and hemoglobin blood substitutes is to transport oxygen from the lung to the tissues. However, a recent exciting finding (Jia et al, 1996, Stamler,1996) seems to suggest that hemoglobin in circulating red blood cells may also have an important role in the transport of nitric oxide (NO) and S-nitrosothiols (SNO).
Very briefly, this starts with three simple facts. (1) Oxyhemoglobin has a higher affinity for SNO. (2) Deoxyhemoglobin has a higher affinity for NO. (3) Lung is a good source of SNO. When hemoglobin takes up oxygen in the lung it becomes oxyhemoglobin that has a high affinity for SNO. It therefore also takes up SNO.
(Figure 4. Hemoglobin(Hb) transports S-nitrosothiols(SNO) and nitric oxide (NO)). This way, hemoglobin carries both oxygen and SNO in the arterial blood to the tissues. As oxyhemoglobin releases oxygen to the tissues, hemoglobin becomes deoxyhemoglobin. Since dexoyhemoglobin has a low affinity for SNO and a high affinity for NO, it releases SNO and picks up NO. This moves to the lung. Here, dexoyhemoglobin picks up oxygen to become oxyhemoglobin and releases NO in exchange for SNO. Therefore, the cycle continues.
It has been proposed that this SNO has an important role in the control of vasoactivity(Jia, 1996, Stamler,1996). If this theory is supported by other workers, it may have very important implications in the design of future generations of hemoglobin based blood substitutes. Thus, in addition to the transport of oxygen, we may also have to look into the transport of NO and SNO by modified hemoglobin blood substitutes.
3. REPERFUSION INJURY
Red blood cells contain catalase, superoxide dismutase and other enzymes. However, modified hemoglobin blood substitutes are prepared using ultrapure hemoglobin devoid of all enzyme systems. This is because of the need to remove all traces of endotoxin and other potential contiaminants in the preparation of first generation blood substitutes. There are many groups analyzing the effects of modified hemoglobin on ischemic reperfusions and on nitric oxide. Some examples include those from Alayash(1996), Kim et al (1996), McKenzie et al (1996), Pickelmann et al (1996), Muldoon et al (1996), Vercellotti (1996) and many others.
There are a number of examples where superoxide dismutase and catalase play an important role. The most common example is in reperfusion injury. Lack of oxygen supply from hemorrhagic shock or other causes of inadequate circulation or oxygenation results in ischemia. Ischemia stimulates the production of hypoxanthine. It also activates the enzyme xanthine oxidase. When the tissue is reperfused with oxygen, xanthine oxidase converts hypoxanthine into superoxide. By a number of mechanisms superoxide results in the formation of oxygen radicals. Oxygen radicals can cause tissue injury. (Figure 5. Mechanisms of tissue injury from reperfusion of ischemic tissues). Enzymes in red blood cell help to prevent this to some extent. Thus, superoxide dismutase converts some of the superoxide into hydrogen peroxide which is converted by catalase into water and oxygen. However, the present first generation modified ultrapure hemoglobin does not contain any of these enzymes. This may mean that there is potentially a higher chance for reperfusion injury when using blood substitutes prepared from ultrapure hemolgobin.
In preparing second generation modified hemoglobin, we may want to go a
step further to inlcude enzymes or other antioxidants in the blood substitutes. There are
a number of approaches to counteract this potential problem. Biro's group (Anthony et
al,1996) suggests that activated polyglutamate polymerized hemoglobin may act as a
scavenger of free iron. Hsia (1996) proposes the use of polynitroxylated hemoglobin with
antioxidant activity. Simoni et al (1996) prepared a "novel" hemoglobin based
blood substitutes by modification of the hemoglobin molecule for the same reason. Lemon et
al (1996) constructed recombinant hemoglobin to alter the intrinsic rate of reactivity of
hemoglobin for nitric oxide by mutagenesis of the distal heme pockets. We have been
studying the crosslinking of trace amounts of catalase and superoxide dismutase to
hemoglobin results(D'Agnillo & Chang,1993,1996 Quebec & Chang, 1995,1996, Razack
et al 1996)(See recent 1998 papers by
D'Agnillo & Chang (1) Nature Biotechnology 16:667-671, 1998 (2) Free
Radical Biology & Medicine, 24:906-912,1998)
In-vitro studies comparing polyHb-SOD-catalase to ultrapure polyhemoglobin shows the following results(D'Agnillo & Chang,1993, Quebec & Chang,1995,1996). PolyHb-SOD-catalase is much more effective in removing oxygen radicals and peroxides. It also stabilizes the crosslinked hemoglobin resulting in decrease oxidative iron and heme release. It also reduces the formation of methemoglobin during the preparation of polyhemoglobin. We have also carried out studies using ischemic reperfusion for both the intestine and the limbs (D'Agnillo & Chang,1996, Razack et al 1996) The result of the intestinal ischemic reperfusion study is shown below (Razack et al 1996). Cross-linked ultrapure polyhemoglobin causes the formation of oxygen radicals as measured by an increase in 3,4 dihydroxylbenzoate. This is significantly reduced when we used PolyHb-SOD-catalase for the reperfusion. Figure 6. Polyhemoglobin containing superoxide dismutase and catalase reduces the formation of harmful oxygen radicals in ischemic reperfusion.
ENCAPSULATED HEMOGLOBIN: A THIRD GENERATION MODIFIED HEMOGLOBIN BLOOD SUBSTITUTE?
Crosslinked hemoglobins as described above are simpler and therefore the first modified hemoglobins ready for clinical trials and eventually routine clinical use. However, crosslinked hemoglobin is only a partial substitute for red blood cells. Since hemoglobin is not covered, it has to be ultrapure to avoid adverse reactions. Artificial red blood cells formed by encapsulation of hemoglobin and enzymes are more like red blood cells. On the other hand, they are much more complex and therefore require more time to develop for clinical use.
1. MICROENCAPSULATION OF HEMOLGOBIN AND ENZYMES
The first encapsulation of the contents of red blood cell , including hemoglobin and enzymes, inside artificial red blood cells with artificial membrane was as early as 1957(Chang,1957). This has oxygen dissociation curve comparable to that of red blood cells. Further study was carried out using different membrane materials including crosslinked protein, bilayer lipid complxed to protein or polymer, polymeric membranes including silicone rubber and other (Chang, 1964, 1965, 1966, 1972).
Red blood cell enzymes like carbonic anhydrase (Chang, 1964) and catalase (Chang & Poznansky, 1968) in these microcapsules retained their activities. Encapsulated catalase was used successfully as an antioxidant against the toxic effects of hydrogen peroxide in experimental animals (Chang & Poznansky, 1968). These are acatalesemic mice with an inborn error of metabolism in their catalase enzyme. We replaced this enzyme deficiency in their red blood cells by hemoglobin artificial red blood cells containing catalase (Chang & Poznansky, 1968).
The single major problem is uptake by the reticuloendothelial system. We found for the first time that removal of sialic acid from red blood cell membrane resulted in the rapid removal of rbc from the circulation(Chang, 1965,1972). This observation led us to prepare 1-5 micron diameter artificial red blood cells with modifications of surface properties including negative surface charge (Chang, 1964, 1965, 1966, 1972) and the addition of polysaccharides to the membrane (Chang,1972). This improved the circulation time. However, the circulation time was still not enough for practical applications.
2. HEMOGLOBIN LIPID VESICLES
This is the next major step in the encapsulation of hemoglobin. Djordjevich & Miller (1980) prepared smaller lipid membrane artificial red blood cells of 0.2 micron diameter. These stay much longer in the circulation. Many groups especially those of Rudolph (1994, 1996) and Tsuchida (1994,1996) have been carrying out extensive research and development on bilayer lipid membrane artificial red blood cells. Others earlier groups included Beissinger, Farmer, Hunt, Schmidt, Szebeni, and many others.
Modification of surface properties including surface charge and the use of sialic acid analogues have further improved the circulation time. The average half-time in the circulation is now more than 24 hours. Most recent studies by Philips and Ruldolph has increased the half-time to more than 48 hours. It is possible to replace most of the red blood cells in rats with these artificial red blood cells. Some examples of more recent studies are as follows. Szebeni et al (1996) is studying the interaction with human complement using an in-vitro screening method similar to the one develped earlier by Chang et al(1994). Takaori & Fukui(1996) and Usuba, A, & Motokiwill(1996) used hemoglobin lipid vesicles for the treatment of massive hemorrhage. Other studies included the incorporation of enzymatic reduction system of methemoglobin (Ogata et al, 1996). Tsuchida's group is attempting to solve the problem of methemoglobin by the use of artificial reduction systems (Takeoka et al 1996). Large scale production is now feasible (Rudolph 1994,1996; Tsuchida, 1994,1996). Two groups, Rudolph in the U.S.A and Tsuchida in Japan have made rapid progress and extensive studies are being carried out by many groups using their preparations. Studies by a number of groups show that there are no adverse changes in the histology of brain, heart, kidneys and lungs of experimental animals. It is likely that clinical testings would be carried out in the near future.
3. BIODEGRADABLE POLYMER HEMOGLOBIN NANOCAPSULES
Just as success in crosslinked hemoglobin stimulates research into next generation crosslinked hemoglobin, this is also the case in encapsulated hemoglobin. This is perhaps the time for the next step toward a further generation of encapsulated hemoglobin.
For instance, one can look into how to improve even further the following: (i) increase stability in storage and after infusion. (ii) decrease the potential effects of lipid on the reticuloendothelial systems. (iii) avoid lipid peroxidation. (iv) solve the problem of methemoglobin formation.
We are using our background in biodegradable polymer encapsulation started here in 1976 (Chang, 1976). Polylactides and polyglycolides are degraded in the body into water and carbon dioxide. The rate of degradation can be adjusted by changes in molecular weight and type of polymer or copolymer. It can also varied with particle size. We are now using this to prepare biodegradable polymer membrane hemoglobin nanocapsules (Chang & Yu 1992, 1996, Yu & Chang 1994,1996) to have mean diameter of between 80 to 200 nanometres.
Unlike hemoglobin lipid vesicles, the membrane material is made up mostly of biodegradable polymer. Since polymer is stronger than lipid and is also porous, much less membrane material is required. This is shown in the graph in the next page. (Figure 7. Membrane materials of lipid vesicles and nanocapsules). Polylactide is degraded into lactic acid and then water and carbon dioxide. For a 500 ml suspension, the total lactic acid produced is 83 mEq. This is far less than the normal resting body lactic acid production (1000-1400 mEq/day). This is equivalent to 1% of the capacity of the body to breakdown lactic acid (7080 mEq/day).
(Figure 8. Hemoglobin content in rbc, lipid vesicles and nanocapules). Bovine hemoglobin after encapsulation has the same P50, Bohr and Hill coefficients Figure 9. Hemoglobin nanocapsules containing enzymes to reduce methemoglobin as before encapsulation.The content of hemoglobin can be made higher than hemoglobin lipid vesicles and matching that of red blood cells. Superoxide dismutase and catalase can also be included with the hemgolobin (Chang & Yu, 1996). Nanocapsules may improve on the problem related to methemoglobin reductase enclosed inside lipid vesicles. Since lipid vesicles are not permeable to glucose, the required glucose has to be added in high concentrations into the lipid vesicles. In the case of nanocapsules, the biodegradable polymeric membranes can be made permeable to glucose and other molecules. This allows us to prepare hemoglobin nanocapsules containing the methemoglobin reductase system to function as shown below. External glucose can diffuse into the nanocapsules. (Figure 9. Hemoglobin nanocapsules containing enzymes to reduce methemoglobin). Products of the reaction can diffuse out and therefore do not accumulation in the nanocapsules to inhibit the reaction. In vitro study shows promising results in the conversion of methemoglobin to hemoglobin. Animals have been infused with 1/3 the total blood volume. Studies are being carried out to further increase the circulating time of these hemoglobin nanocapsules.
This article is a discussion of modified hemoglobin red blood cell substitutes. However, there are a number of other promising approaches that should be mentioned.
PERFLUOROCHEMICALS
One important promising approach is the use of perfluorochemicals (Clark,1966). Sloviter & Kamimoto(1967) used this for organ perfusion and Geyer (1968) successfully replaced all blood in mice using fluorocarbon emulsions. This led to the development of fluorocarbons in Japan for clinical trials(Naito & Yokoyama, 1978; Mitsuno & Naito, 1979). There has been much progress since the early 1980's. For example, the use of suitable emulsifiers has solved the problem of complement activation(Reiss, 1994). Developments have also resulted in new and improved perfluorochemicals (Goodin et al 1994, Keipert & Conlan, 1996, Lowe et al, 1996, Lutz et al 1996, Meinert, 1996, Reiss, 1994, 1996,).
Further details are available in another article on this website. Phase II clinical trial is ongoing in imaging, cancer and to avoid donor blood transfusion for surgery and cardiopulmonary bypass (Keipert & Conlan, 1996). Extensive research is being carried out to further increase the oxygen carrying capacity. Improvements are also being made towards the possibility of increasing the total amount which can be infused. One of the advantages of fluorochemicals is in the less complicated and more easily control chemical approach (Chapman et al 1996).
OTHERS
Another future potential is the use of transgenic hemoglobin. Human hemoglobin has already been prepared from transgenic pigs (O'Donnell et al 1992). Stem cell culture technology to produce hemoglobin or human red blood cell with specific blood group is another possibility in the future.
ACKNOWLEDGEMENTS
TMSC acknowledges the grant support and career investigatorship from the Medical Research Council of Canada and the Virage Center of Excellence in Biotechnology from the Quebec Ministry of Education and Science. The grant support from the Bayer/Canadian Red Cross Society Research and Development Fund for the hemoglobin nanocapsule work is also gratefully acknowledged.
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Quebec EA & Chang TMS Superoxide dismutase and catalase crosslinked to polyhemoglobin reduces methemoglobin formation in-vitro. Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, 25: 693-706, 1995
Quebec EA & Chang TMS. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996.
Razack,S, D'Agnillo, F & Chang,TMS. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Vol 24, issue 4, 1996.
Reiss, J (guest editor) Blood Substitutes and Related Products: The Fluorocabon Approach, Artificial Cells, Blood substitutes & Immobilization Biotechnology, An International Journal 22:945-1511,1994.
Riess, JG & Krafft, MP. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Rudolph, AS, Sulpizio,T, Kwasiborski, V, Cliff, R, Rabinovici, R & Feuerstein, G. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Rudolph AS. Encapsulated hemolgobin: Current Issues and Future Goals. Artificial Cells, Blood Substitutes and Immobilization Biotechnology, An International Journal, 22,: 347-360, 1994.
Satzler, RK, Arfors, KE, Tuma R, Ma,L, Timble, CE, Hsia, CJC & Lehr, HA. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Sekiguchi, S. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Shoemaker S, Gerber M, Evans G, Paik L, Scoggin C. Initial Clinical Experience with a Rationally designed Genetically Engineered Recombinant Human Hemoglobin. Artificial Cells, Blood Substitutes and Immobilization Biotechnology, An International Journal, 22,: 457-465,1994.
Shorr, RGL, Viau, AT, Abuchowski, A. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Simoni, J, Simoni, G, Newman, G, Bartsell, A, & Feola, M. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Sloviter H, Kamimoto T. Erythrocyte substitute for perfusion of brain. Nature 216:458, 1967. Stamler,JS, Guest Speaker, VI International Symposium on Blood Substitutes, 1996
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Takaori M, & Fukui, A. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Takeoka, S, Sakai, H, Obgushi, T, Nishide, H, & Tsuchida,E. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Tsuchida E. Stabilized Hemoglobin Vesicles. Artificial Cells, Blood Substitutes and Immobilization Biotechnology, An International Journal, 22: 467-479,1994 Tsuchida, E. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Usuba, A, & Motoki, R. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Vercellottii, GM. Abstract volume for VI International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, Volume 24, issue 4, 1996
Walder JA, Zaugg RH, Walder RY, Steele JM & Klotz IM. Diaspirins that cross-link alpha chains of hemoglobin: Bis(3,5-dibromosalicyl) succinate and bis(3,5-dibormosalicyl)fumarate. Biochemistry 18: 4265-4270, 1979.
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Yu WP & Chang TMS Submicron Biodegradable Polymer Membrane Hemoglobin Nanocapsules as Potential Blood substitutes: A Preliminary Report. Artificial Cells, Blood Substitutes and Immobilization Biotechnology, An International Journal, 22: 889-894, 1994.
Yu WP & Chang TMS Submicron Biodegradable Polymer Membrane Hemoglobin Nanocapsules as Potential Blood substitutes: Preparation and characterization. Artificial Cells, Blood Substitutes & Immobilization Biotechnology, An International Journal, 24:169-184, 1996.