CONTENTS:
Modified Hemoglobin: polyhemoglobin, recombinant Hb,conjugated Hb,encapsulated Hb.
References cited in this article
Selected recent publications on blood substitutes from this centre
Editorial: Is there a role for first generation blood substitutes in hemorrhagic shock?)
New year 2000 Editorials: Is there a need for blood substitutes in the new millennium and what should we expect in the way of safety and efficiency?
INTRODUCTION:
The 2 major approaches for artificial blood are: (i) Modified
hemoglobin and (ii) Perfluorochemicals. More centers and industries
are now carrying research and developments on the use of modified hemoglobin as red blood
cell substitutes. A cover story on Blood Substitutes
has recently appeared in the April, 2000 issue of the United Kingdom Journal, Chemistry
and Industry
1. General:
Research on modified hemoglobin was initiated by the author since 1957
(Chang 1957, 1964, 1965, 1966). However, concentrated research and development on modified
hemoglobin only started after 1987 because of public concerns of AIDS from donor blood.
Modified hemoglobin can be sterilized to remove microorganisms including those responsible
for AIDS, Hepatitis and others. In addition, there are many situations where modified
hemoglobin can be used to substitute red blood cells (5). This includes many types of
major surgery where a large amount of blood is needed. It is also particularly useful in
severe traumatic injuries in traffic accidents and other accidents that result in severe
bleeding. The number for this could be very large in major disasters like earthquakes or
wars (6) . Modified hemoglobin is especially useful in these emergency situations.
Modified hemoglobin does not contain blood group antigens, therefore it can be used
without the need for crossmatching or typing. This would saves much time and facilities
and would permit on the spot transfusion as required, similar to giving intravenous salt
solution. Furthermore, modified hemoglobin can be lyophilized and stored as a stable dried
powder which can be reconstituted with the appropriate salt solution just before use.
2. What are modified hemoglobin ?
Hemoglobin molecules extracted from red blood cells are modified by
microencapsulation or crosslinkage. This stabilizes the hemoglobin molecules and also
allows the sterilization of the products to remove H.I.V. and other microorganisms. Rapid
progress has been made in the last 7 years towards clinical use. A number of
companies working in this area are now carrying out clinical trials in human. Some have
progress to different stages of Phase III clinical trial (see clinical trial). The
following is a more detailed description.
3. Why do we have to modify hemoglobin ?
Hemoglobin is the oxygen carrying protein of red blood cells. Hemoglobin
molecules inside red blood cells are in the form of a tetramers (Fig. 1). Red blood cell
membrane retains the cofactor 2,3-DPG which is needed by hemoglobin for it to readily
release oxygen as required - high P50. Hemoglobin can be extracted from red blood cells by
removing the cell membranes to form stroma-free hemoglobin(7). However, this cannot be
used as blood substitutes. When infused into the circulation each hemoglobin molecule of 4
subunits (tetramer) is rapidly broken down into 2 subunits (dimers) (Fig. 2). The smaller
dimers are rapidly excreted by the kidneys. This also has renal toxicity. Furthermore,
without the required cofactor (2,3-DPG) when outside the red blood cells, hemoglobin
cannot readily release oxygen as required (Fig. 2). Two biotechnological approaches are
used to modify hemoglobin to prevent these problems.
4. Encapsulated hemoglobin: artificial red blood
cells.
The author first reported the preparation of artificial red blood
cells(Chang 1957, 1964)(Fig.3). These have P50 and oxygen dissociation curve similar to
red blood cells, since 2-3-DPG is reatainedn inside (1)(Fig.3). Hemoglobin also stays
inside as tetramers (Fig 3). These artificial red blood cells do not have blood group
antigens on the membrane and therefore do no aggregation in the presence of blood group
antibodies (4). However, the single major problem is the rapid removal of these artificial
cells from the circulation. Much of the studies since that time are to improve survival in
the circulation by decreasing uptake by the reticuloendothelial system. Preparation of
smaller submicron lipid membrane artificial red blood cells resulted in improvements in
circulation time(12). Microencapsulated hemoglobin or artificial red blood cells are now
being extensively explored by many researchers around the world. These recent advances
make possible the following results. The average half-time in the circulation is now up to
20 hours. The uptake is mainly by the reticuloendothelial system. It is possible to
replace 90% of the red blood cells in rats with these artificial red blood cells. The
animals with this percentage of exchange transfusion still remain viable. Studies also
reported effectiveness in hemorrhagic shock. Preliminary study by a number of groups shows
that they are not toxic. There are no changes in the histology of brain, heart, kidneys
and lungs of rats. To further improve stability and biodegradability, we are now using
biodegradable polymer membrane to prepare artificial red blood cells of less than 200
nanometre (less than 0.2 micron) diameter (16).
Microencapsulation of hemoglobin to prepare artificial red blood cell is a rather ambitious approach (1). Although this attempt to mimic red blood cells has resulted in a complete red blood cell substitute, it is rather complicated. Further research is needed and this approach is now considered as a second generation modified hemoglobin. A simpler crosslinked modified hemoglobin has been developed as a first generation modified hemoglobin for more immediate clinical applications.
5. Crosslinked hemolgobin
(i) Polyhemoglobin: Hemoglobin
contains many amino groups most of which are on the surface of the hemoglobin molecule.
The author first reported the use of a bifunctional agent (diacid) to crosslink hemoglobin
(Chang 1964,1965). This was used first to form cross-linked hemoglobin membranes for
artificial red blood cells, but it was found that with decreasing size of artificial cells
all the hemoglobin molecules are crosslinked into polyhemoglobin
(Chang 1964,1965). The reaction is as followed:
| Cl-CO-(CH2)8-CO-Cl | + | HB-NH2 | = | HB-NH-CO-(CH2)8-CO-NH-HB |
| Diacid | Hemoglobin | Crosslinked hemoglobin (Chang 1964) |
Extensive studies have been carried out by many groups especially in the last 7 years on crosslinked hemoglobin.(8-11)(Fig.4). Crosslinking prevents the breakdown of hemoglobin tetramers into dimers (2,3). Smaller polyhemoglobin consisting of 4 to 5 hemoglobin molecules stays longer in the circulation . The addition of a 2,3-DPG analogue, pyridoxal phosphate, to crosslinked hemoglobin improves the P50 (18). This approach have been developed (19-22) so that it is the first crosslinked hemoglobin used in clinical trial (22). Phase III clinical trials by the Northfield group
(Update: Phase III completed and filed for F.D.A. approval) and the Biopure group (Update: Approved for routine use in patients in South Africa) show that up to 10,000ml can be infused with no reported adverse effect. It also replaces the lost red blood cells. The crosslinking reaction used by these two groups is based on a bifunctional crosslinking agent, glutaraldehyde (Chang 1971), very similar to the crosslinkage reported earlier (Chang 1964, 1965) in the above equation:| H-CO-(CH2)3-CO-H | + | HB-NH2 | = | HB-NH-CO-(CH2)3-CO-NH-HB |
| glutaraldehyde | hemoglobin | Crosslinked Hemoglobin (Chang 1971) |
At present, other crosslinkers are also being developed (Table I). These are designed to have the dual function of a bifunctional crosslinker which also acts as a 2,3-DPG analogue. Some of these are based on bifunctional dialdehydes derived from oxidizing the ring structures of sugars or nucleotides.One approach in clinical trial involves the use of a dialdehyde prepared from oxidizing a sugar molecule to form ring-opened raffinose, o-raffinose (23). O- raffinose polymerized hemoglobin has good P50 without the need for additional 2,3-DPG analogue. This group is starting their Phase III clinical trial.
(Update: Phase III in coronary surgery requiring about 1,000 ml has been completed in Canada and the United Kingdom and awaiting regulatory agencies approval)(ii) Intramolecularly crosslinked hemoglobin. The crosslinkers described above can be used for both intermolecular and intramolecular crosslinkage. Studies have been carried out to specifically crosslink hemoglobin molecules intramolecularly(17, 24). A bifunctional agent, 2-Nor-2-formylpyridoxal 5-phosphate which is also a 2,3-DPG analogue can intramolecularly crosslink the 2 beta subunits of the hemoglobin molecules (18). Another 2,3-DPG pocket modifier, bis(3,5-dibromosalicyl) fumarate (DBBF) intramolecularly crosslinks the 2 alpha subunits of the hemoglobin molecule (25). This prevents dimer formation and also improves P50 . Baxter has not continue clinical trial on this intramolecularly crosslinked hemoglobin and is now working on a second generation recombinant hemoglobin. Many other bifunctional 2,3-DPG pocket modifiers are being studied (5-11).
(iii) Conjugated hemoglobin is the crosslinking of hemoglobin to polymers (2-4) . The use of soluble polymers resulted in soluble conjugated hemoglobin with good circulation time(28-30). These are now in clinical trial.
(iv) Recombinant hemoglobin: This is based on genetic engineering of E.coli to result in the prodcution of hemoglobin hemoglobin with good P50 and that retains its tetrameric configuration after infusion. They have successfully prepared a second generation recombinant hemoglobin in which the receptor site for nitric oxide has been blocked. This has resulted in a preparation that does not cause vasoconstriction when infused into experimental animals. (See Lemon's group in Nature Biotechnology, July 1998 issue)
Circulation time of modified hemoglobin:
Removal of polyhemoglobin and conjugated hemoglobin after infusion is mainly by the reticuloendothelial system. The half time of polyhemoglobin in the circulation is about 25 to 30 hours. Conjugated hemoglobin stay in the circulation even longer. Intramolecularly crosslinked hemoglobin escapes more rapidly from the circulation and therefore has a shorter circulation time. Survival time in the circulation depends on the dose and the animal species. This time is much shorter than that of red blood cells. However,this is enough for most of the shorter term uses described earlier. For example, all three types of crosslinked hemoglobin are effective in animal studies of hemorrhagic shock and isovolemic exchange transfusions (8-11)
6. Present status
Crosslinked hemoglobin is likely to be the first modified hemoglobin
ready for routine clinical use. Initial problems which have now been mostly solved
were related to potential toxicity and the problems that animal safety studies do not
reflect exactly human response. Most of these problems have now been solved by extensive
basic studies on hemoglobin (8-11,31-34). In addition, an in-vitro screening test was
developed to bridge the gap between animal safety studies and human response (34). This is
based on testing the effect of adding 0.1 ml of modified hemoglobin to 0.4 ml of human
plasma in a test tube, then analysing C3a to see if there is any complement
activation(34). Earlier results of clinical trials have been described (35-38). The
most recent clinical trial update is in the next section.
Northfield has reported in the August 1998 issue of the Journal of the American College of Surgeons (J. Am Coll Surg 187:113-122, 1998) the result of their prospective, randomized trial to compare directly the therapeutic benefit of PolyHeme with that of allogeneic red blood cells in the treatment of acute blood loss. "The First Randomized Trial of Human Polymerized Hemoglobin as a Blood substitute in Acute Trauma and Emergent Surgery". In the study, 44 trauma surgery patients were randomized to receive either the blood subsitute, PolyHeme, or donated red blood cells to replace the blood they lost. The blood substitutes were able to maintain total hemoglobin concentration and also reduced by nearly half the amount of donated blood the patients needed. In their recent report at the 1999 June ASAIO panel and 1999 September FDA workshop, they are now well into Phase III clinical trial having infused up to 10,000ml with no reported side reactions and with efficacy shown. Northfield has recently files for FDA approval August 2001 (http://www.northfieldlabs.com/polyheme.htm)
Biopure is well into their Phase III clinical trial in human. They have reported in a UK meeting (Jan. 1999) that they have infused large volumes of their blood substitutes including repeated infusion in their clinical trials. Their recent report at the 1999 June ASAIO panel and 1999 Sept FDA Workshop, shows that they have infused up to 10,000ml into patients with no side effects reported and with efficacy. Their vetrenary polyhemoglobin product has been approved by the F.D.A. (U.S.) for routine use in vetrenary medicine for cannine anemia. In a recent publication (J Vasc Surg 2000;31:299-308), LaMuraglia et al reported the use of this HBOC-201 to reduce the allogenic transfusion requirement in aortic surgery in a single blind, multicentrer study involving 72 patients. In a more recent article (New England J Medicine 2000;342:No 22 June 1) Mullon et al reported "Trasfusions of Polymerized Bovine Hemoglobin in a patient with Severe Autoimmune Hemolytic Anemia". There is an editorial in the same issue by H.G.Klein (New England J Medicine 2000;342:No 22 June 1) on "The Prospects for Red-Cell Substitutes" The Biopure Product has been approved for use in human in South Africa for acute anemia in surgery patients around April 2001. Biopure has recently (27 August 2001) described the safey and efficacy results of their pivotal Phase III clinical trial results (http://www.biopure.com)
Hemosol is using its o-raffinose human polyhemoglobin for coronary artery bypass grafting surgery (CABG) and has (March 2000)announced that they have completed their Phase III trials for CABG in Canada and the U.K. and waiting for regulatory approval for routine clinical uses. Their recent (June 2000) analysis of the results confirmed that it was safe in cardiac surgery patients with no clinically limiting side-effects, analysis of clinical efficacy is ongoing. They have also completed Phase II clinical trial on orthopedic surgery and on anemia.
Enzon has reported earlier on their phase II clinical trial using PEG conjugated hemoglobin for sensitizing tumour in cancer treatment
Apex Bioscience (Ajinomoto) is in clinical trial using PHP conjugated hemoglobin for scavenging nitric oxide in septic shock patients and in other applications.
Baxter has not continued with their clinical trials on intramolecularly crosslinked hemoglobin in human. Somatogen has jointed Baxter to develop a new generations of recombinant hemoglobin. They have already successfully prepared a second generation recombinant hemoglobin in which the receptor site for nitric oxide has been blocked. This has resulted in a preparation that does not cause vasoconstriction when infused into experimental animals. (See Nature Biotechnology, July 1998 issue)
7. Is there a role for first generation blood substitutes in hemorrhagic shock?
First generation blood substitutes are prepared mostly from ultrapure hemgolobin containing no red blood cell antioxidant enzymes like superoxide dismutase and catalase. Thus in the use or design of clinical trial for the resuscitation of severe hemorrhagic shock it will be extremely important to take the this into consideration(65). If the resuscitation takes place almost immediately after hemorrhagic shock, then there may not be too much of a problem with ischemia-reperfusion injury. However, if the resuscitation takes place after sustained period of hemorrhagic shock, there may be sufficient sustained ischemia so that resuscitation with the first generation blood substitutes may result in ishemic-reperfusion injuries and causing more harm than without such resuscitation(65). This would also apply to other ischemic conditions including reperfusion in stroke and other situations. In these cases, it would be important to use 2nd and 3rd generation blood substitutes containing antioxidant enzymes(60,61). See editorial on this question
8. Future Development
With the first generation modified hemoglobin blood substitutes being tested in human, studies have already started on further refinements of crosslinked hemoglobin. We are studyging the incorporation of superoxide dismutase and catalase into crosslinked hemoglobin to prevent reperfusion injury due to oxygen radicals (39). The third generation modified hemoglobin which is a more complete red blood cell substitute is also being extensively developed. This is the use of microencapsulated hemglobin or artificial red blood cells (1-4, 8- 16). These are more like red blood cells since hemoglobin is not exposed to the outside environment. Furthermore, multienzyme system can also be enclosed. This includes the entrapment of catalase and superoxide dismutase with hemoglobin. The approach of microencapsulation of multienzyme systems with cofactor recycling (40) is also being used to prevent methemoglobin formation ((11). This brief overview cannot include the numerous ongoing studies and research in this area. All those interested in more details can refer to the many detailed publications, books and symposium volumes in this area (8-11), including an article on this website (click here to access this article). Published articles from here included:
CHANG TMS (1999) "Future Prospectives for Artificial Blood" from Trends in Biotechnology, February , 17:61-67.\
CHANG TMS (2000) " Red Blood Cell
Substitutes"
1. General:
Of the synthetic organic material, silicone and fluorocarbon are known for
their ability to carry oxygen. Thus in the 1960's Clark and Gollan (41) demonstrated that
mice immersed in oxygenated silicone oil or liquid fluorocarbon could breathe in the
liquid. In the same year Chang (42) demonstrated that artificial cells formed from a
hybrid of silicone rubber and hemolysate were very efficient in carrying and releasing
oxygen. However, these solid elastic silicone rubber artificial cells did not survive
sufficiently in the circulation. Sloviter and Kamimoto (43) demonstrated that perfusion
using finely emulsified fluorocarbon could maintain rat brain function for several hours.
Geyer, Monroe and Taylor (44) demonstrated that finely emulsified fluorocarbon could
replace essentially all the blood of rats with the rats surviving and recovering. This
exciting demonstration did not immediately lead to clinical application because
F-Tributylamine available at that time had a long retention time (T11/2 of more
than 800 days) in the RES and therefore could not be used clinically. Extensive
development was carried out in Japan by Naito, Yokoyama(45) resulting in the development
in 1976 of fluosol-DA 20 suitable for clinical testing.
(1) Perfluorocarbons : fluosol-da(20%)
Fluosol-DA is a 20% (w/v) mixture of 7 parts of perfluorodecalin and 3
parts perfluorotripropylamine, with 2.7% pluronic F-68 as an emulsifier and 0.4% of egg
yolk phospholipids to form membrane coating on the emulsion (45-48). The average particle
size of the emulsion is 0.118 um. Unlike the earlier fluorocarbon which has a tissue
retention T1/2 of more than 800 days, perfluorodecalin has a T1/2 of
7.2 days. Unfortunately perfluorodecalin cannot be used to form stable emulsion and
perfluorotripropylamine with a T1/2 of 64.7 days has to be combined to form the
stable emulsion. The much shorter retention time of the fluosol-DA 20 allows its use for
clinical trial and testing. Because of the viscosity of the fluorocarbon emulsion at high
concentrations, the maximum amount used is only 20%. Because of this smaller amount of
fluorocarbon used and also because oxygen can only be dissolved in fluorocarbon and there
is no binding function like hemoglobin, sufficiently oxygen carriage can only take place
when the patients are breathing 70 to 90% oxygen. CO2 also dissolves in the
fluorocarbon and is transported to the lung for excretion. Other problems includes their
rapid removal from the circulation and retention of fluorocarbon in the
reticuloendothelial system (RES) resulting in RES suppression. This will potentially
result in lowered resistance to infection. In addition, side effects were observed in some
patients due to complement activation caused by the Pluronic surfactant used in fluosol.
Infusion of one ml test dose of fluosol produced an immediate transient and small drop in
neutrophil and platelets in some patients. Fluosol-DA has to be stored in a frozen state.
(2) New perfluorochemicals
Two new types of preparations have been developed (50-55). One type is
based on perfluoroctyl bromide (C8F17Br) and perfluorodichoroctane
(C8F16Cl2). Both types allows the use of higher
concentrations of PFC. Oxygent™ (Alliance Pharmaceutical Corp., San Diego)
is prepared from perfluoroctyl bromide (C8F17Br) with egg yolk
lecithin as the surfactant. The use of egg yolk lecithin instead of Pluronic surfactant
has solved the problem of complement activation( Reiss 1991). Another approach,
Oxyfluor (HemoGen, St. Louis) is based on the use of perfluoro-dichoroctane (C8F16Cl2)
with triglyceride and egg yolk lecithin (Goodin et al 1994). The observation of
side effects when the dose is about 1.8 g PFC/ kg means that at least at present, the use
of the new improved preparations of PFC-based blood substitutes is limited to a lower
dosage. Oxygent are being used in Phase II clinical trials (Wahr et al 1994) in
surgical patients breathing 100% oxygen. The use of 0.9g/kg of oxygent appears to be able
to avoid need for the use of 1-2 units of blood. The present emphasis is therefore to
study the use of PFC in surgery to offset the need for this amount of blood during
surgery. This is to be combined with autologous blood predeposition and reinfusion after
surgery.
(3) Other Potential Areas of Applications.
There are a number of other potential applications for perfluorochemicals
(44, 46, 53, 54). At present, these will be limited to the lower dosage level as
described above. Thus in thrombosis or embolism, the small PFC particles and the increased
oxygen pressure may help the affected tissue. Use in patients who because of religious
belief cannot use human blood cells is an important and obvious area. Other applications
not related to its use as blood substitutes are not within the scope of the present
discussion.
(4) Present status & future perspectives of
perfluorochemicals
The biggest advantage of perfluorochemicals is that it they are synthetic
material that can be chemically produced in large amounts without having to depend on
donor blood or other biological sources. Much has been done in the last 10 years to
improve this approach. The earlier problem of complement activation has been solved by
changing the surfactant. Higher concentrations of the new perfluorochemicals can now be
used to increase oxygen carrying capacity. At present this is limited by the rather low
dosage of 0.9g/Kg for human use. This low dosage is partly because of side effects
observed in humans at dosage of 1.8g/Kg. Here , the patients still have to breathe a 100%
oxygen. With further research and development, the problem related to side effects at
higher dosage is likely to be resolved. If this can be resolved then the highest dosage
will only be limited by the dosage which would not cause significant suppression of the
reticuloendothelial system. In this regard, ever improving perfluorochemicals with
decreasing residual time in the reticuloendothelial system are being made available. It is
likely that further improvements in perfluorochemicals may also lead to further
improvements in oxygen carriage thus further reducing the level of oxygen required for
breathing.
III. FUTURE DEVELOPMENTS OF
BLOOD SUBSITUTES - click to access article
REFERENCES
References cited in the above article Click
References on Blood Substitutes by Chang et al (1957- present) Click
Selected recent publications on blood substitutes from Chang's Group Click
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Boston, 1985, pp 169-184. Blood substitute
48 Ohyanagi H, Toshima K, Sekita M, Okamoto M, Itoh T, Mitsuno T, Naito R, Suyama T,
Yokoyama K. Clinical study of perfluorochemical whole blood substitute. Clinical
Therapeutics 2:306, 1979.
49 Faithfull, NS Oxtgen delivery from fluorocarbon emulsions - aspects of convective and
diffusive transport.J. Biomaterials, Artificial Cells and Artificial Organs. 20:797-804,
1992
50 Goodin T. Results of a Phase I clinical trial of a 40 v/v% emulsion of HM351
(OxyfluorTM) in healthy volunteers. 2nd Annual IBC conference on Blood substitutes &
Related Products. Whasington DC (1994)
51 Goodin TH, Grossbard EB, Kaufman RJ, Richard TJ, Kolata RJ, Allen JS, Layton TE.
A Perfluorochemical emulsion for prehospital resuscitation of experimental hemorrhagic
shock: a prospective, randomized controlled study. Crit Care Med 22:680-689(1994)
52 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.
53 Riess JG Fluorocabron-based in vivo oxygen transport and delivery systems. Vox Sang
61:225-239, 1991.
54 Wahr JA, Trouwborst, Spence RK et al. A pilot study of the efficay of an oxtgen
carrying emulsion OxtgentTM, in patients undergoing surgical blood loss. Anesthesiology
80:A397, 1994.
56 Chang TMS Monograph Blood substitutes: principales, methods,products and clinical
trials. Vol I (monograph) Vol II (editor), Karger-Landes System, Basel & Austin.
57. F. D'AGNILLO & TMS CHANG (1997) Production of hydroxyl radical generation in a rat hindlimb model of ischemia-reperfusion injury using crosslinked hemoglobin-superoxide dismutase-catalase. Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an internationaljournal 25:163-180
58. S. RAZACK, F. D'AGNILLO & TMS CHANG (1997 )Crosslinked hemoglobin-superoxide dismutase-catalase scavenges free radicals in a rat model of intestinal ischemia-reperfusion injury. Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an international journal 25: 181-1924.
59. T.M.S. CHANG(1997) Recent And Future Developments in Modified Hemoglobin and Microencapsulated Hemoglobin as Red Blood Cell Substitutes. Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an international journal 25: 1-24
60. D'Agnillo, F & TMS Chang (1998) Absence of hemoprotein-associated free radical events following oxidant challenge of crosslinked hemoglobin-superoxide dismutase-catalase. Free radical Biology and medicine. 24(6):906-912
61. D'AGNILLO,F & TMS CHANG (1998). Crosslinked Hemoglobin-Superoxide Dismuatase-Catalase as a blood substitute with antixoidant properties. NATURE BIOTECHNOLOGY 16(7): 667-671
62. CHANG, TMS(1998) Modified hemoglobin-based blood substitutes: crosslinked, recombinant and encapsulated hemoglobin. Vox Sanguinis 74(suppl. 2):233-241
63. CHANG, TMS (1998) Modified hemoglobin blood substitutes: present status and future perspectives. Biotechnology Annual Review 4:75-112
64. CHANG TMS & WP YU (1998) Nanoencapsulation of hemoglobin and red blood cell enzymes based on nanotechnology and biodegradable polymer . in Volume II, Book on "Blood substitutes: principles, methods, products & clinical trials" Karger, Basel,Switzerland. pp 216-231
65. CHANG TMS (1998) Is there a role for first generation blood substitutes in hemorrhagic shock? Artificial Cells, Blood Substitutes & Immobilization Biotechnology,an international journal 26:(5-6) i-iv
Some recent publications on blood substitutes from Chang's group
1. T.M.S. CHANG (monograph) (1997) Blood Substitutes: Principles, Methods, Products and Clinical Trials. Volume I . Karger-Landes click here.
2. T.M.S. CHANG (editor) (1998) Blood Substitutes: Principles, Methods, Products and Clinical Trials. Volume II. Karger-Landes click here
3. F. D'AGNILLO & TMS CHANG (1997) Production of hydroxyl radical generation in a rat hindlimb model of ischemia-reperfusion injury using crosslinked hemoglobin-superoxide dismutase-catalase. Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an internationaljournal 25:163-180
4. S. RAZACK, F. D'AGNILLO & TMS CHANG (1997 )Crosslinked hemoglobin-superoxide dismutase-catalase scavenges free radicals in a rat model of intestinal ischemia-reperfusion injury. Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an international journal 25: 181-1924.
5. T.M.S. CHANG(1997) Recent And Future Developments in Modified Hemoglobin and Microencapsulated Hemoglobin as Red Blood Cell Substitutes. Artificial Cells, Blood Substitutes & Immobilization Biotechnology, an international journal 25: 1-24
6. Yu WP & TMS CHANG (1996):Submicron polymer membrane hemoglobin nanocapsules as potential blood substitutes: preparation and characterization. Artificial Cells, Blood Substitutes & Immobilization Biotechnology,an international journal 24:169-184
7. D'Agnillo, F & TMS Chang (1998) Absence of hemoprotein-associated free radical events following oxidant challenge of crosslinked hemoglobin-superoxide dismutase-catalase. Free radical Biology and medicine. 24(6):906-912
8. D'AGNILLO,F & TMS CHANG (1998). Crosslinked Hemoglobin-Superoxide Dismuatase-Catalase as a blood substitute with antixoidant properties. NATURE BIOTECHNOLOGY 16(7): 667-671
9. CHANG, TMS(1998) Modified hemoglobin-based blood substitutes: crosslinked, recombinant and encapsulated hemoglobin. Vox Sanguinis 74(suppl. 2):233-241
10. CHANG, TMS (1998) Modified hemoglobin blood substitutes: present status and future perspectives. Biotechnology Annual Review 4:75-112
11. CHANG TMS & WP YU (1998) Nanoencapsulation of hemoglobin and red blood cell enzymes based on nanotechnology and biodegradable polymer . in Volume II, Book on "Blood substitutes: principles, methods, products & clinical trials" Karger, Basel,Switzerland. pp 216-231
12. CHANG TMS (1998) Is there a role for first generation blood substitutes in hemorrhagic shock? Artificial Cells, Blood Substitutes & Immobilization Biotechnology,an international journal 26:(5-6) i-iv
13. CHANG TMS (1999) "Future Prospectives for Artificial Blood" from Trends in Biotechnology, February 1999, 17:61-67
CHANG TMS (2000) " Red Blood Cell
Substitutes"
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