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Blackwell Science, LtdOxford, UKAORArtificial Organs0160-564X2004 International Society for Artificial Organs282167172Original ArticleT.M.S. CHANG
Artificial Organs
28(2):167–172, Blackwell Publishing, Inc.
© 2004 International Society for Artificial Organs
Artificial Cells for Cell and Organ Replacements
Thomas Ming Swi Chang
Artificial Cells and Organs Research Center, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
Abstract: The artificial cell is a Canadian invention
(Chang, Science, 1964). This principle is being actively
investigated for use in cell and organ replacements. The
earliest routine clinical use of artificial cells is in the form
of coated activated charcoal for hemoperfusion for use in
the removal of drugs, and toxins and waste in uremia and
liver failure. Encapsulated cells are being studied for the
treatment of diabetes, liver failure and kidney failure, and
the use of encapsulated genetically-engineered cells is
being investigated for gene therapy. Blood substitutes
based on modified hemoglobin are already in Phase III
clinical trials in patients, with as much as 20 units being
infused into each patient during trauma surgery. Artificial
cells containing enzymes are being developed for clinical
trial in hereditary enzyme deficiency diseases and other
diseases. The artificial cell is also being investigated for
drug delivery and for other uses in biotechnology, chemical engineering and medicine. Key Words: Artificial
cells—Hybrid—Liver—Kidney—Gene therapy—Blood
substitutes.
Artificial cells were first reported by Chang at
McGill University a number of years ago (1–4)
(Fig. 1). Biologically-active materials inside the artificial cells are prevented from coming into direct
contact with external materials like leukocytes,
antibodies or tryptic enzymes. Smaller molecules can
equilibrate rapidly across the ultrathin membrane,
which has a large surface-to-volume relationship. A
number of potential medical applications using artificial cells have been proposed (2–6). The first of
these to be developed successfully for routine clinical
use is hemoperfusion (4). After initial clinical trials
for poisoning, kidney failure and liver failure (5), it
is now in routine clinical use (7,8). Some exciting
recent developments include their use for blood substitutes and for the replacement of the metabolic
functions of cells and organs (6).
After initial clinical trials for poisoning, kidney failure and liver failure (7), it is now in routine clinical
use, especially for the treatment of suicidal or accidental poisoning from medications (8). It is also
being used in combination with the hybrid artificial
liver in clinical trials.
CELL ENCAPSULATION FOR HYBRID
ARTIFICIAL ORGANS
Chang first reported the encapsulation of biological cells in 1966 based on a drop method and proposed that “protected from immunological process,
encapsulated endocrine cells might survive and
maintain an effective supply of hormone” (3,5).
Artificial pancreas, artificial liver and others
Chang approached the Conaught Laboratory to
develop his crosslinking drop method for use in islet
transplantation for diabetes. Sun from Conaught
and his collaborators later developed this drop
method by using milder physical crosslinking (9).
This resulted in alginate-polylysine-alginate (APA)
microcapsules containing cells. They showed that,
after implantation, the islets inside artificial cells
remained viable and continued to secrete insulin to
control the glucose levels of diabetic rats (9). Cell
encapsulation for cell therapy has been extensively
developed by many groups, especially using artificial
HEMOPERFUSION
The first successful use of the artificial cell in routine clinical applications is hemoperfusion (5–8).
2
Received November 2003.
Address correspondence and reprint requests to Thomas
Ming Swi Chang, Artificial Cells and Organs Research Center,
Faculty of Medicine, McGill University, 3655, Promenade SirWilliam-Osler, Montreal, Quebec, Canada H3G 1H6. E-mail:
[email protected]
167
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T.M.S. CHANG
ARTIFICIAL CELLS IN BIOTECHNOLOGY & MEDICINE
Chang (1964) SCIENCE
Chang et al (1966) Can J Physiol Pharm
Chang & Poznansky (1968) NATURE
Chang (1971) NATURE
oxygen,
Nutrients
Substrates
Toxins, drugs
CELLS
HEMOGLOBIN
ENZYMES
BIOREACTANTS
ETC
oxygen,
Wastes metabolites
Products, drugs
Hormones, peptides
ANTIBODY
WBC
TRYPTIC ENZYMES
FIG. 1. The basic principle of artificial cells. (With permission
from Artificial Cells, Blood Substitutes and Immobilization Biotechnology, an international journal 2004;32:1–14.)
cells containing endocrine tissues, hepatocytes and
other cells for cell therapy (9–15) (Fig. 1).
We have been studying the use of the implantation of encapsulated hepatocytes for liver support
(16–24). We found that implantation increases the
survival of rats with acute liver failure (17), maintains
a low bilirubin level in hyperbilirubinemic Gunn rats
(18), and prevents xenograft rejection (19). We
developed a two-step cell encapsulation method to
improve the APA method, resulting in the improved
survival of implanted cells (20,21). Using this twostep method together with the coencapsulation of
stem cells and hepatocytes, we have further increased
the viability of encapsulated hepatocytes both in culture and also after implantation (22,24) (Fig. 2). One
implantation of the coencapsulated hepatocytes and
stem cells into Gunn rats lowered the systemic bilirubin levels and maintained this low level for two
months (24). Implanted encapsulated hepatocytes
can only maintain a low level for one month.
Microencapsulated genetically-engineered cells
Microencapsulated genetically-engineered cells
have been studied by many groups for potential
applications in amyotrophic lateral sclerosis, dwarfism, pain treatment, IgG1 plasmacytosis, hemophilia
B, Parkinsonism and axotomized septal cholinergic
neurons (25,26). One group uses hollow fibers to
macroencapsulate genetically-engineered cells. This
way, the fibers can be inserted and then retrieved
after use, without being retained in the body (26).
To avoid the need for implantation, we studied the
oral use of microencapsulated genetically-engineered nonpathogenic E.coli DH5 cells containing
Artif Organs, Vol. 28, No. 2, 2004
Klebsiella aerogenes urease gene to lower systemic
urea in renal failure rats (27,28). However, these
genetically-engineered micro-organisms are not sufficiently stable in their ability to remove urea. We are
looking at the metabolic induction of lactobacillus,
similar to those used in yogurt, in order not to introduce genetically-engineered cells into the body (29).
ARTIFICIAL RED BLOOD CELLS
Complete artificial red blood cells of
micron dimensions
The original complete artificial red blood cells
(RBC) prepared here containing hemoglobin and
enzymes have all the properties of RBC when tested
in vitro (1,2). However, they did not survive for a
sufficient length of time in the circulation after
infusion.
Polyhemoglobin as a blood substitute
As a result of the above, we used a simpler molecular version based on the use of bifunctional agents,
such as diacid (2,5) or later glutaraldehyde (30), to
crosslink hemoglobin molecules into polyhemoglobin. Due to problems related to human immunodeficiency virus (HIV) in donor blood, there has been
extensive development toward blood substitutes,
starting in the early 1990s (31–34). At present, two of
these are in the final stages of clinical trials and are
waiting for Food and Drug Administration (FDA)
approval. These have been developed independently
by two groups based on our basic principle of gluat-
HEPATOCYTES COENCAPSULATED WITH STEM CELLS
VIABILITY AFTER IMPLANTATION
( Liu & Chang, ACBSIB 2002)
90
80
HViability(%)
168
Encap hepatocytes with stem cells
Encap hepatocytes only
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
T ime (w e e k)
FIG. 2. An experiment showing that coencapsulation with stem
cells increases the viability of hepatocytes after implantation.
(With permission from Artificial Cells, Blood Substitutes and
Immobilization Biotechnology, an international journal 2002;
30:99–112.)
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5
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••
7
aradehyde crosslinked polyhemoglobin (30). One is
pyridoxalated glutaraldehyde human polyhemoglobin (35,36). In a Phase III clinical trial it was shown
that this can successfully replace extensive blood loss
in trauma surgery by maintaining the hemoglobin
level with no reported side-effects (36). Up to 20
units have been infused into individual trauma
surgery patients (35). Another blood substitute is
glutaraldehyde crosslinked bovine polyhemoglobin,
which has been extensively tested in Phase III clinical
trials (37,38). This bovine polyhemoglobin has been
approved for veterinary medicine in the U.S.A. and
for routine clinical use in South Africa. Conjugated
hemoglobin development and Phase II clinical trialsConjugated hemoglobin: development and Phase
II clinical trials The above two polyhemoglobins
have been approved for compassionate use in
humans and they are waiting for regulatory approval
for routine clinical use in humans in North America.
They have a number of advantages when compared
to donor RBC and they are particularly useful in
surgery. However, these are only oxygen carriers and
do not have all of the functions of RBC that may be
needed for certain clinical conditions (39).
Polyhemoglobin crosslinked with RBC
antioxidant enzymes
Reperfusion using an oxygen carrier alone in
sustained severe hemorrhagic shock or sustained
ischemic organs, as in stroke, myocardial infarction
or organ transplantation, may result in the produc-
BRAIN EDEMA IN RATS AFTER ACUTE GLOBAL
CEREBRAL ISCHEMIA & REPERFUSION
(Powanda & Chang ACBSIB 2002 )
PolyHb
PolyHb-SOD-CAT
FIG. 3. This is a rat model of acute global cerebral ischemia
followed by reperfusion with different oxygen-carrying solutions.
Unlike polyhemoglobin, polyHb-CAT-SOD does not cause brain
edema when used in this situation. (With permission from Artificial Cells, Blood Substitutes and Immobilization Biotechnology,
an international journal 2002;30:25–42.)
169
NANO DIMENSION ARTIFICIAL RBC
NANOENCAPSULATED HB & ENZYMES
GLUCOSE
GLUCOSE
ADENINE, INOSINE
ATP
EMBDEN-MEYERHOF
SYSTEM
HEMOGLOBIN
NAD
REDUCING
AGENT
2,3-DPG
NADH
METHB
LACTATE
LACTATE
CARBONCIC ANHYDREASE
CO2
SUPEROXIDE DISMUTASE
SUPEROXIDE
CATALASE
H2O2
FIG. 4. Nanodimension artificial red blood cells (RBC) with a
polyethylene-glyco-polylactide membrane. In addition to hemoglobin, this contains the same enzymes that are normally present
in RBC. Thus, it has the complete function of the RBC. (With
permission from Artificial Cells, Blood Substitutes and Immobilization Biotechnology, an international journal 2003;31:231–248.)
tion of oxygen radicals and tissue injury (31,39). We
are using a crosslinked polyhemoglobin-superoxide
dismutase-catalase (PolyHb-SOD-CAT) (40–43).
Unlike PolyHb, PolyHb-SOD-CAT did not cause a
significant increase in oxygen radicals when it was
used to reperfuse ischemic rat intestines (42). More
recently (43), in a transient global cerebral ischemia
rat model, we found that, after 60 min of ischemia,
reperfusion with polyHb resulted in significant
increases in the blood–brain barrier and the breakdown of the blood–brain barrier (Fig. 3). On the
other hand, polyHb-SOD-CAT did not result in
these adverse changes (43) (Fig. 3).
Nanodimension artificial RBC
Chang’s original idea of a complete artificial RBC
(1,2) is now being developed as a third generation
blood substitute (39). Hemoglobin lipid vesicles is
one of these approaches (44–46). We are using a different approach based on a biodegradable polymer
and nanotechnology, resulting in nanoartificial RBC
of 80–150 nm diameter (47–49). These nanoartificial
RBC contain all of the RBC enzymes needed for the
long-term function of the nanoartificial RBC (49)
(Fig. 4). Our recent studies show that, using a polyethylene-glycol-polylactide copolymer membrane,
we are able to increase the circulation time of these
nanoartificial RBC to double that of polyHb (49).
Artif Organs, Vol. 28, No. 2, 2004
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170
T.M.S. CHANG
ARTIFICIAL CELLS IN ENZYME THERAPY
Enzyme therapy by implantation
We have previously implanted artificial cells containing catalase into acatalesemic mice, animals with
a congenital deficiency in catalase (50). This replaced
the deficient enzymes and prevented the animals
from the damaging effects of oxidants. The artificial
cells protect the enclosed enzyme from immunological reactions (51). It was also shown that artificial
cells containing asparaginase implanted into mice
with lymphosarcoma delayed the onset and growth
of lymphosarcoma (52). The single problem preventing the clinical application of enzyme artificial cells
is the need to repeatedly inject these enzyme artificial cells.
Oral administration to avoid the need
for implantation
To solve this problem, artificial cells were given
orally. As they travel through the intestine, they act
as microscopic dialyzers. By encapsulating enzymes
and other material inside the microcapsules, they can
act as a combined dialyzer–bioreactor. For example,
artificial cells containing urease and ammonia adsorbent were used to lower the systemic urea level (5).
We found that microencapsulated phenylalanineammonialyase given orally can lower the elevated
phenylalanine levels in phenylketonuria (PKU) rats
(53). This is because of our more recent finding of an
extensive recycling of amino acids between the body
and the intestine (54). This is now being developed
for clinical trial in PKU (55,56). In addition to PKU,
other examples from our recent studies show that
oral artificial cells containing tyrosinase are effective
in lowering systemic tyrosine levels in rats (57). We
have also used oral microencapsulated xanthine oxidase to lower the systemic hypoxanthine levels in a
patient with Lesch–Nyhan disease (58).
DRUG DELIVERY SYSTEMS
Our initial use of polylactide biodegradable semipermeable microcapsules containing enzymes, insulin, hormones, vaccines and other biologicals in 1976
(59) is now being extended by many groups. This
includes our studies on the preparation and characterization of polylactic acid microcapsules (60) and
polylactic acid nanocapsules (61) containing ciprofloxacin for controlled release. The nanocapsules
described above which contain high concentrations
of proteins (12 g/dL) and enzymes for blood substitutes (47–49) are also useful for the delivery of
biologically-active proteins and peptides. Other
Artif Organs, Vol. 28, No. 2, 2004
approaches based on nanodimension artificial cells in
the form of liposomes, nanoparticles and nanocapsules are being increasingly used by many groups for
drug delivery, as reviewed in a recent book (62).
CONCLUSION
8
The principle of artificial cells was a very novel
idea when it was first proposed (2). As a result, it
took some time before others started to actively
investigate and extend this principle for use in cell
and organ replacements. In the earliest routine clinical use of artificial cells, a very simple principle of
artificial cells in the form of coated activated charcoal
for hemoperfusion was used in the removal of drugs,
and toxins and waste in uremia and liver failure.
The successful clinical application of this simpler
approach has resulted in the increasing development
of the more complicated approaches of artificial cells.
For example, encapsulated cells are being studied for
the treatment of diabetes, liver failure and kidney
failure, and the use of encapsulated geneticallyengineered cells is being investigated for gene
therapy. Blood substitutes based on modified hemoglobin are already in Phase III clinical trials in
patients, with as much as 20 units being infused into
each patient during trauma surgery. Artificial cells
containing enzymes are being developed for clinical
trial in hereditary enzyme deficiency diseases and
other diseases. The artificial cell is also being investigated for drug delivery and for other uses in biotechnology, chemical engineering and medicine.
REFERENCES
1. Chang TMS. (1957) Hemoglobin Corpuscles. Report of a
research project for Honours Physiology. Quebec: Medical
Library, McGill University. Also reprinted as part of “30 anniversary in Artificial Red Blood Cells Research”. J Biomat Artif
Cells Artif Org 1988;16:1–9.
2. Chang TMS. Semipermeable microcapsules. Science 1964;146:
524–5.
3. Chang TMS, MacIntosh FC, Mason SG. Semipermeable
aqueous microcapsules: I. Preparation and properties. Can J
Physiol Pharmacol 1966;44:115–28.
4. Chang TMS. Semipermeable aqueous microcapsules [artificial
cells]: with emphasis on experiments in an extracorporeal
shunt system. Trans Am Soc Artif Intern Organs 1966;12:13–9.
5. Chang TMS. Artificial Cells. Springfield, IL: C.C. Thomas Publisher. 1972. Available at: http://www.artcell.mcgill.ca.
6. Chang TMS. Artificial cells. In: Dulbecco R, ed. Encyclopedia
of Human Biology. (2nd Edition). San Diego, CA: Academic
Press, Inc, 1997;457–63.
7. Chang TMS. Microencapsulated adsorbent hemoperfusion for
uremia, intoxication and hepatic failure. Kidney Int 1975;7:
S387–92.
8. Winchester JF. Hemoperfusion. In: Maher JF, ed. Replacement of Renal Function by Dialysis. Boston, MA: Kluwer
Academic Publisher, 1988;439–592.
9. Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science, 1980;210:908–9.
9
aor_47343.fm Page 171 Monday, December 15, 2003 6:39 PM
••
10
11
12
10. Chang TMS. Artificial cells with emphasis on bioencapsulation in biotechnology. Biotechnol Annu Rev 1995;1:267–95.
11. Orive G, Hernandez RM, Gascon AR, et al. Cell encapsulation: promise and progress. Nature Med 2003;9:104–7.
12. Kulitreibez WM, Lauza PP, Cuicks WL (eds). Cell Encapsulation Technology and Therapy. Boston, MA: Burkhauser,
1999.
13. Hunkeler D, Prokop A, Cherrington AD, Rajotte R, Sefton
M (eds). Bioartificial organs II: Technology, Medicine and
Material, Ann NY Acad Sci 1999;875:271–9.
14. Chang TMS, Prakash S. Procedure for microencapsulationof
enzymes, cells and genetically engineered microorganisms.
Mol Biotechnol 2001;17:249–60.
15. Dionne KE, Cain BM, Li RH, et al. Transport characterization
of membranes for immunoisolation. Biomaterials 1996;17:
257–66.
16. Chang TMS. Bioencapsulated hepatocytes for experimental
liver support. J Hepatol 2001;34:148–9.
17. Wong H, Chang TMS. Bioartificial liver: implanted artificial
cells microencapsulated living hepatocytes increases survival
of liver failure rats. Int J Artif Organs 1986;9:335–6.
18. Brunis Chang TMS. Hepatocytes immobilized by microencapsulation in artificial cells: effects on hyperbiliru-binemia in
Gunn rats. J Biomat Artif Cells Artif Org 1989;17:403–12.
19. Wong H, Chang TMS. The viability and regeneration of artificial cell microencapsulated rat hepatocyte xenograft transplants in mice. J Biomat Artif Cells Artif Org 1988;16:731–40.
20. Wong H, Chang TMS. Microencapsulation of cells within alginate poly-L-lysine microcapsules prepared with standard single step drop technique: histologically identified membrane
imperfections and the associated graft rejection. Artif Cell
Blood Sub 1991;19:675–86.
21. Wong H, Chang TMS. A novel two-step procedure for immobilizing living cells in microcapsule for improving xenograft
survival. Artif Cell Blood Sub 1991;19:1991.
22. Liu Z, Chang TMS. Effects of bone marrow cells on hepatocytes: when co-cultured or co-encapsulated together. Artif Cell
Blood Sub 2000;28:365–74.
23. Liu ZC, Chang TMS. Transplantation of co-encapsulated
hepatocytes and marrow stem cells into rats. Artif Cell Blood
Sub 2002;30:99–112.
24. Liu ZC, Chang TMS. Coencapsulation of stem sells and hepatocytes: in-vitro conversion of ammonia and in-vivo studies on
the lowering of bilirubin in Gunn rats after transplantation.
Int J Artif Organs 2003;26:491–7.
25. Chang TMS, Prakash S. Therapeutic uses of microencapsulated genetically engineered cells. Mol Med Today 1998;4:221–
7.
26. Aebischer P, Schluep M, Deglon N, et al. Intrathecal delivery
of CNTF using encapsulated genetically modified xenogeneic
cells in amyotrophic lateral sclerosis patients. Nat Med
1996;2:696–9.
27. Prakash S, Chang TMS. Microencapsulated genetically Engineered live E.coli DH5 cells administered orally to maintain
normal plasma urea level in uremic rats. Nat Med 1996;2:883–
7.
28. Chang TMS. Live E.coli cells to treatment uremia: replies to
letters to the editor. Nat Med 1997;3:1997.
29. Chow KM, Liu ZC, Prakash S, Chang TMS. Free and icroencapsulated Lactobacillus and effects of metabolic induction on
urea removal. Artif Cell Blood Sub 2003;4. in press.
30. Chang TMS. Stabilization of enzyme by microencapsulation
with a concentrated protein solution or by crosslinking with
glutaraldehyde. Biochem Biophys Res Com 1971;44:1531–3.
31. Chang TMS. Blood Substitutes. Principles, Methods, Products
and Clinical Trials, Vol. 1. Basel: Karger, 1997. Available at:
http://www.artcell.mcgill.ca.
32. Chang TMS. Artificial blood: a prospective. Trends Biotechnol
1999;17:61–7.
33. Chang TMS. Oxygen carriers. Curr Opin Investig Drug 2002;
3:1187–90.
171
34. Winslow R. Current status of blood substitute research:
towards a new paradigm. J Int Med 2003;253:508–17.
35. Gould SA, Moore EE, Hoyt DB, et al. The life-sustaining
capacity of human polymerized hemoglobin when red cells
might be unavailable. J Am College Surgeons 2002;195:445–52.
36. Gould SA, Sehgal LR, Sehgal HL, DeWoskin R, Moss GS.
The clinical development of human polymerized hemoglobin.
In: Chang TMS, ed. Blood Substitutes: Principles, Methods,
Products and Clinical Trials, Vol. 2 Basel: Karger, 1998;12–28.
37. Sprung J, Kindscher JD, Wahr JA, et al. The use of bovine
hemoglobin glutamer-250 (Hemopure) in surgical patients:
results of a multicenter, randomized, single-blinded trial.
Anesth Analg 2002;94:799–808.
38. Pearce LB, Gawryl MS. Overview of preclinical and clinical
efficacy of Biopure’s Hbocs. In: Chang TMS, ed. Blood Substitutes: Principles, Methods, Products and Clinical Trials, Vol.
2. Basel: Karger, 1998;82–98.
39. Chang TMS. New generations of red blood cell substitutes.
J Intern Meds 2003;253:527–35.
40. D’Agnillo F, Chang TMS. Polyhemoglobin-superoxide dismutase, catalase as a blood substitute with antioxidant properties.
Nature Biotechnol 1998;16:667–71.
41. D’Agnillo F, Chang TMS. Absence of hemoproteinassociated free radical events following oxidant challenge of
crosslinked hemoglobin-superoxide dismutase-catalase. Free
Radic Biol Med 1998;24:906–12.
42. Razack S, D’Agnillo F, Chang TMS. Effects of Polyhemoglobin-catalase-superoxide dismutase on oxygen radicals in an
ischemia-reperfusion rat intestinal model. Artif Cell Blood
Sub 1997;25:181–92.
43. Powanda D, Chang TMS. Cross-linked polyhemoglobinsuperoxide dismutase-catalase supplies oxygen without causing blood brain barrier disruption or brain edema in a rat
model of transient global brain ischemia-reperfusion. Artif
Cell Blood Sub 2002;30:2002.
44. Rudolph AS, Rabinovici R, Feuerstein GZ. (eds) Red Blood
Cell Substitutes. NY: Marcel Dekker, Inc., 1997.
45. Tsuchida E (ed). Blood Substitutes: Present and Future Perspectives. Amsterdam: Elservier, 1998.
46. Philips WT, Klpper RW, Awasthi VD, et al. Polyethylene
glyco-modified liposome-encapsulated hemoglobin: a long
circulating red cell substitute. J Pharm Exp Therapeutics
1999;288:665–70.
47. Yu WP, Chang TMS. Submicron biodegradable polymer
membrane hemoglobin nanocapsules as potential blood substitutes: a preliminary report. Artif Cell Blood Sub (Guest
editor: Winslow R) 1994;22:889–94.
48. Chang TMS, Yu WP. Nanoencapsulation of hemoglobin and
red blood cell enzymes based on nanotechnology and biodegradable polymer. In: Chang TMS, ed. Blood Substitutes: Principles, Methods, Products and Clinical Trials, Vol. 2. Basel:
Karger, 1998;216–31.
49. Chang TMS, Powanda D, Yu WP. Analysis of polyethyleneglycol-polylactide nano-dimension artificial red blood cells in
maintaining systemic hemoglobin levels and prevention of
methemoglobin formation. Artif Cell Blood Sub 2003;31:231–
48.
50. Chang TMS, Poznansky MJ. Semipermeable microcapsules
containing catalase for enzyme replacement in acatalsaemic
mice. Nature 1968;218:242–5.
51. Poznansky MJ, Chang TMS. Comparison of the enzyme kinetics and immunological properties of catalase immobi lized by
microencapsulation and catalase in free solution for enzyme
replacement. Biochim Biophys Acta 1974;334:103–15.
52. Chang TMS. The in vivo effects of semipermeable microcapsules containing 1-asparaginase on 6C3HED lymphosarcoma.
Nature 1971;229:117–8.
53. Bourget L, Chang TMS. Phenylalanine ammonia-lyase immobilized in microcapsules for the depleture of phenylalanine in
plasma in phenylketonuric rat model. Biochim Biophys Acta
1986;883:432–8.
Artif Organs, Vol. 28, No. 2, 2004
13
14
15
aor_47343.fm Page 172 Monday, December 15, 2003 6:39 PM
172
16
17
T.M.S. CHANG
54. Chang TMS, Bourget L, Lister C. New theory of enterorecirculation of amino acids and its use for depleting unwanted
amino acids using oral enzyme-artificial cells, as in removing
phenylalanine in phenylketonuria. Artif Cell Blood Sub
1995;25:1–23.
55. Sarkissian CN, Shao Z, Blain F, et al. A different approach
to treatment of phenylketonuria: phenylalanine degradation
with recombinant phenylalanine ammonia lyase. Proc Natl
Acad Sci, 1999;96:2339–44.
56. Liu J, Jia X, Zhang J, Xiang G, Hu W, Zhou Y. Study on a
novel strategy to treatment of Phenylketonuria. Artif Cell
Blood Sub 2002;30:243–58.
57. Yu BL, Chang TMS. Effects of combined oral administration
and intravenous injection on maintaining decreased systemic
tyrosine levels in rats. Artif Cell Blood Sub 2004;32. in press.
Artif Organs, Vol. 28, No. 2, 2004
58. Palmour RM, Goodyer P, Reade T, Chang TMS. Microencapsulated xanthine oxidase as experimental therapy in LeschNyhan Disease. Lancet 1989;2:687–8.
59. Chang TMS. Biodegradable semipermeable microcapsules
containing enzymes, hormones, vaccines, and other biologicals. J Bioengineering 1976;1:25–32.
60. YuWP, Wong J, Chang TMS. Preparation and characterization of polylactic acid microcapsules containing ciprofloxacin for controlled release. J Microencapsulation 1998;
15:515–23.
61. Yu WP, Wong JP, Chang TMS. Biodegradable polylactic acid
nanocapsules containning ciprofloxacin: preparation and characterization. Artif Cell Blood Sub 1999;27:263–78.
62. Ranade VV, Hollinger MA. Drug Delivery Systems. Boca
Raton, FL: CRC Press, 2003.
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Artificial Organs
Volume 28, 2004
BSA article no: 47343
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