artificial cell bioencapsulation in macro, micro, nano, and molecular dimensions: keynote lecture
TRANSCRIPT
ARTIFICIAL CELLS, BLOOD SUBSTITUTES, AND BIOTECHNOLOGY
Vol. 32, No. 1, pp. 1–23, 2004
Artificial Cell Bioencapsulation in Macro,
Micro, Nano, and Molecular Dimensions:
Keynote Lecture
Thomas Ming Swi Chang, O.C., M.D., C.M., Ph.D.,
F.R.C.P.(C)., F.R.S.C.,*
Artificial Cells and Organs Research Centre, MSSS-FRSQ Research
Group in Transfusion Medicine, and Departments of Physiology,
Medicine and Biomedical Engineering, Faculty of Medicine
McGill University, Montreal, Quebec, Canada
ABSTRACT
Artificial cells now ranges from macro-dimensions, to micron-
dimensions, to nano-dimensions, and to molecular dimensions.
Those in the macro-dimensions are suitable for use in the bio-
encapsulation of cells, tissues, microorganisms, and bioreactants.
Those in the micron-dimensions are suitable for the bioencapsulation
*Correspondence: Professor Thomas Ming Swi Chang, O.C., M.D., C.M.,
Ph.D., F.R.C.P.(C)., F.R.S.C., Artificial Cells and Organs Research Centre,
MSSS-FRSQ Research Group in Transfusion Medicine, and Departments of
Physiology, Medicine and Biomedical Engineering, Faculty of Medicine, McGill
University 3655, Drumond Street, Montreal, Quebec, Canada H3G 1H6; E-mail:
1
DOI: 10.1081/BIO-120028665 1073-1199 (Print); 1532-4184 (Online)
Copyright & 2004 by Marcel Dekker, Inc. www.dekker.com
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of enzymes, microorganisms, peptides, drugs, vaccine, and other
materials. Those in the nano-dimension are being used for blood
substitutes and carriers for enzymes, peptides, drugs, etc. Those in
the molecular-dimensions are used as blood substitutes, crosslinked
enzymes etc.
Key Words: Artificial cells; Bioencapsulation; Nanocapsules;
Microcapsules; Macrocapsules; Encapsulation; Enzyme therapy;
Cell therapy; Gene therapy; Blood substitutes; Drug delivery.
INTRODUCTION
Artificial cells in the micro-dimensions were first reported by Changa number of years ago (Chang, 1957, 1964, 1966, 1972; Chang et al.,1966) (Fig. 1). Biologically active materials inside the artificial cells areprevented from coming into direct contact with external materials likeleucocytes, antibodies, or tryptic enzymes. Smaller molecules canequilibrate rapidly across the ultrathin membrane with large surface tovolume relationship. A number of potential medical applications usingartificial cells have been proposed (Chang, 1964, 1966, 1972; Chang et al.,1966). However, even at the very beginning, it was necessary to increasethis to the macro-dimensions for certain type of bioencapsulation. Oneexample is cell encapsulation (Chang, 1972; Chang et al., 1966) andanother example is the encapsulation of adsorbents for usein hemoperfusion (Chang, 1966, 1972, 1975; Winchester, 1988).Artificial cells (Chang, 1997a) bioencapsulation now range frommacro-dimensions, to micron-dimensions, to nano-dimensions, and to
Figure 1. Basic principle of artificial cells.
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molecular-dimensions (Fig. 2). This paper describes some examples fromour research using those in the micron-dimensions for the bioencapsula-tion of enzymes, peptides, drugs, vaccine, and other materials; those inthe macro-dimensions for cell encapsulation and encapsulation ofbioreactants; those in the molecular-dimensions are used as bloodsubstitutes, crosslinked enzymes etc.; and those in the nano-dimensionfor blood substitutes, enzymes, peptides, drugs etc.
ARTIFICIAL CELLS IN THE MICRON-DIMENSIONS
Enzyme Therapy by Implantation (Fig. 3)
We have earlier implanted artificial cells containing catalase intoacatalesemic mice, animals with a congenitical deficiency in catalase(Chang and Poznansky, 1968). This replaces the deficient enzymes andprevented the animals from the damaging effects of oxidants. Theartificial cells protect the enclosed enzyme from immunological reactions(Poznansky and Chang, 1974). It was also showed that artificial cellscontaining asparaginase implanted into mice with lymphosarcomadelayed the onset and growth of lymphosarcoma (Chang, 1971a). Thesingle problem preventing the clinical application of enzyme artificialcells is the need to repeatedly inject these enzyme artificial cells.
Figure 2. Artificial cells in the macro, micro, nano, and molecular dimensions.
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Oral Administration to Avoid the Need for
Implantation (Fig. 4)
To solve this problem, artificial cells were given orally. As they travel
through the intestine, they act as microscopic dialyzers (Fig. 4). By
encapsulating enzymes and other material inside the microcapsules, they
can act as combined dialyzer–bioreactor (Fig. 4). For example, artificial
cells containing urease and ammonia adsorbent were used to lower
the systemic urea level (Chang, 1972). We found that microencapsu-
lated phenylalanine ammonia lyase given orally can lower the elevated
phenylalanine levels in phenylketonuria (PKU) rats (Bourget and Chang,
1986) (Fig. 5). This is because of our more recent finding of an extensive
recycling of amino acids between the body and the intestine (Chang et al.,
1995) (Fig. 6). This is now being developed for clinical trial in PKU (Liu
et al., 2002; Sarkissian et al., 1999). In addition to PKU other examples
our recent studies shows that oral artificial cells containing tyrosinase
is effective in lowering systemic tyrosine levels in rats (Yu and Chang,
2004) (Fig. 7). We have also used oral microencapsulated xanthine
oxidase to lower the systemic hypoxanthine levels in a patient with Lesch-
Nyhan Disease (Palmour et al., 1989) (Fig. 8).
Figure 3. Basic principle of the use of enzyme artificial cells for implantation in
inborn errors of metabolism (e.g., acatelasemia) and cancer (lymphosarcoma).
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Drug Delivery Systems
Our initial use of polylactide biodegradable semipermeable micro-capsules containing enzymes, insulin, hormones, vaccines, and other bio-logicals in 1976 (Chang, 1976) is now being extended by many groups.
Figure 5. Oral artificial cells containing phenylalanine ammonia lyase for the
removal of systemic phenylalanine in the most common inborn error of
metabolism, phenylketonuria.
Figure 4. Our recent approach of giving artificial cells orally to avoid the need
for implantation. This way, each artificial cell as it travels through the intestine,
acts as a microscopic dialyzer. By placing enzyme or other bioreactants inside the
artificial cells, they can act as combined dialyzer–bioreactors on their passage
through the intestine. After carrying out their function, they are excreted in the
stool. Thus there is no accumulation in the body.
Artificial Cell Bioencapsulation 5
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Figure 6. Our new finding that there is an extensive recirculation of amino acids
between the body and the intestine. Pancreatic, gastric, intestinal, and other
secretions from the body contains a large amount of protein, peptides, and other
sources of amino acids. These are digested in the intestine into amino acids that
are reabsorbed into the body. Artificial cells containing one enzyme to break
down one amino acid can thus break the recirculation for this particular amino
acid resulting in decreasing its concentration in the body.
Figure 7. Oral artificial cells containing tyrosinase can selectively lower the
systemic tyrosine level.
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This includes our studies on the preparation and characterization ofpolylactic acid microcapsules containing ciprofloxacin for controlledrelease (Yu et al., 1998).
ARTIFICIAL CELLS IN THE MACRO-DIMENSIONS
Hemoperfusion
The first successful use of artificial cell in routine clinical use ishemoperfusion (Chang, 1972, 1975; Winchester, 1988). After initialclinical trails for poisoning, kidney failure, and liver failure (Chang,1975), it is now in routine clinical uses (Winchester, 1988).
Cell Encapsulation of Hepatocytes, Islets and
Other Endocrine Cells, and Stem Cells
Chang et al. reported the encapsulation of biological cells in1966 based on a drop method and proposed that ‘‘protected from
Figure 8. Unlike amino acid, hypoxanthine is very lipid soluble and can thus
equilibrate rapidly with the intestinal content. Artificial cells containing xanthine
oxidase can therefore lower the elevated systemic hypoxanthine level in a patient
with Lesch-Nyhan disease.
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immunological process, encapsulated endocrine cells might survive and
maintain an effective supply of hormone’’ (Chang 1972; Chang et al.,
1966) (Fig. 9). Chang approached Conaught Laboratory to develop this
for use in islet transplantation for diabetes. Sun from Conaught and
his collaborators have later developed this drop-method by using milder
physical crosslinking (Lim and Sun, 1980). This resulted in alginate-
polylysine-alginate (APA) microcapsules containing cells. They show that
after implantation, the islets inside artificial cells remain viable and
continued to secrete insulin to control the glucose levels of diabetic rats
(Lim and Sun, 1980).Cell encapsulation for cell therapy has been extensively developed by
many groups especially using artificial cells containing endocrine tissues,
hepatocytes, and other cells for cell therapy (Chang, 1995; Chang and
Prakash, 2001; Dionne et al., 1996; Hunkeler et al., 1999; Kulitreibez
et al., 1999; Lim and Sun, 1980; Orive et al., 2003) (Fig. 9). We have been
studying the use of implantation of encapsulated hepatocytes for liver
support (Brunis and Chang, 1989; Chang, 2001; Wong and Chang, 1986,
1988, 1991a, 1991b; Liu and Chang, 2000, 2002, 2003). We found that
implantation increases the survival of rats in acute liver failure (Wong
and Chang, 1986); maintains a low bilirubin level in hyperbilirubinemic
ARTIFICIAL CELLS INCELL ENCAPSULATION
Chang et al (1966) Can J Physiol PharmChang (1972) Monograph “Artificial Cells”
CELLS
ANTIBODYWBCTRYPTIC ENZYMES
Figure 9. Basic principle of artificial cells for cell encapsulation. The
encapsulated cells are retained inside the microcapsules and thus isolated from
the external environment. This prevents immunological rejection of the
encapsulated cells. However, the membrane can be made permeable to oxygen
and nutrients needed by the cell. Also, the products of the cells like insulin,
peptide, and other materials can leave the microcapsules.
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Gunn rats (Brunis and Chang, 1989); prevents xenograft rejection (Wongand Chang, 1988). We developed a two step cell encapsulation method toimprove the APA method resulting in improved survival of implantedcells (Wong and Chang, 1991a, 1991b). Using this two step methods plusthe use of co-encapsulation of stem cells and hepatocytes (Fig. 10) wehave further increased the viability of encapsulated hepatocytes both inculture and also after implantation (Liu and Chang, 2000, 2002) (Fig. 11).One implantation of the co-encapsulated hepatocytes-stem cells intoGunn rats lowered the systemic bilirubin levels and maintained this lowlevel for 2 months (Liu and Chang, 2003). Implanted encapsulatedhepatocytes can only maintain a low level for 1 month.
Microencapsulated Genetically Engineered Cells
This has been studied out by many groups for potential applicationsin amyotrophic lateral sclerosis, Dwarfism, pain treatment, IgG1
plasmacytosis, Hemophilia B, Parkinsonism, and axotomized septalcholinergic neurons (Aebischer et al., 1996; Chang and Prakash, 1998)(Fig. 12). One group uses hollow fibers to macroencapsulated geneticallyengineered cells. This way, the fibers can be inserted and then retrievedafter use without being retained in the body (Aebischer et al., 1996).
To avoid the need for implantation, we studied the oral use (Fig. 5)of microencapsulated genetically engineered nonpathogenic E. coliDH5 cells containing Klebsiella aerogenes urease gene to lower systemicurea in renal failure rats (Chang, 1997b; Prakash and Chang, 1996).
Hepatocytesplus
Stem cells
ANTIBODYWBC
COENCAP OF CELL AND ADULT STEM CELLS
Figure 10. Co-encapsulation of cells with stem cells to increase the viability of
the encapsulated cells.
Artificial Cell Bioencapsulation 9
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BIOENCAPSULATION OF GENETICALLY ENGINEERED CELLS
GENETICALLYENGINEERED
CELLS
ANTIBODYWBCTRYPTIC ENZYMES
substrates products
Figure 12. Basic principle of bioencapsulation of genetically engineered cells is
the same as for cell encapsulation.
Figure 11. Experiment showing that co-encapsulation with stem cells increases
the viability of hepatocytes after implantation.
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However, these genetically engineered microorganism are not sufficientlystable in their ability to remove urea. We are looking at the metabolicinduction of lactobacillus similar to those use in yourgart, in order not tointroduce genetically engineered cells into the body (Chow et al., 2003).
ARTIFICIAL CELLS IN THE MOLECULAR
DIMENSIONS
Polyhemoglobin as Blood Substitutes
Chang has extended his original approach of artificial cells con-taining hemoglobin and enzymes (Chang, 1957, 1964) to form poly-hemoglobin—a molecular version of artificial cells. This is based on hisuse of bifunctional agents like diacid (Chang, 1964, 1972) or laterglutaraldehyde (Chang, 1971b) to crosslink hemoglobin molecules intopolyhemoglobin (Fig. 13). With problem related to H.I.V. in donorblood, there has been extensive development towards blood substitutesstarting in the early 1990’s (Chang, 1997c, 1999, 2002, 2003; Winslow,2003). At present, two of these are in the final stages of clinical trials andwaiting for F.D.A. approval. These are developed independently by twogroups based on Chang’s basic principle of gluataradehyde crosslinkedpolyhemoglobin (Chang, 1971b). One is pyridoxalated glutaraldehydehuman polyhemoglobin (Gould et al., 1998, 2002). They show in Phase
Figure 13. Molecular dimension red blood cell substitutes in the form of
polyhemoglobin. This is formed by the intermolecular crosslinking of hemoglobin
into a soluble complex. In this form, they are retained in the circulation.
Artificial Cell Bioencapsulation 11
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III clinical trial that this can successfully replace extensive blood loss intrauma surgery by maintaining the hemoglobin level with no reportedside effects (Gould et al., 1998, 2002). They have infused up to 20 unitsinto individual trauma surgery patients (Gould et al., 2002). Anotherone is glutaraldehyde crosslinked bovine polyhemoglobin that has beenextensive tested in Phase III clinical trials (Pearce and Gawryl, 1998;Sprung et al., 2002). This bovine polyhemoglobin has been approved forveterinary medicine in the U.S. and for routine clinical use in SouthAfrica. The above two polyhemoglobins have been approved forcompassionate uses in human and they are waiting for regulatoryapproval for routine clinical uses in human in North America. They havea number of advantages when compared to donor red blood cells (Fig. 14)and they are particularly useful for use in surgery. However, these areonly oxygen carriers and do not have all the functions of red blood cellsthat may be needed for certain clinical conditions (Chang, 2003).
Polyhemoglobin Crosslinked with RBC Antioxidant Enzymes
Reperfusion using oxygen carrier alone in sustained severe hemor-rhagic shock or sustained ischemic organs as in stroke, myocardial
Figure 14. Comparison of polyhemoglobin with donor red blood cells.
Polyhemoglobin has many advantages over red blood cells and is useful for use
during surgery. However, it cannot be used in a number of other clinical
conditions. This is because unlike red blood cells (RBC), polyhemoglobin is only
an oxygen carrier. It does not have RBC enzymes needed for many functions
including the removal of oxygen radicals. Furthermore, its circulation time is
much shorter than that of RBC.
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infarction, or organ transplantation may result in the production ofoxygen radicals and tissue injury (Chang, 1997c, 2002; Gould et al.,2002). We are using a crosslinked polyhemoglobin-superoxide dismutase-catalase (PolyHb-SOD-CAT) (D’Agnillo and Chang, 1998a, 1998b,Powanda and Chang, 2002, Razack et al., 1997) (Fig. 15). UnlikePolyHb, PolyHb-SOD-CAT did not cause a significant increase inoxygen radicals when it is used to reperfuse ischemic rat intestine (Razacket al., 1997). More recently (Powanda and Chang, 2002), in a transientglobal cerebral ischemia rat model, we found that after 60 min ofischemia, reperfusion with polyHb resulted in significant increases in
Figure 15. Crosslinking of hemoglobin with two RBC enzymes to form
polyhemoglobin-catalase-superoxide dismutase (PolyHb-CAT-SOD). Unlike
polyhemoglobin, this has RBC enzymes that can remove oxygen radicals.
ISCHEMIAHYPOXANTHINE
XANTHINEOXIDASE
O2 REPERFUSIONPOLYHb-SOD-CATALASE
SUPEROXIDE
Superoxidedismutase
catalase
H2O2
H2O
OH+
OXYGENRADICALS
LESS TISSUE INJURY
Figure 16. In conditions like severe sustained hemorrhagic shock, stroke,
myocardial infarction and organ transplantation, reperfusion with polyhemoglo-
bin can sometimes result in oxygen radicals that causes tissue injury. PolyHb-
CAT-SOD can supply oxygen and at the same time significantly lower any
oxygen radicals formed.
Artificial Cell Bioencapsulation 13
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blood–brain barrier and the breakdown of blood–brain barrier (Fig. 17).On the other hand, polyHb-SOD-CAT did no result in these adversechanges (Powanda and Chang, 2002) (Fig. 17).
Polyhemoglobin Crosslinked with Tyrosinase
Crosslinking tyrosinase with hemoglobin results in a solublepolyhemoglobin-tyrosinase complex. This has the dual function oflowering systemic tyrosine that has the potential to slow the growth ofmelanoma (Yu and Chang, 2004). At the same time, polyhemoglobin,being in solution, can more readily perfuse the under perfuse melanomablood vessels bring more oxygen for more effective radiation therapy(Yu and Chang, 2004).
ARTIFICIAL CELLS IN THE NANO-DIMENSIONS
Blood Substitutes
Chang’s original idea of a complete artificial red blood cell (Chang,1957, 1964) is now being developed as third generation blood substitute(Chang, 2003). Hemoglobin lipid vesicles is one of these approaches
Figure 17. 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.
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(Philips et al., 1999; Rudolph et al., 1997; Tsuchida, 1998). We are using adifferent approach based on biodegradable polymer and nanotechnologyresulting in nano artificial RBC of 80 to 150 nanometre diameter (Changand Yu, 1998; Chang et al., 2003; Yu and Chang, 1994). These nanoartificial rbc contain all the red blood cell enzymes needed for the longterm function of the nano artificial RBC (Chang et al., 2003) (Fig. 18).Our recent studies show that using a polyethylene-glycol-polylactidecopolymer membrane we are able to increase the circulation time of thesenano artificial rbc to double that of polyHb (Chang et al., 2003) (Fig. 19).
Drug Delivery
We have been studying the preparation and characterization ofpolylactic acid nanocapsules containing ciprofloxacin for controlledrelease (Yu et al., 1999). The nanocapsules described above (Changand Yu, 1998; Chang et al., 2003; Yu and Chang, 1994) are also usefulfor the delivery of biologically active proteins and peptides. Otherapproaches based on nanodimension artificial cells in the form ofliposomes, nanoparticles, and nanocapsules are being increasing used bymany groups for drug delivery.
Figure 18. Nanodimension artificial red blood cells with polyethylene-glyco-
polylactide membrane. In addition to hemoglobin, this contains the same enzymes
that are normally present in red blood cells. Thus, it has the complete function
of the red blood cells.
Artificial Cell Bioencapsulation 15
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GENERAL
This paper only briefly summarizes some of the research from thisgroup on artificial cells in the macro, micro, nano, and molecular
Figure 19. Nanodimension artificial red blood cells with polyethylene-glyco-
polylactide membrane. The circulation time is double that of polyhemoglobin.
The circulation half time of polyhemoglobin in human is about 24 hours. This
means that the nanodimension artificial RBC may have a circulation time of
about 48 h in human.
ARTIFICIAL CELL BIOENCAPSULATIONExamples of application
• Artificial Organs: hemoperfusion
• Drug delivery systems: biodegradable micro & nano particles, liposomes etc.
• Red blood Cell Substitutes & oxygen therapeutics
• Enzyme encapsulation in enzyme therapy
• Encapsulation of cells including islets, hepatocytes, genetically engineered cells, stem cells etc
• Applications in Agriculture and Industry
Figure 20. Some examples of areas of application for artificial cells.
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dimensions. Bioencapsulation of biosorbent is rather simple and there-fore has been in routine clinical uses after only a few years of research anddevelopment (Chang, 1975; Winchester, 1988). Some areas of micro-encapsulation of drugs for delivery are also straight forward and are inclinical applications. However, in the more complicated areas like cellencapsulation and blood substitutes one cannot expect that clinicalapplication will come after just a few years of research and development.There are many areas of applications being explored by many groupsaround the world (Fig. 20). The promise and potential of artificial cellsalso comes with the need for further development towards actualapplications. Much needs to be done in order to move the more advanceuses of artificial cells to fruitful applications (Fig. 21).
Artificial cell is a rapidly evolving area and rapidly updating andlinks to other groups around the world can be found on our McGillUniversity website: www.artcell.mcgill.ca.
ACKNOWLEDGMENTS
This author acknowledges the supports of the Canadian Institutes ofHealth Research, the ‘‘Virage’’ Centre of Excellence in Biotechnologyfrom the Quebec Ministry, the MSSS-FRSQ Research Group award onBlood Substitutes in Transfusion Medicine from the Quebec Ministry of
BIOENCAPSULATION INMEDICINE, ARGRICULTURE & INDUSTRY
Further interdisciplinary group efforts
1. New or Improved membrane materials
2. New or Improved methods of Bioencapsulation
3. Characterization of material prepared frombioencapsulation: membrane & content
4. Functional properties: In-vitro and in-vivo
5. Scale up of optimized systems: process and method
6. Collaboration and Transfer of Technology to Industries
Figure 21. With the promise and progress in artificial cells, also come with the
need for further development towards actual applications. Much needs to be done
in order to move the more advanced application of artificial cells to fruitful
applications.
Artificial Cell Bioencapsulation 17
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Health and the Research Fund of the Bayer/Canadian Blood Agency/Hema Quebec/Canadian Institutes of Health.
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