biomaterials science || artificial cells

ChapTEr II.5.4 Artificial Cells 811

Weiss, W. J., Rosenberg, G., Snyder, A. J., Pierce, W. S., Pae, W. E., Kuroda, H., Rawhouser, M. A., Felder, G., Reibson, J. D., Cleary, T. J., Ford, S. K., Marlotte, J. A., Nazarian, R. A., & Hicks, D. L. (1999). Steady state hemodynamic and energetic characterization of the Penn State/3M Health Care Total Arti-ficial Heart. ASAIO J., 45, 189193.

Westaby, S., Katsumata, T., Houel, R., Evans, R., Pigott, D., Frazier, O. H., & Jarvik, R. (1998). Jarvik 2000 Heart:

Potential for bridge to myocyte recovery. Circulation, 98, 15681574.

Wilhelm, C. R., Ristich, J., Knepper, L. E., Holubkov, R., Wis-niewski, S. R., Kormos, R. L., & Wagner, W. R. (1999). Measurement of hemostatic indices in conjunction with transcranial doppler sonography in ventricular assist device patients. Stroke, 30, 25542561.

CHAPTER II.5.4 ARTIFICIAL CELLS

Thomas Ming Swi ChangDirector, Artificial Cells and Organs Research Centre, Departments of Physiology, Medicine and Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, QC, Canada

BASIC FEATURES OF ARTIFICIAL CELLS

The initial research on artificial cells (Chang, 1964) forms the basic principle that has been extended for use in many areas by many groups (Chang, 2005a, 2007, 2010; Liu and Chang, 2010). Very brief examples of some of these basic features include (Figure II.5.4.1):

1. Artificial cells can contain the same biological material as biological cells. In addition, they are more versa-tile since adsorbents, magnetic materials, drugs, cells, stem cells, enzymes, multienzyme systems, hemoglo-bin, microorganisms, vaccines, gene-for-gene therapy, genetically engineered cells, hormones, peptides, and many other materials can also be included separately or in combination (Figure II.5.4.1).

2. The membrane of an artificial cell separates its content from the outside, but at the same time the membrane can be prepared to selectively allow different types of molecules to cross (Figure II.5.4.1). For example, one can prepare artificial cell membranes that selec-tively allow the movement of molecules according to molecular size, lipid solubility, affinity to carrier mechanisms, etc. By selecting the proper membrane material, the permeability can range from a mem-brane that does not allow any molecules to cross, to those that allow even very large molecules such as proteins to cross. The membrane material includes polymer, biodegradable polymer, lipid, cross-linked protein, lipidpolymer complex, lipidprotein com-plex, and membrane with transport carriers.

3. Surface properties of artificial cell membrane can be varied by: (1) incorporation of negative or posi-tive charge; (2) incorporation of albumin to increase blood compatibility; (3) incorporation of antigens to bind antibodies or antibodies to bind antigen; (4) incorporation of polysaccharide such as heparin or polyethylene glycol (PEG) to increase compatibility or retention time in circulation.

4. In addition to being of cellular dimensions in the micron range, they can also be in the macro range, in the nano range or in the nanobiotechnological range (Figure II.5.4.1).

5. The artificial cell membranes can be ultrathin and yet strong. There is a large surface area to volume rela-tionship. For example, 10 ml of 20 m diameter arti-ficial cells has a total surface area of 2500 cm2, which is the same as that in an artificial kidney machine. Since the artificial cell membrane is also 100 times thinner, permeant molecules can potentially move across 10 ml of 20 m diameter artificial cells 100 times faster than across the artificial kidney machine (Chang, 1966). In addition, the microscopic size of artificial cells allows material to diffuse rapidly inside the artificial cells.

RESEARCH INTO THE APPLICATIONS OF ARTIFICIAL CELLS

This includes hemoperfusion, immunosorbents, drug delivery, blood substitutes, enzyme therapy, cell and stem cell therapy, biotechnology and nanobiotechnol-ogy, nanomedicine, regenerative medicine, agriculture, industry, aquatic culture, nanocomputers, nanorobot-ics, nanosensors, and other areas (Table II.5.4.1). The following is a brief overview of some examples of artificial cells.

ARTIFICIAL CELLS IN HEMOPERFUSION

This is the first large-scale US Food and Drug Admin-istration (FDA) approved routine clinical application in patients based on artificial cells. As mentioned above, the microscopic dimensions of artificial cells result in a large surface-to-volume relationship. This, together with the ultrathin membranes, allows artificial cells contain-ing bioadsorbents to be much more effective when com-pared to standard hemodialysis in removing toxins and drugs from the blood of patients (Chang, 1966, 1969, 1972a, 1975, 2007; Chang et al., 1973). In this method, thousands of 90 micron diameter artificial cells contain-ing adsorbents are retained by screens at either end, inside a column the size of a tea cup (Figure II.5.4.2). Blood from the patients containing toxins or drug can perfuse through the screens to come into contact with the

812 SECTION II.5 Applications of Biomaterials

artificial cells. Toxins or drugs diffusing into the artificial cells are removed by the adsorbents inside artificial cells. This cleanses the blood that returns to the patient.

routine Clinical Uses in patients with Suicidal Drug Overdose

The most common routine use of this approach is the use of microscopic polymeric artificial cells encapsulat-ing activated charcoal (Chang, 1969, 1975, 2007; Chang et al., 1973) (Figure II.5.4.2). This solves the major

problems of release of embolizing particles and dam-age to blood cells when bioadsorbents are used with-out artificial cell membranes. This was first successfully used in suicidal overdose patients (Chang et al., 1973) (Figure II.5.4.3). Since then, this has been become a rou-tine treatment worldwide for acute poisoning in adults and children, especially in suicidal overdose (Chang, 1975; Chang et al., 1973; Winchester, 1996; Kawasaki et al., 2000; Lopez Lago et al., 2002; Lin et al., 2002, 2004; Singh et al., 2004; Peng et al., 2004). This is par-ticularly useful in places where dialysis machines are not

extracellular

Basic principle of early Artificial Cells

(Chang 1957,1964)

intracellular

Ultrathin artificial membrane

Like biological cells: (1) content retained inside to(2) act on permeant molecules (3) release products (4) Intracellular environment(5) Extracellular environment

extracellular

Types of early Artificial Cells

(Chang 1957 to 1966)

cellsenzymes

hemoglobinmagnetic materials

adsorbentsproteins

Ultrathin artificial membrane

Present status of Artificial Cells

(Chang 2005 Nature Rev Drug Disc)

Membrane:

PolymericBiodegradableLipid Xlinked proteinConjugatedCarriers etc

CellsStem cellsEnzymes

Multienzyme systemshemoglobin

magnetic materialsMicroorganism

vaccinesgene for gene therapy

genetically engineered cellsbiotechnological products

adsorbentsdrug releasehormonespeptidesproteins

Macro

Micron

Nano

dimensions

Contents

FIGURE II.5.4.1 Upper: Basic principle of early artificial cells. Center: Different types of early artificial cells based on this basic principle. Lower: Present status of artificial cells with wide variations in contents, membrane material, and dimensions. (Figure from Chang (2007), Monograph with permission from World Scientific Publisher.)


easily available, and the hemoperfusion devices are less costly, especially outside North America. Hemoperfusion has saved the lives of thousands of suicidal drug over-dose patients in some countries outside North America.

Immunosorbents

The success in the clinical uses of artificial cells contain-ing bioadsorbents for detoxification has led to increas-ing interest in research and development in many other

areas. One of these is in artificial cells containing immu-noadsorbents (Chang, 1980).

Albumin can bind tightly to the ultrathin collodion membrane of adsorbent artificial cells (Chang, 1969). This was initially used to increase the blood compatibility of the adsorbent artificial cells for hemoperfusion (Chang, 1969). We also applied this albumin coating to synthetic immu-nosorbents resulting in blood compatible synthetic blood group immunoadsorbents (Chang, 1980). This albumin-coated synthetic adsorbent has been applied clinically for removing blood group antibodies from plasma for bone marrow transplantation (Bensinger et al., 1981). In addi-tion, albumin-coated collodion activated charcoal (ACAC) was found to effectively remove antibodies to albumin in animal studies (Terman et al., 1977). This has become the basis of one line of research in which other types of anti-gens or antibodies are applied to the collodion coating of the activated charcoal to form immunoadsorbents. Other immunoadsorbents based on this principle have also been developed for the treatment of human systemic lupus ery-thrematosus, removal of antiHLA antibodies in transplant candidates, treatment of familial hypercholesterolemia with monoclonal antibodies to low-density lipoproteins, and other conditions (Terman et al., 1979; Hakim et al., 1990; Wingard et al., 1991; Yang et al., 2004).

NANOBIOTECHNOLOGY FOR PARTIAL ARTIFICIAL RED BLOOD CELLS

There is much recent interest in nanobiotechnology and nanomedicine. This is a large and complex area that embraces many diverse approaches. One of these is to make the original artificial cells smaller, using the same basic principle and methods. This includes biodegradable nanoparticles, nanosphere, and nanocapsules. Examples include nano-artificial red blood cells with lipid mem-brane or biodegradable polymeric membranes. A later section will summarize other examples used in drug delivery systems. Much smaller nanobiotechnological

Blood + toxins

PATIENT

Blood

ADSORBENTS

PLATELETS

RBC, WBC

Toxins, drugs

metabolites

HEMOPERFUSION USING

ADSORBENT ARTIFICIAL CELL

Thousands of 90 micron diameter artificial cells containing adsorbents are retained inside the container by screens at either end. Blood containing toxins or drug can perfuse through the screen to come in contact with artificial cells. Toxin or drugs diffusing into the artificial cells are removed by the adsorbent inside artificial cells. This cleans the blood that returns to the patient.

membrane

A typical adsorbent artificial cell

FIGURE II.5.4.2 Upper right: A hemoperfusion device held in the hand. Left: Schematic representation of the hemoperfusion device. Right: Schematic representation of an artificial cell containing adsor-bent. (Figure from Chang (2007), with copyright permission from World Scientific Publisher.)

0

10

0 40TIME AFTER ADMISSION (HOURS)

Methyprylon(mg/dl)

Comatose

Hypotensive

Cardiac arrest

Normo-

Tensive

Pain reflex

Spontaneous

movementFully recovered

HP

FIGURE II.5.4.3 Clinical and laboratory results of hemoper-fusion in a patient with severe suicidal methyprylon overdose. ( Figure from Chang (2007), with copyright permission from World Scientific Publisher.)

TaBLE I I .5.4.1 Artificial Cell: Applications

Hemoperfusion Immunosorbents Drug delivery Blood substitutes Enzyme and gene therapy Cell and stem cell therapy Biotechnology and nanobiotechnology Nanomedicine Regenerative medicine Agriculture, industry, aquatic culture Nanocomputers and nanorobotics Nanosensors, etc.

Chang, 2005aChang, 2007Chang, 2009www.artcell.mcgill.ca


complexes can be prepared by the assembling of biologi-cal molecules. Examples include the assembling of hemo-globin molecules into soluble polyhemoglobin, and the assembling of hemoglobin molecules and enzymes into soluble polyhemoglobinenzyme complexes.

Nanobiotechnology and artificial Cells

Nanobiotechnology is the assembling of biological mol-ecules into nanodimension complexes of different config-urations. These include nanostructures with nano-range diameters, membranes with nanodimension thickness or nanotubules with nanodimension diameters.

The first nanobiotechnology approach reported is the cross-linking of hemoglobin into an ultrathin poly-hemoglobin (PolyHb) membrane with nanodimension thickness (Chang, 1964, 1965) (Figure II.5.4.4). This is used to form the membrane of artificial red blood cells (Chang, 1964, 1965, 1972a). If the emulsion is made very small, then the whole submicron artificial cells can be cross-linked into PolyHb of nanodimension. Glutar-aldehyde can cross-link hemoglobin into soluble PolyHb of nanodimension, each consisting of an assembly of 45 hemoglobin molecules (Chang 1972b; Keipert and Chang, 1985, 1987) (Figure II.5.4.4).

Two groups have independently developed this 1971 (Chang, 1971b) basic method of glutaraldehyde cross-linking for clinical use. One is glutaraldehyde human PolyHb (PolyHb) (Gould et al., 1998, 2002). Their phase III clinical trial shows that this can replace blood lost in trauma surgery by keeping the blood hemoglobin at an acceptable level. More recently they have carried out clinical trials in prehospital ambulance patients (Moore et al., 2009). The second PolyHb is glutaraldehyde-cross-linked bovine PolyHb which has been tested in phase III clinical trials (Pearce and Gawryl, 1998; Sprung et al., 2002; Pearce et al., 2006; Jahr et al., 2008). South Africa has approved this for routine clinical use in patients. Unlike red blood cells, there is no blood group in PolyHb, and thus PolyHb can be given on the spot, without wait-ing for typing and cross-matching in the hospital. PolyHb is also free from infective agents such as HIV, hepatitis C, bacteria, parasites, and so on. Furthermore, whereas donor blood has to be stored at 4C and is only good for 42 days, PolyHb can be stored at room temperature for more than one year. Thus, PolyHb can have important uses in a number of clinical conditions, notably for sur-gery when there is no potential for ischemia-reperfusion, as will be discussed below (Chang, 2006a, 2009).

Nanobiotechnology and the assembling of hemoglobin with antioxidant Enzyme

As PolyHb can be kept at room temperature and used immediately, it can have potential for treating severe bleeding (hemorrhagic shock). However, this has to be done as soon as possible, since if there is much delay

PolyHb alone might result in the production of oxygen radicals that cause tissue injury (ischemia-reperfusion injuries). Antioxidant enzymes normally present in red blood cells are insufficient to prevent this problem. We use glutaraldehyde cross- linking to assemble a soluble nano-biotechnology complex by cross-linking hemoglobin, superoxide dismutase, and catalase into PolyHb-SOD-CAT (DAgnillo and Chang, 1998; Chang 2006b, 2007, 2008) (Figure II.5.4.4). This way, one can increase the anti-oxidant enzymes to much higher levels than those in red blood cells.

Obstruction of arteries due to clots or other causes can result in stroke (cerebral ischemia) or heart attack (myocardial infarction). As it is a solution, PolyHb can more easily perfuse partially obstructed vessels. How-ever, if there is prolonged lack of oxygen, reperfusion with PolyHb alone may result in damaging oxygen radi-cals resulting in ischemia-reperfusion injuries. Thus, in patients with coronary heart disease and myocardial ischemia, infusion with polyhemoglobin can result in ischemia-reperfusion injury that can be severe enough to result in heart attack (myocardial infarction). In severe trauma, one is often faced with hemorrhagic shock from severe bleeding, in addition to cerebral ischemia due to head injuries. In a hemorrhagic shock and stroke rat model, after 60 minutes of ischemia, reperfusion with PolyHb resulted in significant increase in the break-down of the bloodbrain barrier, and an increase in brain water (brain edema) (Powanda and Chang, 2002). On the other hand, PolyHb-SOD-CAT did not result in these adverse changes (Powanda and Chang, 2002). In sustained hemorrhagic shock due to severe loss of blood, ischemia-reperfusion can result in irreversible shock due to leakage of bacteria and endotoxins from the intestine into the circulating blood. Our study shows that in an ischemia-reperfusion rat inestinal model, polyhemoglo-bin resulted in significant increases in oxygen radicals. On the other hand, the use of PolyHb-SOD-CAT did not result in any significant increases in oxygen radicals (Razack et al., 1997; Chang, 2007).

Nanobiotechnology for the assembling of hemoglobin with Other Enzymes

The microcirculation structure in tumors is abnormal, and as a result there is a decrease in perfusion by oxygen carrying red blood cells. This results in a lower oxygen tension in the tissue of many types of tumors (Pearce and Gawryl, 1998). It is known that radiation therapy and some chemotherapy can work better when the tissue oxygen tension is higher. PolyHb can more easily perfuse the abnormal microcirculation of tumors to supply oxy-gen needed for chemotherapy or radiation therapy. With a circulation half-time of 24 hours, the effect is not long-term and can be adjusted to the duration of chemotherapy or radiation therapy. When used together with chemo-therapy, PolyHb decreases tumor growth and increases the lifespan in a rat model of gliosarcoma brain tumor


SOD CAT tyrosinase

Polyhemoglobin (PolyHb)Chang, Science 64, BBC 71 PolyHb-catalase

Chang BBC 1971

PolyHb-CAT-SODDAgnillo & Chang, Nature Med 1998

PolyHb-tyrosinaseBLYu & Chang,

Melanoma Res 2004

Hb

Figure from Chang 1965

Figure from Chang 1965

NANOBIOTECHNOLOGY:

Definition: assembling of biological molecules into nanodimension structures(membrane thickness, nanotubule diameter or diameter of nanostructures)

POLYHEMOGLOBIN

1964 Chang - Diacid1971 Chang - Glutaraldehyde

1 2

1 2

2

1

1 2

1 2

1 2

1 2

1 2

1 2

1 2

1 2

CONJUGATED HB

1964 Chang: polyamide 1968 Wong: dextran 1970 Davis: PEGylation1980 Iwashita: polyethylene glycol

1 2

1 2

CAT

FIGURE II.5.4.4 Nanoartificial cells can be prepared in the nanodimensions as membrane enclosed nanoartificial cells or by the use of nanobiotechnology to assemble biological molecules together into nanodimension structures. Upper: An example of assembling of biological molecules to form PolyHb and conjugated Hb. Lower: Examples of different types of nanobiotechnology based PolyHb-enzymes. (Figure from Chang (2007), with copyright permissions from World Scientific Publisher.)


(Pearce and Gawryl, 1998). We have recently cross-linked tyrosinase with hemoglobin to form a soluble PolyHb- tyrosinase complex (Yu and Chang, 2004) (Figure II.5.4.4). This has the dual function of supplying the needed oxy-gen, and at the same time lowering the systemic levels of tyrosine needed for the growth of melanoma. Intra-venous injections delayed the growth of the melanoma without causing adverse effects in the treated animals (Yu and Chang, 2004).

polyhemoglobin-Fibrinogen

Blood is a multifunctional fluid. As a blood substitute, PolyHb is limited by its lack of platelets and/or coagula-tion properties. In situations of high blood volume loss, large volumes of PolyHb have to be infused to replace the lost blood. Replacing the loss of red blood cells without replacing the lost platelets and coagulation factors can result in the inability of the blood at the injured sites to clot, resulting in continued severe bleeding. As a result, platelets and coagulation factors have to be infused in these situations. Unfortunately, donor platelets are very hard to obtain. There has been development in platelet substitutes to combat thrombocytopenia, but with lim-ited success. We therefore prepared a novel blood substi-tute that is an oxygen carrier with platelet-like activity. This is formed by cross-linking fibrinogen to hemoglobin to form polyhemoglobin-fibrinogen (PolyHb-Fg) (Wong and Chang, 2007). This was studied and compared to PolyHb for its effect on coagulation, both in vitro and in vivo. In the in vitro experiments PolyHb-Fg showed similar clotting times as whole blood, whereas PolyHb showed significantly higher clotting times. This result was confirmed in in vivo experiments using an exchange transfusion rat model. Using PolyHb, exchange transfu-sion of 80% or more increased the normal clotting time (12 mins) to >10 minutes. Partial clots formed with PolyHb did not adhere to the tubing wall. With PolyHb-Fg, a normal clotting time (12 mins) is maintained even with 98% exchange transfusion.

NANOBIOTECHNOLOGY FOR COMPLETE ARTIFICIAL RED BLOOD CELLS

Original Micron Dimension Complete artificial red Blood Cells

The first artificial red blood cells (RBCs) prepared in 1957 (Chang, 1957) have an oxygen dissociation curve simi-lar to red blood cells. We continued to study a number of artificial cell membranes including cellulose, sili-cone rubber, 1,6-hexamethylenediamine, cross-linked protein, phospholipidcholesterol complexes on cross-linked protein membrane or polymer (Chang, 1964, 1965, 1972a). However, these artificial RBCs, even with a diameter down to one micron, survived for a short time in the circulation after intravenous injection. Our

study showed that the long circulation time of RBCs is due to the presence of neuraminic acid on the membrane (Chang, 1965, 1972a). This led us to study the effects of changing surface properties of the artificial RBCs (Chang, 1964, 1965, 1972a). This has resulted in signifi-cant increases in circulation time, but is still insufficient for clinical use.

Submicron hemoglobin Lipid Vesicles as artificial red Blood Cells

We prepared larger artificial cells with lipid membrane by supporting the lipid in the form of lipidprotein mem-brane and lipidpolymer membrane (Chang, 1972a). Others later reported the preparation of submicron (0.2 micron) diameter artificial RBCs using lipid mem-brane vesicles to encapsulate Hb (Djordjevich and Miller, 1980). This increased the circulation time significantly, although it was still rather short. Many investigators have since carried out research to improve the prepara-tion and circulation time. The most successful approach to improve the circulation time is to incorporate polyeth-ylene glycol (PEG) into the lipid membrane of artificial RBCs, resulting in a circulation half-time of more than 30 hours (Philips et al., 1999). Another group in Japan has for many years carried out extensive research and development, commercial development, and preclinical animal studies. Their extensive studies are available in many publications and reviews (Kobayashi et al., 1997; Sakai et al., 2004; Tsuchida, 1998).

Biodegradable polymeric Nanodimension Completely artificial red Blood Cells

We have used a biodegradable polymer, polylactic acid (PLA), for the microencapsulation of Hb, enzymes, and other biologically active material since 1976 (Chang, 1976). More recently, we started to prepare artificial RBCs of 100 nanometer mean diameter using PLA, PEGPLA membrane, and other biodegradable polymers (Yu and Chang, 1994, 1996; Chang, 1997a, 2006c, 2007; Chang et al., 2003). A typical electron micrograph for the biodegradable polymer Hb nanocapsules prepared with d,l-PLA shows that they are spherical and homo-geneous. Their diameter ranges from 40120 nm, with a mean diameter of 80 nm. The membrane thickness is 515 nm. We have replaced most of the 6 g/dl of lipid membrane in Hb lipid vesicles with 1.6 g/dl of biode-gradable polymeric membrane material (Figures II.5.4.5 and II.5.4.6). This marked decrease in the lipid com-ponent would lessen the effects on the reticuloendo-thelial system (RES), and lessen lipid peroxidation in ischemia-reperfusion.

We can increase the Hb content in the PLA nano-artificial cell suspension from 3 g/dl to 15 g/dl (the same as whole blood). This has normal P50, Hills coeffi-cient, and Bohr coefficient. Nanocapsules can be prepared


with up to 15 g/dl Hb concentration. The preparation of PLA nano-artificial RBCs does not have adverse effects on the Hb molecules. This is shown by the following experimental results. There is no significant difference in the oxygen affinity (P50) of Hb nano-artificial RBC and the original Hb used for the preparation, the Hill coef-ficient is 2.4 to 2.9 and the Bohr effect is 0.22 to 0.24 (Yu and Chang, 1994, 1996; Chang, 1997a, 2006c, 2007; Chang et al., 2003).

We have earlier carried out basic research on artifi-cial cells containing multienzyme systems with cofactor recycling (Chang, 1987). A number of enzymes such as carbonic anhydrase, catalase, superoxide dismutase and the MetHb reductase system, normally present in RBC have been encapsulated within nano-artificial RBC and

retain their activities (Figure II.5.4.5) (Chang et al., 2003; Chang, 2007). Unlike lipid membrane, biodegradable polymeric membrane is permeable to glucose. Thus, the inclusion of an RBC MetHb reductase enzyme system prevents MetHb formation even at 37C, and we can also convert MetHb to Hb at 37C. In addition, unlike lipid membrane, the nanocapsule membrane allows plasma factors such as ascorbic acid to enter the nano-capsules to prevent MetHb formation (Figure II.5.4.5).

In order to increase the circulation time, we synthe-sized a new PEGPLA copolymer for the artificial RBC membrane (Chang et al., 2003). After a 30% blood volume toploading using PolyHb (10 g/dl), the best PolyHb can only attain a maximal Hb concentration of 3.35 g/dl. The best PEGPLA nano-artificial RBC,

EMBDEN-MEYERHOF SYSTEM

glucose Adenine inosine

NAD

NADHMETHB

HEMOGLOBIN

2,3-DPG

ATP

lactate

glucose

lactate

CATALASESUPEROXIDE DISMUTASE superoxide

H2O2

REDUCING AGENTSfrom plasma

CARBONIC ANHYDREASE CO2

O2

ARTIFICIAL RBC

(Chang 1957,1964) 1 or larger

RBC MEMBRANE ARTIFICIAL MEMBRANE

RED BLOOD CELLS (RBC)

7-8

LIPID MEMBRANE NANO RBC

Djordjevich & Miller 1980)0.2-0.4 (200-400 nm)

BIODEGRADABLE POLYMERIC

MEMBRANE NANO RBC

(Chang & WPYu, 1994)80-200 nm

FIGURE II.5.4.5 Upper: Composition of red blood cell and artificial red blood cells. Lower Left: Red blood cells. Lower Right: First artificial RBC of 1 micron or larger diameter, first lipid membrane nanodimension artificial RBC, first nanodimension biodegradable polymeric membrane artificial RBC. (Figure from Chang (2007), with copyright permissions from World Scientific Publisher.)


on the other hand, can reach a maximal Hb concentra-tion of 3.60 g/dl. After extensive research, we now have a circulation time in rats that is double that of glutaraldehye cross-linked PolyHb. Since the RES in rat is much more efficient in removing particulate mat-ter compared to humans, it is likely that the half-time would be longer in humans. Recent long-term studies in rats show no adverse effects in the kidney and the reticuloendothelial systems (liver and spleen) (Liu and Chang, 2008).

CELLS, ISLETS, STEM CELLS, GENETICALLY ENGINEERED CELLS, AND MICROORGANISMS

Introduction

The first artificial cells containing intact biological cells were reported in 1964 based on a drop method (Chang, 1964), and it was proposed that: protected from immu-nological process, encapsulated endocrine cells might survive and maintain an effective supply of hormone (Chang et al., 1966) (Figure II.5.4.1).

artificial Cells Containing Islets, hepatocytes, and Other Cells

With Changs encouragement and initial consultation, Sun from Conaught Laboratory and his collaborator developed Changs original drop method (Chang, 1964,

1965, 1972a; Chang et al., 1966) by changing to alginate-polylysine-alginate (APA) as the artificial cell membrane (Lim and Sun, 1980). They showed that after implan-tation, the insulin secreting islets inside artificial cells indeed remained viable and continued to secrete insulin to control the glucose levels of diabetic rats (Lim and Sun, 1980; Sun et al., 1996). Extensive research has been carried out by many laboratories around the world since that time and is available in reviews (Calafiore, 1999; De Vos et al., 2002; Orive et al., 2003, 2004; Hunkeler, 2003; Chang, 2005a,b, 2007). The major hurdle is the need for long-term function after implantation.

We have been studying the use of artificial cells con-taining liver cells (hepatocytes) for liver support. Implant-ing these increases the survival of acute liver failure rats (Wong and Chang, 1986); lowers the high bilirubin level in congenital Gunn rats (Bruni and Chang, 1989); and prevents xenograft rejection (Wong and Chang, 1988). We developed a two-step cell encapsulation method to improve the APA method, resulting in improved survival of implanted cells (Wong and Chang, 1991). Artificial cells containing hepatocytes effectively lower the sys-temic bilirubin in hyperbilirubinemia Gunn rats (Bruni and Chang, 1989, 1995). We used the two-step method plus the co-encapsulation of stem cells and hepatocytes into artificial cells (Liu and Chang, 2000). This results in further increases in the viability of encapsulated hepa-tocytes, both in culture and after implantation (Liu and Chang, 2002). One implantation of artificial cells con-taining both hepatocytes and stem cells into Gunn rats

MEMBRANE MATERIAL

HB LIPID VESICELS

lipidlipidlipidlipidlipidlipidlipidlipid

biodegradable polymerbiodegradable polymerbiodegradable polymerbiodegradable polymerbiodegradable polymerbiodegradable polymerbiodegradable polymerbiodegradable polymerbiodegradable polymer

2

4

6

0NANO ARTIFICIAL RBCs

g/dl

POLYLACTIDE ACID

FATE OF POLYLACTIDE MEMBRANE

LACTIC ACID

CO2+ H

20

Lactic

Acid

(meq)

Nano rbc

83meq/500 ml

Resting Body

Production

1200meq/ day

Max body

Removal

7680meq/day

FIGURE II.5.4.6 Top: Amount of membrane material in hemoglobin lipid vesicles compared to polylactide membrane nano RBC. Bottom: Fate of polylactide membrane in PLA nano RBC compared to PLA metabolism in the body. (Figure from Chang (2007), with copyright permis-sion from World Scientific Publisher.)


lowers the systemic bilirubin levels and maintains this low level for two months (Liu and Chang, 2003). With-out stem cells, implanted hepatocytes in artificial cells can only maintain a low level for one month.

Genetically Engineered Cells and Microorganisms

Many groups have carried out extensive research on arti-ficial cells containing genetically engineered cells. This is a very important area which includes potential applica-tions in amyotrophic lateral sclerosis, Dwarfism, pain treatment, IgG1 plasmacytosis, Hemophilia B, Parkinson-ism and axotomized septal cholinergic neurons, tumor suppression, and other areas (Winn et al., 1994; Saitoh et al., 1995; Aebischer et al., 1996; Al-Hendy et al., 1996; Basic et al., 1996; Tan et al., 1996; Hagihara et al., 1997; Dalle et al., 1999; Bachoud-Lvi et al., 2000; Lorh et al., 2001; Xu et al., 2002; Cirone et al., 2002; Bloch et al., 2004). To avoid the need for implantation, we studied the oral use of artificial cells containing microorganisms for the removal of cholesterol (Garofalo and Chang, 1991) or conversion of phenol to tyrosine (Lloyd-George and Chang, 1995). We have also studied the use of genetically engineered nonpathogenic E. coli DH5 cells to lower systemic urea in renal failure rats (Prakash and Chang, 1993, 1996; Chang, 1997b, 2005a,b). Genetically engineered E. coli allows for proof of principle. For safe use, we have studied the use of a modified Lactobacillus, similar to those used in yoghurt, for enclosure in artificial cells to remove urea (Chow et al., 2003).

General

Cell bioencapsulation for cell therapy has been exten-sively developed by many groups, especially using arti-ficial cells containing endocrine tissues, hepatocytes, genetically engineered cells, and stem cells (Chang, 1972a; Orive et al., 2003; Chang, 2005a,b, 2007). The major hurdle is the need to develop systems that can function on a long-term basis after implantation.

ARTIFICIAL CELLS CONTAINING STEM CELLS IN REGENERATIVE MEDICINE

The above examples of artificial cells containing islets, cells, genetically engineered cells, and other cells require long-term treatment. Developments are needed before artificial cells are ready for long-term implantation use. To allow for more immediate use, one area of research in this laboratory is to look at the use of artificial cells in regenerative medicine. For this we have been study-ing the use of artificial cells containing bone marrow stem cells for liver regeneration. Marked decrease in liver function due to fulminant hepatic failure or exten-sive liver resection for metastatic cancer or severe injury can result in the death of the patient. Since liver can

regenerate if given the required time and conditions, we therefore studied whether artificial cells containing bone marrow stem cells can maintain the experimental animal alive long enough to allow the liver to regenerate and the animal to recover (Figure II.5.4.7).

artificial Cells Containing Bone Marrow Stem Cells in 90% hepatectomized rats

In the present study we injected intraperitoneally artificial cells containing bone marrow stem cells into 90% hepa-tectomized rats (Liu and Chang, 2005, 2006) (Figure II.5.4.7). In the hepatectomized groups that received no treatment, only 20% of the animals survived by the sec-ond day. In the hepatectomized animals receiving free bone marrow stem cells, only 20% of the animals sur-vived on the seventh day. There was no significant differ-ence in survival rate between the normal control group (with no hepatectomy) and the group receiving artificial cells containing bone marrow stem cells. The survival for both groups was 100% when followed for 14 days. Thus it would appear that in rats, artificial cells contain-ing bone marrow stem cells are effective in improving the survival rates of 90% hepatectomized rats. For those rats that survived at week 2 post hepatectomy, the remnant livers were removed and the weights were measured. In the 90% hepatectomized rats that received artificial cells containing bone marrow stem cells, the liver wet weights had recovered to the same size as that of the normal con-trol group (Liu and Chang, 2006). On the other hand, those few animals in the other groups that survived had very low liver weights.

0 14

20

100

RECOVERY RATES %

Free BM stem cells

DAYS AFTER 90% HEPATECTOMY

hepatectomy

Encap BM stem cells

No hepatectomy

** ** ** **

*

FIGURE II.5.4.7 Survival rates of rats with 90% of liver surgi-cally removed, hepatectomy, compared to those without removal of liver tissues, no hepatectomy. One peritoneal injection of arti-ficial cells containing bone marrow stem cells resulted in the sur-vival of the 90% hepatectomized rats. On the other hand, free bone marrow stem cells did not significantly increase the survival rates (Liu and Chang, 2006). (Figure from Chang (2007), with permission from World Scienctific Publisher.)


plasma hepatic Growth Factor (hGF) Levels

In the hepatectomized groups that received artificial cells containing bone marrow stem cells, the blood levels of HGF peaked at day 2 and 3 post-surgery, and were sig-nificantly higher than the other groups (Liu and Chang, 2006). After this, the levels decreased and returned to pre-surgery levels on day 14. In the hepatectomized groups that received: (1) no treatment; or (2) free bone marrow stem cells, the blood HGF levels were much lower.

Laparotomy, histology, and Immunocytochemistry

Two weeks after implantation, laparotomy shows that in the group that received artificial cells containing bone marrow stem cells, the artificial cells remained in the peritoneal cavity. They were found freely dissemi-nated throughout the peritoneal cavity, aggregated behind the liver or under the spleen, or attached to the large omentum. Histological examination of bone marrow stem cells in the artificial cells showed that before transplantation most cells were polygonal, star-like, some with tail shaped cytoplasm. When they were retrieved two weeks after transplantation, most cell morphology transformed to round or oval (Liu and Chang, 2006).

Before implantation, the bone marrow stem cells recovered from artificial cells did not show positive immunochemistry stain. However, when bone marrow stem cells were recovered from artificial cells at week 2 after implantation, there were scattered cells positively stained with hepatocyte markers CK8 and CK18, and also albumin production. There were also cells that stained positively with AFP (Liu and Chang, 2006). In the bone marrow stem cells recovered from artificial cells retrieved 2 weeks post-transplantation, there were cells stained positive for PAS, indicating that there were also cells capable of glycogen production.

possible Mechanisms responsible for the recovery of 90% hepatectomized rat Model

What are the mechanisms responsible for the survival and recovery of the 90% hepatectomized rat model in our present study? Most likely this is due to two com-bined mechanisms (Chang, 2007). 1. Transdifferentiation of bone marrow stem cells in the

artificial cells into hepatocytes. This is supported by our immunochemistry stain studies which show that some of the bone marrow stem cells recovered from artificial cells transdifferentiated into hepatocyte-like cells that expressed ALB, CK8, CK18, and AFP, which are typical markers of hepatocytes. They also produced albumin and glycogen. However, this is a

slow process that takes time, and is most likely only responsible for the later phases of the recovery of the animals.

2. Hepatic growth factor (HGF) is an important factor in liver regeneration (Rokstad et al., 2002) and also in stimulating the transdifferentiation of bone mar-row cells into hepatocytes (Spangrude et al., 1988). There are two subgroups of HGF, one >100,000 Da the other 64,000 Da (Ito and Chang, 1992). HGF of >100,000 mw secreted by hepatocytes are retained and accumulated in the artificial cells, thus helping to increase the regeneration of hepatocytes in the artificial cells (Kashani and Chang, 1988, 1991) and also the transdifferentiation of BMCs. The smaller molecule weight HGF of


injection (Figure II.5.4.1). Implanted urease artificial cells convert systemic urea into ammonia (Chang, 1964, 1965). Implanting artificial cells containing catalase replaces the defective enzyme in mice with a congenital defect in catalase acatalasemia (Chang and Poznansky, 1968). The artificial cells protect the enclosed enzyme from immu-nological reactions (Poznansky and Chang, 1974). Artificial cells containing asparaginase implanted into mice delay the onset and growth of lymphosarcoma (Chang, 1971b).

Giving enzyme artificial cells by mouth avoids the need for repeated injection. For example, artificial cells con-taining urease and ammonia adsorbent can lower the sys-temic urea level (Chang, 1972a). In Lesch-Nyhan disease, enzyme defect results in the elevation of hypoxanthine to toxic levels. Given by mouth, artificial cells contain-ing xanthine oxidase lower the toxic systemic hypoxan-thine levels in an infant with this disease (Chang, 1989; Palmour et al., 1989). Phenylketonuria is a more com-mon congenital enzyme defect. Artificial cells containing phenylalanine ammonia lyase given by mouth lower the systemic phenylalanine levels in phenylketonuria (PKU) rats (Bourget and Chang, 1985, 1986). This has led to investigation into recombinant sources of this enzyme (Safos and Chang, 1995; Sarkissian et al., 1999; Liu et al., 2002) that are now being tested in clinical trials.

Multienzyme Systems with Cofactor recycling

Most enzymes in the body function as multienzyme sys-tems with cofactor recycling. After basic research on arti-ficial cells containing multienzyme systems (Campbell and Chang, 1976; Cousineau and Chang, 1977; Yu and Chang, 1982; Chang 1985; Ilan and Chang, 1986) we looked into their possible use. Thus, artificial cells contain-ing three different enzymes can convert metabolic waste such as urea and ammonia into essential amino acids (Gu and Chang, 1988, 1990). The needed cofactor, NADH, can be recycled and retained inside the artificial cell by cross-linking to dextran or by the use of a lipidpolymer membrane. As discussed earlier, under artificial red blood cells, all the multienzyme system in red blood cells can be incuded inside nanodimensional artificial red blood cells (Chang, 2007; Chang et al., 2003) (Figure II.5.4.5).

DRUG DELIVERY

Gene and enzyme therapy is a form of drug delivery using artificial cells. However, the genes and enzymes are retained inside the artificial cells at all times, and act on substrates diffusing into the artificial cells. Artifi-cial cells in different modified forms have also been used in drug delivery. However, these are used to separate the drug from the external environment, and to release the drug at a specific site and a specific rate when and where it is needed. Drug delivery is an extremely large and wide area, and many excellent reviews and books

are available. In this chapter, only artificial cell-related systems will be very briefly summarized.

polymeric Membrane artificial Cells of Microscopic Dimensions

Luzzi (1970) used nylon membrane artificial cells, microcapsules, prepared as reported (Chang, 1964), to microencapsulate drugs for slow release for oral admin-istration. Others have also extended this approach. How-ever, the modern approaches in drug delivery systems are based on injectable biodegradable systems.

Biodegradable polymeric artificial Cells (Microparticles, Nanoparticles, Microcapsules, Nanocapsules)

Biodegradable polylactide membrane artificial cells have been prepared to contain enzymes, hormones, vaccines, and other biologicals (Chang, 1976). The polylactide polymer can degrade in the body into lactic acid, and finally into water and carbon dioxide. Variations in preparation can result in artificial cells that release insu-lin at different rates (Chang, 1976) (Figure II.5.4.8).

We have also used these for the slow release of pros-taglandin E2 (Zhou and Chang, 1988) and ciprofloxa-cin (Yu et al., 1998, 2000). Biodegradable drug delivery systems are now used widely in different forms, rang-ing from microscopic to nanodimensions. They are also known as nanoparticles, nanocapsules, polymersomes, nanotubules, etc. Langers group has written an excellent review on this topic (LaVan et al., 2002). Copolymers of polyethylene glycol (PEG) and polylactic acid (PLA) have been used to increase the circulation time of nanodimen-sional artificial cells. As described earlier in this chapter, this also forms the basis for preparing nanodimension PEGPLA membrane artificial red blood cells (Yu and Chang, 1996; Chang et al., 2003; Chang, 2005a, 2006a).

Liposomes Evolved into Lipid Membrane artificial Cells and Then Back into polymeric Membrane artificial Cells

Bangham (1965) reported the preparation of liposomes each consisting of microspheres of hundreds of concen-tric lipid bilayers multi-lamellar. This was initially used as membrane models in basic research. Gregoria-dis (1976) first reported the use of liposomes as drug delivery systems. However, the large amount of lipid in the multi-lamellar liposome limits the amount of water-soluble drugs that can be enclosed. Thus, the basic principle and method of preparing artificial cells using ether as the dispersing phase (Chang, 1957, 1964) was extended by researchers into what they call an ether evaporation method to form single bilayer (unilamel-lar) lipid membrane liposomes (Deamer and Bangham, 1976). These lipid membrane artificial cells have since


been extensively studied for use as drug delivery systems (Torchilin, 2005). Surface charges have also been incor-porated into liposomes for possible targeting of drug, and more recently the use of positively charged lipid to complex with DNA. Polyethylene glycol (PEG) has also been incorporated to the liposome surface to result in longer circulation time. Thus, lipid vesicles are becoming more like the lipidpolymer membrane arti-ficial cells (Chang, 1972a), and are no longer pure lipid vesicles. Further development led to the incorporation of antibodies into the lipid membrane, to allow for targeting to cells with the corresponding antigens. The principle of loading magnetic particles into artificial cells (Chang, 1966) has also been used for loading into liposome, allowing for magnetic targeting. One major advantage of lipid vesicles is in their ability to fuse with cellular membrane or membranes of intracellular organ-elles. This allows for much versatility in their ability to deliver drugs to different sites of the cells. A number of drugs in PEGlipid vesicles have already been approved for clinical use or are in clinical trial (Torchilin, 2005). Dischers group (Discher et al., 1999; Photos et al., 2003) tried to increase the strength of the PEGlipid membrane artificial cells by using self-assembling of copolymer to form a membrane of PEG polymer. This significantly increased the circulation time and strength when com-pared to PEGlipid membrane artificial cells Thus, mul-tilamellar liposome has evolved into lipid membrane artificial cells, then polymer(PEG)lipid membrane arti-ficial cells, and finally back to the original polymeric membrane artificial cells (Chang, 1964) now called polymersomes.

OTHER AREAS OF ARTIFICIAL CELLS

artificial Cells Containing Magnetic Materials

When magnetic material is included in artificial cells (ACs) containing bioactive materials, one can use an external magnetic field to direct the artificial cells (Chang, 1966). This way, the magnetic field can: (1) direct the movement of the AC; (2) remove the AC after reaction; (3) retain the AC at specific site of action; (4) stir or agitate the AC as in bioreactors. This principle is now being used very extensively in bioreactors, in removing specific materials from a mixture as in diag-nositcs kits, in drug delivery systems, and in other areas of application.

Nanobiosensors

Nanobiosensors is an area that is of increasing interest, and different approaches are being investigated. One of the many approaches is to use a biosensor where a lipid bilayer is tethered on ultrathin polymeric sup-port to form a lipidpolymer complex. In this form, dif-ferent channels can be inserted into the membrane to allow for selective movement of specific solute for detection, somewhat similar to the principle reported earlier (Chang, 1969, 1972a; Rosenthal and Chang, 1971, 1980). Another approach that is also possible is to encapsulate enzymes inside artificial cells of micro-scopic or nanodimension. This way, the product of enzymatic reaction can be followed by fluorescence or

Fast release

slower release

Very slow release

0 10 20TIME (HRS)

% R

ELEA

SE

100

50

0

Polylactide artificialcells containing insulin

FIGURE II.5.4.8 Biodegradable membrane artificial cells have been prepared to contain enzymes, hormones, vaccines, and other biologicals (Chang, 1976). This figure summarizes the result of polylactide artificial cells prepared by double emulsion method. Variations in the molecular weight of polylactide and the thickness of the membrane can result in artificial cells that release insulin at different rates. Faster release comes from the encapsulation of insulin solution at high concentration. Very slow release comes from the encapsulation of insulin crystals (Chang, 1976). (Figure from Chang (2007), with copyright permission of World Scientific Publisher.)


other methods. The ability to prepare artificial cells with intracellular compartmentation (Chang, 1965, 1972a; Chang et al., 1966) would allow multi-step enzyme reaction to occur and be detected separately. Depending on the type of reaction being followed, one can use either polymeric membrane artificial cells, lipid membrane artificial cells or lipidpolymer membrane artificial cells.

artificial Cells Containing radioisotopes or radio-Opaque Material

The general principle of artificial cells could be explored in many other areas. Thus, artificial cells containing radioactive isotopes or antimetabolites might be used for intra-arterial injection into tumors. In this case, some of the microcapsules might lodge at the tumor site, while others would be carried by lymphatic channels to metas-tases in regional lymph nodes. Artificial cells containing radio-opaque material would provide a contrast medium. Provided they can circulate readily in the bloodstream, they might be used as vehicles for contrast materials in angiography.

Nonmedical Uses of artificial Cells

There are many developments and uses of the principle of artificial cells for agriculture, bioreactors, cosmetics, food production, aquatic culture, nanocomputers, and nanorobotics. However, these are not within the scope of this chapter.

THE FUTURE OF ARTIFICIAL CELLS

The 1972 monograph Artificial Cells (Chang, 1972a) predicted that: Artificial Cell is not a specific physical entity. It is an idea involving the preparation of artificial structures of cellular dimensions for possible replace-ment or supplement of deficient cell functions. It is clear that different approaches can be used to demonstrate this idea. This prediction is already out of date since, in the last 50 years (Chang, 1957), artificial cells have progressed way beyond this 1972 prediction. Artificial cells can now be of macro-, micro-, nano-, and molecular dimensions. There are also unlimited possibilities in variations for artificial cell membranes and contents (Figure II.5.4.1). We have just touched the surface of the potential of arti-ficial cells.

ACKNOWLEDGMENTS

The ongoing support of the Canadian Institutes of Health Research (whose original name was the Medical Research Council of Canada) over the last many years is gratefully acknowledged. Other support included the Quebec Hemovigillance and Transfusion Medicine Pro-gram under FRSQ.

BIBLIOGRAPHY

Many of the references and books from the authors are avail-able free from the McGill University public service website at www.artcell.mcgill.ca.

Aebischer, P., Pochon, N. A., Heyd, B., Dglon, N., Joseph, J. M., et al. (1996). Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer encapsulated xenogenic cell line engi-neered to secrete hCNTF. Hum. Gene. Ther., 1, 851860.

Al-Hendy, A., Hortelano, G., Tannenbaum, G. S., & Chang, P. L. (1996). Growth retardation: An unexpected outcome from growth hormone gene therapy in normal mice with microen-capsulated myoblasts. Hum. Gene. Ther., 7, 6170.

Bachoud-Lvi, A. C., Dglon, N., Nguyen, J. P., Bloch, J., Bourdet, C., et al. (2000). Neuroprotective gene therapy for Hunting-tons disease using a polymer encapsulated BHK cell line engineered to secrete human CNTF. Hum. Gene. Ther., 11, 17231729.

Bangham, A. D. (1965). Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol., 13, 238252.

Basic, D., Vacek, I., & Sun, A. M. (1996). Microencapsulation and transplantation of genetically engineered cells: A new approach to somatic gene therapy. Artif. Cells Blood Substit. Immobil. Biotechnol., 24, 219255.

Bensinger, W., Baker, D. A., Buckner, C. D., Clift, R. A., & Thomas, E. D. (1981). Immunoadsorption for removal of A and B blood-group antibodies. New Engl. J. Med., 314, 160162.

Bloch, J., Bachoud-Lvi, A. C., Dglon, N., Lefaucheur, J. P., Winkel, L., et al. (2004). Neuroprotective gene therapy for Huntingtons disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: Results of a phase I study. Hum. Gene. Ther., 15, 968975.

Bourget, L., & Chang, T. M. S. (1985). Phenylalanine ammonia-lyase immobilized in semipermeable microcapsules for enzyme replacement in phenylketonuria. FEBS Lett., 180, 58.

Bourget, L., & Chang, T. M. S. (1986). Phenylalanine ammonia-lyase immobilized in microcapsules for the depleture of phe-nylalanine in plasma in phenylketonuric rat model. Biochim. Biophys. Acta., 883, 432438.

Bruni, S., & Chang, T. M. S. (1989). Hepatocytes immobilized by microencapsulation in artificial cells: Effects on hyperbiliru-binemia in Gunn rats. J. Biomater. Artif. Cells Artif. Organs., 17, 403412.

Bruni, S., & Chang, T. M. S. (1995). Kinetics of UDP-glucurono-syltransferase in bilirubin conjugation by encapsulated hepa-tocytes for transplantation into Gunn rats. J. Artif. Organs., 19, 449457.

Calafiore, R. (1999). Transplantation of minimal volume micro-capsules in diabetic high mammalians. Ann. NY. Acad. Sci., 875, 219232.

Campbell, J., & Chang, T. M. S. (1976). The recycling of NAD+ (free and immobilized) within semipermeable aqueous micro-capsules containing a multi-enzyme system. Biochem. Biophys. Res. Commun., 69, 562569.

Chang, E. J., Lee, T. H., Mun, K. C., Kim, H. C., Suh, S. I., et al. (2004a). Effects of polyhemoglobin-antioxidant enzyme com-plex on ischemia-reperfusion in kidney. Transplant Proc., 36, 19521954.

Chang, E. J., Lee, S. H., Mun, K. C., Suh, S. I., Bae, J. H., et al. (2004b). Effect of artificial cells on hepatic function after ischemia- reperfusion injury in liver. Transplant Proc., 36, 19591961.

Chang, T. M. S. (1957). Hemoglobin corpuscles. Report of a research project for Honours Physiology, Medical Library, McGill University. Also reprinted 1988 as part of 30th Anni-versary in Artificial Red Blood Cells Research. Biomater. Artif. Cells Artif. Organs., 16, 19.

Chang, T. M. S. (1964). Semipermeable microcapsules. Science, 146, 524525.


Chang, T. M. S. (1965). Semipermeable Aqueous Microcapsules. Ph.D. thesis, McGill University.

Chang, T. M. S. (1966). Semipermeable aqueous microcapsules (artificial cells): With emphasis on experiments in an extra-corporeal shunt system. Trans. Am. Soc. Artif. Intern. Organs., 12, 1319.

Chang, T. M. S. (1969). Removal of endogenous and exogenous toxins by a microencapsulated absorbent. Can. J. Physiol. Pharmacol., 47, 10431045.

Chang, T. M. S. (1971a). The in vivo effects of semipermeable microcapsules containing L-asparaginase on 6C3HED lym-phosarcoma. Nature, 229, 117118.

Chang, T. M. S. (1971b). Stabilisation of enzymes by microen-capsulation with a concentrated protein solution or by micro-encapsulation followed by cross-linking with glutaraldehyde. Biochem. Biophys. Res. Commun., 44, 15311536.

Chang, T. M. S. (1972a). Artificial Cells. Springfield, IL: Charles C. Thomas (out of print but available for free online viewing at www.artcell.mcgill.ca).

Chang, T. M. S. (1972b). Haemoperfusions over microencapsu-lated adsorbent in a patient with hepatic coma. Lancet, 2, 13711372.

Chang, T. M. S. (1974). Plateletsurface interaction: Effect of albumin coating or heparin complexing on thrombogenic sur-faces. Can. J. Physiol. Pharmacol., 52, 275285.

Chang, T. M. S. (1975). Microencapsulated adsorbent hemoperfu-sion for uremia, intoxication and hepatic failure. Kidney Int., 7, S387S392.

Chang, T. M. S. (1976). Biodegradable semipermeable microcap-sules containing enzymes, hormones, vaccines, and other bio-logicals. J. Bioeng., 1, 2532.

Chang, T. M. S. (1980). Blood compatible coating of synthetic immunoadsorbents. Trans. Am. Soc. Artif. Intern. Organs., 26, 546549.

Chang, T. M. S. (1985). Artificial cells with regenerating multien-zyme systems. Meth. Enzymol., 112, 195203.

Chang, T. M. S. (1987). Recycling of NAD(P) by multienzyme systems immobilised by microencapsulation in artificial cells. Meth. Enzymol., 136, 6782.

Chang, T. M. S. (1989). Preparation and characterization of xan-thine oxidase immobilized by microencapsulation in artificial cells for the removal of hypoxanthine. Biomater. Artif. Cells Artif. Organs., 17, 611616.

Chang, T. M. S. (1997a). Red Blood Cell Substitutes: Principles, Meth-ods, Products and Clinical Trials. (Vol. I (Monograph)). Basel, Switzerland: Karger/Landes Systems. (Available for free online viewing at www. artcell.mcgill.ca or www.artificialcell.info.)

Chang, T. M. S. (1997b). Artificial cells. In D. Renalo (Ed.), Ency-clopedia of Human Biology (2nd ed., pp. 457463). San Diego, CA: Academic Press, Inc.

Chang, T. M. S. (2001). Bioencapsulated hepatocytes for experi-mental liver support. J. Hepatol., 34, 148149.

Chang, T. M. S. (2005a). Therapeutic applications of polymeric artificial cells. Nat. Rev: Drug Discov., 4, 221235.

Chang, T. M. S. (2005b). Methods for microencapsulation of enzymes and cells. Meth. Biotechnol., 17, 289306.

Chang, T. M. S. (2006a). Blood substitutes based on bionanotech-nology. Trends Biotechnol., 24, 372377.

Chang, T. M. S. (2006b). Polyhemoglobin-enzyme complexes. In R. Winslow (Ed.), Blood Substitutes (pp. 451459). London, UK: Elsevier Academic Press.

Chang, T. M. S. (2006c). PEG-PLA biodegradable hemoglobin nanocapsules as RBC substitutes. In R. Winslow (Ed.), Blood Substitutes (pp. 523531). London, UK: Elsevier Academic Press.

Chang, T. M. S. (2007). Artificial Cells: Biotechnology, Nano-technology, Blood Substitutes, Regenerative Medicine, Bioen-capsulation, Cell/Stem Cell Therapy. Singapore and London: World Scientific Publisher/Imperial College Press. 435 pages.

Chang, T. M. S. (2008). Nanobiotechnological modification of hemoglobin and enzymes from this laboratory. Biochimica. et. Biophysica. Acta: Proteins and Proteomics, 1784, 14351144.

Chang, T. M. S. (2009). Nanobiotechnology for hemoglobin based blood substitutes. Critical Care Clinics, 25, 373382.

Chang, T. M. S. (2010). Blood replacement with nanobiotechno-logically engineered hemoglobin and hemoglobin nanocap-sules. Wiley. Interdiscip. Rev. Nanomed. Nanobiotechnol., 2, 418430.

Chang, T. M. S., & Malave, N. (1970). The development and first clinical use of semipermeable microcapsules (artificial cells) as a compact artificial kidney. Trans. Am. Soc. Artif. Intern. Organs., 16, 141148.

Chang, T. M. S., & Poznansky, M. J. (1968). Semipermeable microcapsules containing catalase for enzyme replacement in acatalsaemic mice. Nature, 218, 242245.

Chang, T. M. S., & Prakash, S. (1998). Microencapsulated geneti-cally engineered cells: Comparison with other strategies and recent progress. Mol. Med. Today., 4, 221227.

Chang, T. M. S., & Yu, W. P. (1992). Biodegradable polymer mem-brane containing hemoglobin as potential blood substitutes. British Provisional Patent No. 9219426.5 (issued Sept. 14, 1992).

Chang, T. M. S., Macintosh, F. C., & Mason, S. G. (1966). Semi-permeable aqueous microcapsules: I. Preparation and proper-ties. Can. J. Physiol. Pharmacol., 44, 115128.

Chang, T. M. S., Coffey, J. F., Barre, P., Gonda, A., Dirks, J. H., et al. (1973). Microcapsule artificial kidney: Treatment of patients with acute drug intoxication. Can. Med. Assoc. J., 108, 429433.

Chang, T. M. S., Langer, R., Sparks, R. E., & Reach, G. (1988). Drug delivery systems in biotechnology. J. Artif. Organs., 12, 248251.

Chang, T. M. S., Bourget, L., & Lister, C. (1995). A 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. Cells Blood Substit. Immobil. Biotechnol., 25, 123.

Chang, T. M. S., DAgnillo, F., & Razack, S. (1998). Cross-linked hemoglobin superoxide dismutase-catalase: A second genera-tion hemoglobin based blood substitute with antioxidant activ-ities. In T. M. S. Chang (Ed.), Blood Substitutes: Principles, Methods, Products and Clinical Trials. (Vol. 2, pp. 178196). Basel, Switzerland: Karger.

Chang, T. M. S., DAgnillo, F., Yu, W. P., & Razack, S. (2000). New generations of blood subsitutes based on polyhemoglo-bin-SOD-CAT and nanoencapsulation. Adv. Drug Delivery Rev., 40, 213218.

Chang, T. M. S., Powanda, D., & Yu, W. P. (2003). Analysis of polyethyleneglycolpolylactide nano-dimension artificial red blood cells in maintaining systemic hemoglobin levels and pre-vention of methemoglobin formation. Artif. Cells Blood Sub-stit. Biotechnol., 31, 231248.

Chow, K. M., Liu, Z. C., Prakash, S., & Chang, T. M. S. (2003). Free and microencapsulated Lactobacillus and effects of meta-bolic induction on Urea Removal Artificial Cells. Blood Substit. and Biotechnol., 4, 425434.

Cirone, P., Bourgeois, M., Austin, R. C., & Chang, P. L. (2002). A novel approach to tumor suppression with microencapsulated recombinant cells. Hum. Gene. Ther., 13, 11571166.

Cousineau, J., & Chang, T. M. S. (1977). Formation of amino acid from urea and ammonia by sequential enzyme reaction using a microencapsulated multienzyme system. Biochem. Biophys. Res. Commun., 79, 2431.

DAgnillo, F., & Chang, T. M. S. (1998). Polyhemoglobin-super-oxide dismutasecatalase as a blood substitute with antioxidant properties. Nature Biotechnol., 16, 667671.

Dalle, B., Payen, E., Regulier, E., Deglon, N., Rouyer-Fessard, P., et al. (1999). Improvement of the mouse -thalassemia upon erythropoietin delivery by encapsulated myoblasts. Gene. Ther., 6, 157161.


De Vos, P., Hamel, A. F., & Tatarkiewicz, K. (2002). Consider-ations for successful transplantation of encapsulated pancre-atic islets. Diabetologia, 45, 159173.

Deamer, D. W., & Bangham, A. D. (1976). Large-volume lipo-somes by an ether vaporization method. Biochim. Biophys. Acta., 443, 629634.

Discher, B. M., Won, Y., & David, S. E. (1999). Polymersomes: Tough vesicles made from diblock copolymers. Science, 284, 11431144.

Djordjevich, L., & Miller, I. F. (1980). Synthetic erythrocytes from lipid encapsulated hemoglobin. Exp. Hematol., 8, 584.

Garofalo, F. A., & Chang, T. M. (1991). Effects of mass transfer and reaction kinetics on serum cholesterol depletion rates of free and immobilized. Pseudomonas pictorum. Appl. Biochem.Biotech., 27, 7591.

Gould, S. A., et al. (1998). The clinical development of human polymerized hemoglobin. In T. M. S. Chang (Ed.), Blood Substitutes: Principles, Methods, Products and Clinical Trials (Vol. 2, pp. 1228). Basel, Switzerland: Karger.

Gould, S. A., Moore, E. E., Hoyt, D. B., Ness, P. M., Norris, E. J., et al. (2002). The life-sustaining capacity of human polymer-ized hemoglobin when red cells might be unavailable. J. Am. Coll. Surg., 195, 445452.

Greenburg, A. G., & Kim, H. W. (1992). Evaluating new red cell substitutes: A critical analysis of toxicity models. Biomater. Artif. Cells Immobil. Biotechnol., 20, 575581.

Gregoriadis, G. (1976). Drug Carriers in Biology and Medicine. New York, NY: Academic Press, Inc.

Gu, G., & Chang, T. M. S. (2009). Extraction of erythrocyte enzymes for the preparation of Polyhemoglobin-catalase-superoxide dismutase. Artif. Cells, Blood Substit. and Biotech-nol., 37, 6977.

Gu, K. F., & Chang, T. M. S. (1988). Conversion of alpha-keto-glutarate into L-glutamic acid with urea as ammonium source using multienzyme system and dextran-NAD+ immobilised by microencapsulation with artificial cells in a bioreactor. J. Bio-eng. Biotechnol., 32, 363368.

Gu, K. F., & Chang, T. M. S. (1990). Production of essential L-branched-chained amino acids, in bioreactors containing artificial cells immobilized multienzyme systems and dextran-NAD+. Appl. Biochem. Biotechnol., 26, 263269.

Hagihara, Y., Saitoh, Y., Iwata, H., Taki, T., Hirano, S., et al. (1997). Transplantation of xenogeneic cells secreting beta-endorphin for pain treatment: Analysis of the ability of compo-nents of complement to penetrate through polymer capsules. Cell Transplan., 6, 527530.

Hakim, R. M., Milford, E., Himmelfarb, J., Wingard, R., Lazarus, J. M., et al. (1990). Extracorporeal removal of antiHLA antibodies in transplant candidates. Am. J. Kidney. Dis., 16, 423.

Hunkeler, D. L. (2003). Bioartificial organ grafts: A view at the beginning of the third millennium. Artif. Cells Blood Substit. Immobil. Biotechnol., 31, 365382.

Ilan, E., & Chang, T. M. S. (1986). Modification of lipidpoly-amide microcapsules for immobilization of free cofactors and multienzyme system for the conversion of ammonia to gluta-mate. Appl. Biochem. Biotechnol., 13, 221230.

Ito, Y., & Chang, T. M. S. (1992). In vitro study of multicellu-lar hepatocytes spheroid formed in microcapsules. J. Artif. Organs., 16, 422426.

Jahr, J. S., Mackenzie, C., Pearce, L. B., Pitman, A., & Green-burg, A. G. (2008). HBOC-201 as an alternative to blood transfusion: Efficacy and safety evaluation in a multicenter phase III trial in elective orthopaedic surgery. J. Trauma., 64, 14841497.

Kashani, S. A., & Chang, T. M. S. (1988). Release of hepatic stimu-latory substance from cultures of free and microencapsulated hepatocytes: Preliminary report. J. Biomater. Artif. Cells Artif. Organs., 16, 741746.

Kashani, S., & Chang, T. M. S. (1991). Effects of hepatic stimu-latory factor released from free or microencapsulated hepa-tocytes on galactosamine induced fulminant hepatic failure animal model. Biomater. Artif. Cells Immobil. Biotechnol., 19, 579598.

Kawasaki, C., Nishi, R., Uekihara, S., Hayano, S., & Otagiri, M. (2000). Charcoal hemoperfusion in the treatment of phenytoin overdose. Am. J. Kidney Dis., 35, 323326.

Keipert, P. E., & Chang, T. M. S. (1985). Pyridoxylated polyhe-moglobin as a blood substitute for resuscitation of lethal hem-orrhagic shock in conscious rats. Biomater. Med. Dev. Artif. Organs, 13, 115.

Keipert, P. E., & Chang, T. M. S. (1987). In vivo effects of total and partial isovolemic exchange transfusion in fully conscious rats using pyridoxylated polyhemoglobin solution as a colloidal oxygen-delivery blood substitute. Vox Sang., 53, 714.

Kobayashi, K., Izumi, Y., Yoshizu, A., Horinuchi, H., Park, S. I., et al. (1997). The oxygen carrying capability of hemoglobin vesicles evaluated in rat exchange transfusion models. Artif. Cells Blood Substit. Immobil. Biotechnol., 25, 357366.

LaVan, D. A., Lynn, D. M., & Langer, R. (2002). Moving smaller in drug discovery and delivery. Nat. Rev. Drug Discov., 1, 7784.

Lim, F., & Sun, A. M. (1980). Microencapsulated islets as bioarti-ficial endocrine pancreas. Science, 210, 908909.

Lin, C. C., Chou, H. L., & Lin, J. L. (2002). Acute aconitine poisoned patients with ventricular arrhythmias successfully reversed by charcoal hemoperfusion. Am. J. Emerg. Med., 20, 6667.

Lin, C. C., Chan, T. Y., & Deng, J. F. (2004). Clinical features and management of herb induced aconitine poisoning. Ann. Emerg. Med., 43, 574579.

Liu, J., Jia, X., Zhang, J., Hu, W., & Zhou, Y. (2002). Study on a novel strategy to treatment of phenylketonuria. Artif. Cells Blood Substit. Immobil. Biotechnol., 30, 243258.

Liu, Z. C., & Chang, T. M. S. (2000). Effects of bone marrow cells on hepatocytes: When cocultured or co-encapsulated together. Artif. Cells Blood Substit. Immobil. Biotechnol., 28, 365374.

Liu, Z. C., & Chang, T. M. S. (2002). Increased viability of trans-plantation hepatocytes when coencapsulated with bone mar-row stem cells using a novel method. Artif. Cells Blood Substit. Immobil. Biotechnol., 30, 9911.

Liu, Z. C., & Chang, T. M. S. (2003). Coencapsulation of stem sells and hepatocytes: In vitro conversion of ammonia and in vivo studies on the lowering of bilirubin in Gunn rats after trans-plantation. Int. J. Artif. Organs., 26, 491497.

Liu, Z. C., & Chang, T. M. S. (2005). Transplantation of bioen-capsulated bone marrow stem cells improves hepatic regenera-tion and survival of 90% hepatectomized rats: A preliminary report. Artif. Cells Blood Substit. Biotechnol., 33, 405410.

Liu, Z. C., & Chang, T. M. S. (2006). Transdifferentiation of bio-encapsulated bone marrow cells into hepatocyte-like cells in the 90% hepatectomized rat model. J. Liver Transplant., 12, 566572.

Liu, Z. C., & Chang, T. M. S. (2008). Long term effects on the histology and function of livers and spleens in rats after 33% toploading of PEG-PLA-nano-artificial red blood cells. Artif.Cells, Blood Substit. and Biotechnol., 36, 513524.

Liu, Z. C., & Chang, T. M. S. (2009). Preliminary study on intras-plenic implantation of artificial cell bioencapsulated stem cells to increase the survival of 90% hepatectomized rats. Artif. Cells, Blood Substit. and Biotechnol., 37(1), 5355.

Liu, Z. C., & Chang, T. M. S. (2010). Artificial cell microencap-sulated stem cells in regenerative medicine, tissue engineering and cell therapy. Adv. Exp. Med. Biol., 670, 6879.

Lloyd-George, I., & Chang, T. M. S. (1995). Characterization of free and alginate polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate and phenol into L-tyrosine and L-DOPA. J. Bioeng. Biotech-nol., 48, 706714.


Lopez Lago, A. M., Rivero Velasco, C., Galban Rodrguez, C., Mario Rozados, A., Piero Sande, N., et al. (2002). Paraquat poisoning and hemoperfusion with activated charcoal. Ann. Intern. Med., 19, 310312.

Lorh, M., Hoffmeyer, A., & Kroger, J. C. (2001). Microencapsu-lated cell-mediated treatment of inoperable pancreatic carci-noma. Lancet, 357, 15911592.

Luzzi, L. A. (1970). Preparation and evaluation of the prolonged release properties of nylon microcapsules. J. Pharm. Sci., 59, 338.

Michalopoulos, G., Houck, K. D., Dolan, M. L., & Luetteke, N. C. (1984). Control of hepatocyte replication by two serum fac-tors. Cancer Res., 44, 44144419.

Moore, E. E., Moore, F. A., Fabian, T. C., Bernard, A. C., Fulda, G. J., et al. (2009). Human polymerized hemoglobin for the treatment of hemorrhagic shock when blood is unavailable: The USA Multicenter Trial. J. Am. Coll. Surg., 208, 113.

Orive, G., Hernndez, R. M., Gascn, A. R., Calafiore, R., Chang, T. M., et al. (2003). Cell encapsulation: Promise and progress. Nat. Med., 9, 104107.

Orive, G., Hernndez, R. M., Gascn, A. R., Calafiore, R., Chang, T. M., et al. (2004). History, challenge and perspectives of cell microencapsulation. Trends Biotechnol., 22, 8792.

Palmour, R. M., Goodyer, P., Reade, T., & Chang, T. M. S. (1989). Microencapsulated xanthine oxidase as experimental therapy in Lesch-Nyhan disease. Lancet, 2, 687688.

Pearce, L. B., & Gawryl, M. S. (1998). Overview of preclinical and clinical efficacy of Biopures HBOCs. In T. M. S. Chang (Ed.), Blood Substitutes: Principles, Methods, Products and Clinical Trials (Vol. 2, pp. 8298). Basel, Switzerland: Karger.

Pearce, L. B., Gawryl, M. S., Rentko, V. T., et al. (2006). HBOC-201 (Hb Glutamer-250 (Bovine), Hemopure): Clinical studies. In R. Winslow (Ed.), Blood Substitutes (pp. 437450). San Diego, CA: Academic Press.

Peng, A., Meng, F. Q., Sun, L. F., Ji, Z. S., & Li, Y. H. (2004). Thera-peutic efficacy of charcoal hemoperfusion in patients with acute severe dichlorvos poisoning. Acta. Pharmacol. Sin., 25, 1521.

Philips, W. T., Klipper, R. W., Awasthi, V. D., Rudolph, A. S., Cliff, R., et al. (1999). Polyethylene glyco-modified liposome-encapsulated hemoglobin: A long circulating red cell substi-tute. J. Pharm. Exp. Ther., 288, 665670.

Photos, P. J., Bacakova, L., Discher, B., & Bates, F. S. (2003). Poly-mer vesicles in vivo: Correlations with PEG molecular weight. J. Control Release., 90, 323334.

Powanda, D., & Chang, T. M. S. (2002). Cross-linked polyhemo-globin-superoxide dismutase-catalase supplies oxygen without causing blood brain barrier disruption or brain edema in a rat model of transient global brain ischemia-reperfusion. Artif. Cells Blood Substit. Immobil. Biotechnol., 30, 2542.

Poznansky, M. J., & Chang, T. M. S. (1974). Comparison of the enzyme kinetics and immunological properties of catalase immo-bilized by microencapsulation and catalase in free solution for enzyme replacement. Biochim. Biophys. Acta., 334, 103115.

Prakash, S., & Chang, T. M. S. (1993). Genetically engineered E. coli cells containing K. aerogenes gene, microencapsulated in artificial cells for urea and ammonia removal. Biomater Artif. Cells Immobil. Biotechnol., 21, 629636.

Prakash, S., & Chang, T. M. S. (1996). Microencapsulated geneti-cally engineered live E. coli DH5 cells administered orally to maintain normal plasma urea level in uremic rats. Nature Med., 2, 883887.

Razack, S., DAgnillo, F., & Chang, T. M. S. (1997). Cross-linked hemoglobin superoxide dismutase-catalase scavenges free radi-cals in a rat model of intestinal ischemia-reperfusion injury. Artif. Cells Blood Substit. Immobil. Biotechnol., 25, 181192.

Rokstad, A. M., Holtan, S., Strand, B., Steinkjer, B., Ryan, L., et al. (2002). Microencapsulation of cells producing therapeu-tic proteins: Optimizing cell growth and secretion. Cell Trans-plant, 11, 313324.

Rosenthal, A. M., & Chang, T. M. S. (1971). The effect of valino-mycin on the movement of rubidium across lipid coated semi-permeble microcapsules. Proc. Canad. Fed. Biol. Soc., 14, 44.

Rosenthal, A. M., & Chang, T. M. S. (1980). The incorporation of lipid and Na+-K+-ATPase into the membranes of semiper-meable microcapsules. J. Membrane Sciences, 6(3), 329338.

Safos, S., & Chang, T. M. S. (1995). Enzyme replacement therapy in ENU2 phenylketonuric mice using oral microencapsulated phenylalanine ammonialyase: A preliminary report. Artif. Cells Blood Substit. Immobil. Biotechnol., 23, 681692.

Saitoh, Y., Taki, T., Arita, N., Ohnishi, T., & Hayakawa, T. (1995). Cell therapy with encapsulated xenogeneic tumor cells secreting beta-endorphin for treatment of peripheral pain. Cell Transplant, S1, S13S17.

Sakai, H., Masada, Y., Horinouchi, H., Yamamoto, M., Ikeda, E., et al. (2004). Hemoglobin-vesicles suspended in recombinant human serum albumin for resuscitation from hemorrhagic shock in anesthetized rats. Crit. Care. Med., 32, 539545.

Sarkissian, C. N., Shao, Z., Blain, F., Peevers, R., Su, H., et al. (1999). A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase. Proc. Natl. Acad. Sci., 96, 23392344.

Singh, S. M., McCormick, B. B., Mustata, S., Thompson, M., & Prasad, G. V. (2004). Extracorporeal management of valproic acid overdose: A large regional experience. J. Nephrol., 17, 4349.

Spangrude, G. J., Heimfeld, S., & Weissman, I. L. (1988). Purifica-tion and characterization of mouse hematopoietic stem cells. Science, 241, 5862.

Sprung, J., Kindscher, J. D., Wahr, J. A., Levy, J. H., Monk, T. G., et al. (2002). The use of bovine Hb glutamer-250 (Hemopure) in surgical patients: Results of a multicenter, randomized, sin-gleblinded trial. Anesth. Analg., 94, 799808.

Sun, Y. L., Ma, X. J., & Zhou, D. B. (1996). Normalization of diabetes in spontaneously diabetic cynomologus monkeys by xenografts of microencapsulated porcine islets without immu-nosuppression. J. Clin. Invest., 98, 14171422.

Tan, S. A., Dglon, N., Zurn, A. D., Baetge, E. E., Bamber, B., et al. (1996). Rescue of motoneurons from axotomy-induced cell death by polymer encapsulated cells genetically engineered to release CNTF. Cell Transplant, 5, 577587.

Terman, D. S., Tavel, T., Petty, D., Racic, M. R., & Buffaloe, G. (1977). Specific removal of antibody by extracorporeal circula-tion over antigen immobilized in colodion charcoal. Clin. Exp. Immunol., 28, 180188.

Terman, D. S., Buffaloe, G., Mattioli, C., Cook, G., Tiilquist, R., et al. (1979). Extracorporeal immunoabsorption: Initial expe-rience in human systemic lupus erythematosus. Lancet, 2, 824.

Torchilin, V. P. (2005). Recent advances with liposomes as phar-maceutical carriers. Nat. Rev. Drug Discov., 4, 145160.

Tsuchida, E. (Ed.), (1998). Present and Future Perspectives. Blood Substitutes (Vol. 1, pp. 267). Amsterdam: Elsevier.

Winchester, J. F. (Ed.), (1996). Replacement of Renal Function by Dialysis (4th ed.). Boston, MD: Kluwer Academic Publishers.

Wingard, R. L., Lee, W. O., & Hakim, R. O. (1991). Extracorporeal treatment of familial hypercholesterolemia with monoclonal anti-bodies to low-density lipoproteins. Am. J. Kidney Dis., 18, 559.

Winn, S. R., Hammang, J. P., Emerich, D. F., Lee, A., Palmiter, R. D., et al. (1994). Polymer-encapsulated cells genetically modi-fied to secrete human nerve growth factor promote the survival of axotomized septal cholinergic neurons. Proc. Natl. Acad. Sci. USA, 91, 23242328.

Wong, H., & Chang, T. M. S. (1986). Bioartificial liver: Implanted artificial cells microencapsulated living hepatocytes increases survival of liver failure rats. Int. J. Artif. Organs., 9, 335336.

Wong, H., & Chang, T. M. S. (1988). The viability and regen-eration of artificial cell microencapsulated rat hepatocyte xenograft transplants in mice. J. Biomater Artif. Cells Artif. Organs., 16, 731740.

ChapTEr II.5.5 Extracorporeal Artificial Organs 827

Wong, H., & Chang, T. M. S. (1991). A novel two-step procedure for immobilizing living cells in microcapsule for improving xenograft survival. Biomater Artif. Cells Immobil. Biotech-nol., 19, 687698.

Wong, N., & Chang, T. M. S. (2007). Polyhemoglobin-fibrinogen: A novel blood substitute with platelet-like activity for extreme hemodilution. Artif. Cells, Blood Substit. and Biotechnol., 35, 481489.

Xu, W., Liu, L., & Charles, I. G. (2002). Microencapsulated iNOS-expressing cells cause tumor suppression in mice. FASEB J., 16, 213215.

Yang, L., Cheng, Y., Yan, W. R., & Yu, Y. Y. (2004). Extracor-poreal whole blood immunoadsorption of autoimmune myas-thenia gravis by cellulose tryptophan adsorbent. Artif. Cells, Blood Substit. Biotechnol., 32, 519518.

Yu, B. L., & Chang, T. M. S. (2004). In vitro and in vivo effects of poly hemoglobintyrosinase on murine B16F10 melanoma. Melanoma. Res. J., 14, 197202.

Yu, W. P., & Chang, T. M. S. (1994). Submicron biodegradable polymer membrane hemoglobin nanocapsules as potential blood substitutes: A preliminary report. J. Artif. Cells Blood Substit. Immobil. Biotechnol., 22, 889894.

Yu, W. P., & Chang, T. M. S. (1996). Submicron polymer mem-brane hemoglobin nanocapsules as potential blood substitutes: Preparation and characterization. Artif. Cells Blood Substit. Immobil. Biotechnol., 24, 169184.

Yu, W. P., Wong, J., & Chang, T. M. S. (1998). Preparation and characterization of polylactic acid microcapsules containing ciprofloxacin for controlled release. J. Microencapsul., 15, 515523.

Yu, W. P., Wong, J. P., & Chang, T. M. S. (2000). Sustained drug release characteristics of biodegradable composite poly(d, l)lactic acid poly(l)lactic acid microcapsules containing cipro-floxacin. Artif. Cells Blood Substit. Immobil. Biotechnol., 28, 3956.

Yu, Y. T., & Chang, T. M. S. (1982). Immobilization of mul-tienzymes and cofactors within lipid-polyamide membrane microcapsules for the multistep conversion of lipophilic and lipophobic substrates. Enzyme. Microb. Technol., 4, 327331.

Zhou, M. X., & Chang, T. M. S. (1988). Control release of prostaglandin E2 from polylactic acid microcapsules, mic-roparticles and modified microparticles. J. Microencapsul., 5, 2736.

CHAPTER II.5.5 EXTRACORPOREAL ARTIFICIAL ORGANS

Alastair Campbell RitchieDepartment of Mechanical, Materials and Manufacturing Engineering, University of Nottingham, Nottingham, UK

INTRODUCTION

Extracorporeal circulation is, by definition, any proce-dure in which blood is taken from a patient, treated, and then returned. The vast majority of these procedures are performed intermittently, or as a temporary partial or total replacement. In this chapter, we will consider only those artificial organs which undertake mass transfer to and from the blood. This includes devices used for dialysis in renal replacement therapy, plasma separation (plasmapheresis), and extracorporeal oxygenation.

All these procedures involve the contact of blood with biomaterials, mechanical pumping of the blood, and mass transfer to and from the blood. The issues of bio-compatibility are essential to the success or failure of a procedure; readers are advised to refer to the previous chapter on bloodbiomaterials interaction for a full dis-cussion of the effects of bloodbiomaterial contact, as in this chapter only those aspects specific to extracorporeal therapy will be discussed.

renal replacement Therapy

Renal replacement therapy describes treatments such as peritoneal dialysis, hemodialysis, and hemofiltration, used to detoxify blood in cases of renal insufficiency. Renal insufficiency may be caused by acute renal failure, typically of rapid onset and due to external causes or by chronic

renal failure, usually due to damage to the tissues of the kidneys. Chronic renal failure may be caused by infection, toxins or hypertension, and may also arise as a complica-tion in diabetes mellitus. The number of prevalent (i.e., alive and receiving treatment) patients with end-stage renal disease in the USA between 1980 and 2007 is given in Figure II.5.5.1 (USRDS, 2009). As can be seen from the graph, the incidence of chronic renal failure has risen steadily since 1980, and diabetes has become the promi-nent cause of renal failure, accounting for some 37.4% of all cases in 2007, followed by glomerulonephritis, and renal vascular disease. Of the 527,283 surviving end-stage renal disease (ESRD) patients listed by the United States Renal Data System in that year,1 158,739 (30.1% of the total) had received a transplant that was still functioning. The other patients were dependent upon renal replace-ment therapy.

In 2007, there were 368,544 patients receiving renal replacement therapy in the United States, of whom 92.5% received hemodialysis treatment (USRDS, 2009). Blood may be treated by extracorporeal hemodialysis (familiarly known as dialysis), peritoneal dialysis, and extracorporeal hemofiltration (a form of plasmapheresis) (Coles and El Nahas, 2000). Figure II.5.5.2 shows the numbers of patients treated by blood purification thera-pies in the USA between 1980 and 2007 (USRDS, 2009). As can be seen, as the patient population has increased,

1Data from US Renal Data System, USRDS, 2009, Annual Data Report: Atlas of End-Stage Renal Disease in the United States, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2009. The data reported here have been supplied by the United States Renal Data System (USRDS). The interpretation and reporting of these data are the responsibility of the author, and in no way should be seen as an official policy or interpretation of the US government.

II.5.4 ARTIFICIAL CELLSBASIC FEATURES OF ARTIFICIAL CELLSRESEARCH INTO THE APPLICATIONS OF ARTIFICIAL CELLSARTIFICIAL CELLS IN HEMOPERFUSIONNANOBIOTECHNOLOGY FOR PARTIAL ARTIFICIAL RED BLOOD CELLSNANOBIOTECHNOLOGY FOR COMPLETE ARTIFICIAL RED BLOOD CELLSCELLS, ISLETS, STEM CELLS, GENETICALLY ENGINEERED CELLS,AND MICROORGANISMSARTIFICIAL CELLS CONTAINING STEM CELLS IN REGENERATIVE MEDICINEGENE AND ENZYME THERAPYDRUG DELIVERYOTHER AREAS OF ARTIFICIAL CELLSTHE FUTURE OF ARTIFICIAL CELLSACKNOWLEDGMENTSBIBLIOGRAPHY

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