Artificial Cell Bioencapsulation in Macro, Micro, Nano, and Molecular Dimensions: Keynote Lecture

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 bioencapsulation of cells, tissues, microorganisms, and bioreactants. Those in the micron-dimensions are suitable for the bioencapsulation 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.

• 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 Chang a number of years ago (Chang, [[1957]], [[1964]], [[1966]], [[1972]]; Chang et al., [[1966]]) (Fig. 1). Biologically active materials inside the artificial cells are prevented from coming into direct contact with external materials like leucocytes, antibodies, or tryptic enzymes. Smaller molecules can equilibrate rapidly across the ultrathin membrane with large surface to volume relationship. A number of potential medical applications using artificial cells have been proposed (Chang, [[1964]], [[1966]], [[1972]]; Chang et al., [[1966]]). However, even at the very beginning, it was necessary to increase this to the macro-dimensions for certain type of bioencapsulation. One example is cell encapsulation (Chang, [[1972]]; Chang et al., [[1966]]) and another example is the encapsulation of adsorbents for use in hemoperfusion (Chang, [[1966]], [[1972]], [[1975]]; Winchester, [[1988]]). Artificial cells (Chang, [[1997a]]) bioencapsulation now range from macro-dimensions, to micron-dimensions, to nano-dimensions, and to molecular-dimensions (Fig. 2). This paper describes some examples from our research using those in the micron-dimensions for the bioencapsulation of enzymes, peptides, drugs, vaccine, and other materials; those in the macro-dimensions for cell encapsulation and encapsulation of bioreactants; those in the molecular-dimensions are used as blood substitutes, crosslinked enzymes etc.; and those in the nano-dimension for blood substitutes, enzymes, peptides, drugs etc.

Figure 1. Basic principle of artificial cells.

Figure 2. Artificial cells in the macro, micro, nano, and molecular dimensions.

Artificial Cells in the Micron-Dimensions

Enzyme Therapy by Implantation (Fig. 3)

Figure 3. Basic principle of the use of enzyme artificial cells for implantation in inborn errors of metabolism (e.g., acatelasemia) and cancer (lymphosarcoma).

We have earlier implanted artificial cells containing catalase into acatalesemic mice, animals with a congenitical deficiency in catalase (Chang and Poznansky, [[1968]]). This replaces the deficient enzymes and prevented the animals from the damaging effects of oxidants. The artificial cells protect the enclosed enzyme from immunological reactions (Poznansky and Chang, [[1974]]). It was also showed that artificial cells containing asparaginase implanted into mice with lymphosarcoma delayed the onset and growth of lymphosarcoma (Chang, [[1971a]]). The single problem preventing the clinical application of enzyme artificial cells is the need to repeatedly inject these enzyme artificial cells.

Oral Administration to Avoid the Need for Implantation (Fig. 4)

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.

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 microencapsulated 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 5. Oral artificial cells containing phenylalanine ammonia lyase for the removal of systemic phenylalanine in the most common inborn error of metabolism, phenylketonuria.

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.

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.

Drug Delivery Systems

Our initial use of polylactide biodegradable semipermeable microcapsules containing enzymes, insulin, hormones, vaccines, and other biologicals in 1976 (Chang, [[1976]]) is now being extended by many groups. This includes our studies on the preparation and characterization of polylactic acid microcapsules containing ciprofloxacin for controlled release (Yu et al., [[1998]]).

Artificial Cells in the Macro-Dimensions

Hemoperfusion

The first successful use of artificial cell in routine clinical use is hemoperfusion (Chang, [[1972]], [[1975]]; Winchester, [[1988]]). After initial clinical 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 in 1966 based on a drop method and proposed that “protected from immunological process, encapsulated endocrine cells might survive and maintain an effective supply of hormone” (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]]).

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.

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 Gunn rats (Brunis and Chang, [[1989]]); 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, [[1991a]], [[1991b]]). Using this two step methods plus the use of co-encapsulation of stem cells and hepatocytes (Fig. 10) we have further increased the viability of encapsulated hepatocytes both in culture and also after implantation (Liu and Chang, [[2000]], [[2002]]) (Fig. 11). One implantation of the co-encapsulated hepatocytes-stem cells into Gunn rats lowered the systemic bilirubin levels and maintained this low level for 2 months (Liu and Chang, [[2003]]). Implanted encapsulated hepatocytes can only maintain a low level for 1 month.

Figure 10. Co-encapsulation of cells with stem cells to increase the viability of the encapsulated cells.

Figure 11. Experiment showing that co-encapsulation with stem cells increases the viability of hepatocytes after implantation.

Microencapsulated Genetically Engineered Cells

This has been studied out by many groups for potential applications in amyotrophic lateral sclerosis, Dwarfism, pain treatment, IgG₁ plasmacytosis, Hemophilia B, Parkinsonism, and axotomized septal cholinergic neurons (Aebischer et al., [[1996]]; Chang and Prakash, [[1998]]) (Fig. 12). One group uses hollow fibers to macroencapsulated genetically engineered cells. This way, the fibers can be inserted and then retrieved after use without being retained in the body (Aebischer et al., [[1996]]).

Figure 12. Basic principle of bioencapsulation of genetically engineered cells is the same as for cell encapsulation.

To avoid the need for implantation, we studied the oral use (Fig. 5) of microencapsulated genetically engineered nonpathogenic E. coli DH5 cells containing Klebsiella aerogenes urease gene to lower systemic urea in renal failure rats (Chang, [[1997b]]; Prakash and Chang, [[1996]]). However, these genetically engineered microorganism are not sufficiently stable in their ability to remove urea. We are looking at the metabolic induction of lactobacillus similar to those use in yourgart, in order not to introduce 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 containing hemoglobin and enzymes (Chang, [[1957]], [[1964]]) to form polyhemoglobin—a molecular version of artificial cells. This is based on his use of bifunctional agents like diacid (Chang, [[1964]], [[1972]]) or later glutaraldehyde (Chang, [[1971b]]) to crosslink hemoglobin molecules into polyhemoglobin (Fig. 13). With problem related to H.I.V. in donor blood, there has been extensive development towards blood substitutes starting 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 and waiting for F.D.A. approval. These are developed independently by two groups based on Chang's basic principle of gluataradehyde crosslinked polyhemoglobin (Chang, [[1971b]]). One is pyridoxalated glutaraldehyde human polyhemoglobin (Gould et al., [[1998]], [[2002]]). They show in Phase III clinical trial that this can successfully replace extensive blood loss in trauma surgery by maintaining the hemoglobin level with no reported side effects (Gould et al., [[1998]], [[2002]]). They have infused up to 20 units into individual trauma surgery patients (Gould et al., [[2002]]). Another one is glutaraldehyde crosslinked bovine polyhemoglobin that has been extensive tested in Phase III clinical trials (Pearce and Gawryl, [[1998]]; Sprung et al., [[2002]]). This bovine polyhemoglobin has been approved for veterinary medicine in the U.S. and for routine clinical use in South Africa. The above two polyhemoglobins have been approved for compassionate uses in human and they are waiting for regulatory approval for routine clinical uses in human in North America. They have a number of advantages when compared to donor red blood cells (Fig. 14) and they are particularly useful for use in surgery. However, these are only oxygen carriers and do not have all the functions of red blood cells that may be needed for certain clinical conditions (Chang, [[2003]]).

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.

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.

Polyhemoglobin Crosslinked with RBC Antioxidant Enzymes

Reperfusion using oxygen carrier alone in sustained severe hemorrhagic shock or sustained ischemic organs as in stroke, myocardial infarction, or organ transplantation may result in the production of oxygen 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). Unlike PolyHb, PolyHb-SOD-CAT did not cause a significant increase in oxygen radicals when it is used to reperfuse ischemic rat intestine (Razack et al., [[1997]]). More recently (Powanda and Chang, [[2002]]), in a transient global cerebral ischemia rat model, we found that after 60 min of ischemia, reperfusion with polyHb resulted in significant increases in blood–brain barrier and the breakdown of blood–brain barrier (Fig. 17). On the other hand, polyHb-SOD-CAT did no result in these adverse changes (Powanda and Chang, [[2002]]) (Fig. 17).

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.

Figure 16. In conditions like severe sustained hemorrhagic shock, stroke, myocardial infarction and organ transplantation, reperfusion with polyhemoglobin 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.

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.

Polyhemoglobin Crosslinked with Tyrosinase

Crosslinking tyrosinase with hemoglobin results in a soluble polyhemoglobin-tyrosinase complex. This has the dual function of lowering systemic tyrosine that has the potential to slow the growth of melanoma (Yu and Chang, [[2004]]). At the same time, polyhemoglobin, being in solution, can more readily perfuse the under perfuse melanoma blood 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 (Philips et al., [[1999]]; Rudolph et al., [[1997]]; Tsuchida, [[1998]]). We are using a different approach based on biodegradable polymer and nanotechnology resulting in nano artificial RBC of 80 to 150 nanometre diameter (Chang and Yu, [[1998]]; Chang et al., [[2003]]; Yu and Chang, [[1994]]). These nano artificial rbc contain all the red blood cell enzymes needed for the long term function of the nano artificial RBC (Chang et al., [[2003]]) (Fig. 18). Our recent studies show that using a polyethylene-glycol-polylactide copolymer membrane we are able to increase the circulation time of these nano artificial rbc to double that of polyHb (Chang et al., [[2003]]) (Fig. 19).

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.

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.

Drug Delivery

We have been studying the preparation and characterization of polylactic acid nanocapsules containing ciprofloxacin for controlled release (Yu et al., [[1999]]). The nanocapsules described above (Chang and Yu, [[1998]]; Chang et al., [[2003]]; Yu and Chang, [[1994]]) are also useful for the delivery of biologically active proteins and peptides. Other approaches based on nanodimension artificial cells in the form of liposomes, nanoparticles, and nanocapsules are being increasing used by many groups for drug delivery.

General

This paper only briefly summarizes some of the research from this group on artificial cells in the macro, micro, nano, and molecular dimensions. Bioencapsulation of biosorbent is rather simple and therefore has been in routine clinical uses after only a few years of research and development (Chang, [[1975]]; Winchester, [[1988]]). Some areas of microencapsulation of drugs for delivery are also straight forward and are in clinical applications. However, in the more complicated areas like cell encapsulation and blood substitutes one cannot expect that clinical application will come after just a few years of research and development. There are many areas of applications being explored by many groups around the world (Fig. 20). The promise and potential of artificial cells also comes with the need for further development towards actual applications. Much needs to be done in order to move the more advance uses of artificial cells to fruitful applications (Fig. 21).

Figure 20. Some examples of areas of application for artificial cells.

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 is a rapidly evolving area and rapidly updating and links to other groups around the world can be found on our McGill University website: www.artcell.mcgill.ca.

Acknowledgments

This author acknowledges the supports of the Canadian Institutes of Health Research, the “Virage” Centre of Excellence in Biotechnology from the Quebec Ministry, the MSSS-FRSQ Research Group award on Blood Substitutes in Transfusion Medicine from the Quebec Ministry of Health and the Research Fund of the Bayer/Canadian Blood Agency/Hema Quebec/Canadian Institutes of Health.