Artificial Cell and Red Blood Cell Substitutes

The artificial cell evolved from Chang's attempts to prepare artificial structures for possible replacement or supplement of deficient cell functions [675-679]. Artificial cells for pharmaceutical and therapeutic applications started as microen-capsulation on the micrometer scale. This has expanded up to the higher range of macrocapsules and down to the nanometer range of nanocapsules and even to the macro-molecular range of cross-linked hemoglobin as a blood substitute. Artificial cells are now being prepared by bioen-capsulation in the laboratory for medical and biotechno-logical applications [680-685]. Some exciting developments include research and clinical trials on modified hemoglobin for blood substitutes and the use of artificial cells for enzyme therapy, cell therapy, and gene therapy [686-691]. Like natural cells, biologically active materials inside the artificial cells are retained and prevented from coming into contact with external materials like leucocytes, antibodies, or tryp-tic enzymes. Molecules smaller than protein can equilibrate rapidly across the ultrathin membrane with a large surface to volume relationship. A number of potential medical applications using artificial cells have been proposed [692, 693]. One of these is hemoperfusion for uremia, intoxication, and hepatic failure [692], which has been developed successfully for routine clinical use [693]. Artificial cells containing enzymes have been used for inborn errors of metabolism and other conditions.

Semipermeable microcapsules containing catalase were implanted into acatalesemic mice, animals with a congenital deficiency in catalase [694], which replaced the deficient enzymes and prevented the animals from the damaging effects of oxidants. The artificial cells also protected the enclosed enzyme from immunological reactions [695]. The artificial cells containing asparaginase implanted into mice with lymphosarcoma delay the onset and growth of lym-phosarcoma [11, 696]. The microencapsulated phenylalanine ammonia lyase given orally lowered the elevated pheny-lalanine levels in phenylketonuria rats [697], because of an extensive recycling of amino acids between the body and the intestine [698], which was developed for clinical trial in phenylketonuria [699].

A drop method was developed for the encapsulation of biological cells [677, 679] by using milder physical cross-linking [700], resulting in alginate-polylysine-alginate (APA) microcapsules containing cells. The preparative procedures and properties of microcapsule artificial kidneys were studied [701, 702]. Microencapsulated xanthine oxidase was used for experimental therapy in Lesch-Nyhan disease [703]. Microencapsulated islets were used as a bioartifi-cial endocrine pancreas [700]. The cell encapsulation was developed for cell therapy, including artificial cells containing endocrine tissues, hepatocytes, and other cells for cell therapy [686, 688, 704-707], for potential applications in amyotrophic lateral sclerosis, dwarfism, pain treatment, IgG1 plasmacytosis, Hemophilia B, Parkinsonism, and axo-tomized septal cholinergic neurons. The effects on hyper-bilirubinemia in Gunn rats were discussed for hepatocytes immobilized by microencapsulation in artificial cells [708]. Daka and Chang reported bilirubin removal by the pseu-doperoxidase activity of free and immobilized hemoglobin and hemoglobin coimmobilized with glucose oxidase [709]. Preparation and characterization of xanthine oxidase immobilized by microencapsulation in artificial cells were reported for the removal of hypoxanthine [710]. Even for more immediate clinical applications [711], the ingenious use of capillary fiber to encapsulate cells allows one to implant cells followed by retrieval and reimplantation in clinical trials [711].

Advances in molecular biology have resulted in the availability of nonpathogenic genetically engineered microorganisms that can effectively use uremic metabolites for cell growth. Prakash and Chang studied the oral use of microencapsulated genetically engineered nonpathogenic E. coli DH5 cells containing the Klebsiella aerogenes urease gene in renal failure rats [712-714]. In vitro removal of urea, ammonium, electrolytes, and other metabolites was studied [714-717]. Daily oral administration to partially nephrec-tomized rats including survival studies was investigated [712, 718-720]. Clinical potentials of oral encapsulated E. coli DH5 cells were studied [702, 721-723].

Polyhemoglobin has been used as a blood substitute. Native hemoglobin [tetramer] breaks down into half molecules [dimers] after infusion causing renal toxicity and other adverse effects. Chang extended his original approach of artificial cells containing hemoglobin and enzymes [675-677] to form polyhemoglobin—a molecular version of artificial cells which was based on the use of bifunctional agents like diacid [675-677] or glutaraldehyde [724, 725] to cross-link hemoglobin molecules into polyhe-moglobin. This gluataradehyde cross-linked polyhemoglobin approach has been extensively developed more recently [726-734].

Although polyhemoglobin is in the most advanced stages of clinical trial, there are other modified hemoglobins such as recombinant human hemoglobin [728, 735]. Unlike poly-hemoglobin these are single tetrameric hemoglobin formed by intramolecular cross-linkage [736, 737] or recombinant human hemoglobin [738]. Clinical trials on these show vasoactivities and other effects of nitrate oxide removal [736-738]. A recombinant human tetrameric hemoglobin with markedly decreased affinity for nitric oxide was developed [739]. Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin. When infused into experimental animals, this did not cause vasoactivity.

There are other new generations of modified hemoglobin blood substitutes: Polyhemoglobin stays in the circulation with a half-time of only up to 27 hours. In order to increase this circulation time, Chang's original idea of a complete artificial red blood cell [675-677] is now being developed as a third generation blood substitute. Thus submicrom-eter lipid membrane microencapsulated hemoglobin [740] has been explored [728, 741, 742]. The surface properties were modified to result in a circulation half-time of about 50 hours [743]. A system was developed based on biodegradable polymer and nanotechnology resulting in polylactide membrane hemoglobin nanocapsules of about 150 nm diameter [744, 745]. This was smaller than the lipid vesicles and contained negligible amounts of lipids. The superoxide dis-mutase, catalase, and also multienzyme systems were used to prevent the accumulation of methemoglobin [746].

For detailed progress on artificial cell and red blood cell substitutes, refer to the literature [747-805].

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