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The oxidation reactions have become the main obstacle of development of bovine hemoglobin-derivates products. Herein, the effects of vitamin C (Vc), a easily available natural antioxidant reagent, on the redox reaction of bovine hemoglobin were systematically investigated through methemoglobin (MetHb) formation and spectrophotometric analysis and oxygen affinity monitoring of hemoglobin. The results showed that Vc presented antioxidant effects in the initial stage of reaction and then could accelerated the MetHb content increasing by production of hydrogen peroxide, which can be indirectly characterized by the formation of choleglobin in the following side reactions. The dual effects of Vc include antioxidant and pro-oxidant effects could be confirmed by the spectrophotometric spectrums analysis in this research. The results of this research supplied the novel insight into understanding of redox properties of bovine hemoglobin and also revealed the main obstacle in exploration of Vc application in the future development of bovine hemoglobin-derivates products.

The first reports on biotherapeutic artificial red blood cells (RBC) were published some time ago [1,2]. Most people thought that artificial RBC was a simple matter that could be quickly developed ...

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (Poly-[Hb-SOD-CAT-CA]) contains all three major functions of red blood cells (RBCs) at an enhanced level. It transports oxygen, removes oxygen radicals and transports carbon dioxide. Our previous studies in a 90-min 30 mm Hg Mean Arterial Pressure (MAP) sustained hemorrhagic shock rat model shows that it is more effective than blood in the lowering of elevated intracellular pCO2, recovery of ST-elevation and histology of the heart and intestine. This paper is to analyze the storage and temperature stability. Allowable storage time for RBC is about 1 d at room temperature and 42 d at 4 °C. Also, RBC cannot be pasteurized to remove infective agents like HIV and Ebola. PolyHb can be heat sterilized and can be stored for 1 year even at room temperature. However, Poly-[Hb-SOD-CAT-CA] contains both Hb and enzymes and enzymes are particularly sensitive to storage and heat. We thus carried out studies to analyze its storage stability at different temperatures and heat pasteurization stability. Results of storage stability show that lyophilization extends the storage time to 1 year at 4 °C and 40 d at room temperature (compared to respectively, 42 d and 1 d for RBC). After the freeze-dry process, the enzyme activities of Poly-[SFHb-SOD-CAT-CA] was 100 ± 2% for CA, 100 ± 2% for SOD and 93 ± 3.5% for CAT. After heat pasteurization at 70 °C for 2 h, lyophilized Poly-[Hb-SOD-CAT-CA] retained good enzyme activities of CA 97 ± 4%, SOD 100 ± 2.5% and CAT 63.8 ± 4%. More CAT can be added during the crosslinking process to maintain the same enzyme ratio after heat pasteurization. Heat pasteurization is possible only for the lyophilized form of Poly-[Hb-SOD-CAT-CA] and not for the solution. It can be easily reconstituted by dissolving in suitable solutions that continues to have good storage stability though less than that for the lyophilized form. According to the P50 value, Poly-[SFHb-SOD-CAT-CA] retains its oxygen carrying ability before and after long-term storage.

Even though erythrocytes transport both oxygen and carbon dioxide, research on blood substitutes has concentrated on the transport of oxygen and its vasoactivity and oxidative effects. Recent study in a hemorrhagic shock animal model shows that the degree of tissue PCO(2) elevation is directly related to mortality rates. We therefore prepared a novel nanobiotechnological carrier for both O(2) and CO(2) with enhanced antioxidant properties. This is based on the use of glutaraldehyde to crosslink stroma free hemoglobin (SFHb), superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase (CA) to form a soluble PolySFHb-SOD-CAT-CA. It was compared to blood and different resuscitation fluids on the ability to lower elevated tissue PCO(2) in a 2/3 blood volume loss rat hemorrhagic shock model. Sixty minutes of sustained hemorrhagic shock at 30 mm Hg resulted in the increase of tissue PCO(2) to 95 mm ± 3 mmHg from the control level of 55 mm Hg. Reinfusion of whole blood (Hb 15 g/dL with its RBC enzymes) lowered the tissue PCO2 to 72 ± 4.5 mmHg 60 minutes after reinfusion. PolySFHb-SOD-CAT-CA (SFHb 10 g/dL plus additional enzymes) was more effective than whole blood in lowering PCO(2) lowering this to 66.2 ± 3.5 mmHg. Ringer's Lactated solution or polyhemoglobin lowered the elevated PCO2 only slightly to 87 ± 4.5 mmHg and 84.8 ± 1.5 mmHg, respectively. Moreover, ST-elevation for whole blood (Hb 15 g/dL) and PolySFHb-SOD-CAT-CA (Hb 10 g/dL) was respectively 12.8% ± 4% and 13.0% ± 2% of the control 60 minutes after reinfusion. Both are significantly better than those in the Ringer's lactated group and the PolyHb group. In conclusion, this novel approach for blood substitute design has resulted in a novel nanobiotechnological carrier for both O(2) and CO(2) with enhanced antioxidant properties.

We have prepared a novel nanobiotherapeutic, Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase], which not only transports both oxygen and carbon dioxide but also a therapeutic antioxidant. Our previous study in a severe sustained 90 min hemorrhagic shock rat model shows that it has a hepatoprotective effect. We investigate its hepatoprotective effect further in this present report using an alcohol-damaged primary hepatocyte culture model. Results show that it significantly reduced ethanol-induced AST release, lipid peroxidation, and ROS production in rat primary hepatocytes culture. It also significantly enhanced the viability of ethanol-treated hepatocytes. Thus, the result shows that Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] also has some hepatoprotective effects against alcohol-induced injury in in vitro rat primary hepatocytes cell culture. This collaborate our previous observation of its hepatoprotective effect in a severe sustained 90-min hemorrhagic shock rat model.

Unlike donor red blood cells (RBCs), blood substitutes can be treated to remove infective agents and can be used on the spot or in the ambulance in emergency without the time-consuming typing and cross-matching. Donor RBC requires storage at 4 degrees and is only good for 42 days, but blood substitutes can be stored for much longer time. For example, a bovine polyhemoglobin (PolyHb) can be stored at room temperature for more than 1 year. It has been shown as far back as 1957 that artificial RBC can be prepared with ultrathin polymer membranes of nanodimension thickness. To increase the circulation time, the first-generation engineered hemoglobin (Hb) is formed by using glutaraldehyde to crosslink Hb into soluble nanodimension PolyHb that has been tested clinically in patients. Further extension includes conjugated Hb, intramolecularly crosslinked Hb and recombinant Hb. For certain clinical uses, in addition to engineered Hb, we also need antioxidants to remove oxygen radicals to prevent injury from ischemia reperfusion. Thus, we use nanobiotechnology to prepare second-generation engineered Hb by assembling Hb together with superoxide dismutase (SOD) and catalase (CAT) to form a nanodimension soluble complex of polyhemoglobin (PolyHb)-CAT-SOD. A third generation system is to prepare nanodimension complete artificial RBCs that can circulate for sufficient length of time after infusion. One approach uses lipid vesicles to encapsulate hemoglobin (Hb). Another approach is to use biodegradable polymer-like polylactic acid or a copolymer of polyethylene glycol-polylactide (PEG-PLA) to form the membrane of nanodimension complete artificial RBC (www.artcell.mcgill.ca).

In sustained severe ischemia, reperfusion with oxygen carriers may result in ischemia-reperfusion injuries because of the release of damaging oxygen radicals. A nanobiotechnology-based polyhemogloin-calatase-superoxide dismutase can prevent this because the oxygen carrier, polyhemoglobin, is linked to antioxidant enzymes, catalase and superoxide dismutase. However, these antioxidant enzymes come from nonhuman sources and recombinant human enzymes are expensive. This paper describes our study on extracting these enzymes from red blood cells and analyzing the amount of enzymes needed for adequate protection from ischemia-reperfusion.

Artificial cells consist of a hybrid system combining biological and synthetic materials. It has been investigated for enzyme therapy, erythrocyte substitution, hemoperfussion, artificial kidney, artificial liver, detoxification, immunoadsorbents and other applications.

The first artificial cells were prepared 35 years ago. They contain biologically active materials. They are now being used in medicine and biotechnology. Artificial cells containing adsorbents are already a routine form of treatment in hemoperfusion. This includes treatment for acute poisoning, high blood aluminum and iron, kidney failure, some types of acute liver failure, and other conditions. Artificial cells are being tested for use as red blood cell substitutes. Artificial cells containing cell culture are being tested in animals for the treatment of diabetes, liver failure, and others. Artificial cells containing enzymes are being tested for treatment in hereditary enzyme deficiency diseases and other diseases. Artificial cells containing complex enzyme system can convert wastes like urea and ammonia into useful amino acids. In biotechnology, artificial cells are being used for the production of monoclonal antibodies, interferons, and other biotechnological products. They are also being investigated for use in other applications in biotechnology, chemical engineering, and medicine.

Artificial liver support requires more than a detoxification system. We have investigated additional approaches. Microencapsulated hepatocytes increased the survival time of fulminant hepatic failure (FHF) rats. They also lowered the bilirubin in Gunn rats. Xenograft of microencapsulated rat hepatocytes into mice are immunoisolated. The viability of hepatocyte increased from 62 to 100% after 29 days. This is because of accumulation of a hepatic stimulatory factor (> 100,000 D) secreted by the hepatocytes in the artificial cells. A novel two-step method of cell encapsulation greatly improved immu-noisolation and biocompatibility. Other metabolic approaches included a multienzyme system for conversion of ammonia to essential amino acid, and removal of bilirubin.

Polyhemoglobin is formed by the nanobiotechnological assembling of hemoglobin molecules into soluble nanodimension complex. A further step involves the nanobiotechnological assembly of hemoglobin, catalase and superoxide dismutase into a soluble nanodimension complex. This acts both as oxygen carrier and antioxidant to prevent the oxidative effects of hemoglobin. A further step is the preparation of nanodimension artificial red blood cells that contain hemoglobin and all the enzymes present in red blood cells. Other approaches include a polyhemoglobin–fibrinogen that acts as an oxygen carrier with platelet-like activity, and a polyhemoglobin–tyrosinase to retard the growth of a fatal skin cancer, melanoma.

This study is to investigate the long-term effects of nanodimension PEG-PLA artificial red blood cells containing hemoglobin and red blood cell enzymes on the liver and spleen after 1/3 blood volume top loading in rats. The experimental rats received one of the following infusions: Nano artificial red blood cells in Ringer lactate, Ringer lactate, stroma-free hemoglobin, polyhemoglobin, and autologous rat whole blood. Blood samples were taken before infusions and on days 1, 7, and 21 after infusions for analysis. Nano artificial red blood cells, polyhemoglobin, Ringer lactate and rat red blood cells did not have any significant adverse effects on alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine kinase, amylase and creatine kinase. On the other hand, stroma-free hemoglobin induced significant adverse effects on liver as shown by elevation in alanine aminotransferase and aspartate aminotransferase throughout the 21 days. On day 21 after infusions rats were sacrificed and livers and spleens were excised for histological examination. Nano artificial red blood cells, polyhemoglobin, Ringer lactate and rat red blood cells did not cause any abnormalities in the microscopic histology of the livers and spleens. In the stroma-free hemoglobin group the livers showed accumulation of hemoglobin in central veins and sinusoids, and hepatic steatosis. In conclusion, injected nano artificial red blood cells can be efficiently metabolized and removed by the reticuloendothelial system, and do not have any biochemical or histological adverse effects on the livers or the spleens.

We report a novel method to simultaneously extract superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) from the same sample of red blood cells (RBCs). This avoids the need to use expensive commercial enzymes, thus enabling a cost-effective process for large-scale production of a nanobiotechnological polyHb-SOD-CAT-CA complex, with enhancement of all three red blood cell functions. An optimal concentration of phosphate buffer for ethanol-chloroform treatment results in good recovery of CAT, SOD, and CA after extraction. Different concentrations of the enzymes can be used to enhance the activity of polyHb-SOD-CAT-CA to 2, 4, or 6 times that of RBC.

Polyhemoglobin is already well into the final stages of clinical trials in humans with one approved for routine clinical use in South Africa. Conjugated hemoglobin is also in ongoing clinical trials. Meanwhile, recombinant Hb has been modified to modulate the effects of nitric oxide. Other systems contain antioxidant enzymes for those clinical applications that may have potential problems related to ischemia-reperfusion injuries. Other developments are based on hemoglobin-lipid vesicles and also the use of nanotechnology and biodegradable copolymers to prepare nanodimension artificial red blood cells containing hemoglobin and complex enzyme systems.

Backgrounds/aims This study investigates the ammonia removal capacity of coencapsulated hepatocytes and bone marrow stem cells in culture, and the treatment effect on hyperbilirubinemia Gunn rats when transplanted. Methods The hepatocytes and bone marrow stem cells isolated from Wistar rats were encapsulated alone or coencapsulated. In vitro, the encapsulated cells were cultured in media supplemented with 2.4 mMol/L concentration of ammonium chloride and the ammonia removal and urea synthesis were evaluated. In vivo, the encapsulated cells were transplanted intraperitoneally into hyperbilirubinemia Gunn rats and plasma bilirubin levels were measured before and after transplantation at intervals of 85 days. Results The ammonia removal capacity was maintained longer in the different ammonia concentration media in the coencapsulated hepatocytes and bone marrow cells culture. In the coencapsulation transplantation group, the plasma bilirubin levels were significantly lower than those in the group of hepatocytes encapsulation transplantation during the period of 3 to 10 weeks posttransplantion. Conclusions The coencapsulated heaptocytes and bone marrow cells when compared to encapsulated hepatocytes could improve the maintenance of hepatocyte function both in vitro of ammonia removal in culture, and in vivo of the lowering the Gunn rats blood total bilirubin when transplanted.

We have previously reported the experimental use of genetically engineered Escherichia coli with microencapsulation to lower nitrogenous waste. Concern has surfaced, nonetheless, about safety of genetically engineered product. The purpose of this study is to explore the alternative use of probiotics in removal of plasma urea. After repeated cycles of exposure of Lactobacillus delbrueckii in urea-rich medium under anaerobic environment, the organisms were demonstrated to lower plasma urea concentration in vitro. Suspension of Lactobacillus in uremic plasma reduced the urea nitrogen levels from 51.5 +/- 5.2 mg/dL to 44.3 +/- 3.9 mg/dL (P = 0.02) after 24 hours. With microencapsulation of Lactobacillus (inside semipermeable alginate-polylysine-alginate polymeric membrane), further lowering of urea nitrogen levels was achieved (35.4 +/- 0.8 mg/dL, P = 0.03) at 24 hours. These preliminary data show that expression of certain enzymes could be induced in Lactobacillus delbrueckii and thus capable of lowering plasma urea. Further studies and molecular analysis would be indicated to explore and refine the techniques.

In this study, we prepared microencapsulated tyrosinase and studied its enzyme kinetics and its stability at different pH and temperature. The Km for both free and microencapsulated tyrosinase is 465 microM. The Vmax for the microencapsulated tyrosinase is 49.02 mg/dl x min, whereas that of tyrosinase in free solution is 114.95 mg/dl x min. At the extreme pH of pH 2 and pH 10, free tyrosinase lost all its enzyme activity, whereas microencapsulated tyrosinase retained 14% of its original activity at pH 2 and 17% of its original activity at pH 10. Further in vitro studies were carried out to study the pH optimum of the microencapsulated enzyme at different pH values corresponding to those along the gastrointestinal tract. In temperature stability studies at 4 degrees C and 37 degrees C, higher enzyme activity remained in microencapsulated tyrosinase solution than that of free enzyme solution. Therefore, our results show that microencapsulated tyrosinase is significantly more stable than free tyrosinase. In subsequent studies, we show that daily oral administration of microencapsulated tyrosinase significantly lowers systemic tyrosinase after 3 days and maintains this low level as long as oral administration continues. When combined with one intravenous injection of polyhemoglobin-tyrosinase, the systemic tyrosine level can be lowered within one hour.

Hemoglobin molecules extracted from red blood cells are modified by microencapsulation or crosslinking. This stabilizes the hemoglobin molecules and also allows the sterilization of the products to remove HIV and other microorganisms. A number of modified hemoglobins have been developed and clinical trials have been carried out. The results of these clinical trials have led to the elimination of a number of modified hemoglobins, leaving polyhemoglobin and conjugated hemoglobin. Two types of polyhemoglobin have been most extensively studied in Phase III clinical trials. These results have led to South Africa and more recently Russia’s approval of the routine clinical use of one type of polyhemoglobin. These decisions are based on their risk/benefit analysis for their own requirements. In North America and some other countries the requirements are such that they have not yet approved this product for routine clinical uses until further improvements have been carried out. New generations of modified hemoglobin are being actively developed. These include soluble nano dimension complexes of the following: Oxygen carrier with antioxidant properties (polyhemoglobin-catalase-superoxide dismutase and synthetic mimics of this); combined oxygen and carbon dioxide carriers with antioxidant properties (polyhemoglobin-catalase-superoxide dismutase-carbonic anhydrase); oxygen carrier with platelet-like properties (polyhemoglobin-fibrinogen); nano dimension artificial red blood cells with lipid or biodegradable polymeric membrane and others.

Increase in systemic uric acid occurs in renal insufficiency, gout, chemotherapy, and other diseases. Dialysis can lower this metabolite but is expensive. The use of drugs can, sometime, result in side effects. Therefore, a suitable affordable method for this is required. In this article, for the first time, we report the use of artificial cells containing micro encapsulated genetically engineered E. Coli DH5 cells for lowering uric acid in vitro and in vivo. Results show that this novel approach has the ability to significantly lower uric acid from 84.80±3.40 mg/dl to 9.32±0.05 mg/dl in vitro and from the plasma of the experimental animals from the control levels of 71.00±27.49 mg/dl to 20.33+17.92 mg/dl in vivo. Continued daily oral administration maintained the plasma uric acid concentration of experimental uremic rats to the normal plasma uric acid level range during the entire test period.

This paper discusses our research on two new generation blood substitutes. One is based on the crosslinking of hemoglobin, superoxide dismutase(SOD) and catalase (CAT) to form polyhemoglobin-SOD-CAT. This is being investigated for use in conditions with potential problems related to ischemia-reperfusion injuries as in severe hemorrhagic shock, stroke and other conditions. The second one is based on biodegradable polymeric nanocapsules containing hemoglobin and enzymes. In this form, the hemoglobin and enzymes are separated from the external environment. Furthermore, modifications of the polymeric membrane can result in increase in circulation time.

Lowering of plasma Mg, P, Na, Cl, uric acid, cholesterol, and creatinine is required in renal failurte and other diseases. In this preliminary report, we studied the ability of artificial cells microencapsulated genetically engineered E. coli DH5 cells in lower K, Mg, P, Na, Cl, uric acid, cholestrol, creatinine, and billirubin from plasma in-vitro. Result shows that this novel approach has the ability to significantly lower these metabolites from the plasma in-vitro.

Crosslinking hemoglobin with superoxide dismutase and catalase (PolyHb-SOD-CAT) helps to limit free radical reactivity of modified hemoglobin red blood cell substitutes. In the present study, in vitro oxidant challenge experiments were performed with exogenous hydrogen peroxide (H 2 O 2 ) and xanthine oxidase-derived superoxide (O 2 ·− ). PolyHb-SOD-CAT was compared to PolyHb for the presence of secondary hemoprotein-free radical events. PolyHb-SOD-CAT prevents ferrylhemoglobin formation, measured as Na 2 S-induced absorbance at 620 nm. Similarly, PolyHb-SOD-CAT inhibited ferrozine-detectable iron release at high oxidant-heme ratios. The formation of oxygen radicals, monitored by salicylate hydroxylation, was prevented at high oxidant–heme ratios with PolyHb-SOD-CAT. The peroxidation of liposomal membranes was also inhibited in PolyHb-SOD-CAT mixtures subject to oxidant challenge. These results show that PolyHb-SOD-CAT prevents secondary hemoprotein-associated free radical events. This new type of modified hemoglobin oxygen carrier with antioxidant activity may reduce the potential toxicity of hemoglobin-based substitutes in certain applications, especially during reperfusion of ischemic tissues.

Poly(ethylene glycol)-Poly(lactic acid) block-copolymer (PEG-PLA) was prepared and characterized using Fourier transform infrared spectrophotometer (FTIR). Glutaraldehyde was used to crosslink stroma-free hemoglobin (SFHb), superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) into a soluble complex of PolySFHb-SOD-CAT-CA. PEG-PLA was then used to nanoencapsulated PolySFHb-SOD-CAT-CA by oil in water emulsification. This resulted in the formation of PLA-PEG-PolySFHb-SOD-CAT-CA nanocapsules that have enhanced antioxidant activity and that can transport both O2 and CO2. These are homogeneous particles with an average diameter of 100 nm with good dispersion and core shell structure, high entrapment efficiency (EE%), and nanocapsule percent recovery. A lethal hemorrhagic shock model in rats was used to evaluate the therapeutic effect of the PLA-PEG-PolySFHb-SOD-CAT-CA nanocapsules. Infusion of this preparation resulted in the lowering of the elevated tissue PCO2 and also recovery of the mean arterial pressure (MAP).

Hemoglobin (Hb), a tetrameric protein, is responsible for the transport of oxygen in red blood cells (Perutz 1980). Attempts to use hemolysate (Amberson 1937) and stroma-free Hb as Hb based oxygen carrier (Rabiner et al. 1967) resulted in nephrotoxicity and cardiovascular adverse effects (Savitsky et al. 1978). Thus, nanobiotechnological modification is needed before it can be used (Chang 1964)

We report a novel approach for plasma urea and ammonia removal using artificial cells microencapsulated genetically engineered bacteria E. coli DH5 cells. This has been evaluated for use in a column bioreactor for removing plasma urea and ammonia. It has also been evaluated in uremic rats for urea removal by oral administration. In 30 minutes, microencapsulated E. coli DH5 in a column bioreactor in-vitro lowered plasma urea to 10.47 +/- 3.45 mg/dl from 45.85 +/- 2.98 mg/dl and lowered plasma ammonia concentration to 46.00 +/- 4.00 microM from 679 +/- 32 microM/1. The efficiency of this bioreactor for plasma urea and ammonia removal is much higher than any other available methods. Initial plasma urea and ammonia concentration does not affect the plasma urea and ammonia removal efficiency of the column bioreactor. In in-vivo studies of oral administration to uremic rats, both free and encapsulated bacteria both lowered systemic urea from the initial 52.08 (S.D.2.37) mg/dl to 10.58 (S.D.0.85) mg/dl. Unlike free bacteria, microencapsulated bacteria was not retained in the intestine.

Kinetic analysis of the enzyme UDP-glucuronosyltransferase (UDPGT), responsible for the conjugation of bilirubin, suggests that it is a multisubunit enzyme in which there is cooperative binding of the substrate to the subunits. The binding of bilirubin to UDPGT shows positive cooperativity with an apparent Hill coefficient of 2.9. The binding of UDP-glucuronic acid (UDPGA) exhibits kinetics with mixed cooperativity with an apparent Hill coefficient of 4.028. Homogenized rat hepatocytes, intact hepatocytes, and hepatocytes encapsulated in alginate-polylysine-alginate artificial cells, when incubated with bilirubin (1.6 mM) and UDPGA (20 mM), can form monoconjugated and diconjugated bilirubin. However, the presence of the artificial membrane offers some mass transfer resistance. The intraperitoneal transplantation into the Gunn rat of free and microencap-sulated Wistar rat hepatocytes shows that both are equally effective in lowering the serum bilirubin. Thus, the membrane did not contribute to a lowering of efficacy after transplantation.

The first experimental artificial red blood cells have all three major functions of red blood cells (rbc). However, the first practical one is a simple polyhemoglobin (PolyHb) that only has an oxygen-carrying function. This is now in routine clinical use in South Africa and Russia. An oxygen carrier with antioxidant functions, PolyHb-catalase-superoxide dismutase, can fulfill two of the three functions of rbc. Even more complete is one with all three functions of rbc in the form of PolyHb-catalase-superoxide dismutase-carbonic anhydrase. The most advanced ones are nanodimension artificial rbc with either PEG-lipid membrane or PEG-PLA polymer membrane. Extensions into oxygen therapeutics include a PolyHb-tyrosinase that suppresses the growth of melanoma in a mice model. Another is a PolyHb-fibrinogen that is an oxygen carrier with platelet-like function. Research has now extended well beyond the original research on artificial rbc into many areas of artificial cells. These include nanoparticles, nanotubules, lipid vesicles, liposomes, polymer-tethered lipid vesicles, polymersomes, microcapsules, bioencapsulation, nanocapules, macroencapsulation, synthetic cells, and others. These are being used in nanotechnology, nanomedicine, regenerative medicine, enzyme/gene therapy, cell/stem cell therapy, biotechnology, drug delivery, hemoperfusion, nanosensers, and even by some groups in agriculture, industry, aquatic culture, nanocomputers, and nanorobotics.

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (PolyHb-SOD-CAT-CA) is a therapeutic antioxidant that also transports both oxygen and carbon dioxide. This is formed by crosslinking Hb with SOD, CAT, and CA using glutaraldehyde. Crosslinking stroma free Hb from red blood cell (rbc) reduces CA activity to 55%. Addition of more CA resulted in a preparation with the same CA activity as RBC. PolyHb in the complex acts as a buffer to prevent large pH changes as carbon dioxide is converted to carbonic acid. We then prepare and optimize a novel PolyHb-SOD-CAT-CA, a therapeutic antioxidant that also transports both oxygen and carbon dioxide.

Erwinia herbicola (ATCC 21434) was grown in a medium which caused the cells to induce tyrosine phenol-lyase (TPL) activity. Whole cells of Erwinia herbicola were then microencapsulated within alginate-poly-L-lysine-alginate membraned microcapsules (diameter 800 microns). In a rotary shaker-incubator with a 1.9 cm horizontal throw, an agitation rate of at least 240 revolutions per minute (rpm) was required before the TPL activity of the microencapsulated cells was equal to that of the free cells. The TPL activity of the cells, whether free or microencapsulated, could be used for the conversion of ammonia, pyruvate and phenol into tyrosine at 37 degrees C. The results indicate that free cells and microencapsulated cells effect the conversion of these reactants to tyrosine equally well if the agitation rate is 240 rpm. In liver failure the concentrations of both ammonia, phenol and pyruvate are elevated. Hence the TPL activity of microencapsulated Erwinia herbicola could possibly find application in a novel approach for the removal of toxic phenol and ammonia during liver failure.

Crosslinking of ultrapure hemoglobin, crystalline catalase, and superoxide dismutase resulted in a soluble nanodimensional complex of polyhemoglobin-catalase-superoxide dismutase. A less expensive and more convenient way is to crosslink bovine stroma-free hemolysate (stroma-free hemolysate) that already contains hemoglobin, catalase, and superoxide dismutase into polyhemoglobin with catalase and superoxide dismutase activities (stroma-free polyhemolysate) [21]. The objective of the present study is to evaluate the immunological properties of this stroma-free polyhemolysate. Each of three groups of rats received weekly subcutaneous injections of one of the stroma-free polyhemolysate, stroma-free hemolysate, and saline for four weeks. One week after the four cycles of weekly immunization, serum and plasma were collected for C3a complement activation tests and Ouchterlony antibody-antigen precipitation tests, respectively. Results show that stroma-free polyhemolysate retained significant antioxidant enzyme activity. The C3a complement activation test and Ouchterlony test show that four weekly subcutaneous injections of bovine stroma-free polyhemolysate did not result in any immunological reaction in rats when tested this way.

Nanobiotechnology is the assembling of biological molecules into nanodimension complexes. This has been used for the preparation of polyhemoglobin formed by the assembling of hemoglobin molecules into a soluble nanodimension complex. New generations of this approach include the nanobiotechnological assembly of hemoglobin, catalase, and superoxide dismutase into a soluble nanodimension complex. This acts as an oxygen carrier and an antioxidant for those conditions with potential for ischemiareperfusion injuries. Another recent novel approach is the assembling of hemoglobin and fibrinogen into a soluble nanodimension polyhemoglobin-fibrinogen complex that acts as an oxygen carrier with platelet-like activity. This is potentially useful in cases of extensive blood loss requiring massive replacement using blood substitutes, resulting in the need for the replacement of platelets and clotting factors. A further step is the preparation of nanodimension artificial red blood cells that contain hemoglobin and all the enzymes present in red blood cells.

Pseudomonas pictorum in free or immobilized form can deplete serum cholesterol. The reaction kinetics control the global rate of cholesterol depletion during the initial part of free P. pictorum fermentation. Then, mass transfer takes control of the global rate. In this part of the fermentation, the global rate is first order with respect to the cholesterol concentration. The halftime was 330 min. The global rate is zero order with respect to the bacterial concentration. The activation energy was 83 kJ/mol. These results are consistent with the aqueous cholesterol diffusion model. The open pore agar microcapsule experimental effective diffusivity for lipoproteins at 37 degrees C was 6.7 x 10(-11) m2/s. Mass transfer across the microcapsule was not the limiting factor for the global rate.

This is a study of the effects of stroma-free hemoglobin(Hb) and polyhemoglobin on C3 and C3a levels and on blood cell counts. The effects of membrane stroma from red cells and endotoxins on complement activation were also investigated. Plasma samples from rats were incubated with hemoglobin solutions or control solutions. C3 was measured using anti-rat C3 by nephelometric method. C3a was measured using radioimmunoassay. C3 and C3a were also determined in rats receiving hemoglobin solutions, red cell stroma solutions, or a solution containing complement activators (membrane stroma and bacterial endotoxins). There were no significant differences in C3 or C3a levels between the plasma incubated with hemoglobin solutions and the control plasma incubated with saline (P greater than 0.05). Compared with the saline incubated plasma, C3 was significantly lower and C3a significantly higher in the zymosan treated plasma (P less than 0.01). In hemoglobin infused rats, there was no significant difference between pre-versus post-infusion values in C3 and C3a. There was a significant increase in C3a in the rats receiving stroma suspension (P less than 0.05) or stroma and endotoxins (P less than 0.02). Polyhemoglobin and stroma-free hemoglobin did not cause significant changes in total leucocyte, differential, or platelet counts. Our study suggests that purified stroma-free Hb and polyHb do not activate complement, and that C3a is a more sensitive parameter for monitoring complement activation than C3. On the other hand, contaminates in hemoglobin preparations, such as membrane stroma or endotoxins, activate the complement system.

The original artificial red blood cells have evolved into oxygen carriers in the form of polyhemoglobin and conjugated hemoglobin. Clinical conditions requiring only oxygen carriers are responding well to these types of oxygen carriers without the need for a complete artificial red blood cell. For those conditions requiring more than just oxygen carriers, new generations of polyhemoglobin containing antioxidant enzymes are being developed. Though a complete artificial red blood cell comparable to red blood cell is still a dream, development in lipid membrane artificial red blood cells and biodegradable polymeric nano artificial red blood cells are steps towards this possibility. The many years of neglect on basic research in the area of blood substitutes have resulted in the lack of important basic knowledge needed for the rapid development of blood substitutes suitable for clinical use. This is further hampered by the mistaken conception that blood substitute is a single entity. We need to look at blood substitutes as consisting of progressively more complicated entities, e.g. oxygen carriers, oxygen carriers with antioxidant activity, and complete red blood cell substitutes. Each of these entities is not applicable to all clinical conditions, but is suitable for specific applications.

Polyhemoglobin is already well into the final stages of clinical trials in humans. One has been approved for routine clinical use in South Africa. Perfluorochemicals are chemical oxygen carriers that are being actively developed with some in the advanced stages of clinical trials. Meanwhile, new generations of modified Hb are being developed that can modulate the effects of nitric oxide. Other systems are also being developed to include antioxidant properties for those clinical applications that may have potential problems related to oxygen radicals. Other products in advanced stages of animal testing are based on hemoglobin-lipid vesicles, heme-albumin and heme-lipid vesicles. A further development is the use of nanotechnology and biodegradable copolymers to prepare nano-dimension artificial red blood cells containing hemoglobin and complex enzyme systems.

We have recently reported our study on novel nano-dimension red blood cell (rbc) substitute based on ultrathin PEG-PLA membrane nanocapsules (80-150 nanometer diameter) containing hemoglobin (Hb) and enzymes. These have a markedly increased the circulation half-times as compared to our earlier PLA membrane nanocapsules. In the present study to be reported here, instead of looking at this from a pharmacodynamic point of view, we design the Hb nanocapsules from the point of view of transfusion medicine. For instance, the maximal levels of systemic non-red blood cell (rbc) Hb that can be attained after one infusion of 30% blood volume of 10 gm/dl Hb in the form of different types of PEG-PLA Hb nanocapsules or polyHb. Also the length of time one infusion can maintain a given systemic non-rbc hemoglobin Hb level. Of the two types of polyhemoglobins similar to those in clinical trials but prepared in this laboratory, the maximal levels of Hb reached were 3.35 gm/dl and 3.10 gm/dl respectively. The times for the hemoglobin level to fall to 1.67 gm/dl were 14 hours and 10. hours respectively, corresponding to 24 hours and 17 hours in human. The best PEG-PLA Hb nanocapsules are prepared using a combination of the following 4 factors: use of polymerized Hb, the use of higher M.W. PLA, the use of higher concentrations of PEG-PLA and the crosslinking of the newly formed PEG-PLA Hb nanocapsules. With this, the maximal non-rbc systemic Hb reached was 3.66 gm/dl and the time to reach 1.67 gm/dl was 24.2 hours, or 41.5 hours in human if extrapolated using the results obtained with polyHb in rats.

Polyhaemoglobins (PolyHb) and perfluorochemicals are in advanced phase III clinical trials and conjugated haemoglobins in phase II clinical trial. New recombinant human haemoglobin with no vasoactivity is being developed. A soluble macromolecule of PolyHb-catalase-superoxide dismutase is being studied as an oxygen carrier with antioxidant properties. New artificial red blood cells that are more like RBC are being developed. One is based on haemoglobin lipid vesicles. A more recent one is based on nano-dimension artificial red blood cells containing haemoglobin and RBC enzymes with membrane formed from composite copolymer of polyethylene glycol-polylactic acid. Their circulation time is double that of PolyHb.

The basic principles of artificial cells, encapsulation and immobilization form the asisS for a number of bioartificial organs. Hemoperfusion based on encapsulated adsorbent has been in routine clinical uses for many years to remove toxins or drugs from the circulating blood. Blood substitutes based on crosslinked hemoglobin or encapsulated hemoglobin are being developed and tested in phase II and Phase III clinical trials. Enzyme therapy using microencapsulated enzymes have been studied in animal studies and in a preliminary human study. encapsulation or other ways of immobilization of cells are being developed extensively by many groups. This includes the encapsulation or immobilization of islets, hepatocytes and genetically engineered cells.

This paper describes the growth kinetics of genetically engineered E. coli DH5 cells inside the APA membrane artificial cells. The APA microcapsule membrane found does not significantly affects the growth of the encapsulated E. coli DH5 cells. The total protein production of the E. coli DH5 cells inside the APA microcapsules were not significantly different from that of the bacterial cells grown in the free bacterial media. The result also show that the log phase APA artificial cells containing genetically engineered E. coli DH5 would be highly effective for the conversion of various external metabolites.

This article describes a novel method of urea and ammonia removal using microencapsulated, genetically engineered Escherichia coli DH5 cells. Optimization of bacterial cell encapsulation was carried out. The optimal method consists of alginate 2.00% (w/v) at a flow rate of 0.0724 mL/min and a coaxial air flow rate of 2.00 L/min. This produces spherical, alginate-poly-L-lysine-alginate (APA) microcapsules of an average 500 +/- 45 mum diameter. Increasing the concentration of alginate from 1.00% to 1.75% improves the quality of the microcapsules, while cell viability remains unaffected. The APA microcapsules are mechanically stable up to 210-rpm agitation with no bacterial cell leakage. The in vitro performance of urea and ammonia removal by encapsulated bacteria is assessed. One hundred milligrams of bacterial cells in APA microcapsules, in their log phase state of growth, can lower 87.89 +/- 2.25% of the plasma urea within 20 min and 99.99% in 30 min. The same amount of encapsulated bacteria can also lower ammonia from 975.14 +/- 70.15 muM/L to 81.151 +/- 7.37 muM/L in 30 min. There are no significant differences in depletion profiles by free and encapsulated bacteria for urea and ammonia removal. This novel approach using microencapsulated, genetically engineered E. coli cells is significantly more efficient than presently available methods of urea and ammonia removal. For instance, it is 30 times more efficient than the standard urease-ammonium adsorbent system. (c) 1995 John Wiley & Sons, Inc.

Oral binders remove intestinal bile acid and prevent its reabsorption and recycling thereby lowering systemic cholesterol levels. The results in this paper demonstrate the presence of another extensive enterorecirculation for amino acids. Pancreatic and other glandular secretions into the intestine contain large amounts of proteins, enzymes and polypeptides. Tryptic digestion converts these into amino acids which are then reabsorbed back into the body as they pass down the intestine. This paper shows that this forms a large enterorecirculation of amino acids between the body and intestine. The dietary protein source of amino acids is negligible when compared to the endogenous source, since this paper shows that protein-free diet did not alter the intestinal amino acid concentration. This raises the possibility of using this for the selective depletion of specific body amino acids. In this paper we use a phenylketonuria (PKU) model in rats to test the use of this hypothesis. In PKU rats, artificial cells microencapsulated phenylalanine ammonia lyase (PAL) given orally is more effective than a phenylalanine-free diet. The enzyme artificial cells are more efficient in lowering PHE in the intestine, plasma and cerebrospinal fluid. Compared to PKU on PHE-free diet, this has resulted in better weight gain and general physical condition. Preliminary studies also show that artificial cells microencapsulated asparaginase, glutaminase and tyrosinase given orally can deplete the corresponding amino acid from the intestine.

The most common use of artificial cells is for bioencapsulation of biologically active materials. Each artificial cell can contain combinations of materials. The permeability, composition and shape of an artificial cell membrane can be varied using different types of synthetic or biological materials. These possible variations in contents and membranes allow for large variations in the properties and functions of artificial cells. Artificial cells containing adsorbents have been a routine form of treatment in hemoperfusion for patients. This includes acute poisoning, high blood aluminum and iron, and supplement to dialysis in kidney failure. Artificial red blood cell substitutes based on modified hemoglobin are already in Phase I and Phase II clinical trials in patients. Artificial cell encapsulated cell cultures are being studied for the treatment of diabetes, liver failure, gene therapy and other conditions. Research on artificial cells containing enzymes includes their use for treatment in hereditary enzyme deficiency diseases and other diseases. Recent demonstration of extensive enterorecirculation of amino acids in the intestine has allowed oral administration to deplete specific amino acids. One example is phenylketonuria, an inborn error or metabolism resulting in high systemic phenylalanine levels. Preliminary clinical studies in patients using bioencapsulation of cells or enzymes have started. Artificial cells containing complex enzyme systems convert wastes like urea and ammonia into essential amino acids. Artificial cells are being used for the production of monoclonal antibodies, interferon and other biotechnological products. Other areas of biotechnological uses include drug delivery, and other areas of biotechnology, chemical engineering and medicine.

The anaerobic reaction of bovine hemoglobin with divinyl sulfone results in a non-cross-linked intramolecularly-modified new derivative. This chemically-modified hemoglobin is homogeneous with respect to its molecular mass (64 kDa) and electrophoretic properties. The absence of any 32 kDa band from its SDS-PAGE pattern proves the lack of intramolecular cross-linkage, while a single-peak high-performance gel-permeation chromatogram demonstrates the absence of intermolecular cross-linkage. The oxygen binding properties determined at 37 degrees C, 0.15 M Cl- and pH 7.4 display a P50 of 52 mmHg and a Hill coefficient n of 1.9. Under the same experimental conditions the oxygen affinity is not sensitive to chloride anions, suggesting that the covelant modification is in the beta-cleft. The maximum number of Bohr protons released is 0.8/tetramer, which is half that of normal bovine hemoglobin. The retention time in circulation, measured in rats, is similar to that of native bovine hemoglobin. Using a high molar ratio of divinyl sulfone to modified hemoglobin, it is feasible to effect anaerobic intermolecular cross-linkage. The polymerized material, isolated from a 24 h reaction, is a mixture of modified intermolecularly-crosslinked hemoglobins with a molecular mass range from 130 to approx. 500 kDa. The oxygen-transport characteristics of the polymerized material are similar to those of the modified non-cross-linked derivative, whereas its retention time in rats is increased three-fold with respect to native bovine hemoglobin.