Search

The long-term safety and immunological effects of bovine poly-[haemoglobin-catalase-superoxide dismutase-carbonic anhydrase] in rats are studied by four-weekly 5% blood volume top-loading infusions followed by 30% blood volume exchange transfusion. There is no significant difference in growth, biochemistry and blood pressure between the control group receiving lactated ringer solution and those receiving the bovine poly-[haemoglobin-catalase-superoxide dismutase-carbonic anhydrase]. There is no significant change in mean arterial pressures (MAP) before and after each weekly top-loading infusion. After both the four weekly top-loading and the 30% exchange transfusions, the following safety and immune response evaluations are carried out. These include general studies on Ouchterlony double diffusion, total IgG and IgM, and complement activation. This is followed by quantitative measurements of specific antibodies against each of the following bovine components: Hb, CAT, SOD and CA in bovine poly-[haemoglobin-catalase-superoxide dismutase-carbonic anhydrase]. After the four weekly top-loading, each rat received a challenge of 30% blood volume exchange transfusion. The MAP, histamine and tryptase levels are tested before and after the 30% exchange transfusion. There are no anaphylactic reactions as shown by the MAP or histamine and tryptase. The results showed no safety problem nor adverse immune responses. All the rats survived when followed for one week after the 30% exchange transfusion.

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.

Polymerized Porcine Hemoglobin (pPolyHb), a hemoglobin-based oxygen carrier (HBOC), was developed as a potential red blood substitute for clinical applications. Assessment of its effects on the immune system is an important component of the overall safety evaluation of HBOC. For this purpose, we assessed three inflammation indicators, including complement C3a, IL-6, and TNF-α in cultured cells and in a rat model when pPolyHb was incubated or administrated with the cells/animals. Our results suggested that the levels of these three indicators were not statistically changed upon pPolyHb stimulation, indicating that pPolyHb is not immunotoxic to cells and animals in this aspect.

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.

It was 50 years ago that the first “artificial cells” were prepared [1,2]. This was not an attempt to reproduce biological cells, but to use available basic knowledge to prepare simple systems for possible uses in medicine and other areas. The 1957 emulsion method for forming ultrathin polymeric membrane artificial cells containing hemoglobin and red blood cell enzymes (Figure 1) has become the basis for the preparation of other types of microscopic and nanodimension artificial cells. Extensions of the original 1957 drop procedure for forming larger artificial cells (Figure 1) have become the basis for preparing artificial cells to contain islet, hepatocytes, genetic engineered cells, stem cells and other types of cells. Figure 1 Original 1957 method of preparing artificial cells (for details see [1,5]). Upper: drop method for preparing large artificial cells. Principle later extended for use in bioencapsulation of cells, stem cells, genetic engineered cells. Lower: emulsion phase ... There have been increasing and recently explosive interest and research activities around the world on artificial cells, especially in fields related to biotechnology, nanomedicine, nanoscience, bioencapsulation, cell therapy, blood substitutes, advance drug delivery systems, and even nanoscale robotics and others (Table 1). However, instead of the term “artificial cells,” many use other terminologies, such as liposomes, nanoparticles, microcapsules, blood substitutes, bioencapsulation, and so on. Table 1 Examples of areas of application (details in [4,5] As a result, any meaningful literature search for a complete idea of the present status of the whole field of artificial cells is impossible. Furthermore, the fact that papers in this highly interdisciplinary area are published in numerous journals specializing in chemistry, medicine, surgery, bioengineering, nanoscience and others makes a literature search even more difficult. Books in this area are mostly multi-authored, describing very specific and narrow areas. Thus for the 50th anniversary of artificial cells the author has just prepared a monograph on ARTIFICIAL CELLS: Biotechnology, Nanomedicine, Regenerative Medicine, Blood Substitutes and Cell/Stem Cell Therapy [5]. This is now such a large area that it needed more than 1000 references just to summarize the present status and future perspectives of artificial cells.

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.

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.

In strokes, myocardial infarctions, severe sustained hemorrhagic shock, and donor organs, inadequate blood supply results in lack of oxygen to the tissue (ischemia). If ischemia is sustained, reperfusion with the needed oxygen can result in tissue injury (ischemia-reperfusion injury) due to formation of reactive oxygen species. We are studying an oxygen-carrying solution with anitoxidant activity formed by cross-linking hemoglobin, superoxide dismutase, and catalase to form PolyHb-SOD-CAT. The present report studies its effect on the blood-brain barrier and cerebral edema when used in a transient global brain ischemia-reperfusion rat model. We compare this solution to sham-control, oxygenated saline, stroma-free hemoglobin (SF-Hb), polymerized hemoglobin (PolyHb), and a mixture of SF-Hb, SOD, and CAT in free solution. The results show that the cross-linked PolyHb-SOD-CAT solution, unlike the other solutions, can supply oxygen to ischemic tissues without causing reperfusion injury in the transient global brain ischemia-reperfusion model.

This study is to investigate the viability of hepatocytes when transplanted into Wistar rats using co-encapsulated hepatocytes and bone marrow stem cells. Hepatocytes and bone marrow stem cells, isolated from Wistar rats, are co-encapsulated using either the standard single-step method or a novel two-step cell encapsulation method (www.artcell.mcgill.ca). After intraperitoneal transplantation into Wistar rats, the histology, fate of recovered microcapsules and viability of encapsulated hepatocytes are studied. When prepared using the standard method, there is excellent viability but only for up to 3 weeks. After this, there is extensive fibrous coating and severe fibrous adhesion and no microcapsules can be recovered. On the other hand, using the new two-step encapsulation method, the viability of the encapsulated hepatocytes can be followed for more than 4 months after transplantation. Even up to 4 months, there is significantly less host reaction when using the two-step encapsulation method and 50% of the microcapsules can be recovered. Co-encapsulated with bone marrow stem cells resulted in further increase in viability of the hepatocytes when followed up to 4 months after transplantation. This new approach may improve the potential feasibility of using co-encapsulation of hepatocytes and bone marrow stem cells in bio-artificial liver support for the treatment of liver failure, especially for acute liver failure.

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.

Bone marrow cells co-cultured with hepatocytes resulted in hepatocytes that can be maintained in culture for 14-21 days. This is compared to 7-10 days with hepatocytes alone under the same conditions. Similarly, when bone marrow cells are co-encapsulated together with hepatocytes, the viability of hepatocytes in culture medium is prolonged to 28 days. This is compared to 14 days when hepatocytes are encapsulated alone under the same conditions. These results suggest that bone marrow cells can contribute to the viability and maintenance of hepatocytes. It addition, this principle could be applied to other situation as in helping the regeneration of hepatocytes in liver failure and also for other cells and organs.

We implanted artificial cell bioencapsulated bone marrow mesenchymal stem cells into the spleens of 90% hepatectomized (PH) rats. The resulting 14 days survival rate was 91%. This is compared to a survival rate of 21% in 90% hepatectomized rats and 25% for those receiving free MSCs transplanted the same way. Unlike free MSCs, the bioencapsulated MSCs are retained in the spleens and their hepatotrophic factors can continue to drain directly into the liver without dilution resulting in improved hepatic regeneration. In addition, with time the transdifferentiation of MSCs into hepatocyte-like cells in the spleen renders the spleen as a ectopic liver support.

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).

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.

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.

Bioencapsulation was first reported about 30 years ago (Chang 1964). This includes bioencapsulation of cells, enzymes, drugs, magnetic materials, isotopes, absorbents, hemoglobin and other materials (1–5) (Fig. 1). In the last 10 years, explosive interests in biotechnology has led to increasing research on bioencapsulation by many groups. Rapid progress is therefore being made around the world on the use of bioencapsulation in biotechnology (6–8). Studies by other groups are described in details in this symposium volume. This paper is therefore mainly a discussion of some of our own studies on bioencapsulation in biotechnology.

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.

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.

Bioencapsulation of cells is one of the many areas of artificial cells being extensively investigated by centers around the world. This includes the bioencapsulation of hepatocytes. A number of methods have been developed to maintain the specific function and phenotype of the bioencapsulated hepatocytes for in vitro and in vivo applications. These include supplementation of factors in the culture medium; use of appropriate substrates and the co-cultivation of hepatocytes with other type of cells, the so called "feeder cells". These feeder cells can be of liver origin or non-liver origin. We have recently studied the role of bone marrow cells in the maintenance of hepatocytes viability and phenotype by using the coculture of hepatocytes with bone marrow cells (nucleated cells including stem cells), and the coencapsulation of hepatocytes with bone marrow stem cells. This way, the hepatocytes viability and specific function can be maintained significantly longer. In vivo studies of both syngeneic and xenogeneic transplantation show that the hepatocytes viability can be maintained longer when coencapsulated with bone marrow cells. Transplantation of coencapsulated hepatocytes and bone marrow cells enhances the ability of the hepatocytes in correcting congenital hyperbilirubinmia in Gunn rats. Both in vitro and in vivo studies show that bone marrow cells can enhance the viability and phenotype maintenance of hepatocytes. Thus, bone marrow cells play an important role as a new type of feeder cells for bioencapsulated hepatocytes for the cellular therapy of liver diseases.

We transplanted bioencapsulated bone marrow stem cells intraperitoneally into 90% hepatectomized rats and found that this increases both the rates of hepatic regeneration and survival of the animals. Bone marrow cells isolated from Wistar rats were bioencapsulated using alginate-polylysine-alginate method. These bioencapsulated bone marrow cells were transplanted intraperitoneally into 90% hepatectomized syngeneic wistar rats. Control groups included 90% hepatectomized group receiving intraperitoneal injection of either empty microcapsules or free bone marrow cells. Unlike the control groups, transplantation of bioencapsulated bone marrow cells improved the survival of 90% hepatectomized rats, with an efficacy similar to that of bioencapsulated hepatocytes or free hepatocytes. These results suggest that syngeneic bioencapsulated bone marrow stem cells can increase the survival rates of 90% hepatectomized rats. We also discuss the potential for a new alternative to hepatocyte transplantation for cellular therapy of acute liver failure. In particular, bone marrow stem cells can be obtained from the same patient with no immunorejection, whereas in hepatocyte transplant, immunosuppressant will be needed to prevent immunorejection of the donor hepatocytes.

Homogenized rat hepatocytes, whole hepatocytes and encapsulated hepatocytes were incubated with bilirubin and UDP-glucuronic acid to test their ability to form bilirubin conjugates. Bilirubin monoconjugated and diconjugated were detected in all the three preparations by HPLC analysis. The UDP-glucuronosyltransferase (UDPGT) activity of homogenized hepatocytes was 0.002 +/- 0.00006 mM/min per million cells; that of intact hepatocytes was 0.001 +/- 0.00006 mM/min per million cells and that of encapsulated hepatocytes was 0.0005 +/- 0.00002 mM/min per million 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.

In this study, we prepared PolyHb-SOD-catalase (intermolecularly cross-linked hemoglobin, superoxide dismutase (SOD), and catalase). We found that PolyHb-SOD-catalase is effective in scavenging oxygen-derived free radicals. In the xanthine/xanthine oxidase system, the initial rate of cytochrome c reduction was 2.13 +/- 0.26 nmoles cyt. c/min for PolyHb alone. PolyHb- SOD-catalase reduced this to 0.56 +/- 0.08 nmoles cyt. c/min because of its ability to eliminate superoxide (O2-). Addition of PolyHb to 200 microM of hydrogen peroxide (H2O2), changed the H2O2 level slightly to 192 +/- 0.4 microM. Addition of PolyHb-SOD-catalase, on the other hand, lower the level to 41 +/- 0.3 microM. Results also show that both effects were dependent on the concentration of SOD-catalase cross-linked with hemoglobin. Oxidative challenge with H2O2 resulted in minimal changes in the absorbance spectra of PolyHb-SOD-catalase. With PolyHb, there were spectral changes reflecting the formation of methemoglobin and heme degradation. Furthermore, the amount of iron released, after incubation with 250 microM H2O2, was 6.8 +/- 1.8 micrograms/dl for PolyHb-SOD-catalase and 76.6 +/- 1.0 micrograms/dl for PolyHb. These results show that cross-linked SOD-catalase prevents oxidative reactions involving the hemoglobin component of PolyHb-SOD-catalase.

To make highly differentiated encapsulated he-patocytes, we formed hepatocyte spheroids in microcapsules in cultures. Hepatocytes isolated from rats were encapsulated in alginate-poly-L-lysine artificial cells and cultured under different medium conditions. When high molecular weight poly-L-lysine was used to make the capsule membrane, the hepatocytes aggregated and formed spheroids inside microcapsules within 48 hs. We studied the viability and function of encapsulated hepatocyte spheroids; free hepatocyte spheroids; nonaggregated encapsulated hepatocytes; and free hepatocyte monolayers. Cell viability and protein-producing ability were monitored for up to 30 days. Hepatocyte spheroids in the encapsulated or free form remained viable and continued to secrete proteins throughout the 30 days of observation. The viability and function of nonaggregated hepatocytes in the encapsulated or free form declined rapidly. These results suggest that the tridimensional structure of the spheroids may be important in maintaining the viability and function of encapsulated hepatocytes.

Modified hemoglobin consists of (1) encapsulated hemoglobin and (2) cross-linked hemoglobin (polyhemoglobin, intramolecularly cross-linked hemoglobin and conjugated hemoglobin). There have been new advances in all types of modified hemoglobins. Modified hemoglobins are effective in hemorrhagic shock. However, it is important to define hemorrhagic shock models and experimental designs. Important progress has been made in research on vasoactivities, organ perfusion, organ preservation, biodistribution, hematology, complement activation immunology and other areas. A preclinical screening test may bridge the gap between animal safety studies and injection into human. Potential new sources of hemoglobin included bovine hemoglobin, recombinant human hemoglobin and synthetic heme.

Artificial cells contain biologically active materials. 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 cells are being tested for use as red blood cell substitutes. Artificial cells encapsulated cell culture are being tested in animals for the treatment of diabetes and liver failure. A novel 2 step method has prevented xenograft rejection. Artificial cells containing enzymes are being studied for treatment in hereditary enzyme deficiency diseases and other diseases. Recent demonstration of extensive enterorecirculation of amino acids in the intestine has allowed its oral administration to deplete specific amino acids. Artificial cells containing complex enzyme system convert wastes like urea and ammonia into essential amino acids. Artificial cell is being used for the production of monoclonal antibodies, interferons and other biotechnological products. It is also being investigated for drug delivery, and for use in other applications in biotechnology, chemical engineering and medicine.

Repeated subcutaneous immunizing injections into rats of Freund's adjuvant containing rat hemoglobin or o-raffinose rat polyhemoglobin did not result in increase in IgG antibody liters. However, heterologous hemoglobin injected as above is antigenic (49.50 + 6.70 % CPM). Crosslinking heterologous hemoglobin into o-raffinose polyhemoglobin further increased its antigenicity (75.60 + 4.08). Thus, unlike homologous hemoglobin, cross-linking of heterologous hemoglobin increased its antigenicity. Liposome encapsulated homologous hemoglobin injected subcutaneously with or without Freund's adjuvant did not show antigenicity. Encapsulated heterologous hemoglobin resulted in a minimal increase in antibody tilers (10.73 + 4.64) only with Freund's adjuvant, but no increase when injected without Freund's adjuvant. Conjugated heterologous hemoglobin (PEG-Hb) injected intravenously by ilself at weekly inlervals did not result in significant increase in antibody tilers on the 5th week.