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Melanoma is a deadly skin cancer. Surgery is effective for early stages but there may be remnant cells. Treatments of later stages are associated with severe side effects. Moreover, a dangerous type of melanoma cannot be detected early enough for surgery. There is an urgent need for treatment with less severe side effects. We use a novel system of artificial cell polymer-lipid membrane nanocarrier containing a biomolecular nano-system of enzyme-oxygen biotherapeutic. In this report we show (1) its effectiveness and mechanisms in inhibiting the growth of melanoma in a 3D culture collagen medium that is more similar to that in the animal. (2) This allows us to design and carry out animal studies to successfully show that this can inhibit the growth of melanoma in an animal model. This includes following the tumour sizes and body weights every 2 days for 30 days followed by histology of the sites of injection and vital organs. We also analyze the action of the different components of the nanocarrier-nano-biotherapeutic complex. In conclusion, the results show the safety and clinical feasibility of this approach in the animal model and encourages further study towards clinical use.

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 ...

Charged Device Model testing is confronted with high operating frequencies driving CDM to lower voltage levels and by high-density packages with ever smaller ball/pin pitches. A new CDM discharge head design meets these challenges by making DUT contact first and then an internal spark discharge occurs in a controlled environment.

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.

We crosslink hemoglobin (Hb), superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) to form a soluble polyHb-SOD-CAT-CA nanobiotechnological complex. The obtained product is a soluble complex with three enhanced red blood cell (RBC) functions and without blood group antigens. In the present study, 2/3 of blood volume was removed to result in 90-min hemorrhagic shock at mean arterial blood pressure (MAP) of 30 mmHg. This was followed by the reinfusion of different resuscitation fluids, then followed for another 60 min. PolyHb-SOD-CAT-CA maintained the MAP at 87.5 ± 5 mmHg as compared with 3 volumes of lactated Ringer's solution, 43.3 ± 2.8 mmHg; blood, 91.3 ± 3.6 mmHg; polyHb-SOD-CAT, 86.0 ± 4.6 mmHg; poly stroma-free hemolysate (polySFHb), 85.0 ± 2.5 mmHg; and polyHb, 82.6 ± 3.5 mmHg. PolyHb-SOD-CAT-CA was superior to the blood and other fluids based on the following criteria. PolyHb-SOD-CAT-CA reduced tissue pCO2 from 98 ± 4.5 mmHg to 68.6 ± 3 mmHg. This was significantly (p < 0.05) more effective than lactated Ringer's solution (98 ± 4.5 mmHg), polyHb (90.1 ± 4.0 mmHg), polyHb-SOD-CAT (90.9 ± 1.4 mmHg), blood (79.1 ± 4.7 mmHg), and polySFHb (77 ± 5 mmHg). PolyHb-SOD-CAT-CA reduced the elevated ST level to 21.7 ± 6.7% and is significantly (< 0.05) better than polyHb (57.7 ± 8.7%), blood (39.1 ± 1.5%), polySFHb (38.3% ± 2.1%), polyHb-SOD-CAT (27.8 ± 5.6%), and lactated Ringer's solution (106 ± 3.1%). The plasma cardiac troponin T (cTnT) level of polyHb-SOD-CAT-CA group was significantly (P < 0.05) lower than that of all the other groups. PolyHb-SOD-CAT-CA reduced plasma lactate level from 18 ± 2.3 mM/L to 6.9 ± 0.3 mM/L. It was significantly more effective (P < 0.05) than lactated Ringer's solution (12.4 ± 0.6 mM/L), polyHb (9.6 ± 0.7 mM/L), blood (8.1 ± 0.2 mM/L), polySFHb (8.4 ± 0.1 mM/L), and polyHb-SOD-CAT (7.6 ± 0.3 mM/L). PolyHb-SOD-CAT-CA can be stored for 320 days at room temperature. Lyophilized poly-Hb-SOD-CAT-CA can be heat pasteurized at 68F for 2 h. This can be important if there is a need to inactivate human immunodeficiency virus, Ebola virus, and other infectious organisms.

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.

Melanoma is a fatal skin cancer. In this paper, we design a nanobiotechnological approach for the specific depletion of tyrosine that is essential for melanoma growth. We combine two nanobiotechnological methods into a single therapeutic agent in the form of Polylactide (PLA) nanocapsules containing a nanobiotechnological complex of polyhemoglobintyrosinase. Our results show that nanocapsules can enter into the melanoma cells to deplete tyrosine. This leads to the inhibition of tumor growth, migration and colonization in a highly malignant melanoma cell line B16F10. We also analyzed possible mechanisms of action including ROS generation, apoptosis induction and effect on cell cycle

Mesenchymal stem cells (MSCs) derived from bone marrow can secrete cytokines and growth factors and can transdifferentiate into liver cells. We transplanted polymeric membrane bioencapsulated MSCs into the spleens of 90% partial hepatectomized rats. This resulted in 91.6% recovery rates. This is compared to a recovery rate of 21.4% in the 90% hepatectomized rats and 25% in the 90% hepatectomized rats receiving intrasplenic transplantation of free MSCs. After 14 days, the remnant livers in the bioencapsulated MSCs group are not significantly different in weight when compared to the sham control group. From day 1 to day 3 after surgery, in the bioencapsulated MSCs group, the plasma HGF and IL-6 were significantly higher than those in the free MSCs group and control group (P<0.01); plasma TNF-αwas significantly lower (P<0.001). We concluded that the intrasplenic transplantation of bioencapsulated MSCs significantly increases the recovery rates of 90% hepatectomized rats. It is likely that the initial effect is from proliver regeneration factors followed later by the transdifferentiated hepatocyte-like cells. However, histopathological analysis and hepatocyte proliferation study will be needed to better understand the regenerative mechanisms of this result. This study has implications in improving the survival and recovery of patients with very severe liver failure due to hepatitis, trauma, or extensive surgical resection.

We have reported previously that daily intravenous infusions of a soluble nanobiotechnological complex, polyhemoglobin-tyrosinase [polyHb-Tyr], can suppress the growth of murine B16F10 melanoma in a mouse model. In order to avoid the need for daily intravenous injections, we have now extended this further as follows. We have prepared two types of biodegradable nanocapsules containing [polyHb-Tyr]. One type is to increase the circulation time and decrease the frequency of injection and is based on polyethyleneglycol-polylactic acid (PEG-PLA) nanocapsules containing [polyHb-Tyr]. The other type is to allow for intratumoural or local injection and is based on polylactic acid (PLA) nanocapsules containing [polyHb-Tyr]. Cell culture studies show that it can inhibit the proliferation of murine B16F10 melanoma cells in the “proliferation model”. It can also inhibit the attachment of murine B16F10 melanoma cells in the “attachment model.” This could be due to the action of tyrosinase on the depletion of tyrosine or the toxic effect of tyrosine metabolites. The other component, polyhemoglobin (polyHb), plays a smaller role in nanocapsules containing [polyHb-Tyr], and this is most likely by its depletion of nitric oxide needed for melanoma cell growth.

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.

The objective of the present study is to evaluate the pharmacodynamic properties of polymerized porcine hemoglobin (pPolyHb) in an exchange transfusion model. Each of two groups of rats received a volume of pPolyHb or hetastarch that equalled 120–140% of estimated total blood volume (70 ml/kg) exchange transfusion. The results showed pPolyHb retained hemodynamic stability and exhibited superior volume expansion capability. Furthermore, pPolyHb effectively reverse anaerobic metabolism caused by a large amount of volume exchange. In comparison with hetastarch, pPolyHb increased blood oxygen content and tissue oxygenation. All these properties contribute to a higher effectiveness in sustaining the lives of rats in pPolyHb group.

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.

The Labscale tangential flow diafiltration system in conjunction with polyethersulfone Biomax filters (both purchased from Millipore Inc.) were used in the following studies: (1) A crucial step in the preparation of polyhemoglobin (polyHb) is the removal of tetrameric hemoglobin (Hb), which can cause adverse side-effects. The efficiency for this depends on the integrity of the 100 kDa filters. Those with lower integrity were not effective whereas those with a higher integrity of 0.075 ml/min were more effective. A filter with an integrity of 0.075 ml/min can reduce the initial 11.2% tetrameric Hb concentration to 1.3% using a flow rate of 0.2 ml/min. Higher flow rate of 2.2 ml/min was not as effective. (2) Filtration with a 100 kDa filter was quick and efficient in separating stroma-free hemoglobin solutions from cellular debris from hemolysate. (3) Both 5 kDa and 100 kDa filters are efficient in concentrating hemoglobin solutions.

Control trials and statistical analysis were carried out to assess the effects of albumin-collodion microencapsulated activated charcoal (ACAC) hemoperfusion on fulminant hepatic coma. A rat model of galactosamine induced fulminant hepatic coma was used. Rats which did not recover died at 3.0+/-0.6 days after galactosamine injection. Those which survived this period recovered. Forty-eight hours after galactosamine injection, a test group of 21 rats were treated with 1 hour hemoperfusion and compared with an untreated group of 23 rats. 71.4% of the treated group survived as compared to 30.4% of the untreated rats. Statistical analysis (t test) shows a significant increase in recovery for the treated group (less than 0.01). Biochemical and histological results will be discussed.

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.