Lowering of elevated tissue PCO2 in a hemorrhagic shock rat model after reinfusion of a novel nanobiotechnological polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase that is an oxygen and a carbon dioxide carrier with enhanced antioxidant properties.

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.

Novel Nanodimension artificial red blood cells that act as O2 and CO2 carrier with enhanced antioxidant activity: PLA-PEG nanoencapsulated PolySFHb-superoxide dismutase-catalase-carbonic anhydrase.

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).

Nanobiotechnological Nanocapsules Containing Polyhemoglobin-Tyrosinase: Effects on Murine B16F10 Melanoma Cell Proliferation and Attachment.

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.

From artificial red blood cells, oxygen carriers, and oxygen therapeutics to artificial cells, nanomedicine, and beyond.

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.

PEG-PLA Nanocapsules Containing a Nanobiotechnological Complex of Polyhemoglobin-Tyrosinase for the Depletion of Tyrosine in Melanoma: Preparation and In Vitro Characterisation.

Poly (ethylene glycol)-poly (lactic acid) block-copolymer (PEG-PLA) was optimized and characterized using H-NMR spectrum and DSC thermogram. This was then used for the preparation of PEG-PLA nanocapsules containing polyhemoglobin-tyrosinase. Transmission electron microscopic and scanning electron microscopic studies showed round and non-aggregated nanocapsules with a PEG halo around each nanocapsule. Dynamic Light Scattering showed that the Z-average diameter was 65.2 ± 0.5 nm (mean ± SEM) and the polydispersity index was 0.262 ± 0.002. Factors controlling the diameters included the stirring speed of the reaction mixture and the size of the PLA block in the PEG-PLA copolymer. At the body temperature of 37oC, free tyrosinase lost all its enzyme activity after 8 hours. However, Polyhemoglobin-tyrosinase nanocapcules retained 80% of its initial activity after 8 hours. This paper contains the first part of our work on the preparation and in vitro characterisation of PEG-PLA Polyhemoglobin-tyrosinase nanocapsules. Preliminary result in rats shows that 1 intravenous injection lowers the systemic tyrosine level to 10-13% after 5 minutes. The result of the detailed in vitro study and the preliminary animal study in have led to our ongoing detailed animal research to be reported in subsequent papers.

Preliminary study on intrasplenic implantation of artificial cell bioencapsulated stem cells to increase the survival of 90% hepatectomized rats.

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.

Long-term effects on the histology and function of livers and spleens in rats after 33% toploading of PEG-PLA-nano artificial red blood cells.

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.

Effects of PEG-PLA-nano artificial cells containing hemoglobin on kidney function and renal histology in rats.

This study is to investigate the long-term effects of PEG-PLA nano artificial cells containing hemoglobin (NanoRBC) on renal function and renal histology after 1/3 blood volume top loading in rats. The experimental rats received one of the following infusions: NanoRBC in Ringer lactate, Ringer lactate, stroma-free hemoglobin (SFHB), polyhemoglobin (PolyHb), autologous rat whole blood (rat RBC). Blood samples were taken before infusions and on days 1, 7 and 21 after infusions for biochemistry analysis. Rats were sacrificed on day 21 after infusions and kidneys were excised for histology examination. Infusion of SFHB induced significant decrease in renal function damage evidenced by elevated serum urea, creatinine and uric acid throughout the 21 days. Kidney histology in SFHb infusion group revealed focal tubular necrosis and intraluminal cellular debris in the proximal tubules, whereas the glomeruli were not observed damaged. In all the other groups, NanoRBC, PolyHb, Ringer lactate and rat RBC, there were no abnormalities in renal biochemistry or histology. In conclusion, injection of NanoRBC did not have adverse effects on renal function nor renal histology.

Polyhemoglobin-fibrinogen: a novel oxygen carrier with platelet-like properties in a hemodiluted setting.

Polyhemoglobin (polyHb) is currently being assessed in phase III trials under various formulations. At present, none contain clotting factors or platelet substitutes to aid in hemostasis. We have prepared a novel blood substitute that is an oxygen carrier with platelet-like activity. This is formed by crosslinking fibrinogen to hemoglobin to form polyhemoglobin-fibrinogen (polyHb-Fg). This was studied and compared to polyHb for its effect on coagulation both in vitro and in vivo. In the in vitro experiments, PolyHb-Fg showed similar clotting times as whole blood, whereas polyHb showed significantly higher clotting times. This result was confirmed in in vivo experiments using an exchange transfusion rat-model. Using PolyHb, exchange transfusion of 80% or more increased the normal clotting time (1-2 mins) to > 10 mins. Partial clots formed with PolyHb did not adhere to the tubing wall. With PolyHb-Fg, a normal clotting time is maintained, even with 98% exchange transfusion.

Free and microencapsulated Lactobacillus and effects of metabolic induction on urea removal.

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.

Analysis of polyethylene-glycol-polylactide nano-dimension artificial red blood cells in maintaining systemic hemoglobin levels and prevention of methemoglobin formation.

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.

Oxygen carriers.

Three polyhemoglobins, formed by intermolecular cross-linking of hemoglobin molecules are in advanced phase III clinical trials and two conjugated hemoglobins, formed by cross-linking of hemoglobin molecules with soluble polymer, are also undergoing clinical trials. A perflubron-based emulsion is undergoing phase III clinical trials and a new recombinant human hemoglobin that does not bind to nitric oxide is also being developed. New oxygen carriers with antioxidant properties are being developed for conditions with potential for ischemia-reperfusion injuries. Third generation oxygen carriers are based on microencapsulation of hemoglobin and red blood cell enzymes either in liposomes or in biodegradable nanocapsules. This review will briefly discuss lessons learnt from the past, give an overview on the current status of selected oxygen carriers and discuss research areas in need of further development.

Cross-linked polyhemoglobin-superoxide dismutase-catalase supplies oxygen without causing blood-brain barrier disruption or brain edema in a rat model of transient global brain ischemia-reperfusion.

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.