Narrow Results

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 allowing this to be cost effective for large-scale production of a nanobiotechnological polyHb-SOD-CAT-CA with enhancements to all three RBC functions. The best 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 RBCs.

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-minute 30 mm Hg MAP-sustained hemorrhagic shock rat model showed that it was more effective than blood in the lowering of elevated intracellular PCO₂, recovery of ST-elevation and histology of the heart and intestine. This paper analyzes the storage and temperature stability. The allowable storage time for RBCs is about 1 day at room temperature and 42 days at 4°C. Also, RBCs cannot be pasteurized to remove infective agents like HIV and Ebola. PolyHb can be heat sterilized and can be stored for a 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. The results of the storage stability show that lyophillization extends the storage time to a year at 4°C and 40 days at room temperature (compared to 42 days and 1 day, respectively, for RBCs). 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 hours, 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 it in suitable solutions that continue 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.

This study investigates the long-term effects of nano-dimension PEG-PLA artificial red blood cells (RBCs) containing hemoglobin and RBC enzymes on the liver and spleen after a one-third blood volume toploading in rats. The experimental rats received one of the following infusions: Nano-artificial RBCs in Ringer’s lactate solution, stroma-free hemoglobin, polyhemoglobin and autologous rat whole blood. Blood samples were taken before infusions and after infusions on Days 1, 7 and 21 for analysis. Nano-artificial RBCs, polyhemoglobin, Ringer’s lactate solution and rat RBCs 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 the liver, as shown by elevation in alanine aminotransferase and aspartate aminotransferase throughout the 21 days. On Day 21 after the infusions, rats were sacrificed and their livers and spleens were excised for histological examination. Nano-artificial RBCs, polyhemoglobin, Ringer’s lactate solution and rat RBCs 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 the rats’ central veins and sinusoids and hepatic steatosis. In conclusion, injected nano-artificial RBCs can be efficiently metabolized and removed by the reticuloendothelial system, and do not have any biochemical or histological adverse effects on livers or spleens.