ИСТИНА

Numerous disorders from inflammatory to neurodegenerative are accompanied with uncontrolled release of reactive oxygen species – extremely active molecules that destroy cells and tissues. One of perspective weapons against them – antioxidants, in particular, antioxidant enzymes: catalase and superoxide dismutase (SOD). Thus, polyelectrolyte complexes of SOD showed significant therapeutic effect in rats spinal cord injury model. Rats, treated with obtained SOD complexes, improved their BBB-score (physiologic activity scale) up to 19 of 21, while groups, treated with native SOD or placebo, increased only up to 10. However, this incredible result was compromised by complex synthesis, which was affected by numerous factors: concentrations of reagents, time intervals of different stages, etc. The goal of our work was to determine factors that significantly affect the synthesis and optimize the procedure of synthesis SOD nanoparticles. Antioxidant enzyme (SOD), polycation (protamine), polyanion (poly(glutamic acid) – polyethylene glycol) were dissolved in HEPES buffer with 10mM NaCl. Protamine was added to SOD solution and mixed for 30 minutes, then the mixture was supplemented by poly(glutamic acid) – polyethylene glycol and kept without mixing for 30 minutes at 4°C. Then cross-linking agent (glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) was added in molar ratio 5:1, followed by overnight mixing under 4°С. Samples were purified using centrifugation filters (MWCO 50 kDa). Protein yield and specific activity of product were evaluated by BCA assay and pyrogallol assay. In case of EDC cross-linking agent, the time interval between stages was the most significant parameter of synthesis SOD nanoparticles, all other factors were less important. Optimization of procedure allows to increase the protein yield from 20 to 70% and decrease the loss of specific activity from 90 to 40%. Glutaraldehyde is likely to be the most perspective cross-linking agent, providing appropriate size of nanoparticles (diameter about 60 nm), good polydispersity (PdI less than 0.2) and good reproducibility. Moreover, time and other factors did not affect the results in case of glutaraldehyde. Finally, one more step to practical use of antioxidant enzymes in clinical practice has been done. Further research will focus on design of innovative forms for delivery of SOD nanoparticles into the brain. This work was supported by grant from Foundation for assistance to small innovative enterprises in science and technology under the program “UMNIK” and by grant of OPTEK.