Superoxide Dismutase 1 Nanoparticles (Nano-SOD1) as a Potential Drug for the Treatment of Inflammatory Eye Diseases.

Inflammatory eye diseases remain the most common clinical problem in ophthalmology. The secondary processes associated with inflammation, such as overproduction of reactive oxygen species (ROS) and exhaustion of the endogenous antioxidant system, frequently lead to tissue degeneration, vision blurring, and even blindness. Antioxidant enzymes, such as copper-zinc superoxide dismutase (SOD1), could serve as potent scavengers of ROS. However, their delivery into the eye compartments represents a major challenge due to the limited ocular penetration. This work presents a new therapeutic modality specifically formulated for the eye on the basis of multilayer polyion complex nanoparticles of SOD1 (Nano-SOD1), which is characterized by appropriate storage stability and pronounced therapeutic effect without side reactions such as eye irritation; acute, chronic, and reproductive toxicity; allergenicity; immunogenicity; mutagenicity even at high doses. The ability of Nano-SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with a model immunogenic uveitis-the inflammation of the inner vascular tract of the eye. It was shown during preclinical studies that topical instillations of Nano-SOD1 were much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as corneal and conjunctival edema, iris hyperemia, and fibrin clots. Moreover, Nano-SOD1 penetrates into interior eye structures more effectively than SOD itself and retains enzyme activity in the eye for a much longer period of time, decreasing inflammation and restoring antioxidant activity in the eye. Thus, the presented Nano-SOD1 can be considered as a potentially useful therapeutic agent for the treatment of ocular inflammatory disorders.

Covalently immobilized chemically modified lysozyme as a sorbent for bacterial endotoxins (lipopolysaccharides).

Chemical modification of lysozyme was carried out using benzaldehyde and anisaldehyde. It was shown that chemical modification affects only 1-2 amino groups of the protein molecule which does not prevent further covalent immobilization of lysozyme using the remaining free amino groups. The bacteriolytic activity of lysozyme is preserved after chemical modification and after subsequent covalent immobilization. As a result of chemical modification immobilized lysozyme more effectively adsorbs bacterial lipopolysaccharides (endotoxins). Adsorption of immunoglobulin G does not increase after modification. The sorbents obtained in this work can be used for the future development of new medical material for the extracorporeal treatment of sepsis. The proposed scheme for the modification and immobilization of lysozyme can be used with various aldehydes for the preparation of sorbents with different properties.

The bacteriolytic activity of native and covalently immobilized lysozyme against Gram-positive and Gram-negative bacteria is differentially affected by charged amino acids and glycine.

The emergence of new antibiotic-resistant bacterial strains means it is increasingly important to find alternatives to traditional antibiotics, such as bacteriolytic enzymes. The bacteriolytic enzyme lysozyme is widely used in medicine as an antimicrobial agent, and covalent immobilization of lysozyme can expand its range of possible applications. However, information on the effect of such immobilized preparations on whole bacterial cells is quite limited. Here, we demonstrate the differential effects of glycine and charged (basic and acidic) amino acids on the enzymatic lysis of Gram-positive and Gram-negative bacteria by soluble and immobilized lysozyme. Glycine and basic amino acids (histidine, lysine, and arginine) significantly increase the rate of lysis of Gram-negative Escherichia coli cells in the presence of soluble lysozyme, but they do not substantially affect the rate of enzymatic lysis of Gram-positive Micrococcus luteus. Glutamate and aspartate significantly enhance enzymatic lysis of both E. coli and M. luteus. When using immobilized lysozyme, the effects of amino acids on the rate of cell lysis are significantly reduced. For immobilized lysozyme, the presence of an external diffusion mode on cell lysis kinetics at bacterial concentrations below 4 × 108 colony-forming units·mL-1 was shown. The broadening of the pH optimum of lysozyme activity after immobilization has been demonstrated for both Gram-positive and Gram-negative bacteria. The Michaelis constant (Km) values of immobilized lysozyme were increased by 1.5-fold for E. coli cell lysis and 4.6-fold for M. luteus cell lysis compared to soluble enzyme. A greater understanding of the effect of amino acids on the activity of native and immobilized lysozyme is important for both the development of new materials for medical purposes and elucidating the interaction of lysozyme with bacterial cells. Of particular interest is our finding that lysozyme activity against Gram-negative bacteria is enhanced in the presence of glycine and charged amino acids over a wide range of concentrations.