The goal of our study is to develop and characterize mucoadhesive films with entrapped lysozyme based on gelatin/sodium carboxymethyl cellulose as perspective antimicrobial preparation. Lysozyme in mucoadhesive films retains more than 95 % of its initial activity for 3 years of storage. Different physical-chemical and biochemical characteristics of entrapped enzyme were evaluated, such as film thickness, weight, time of dissolution in water, bioadhesive force, in vitro lysozyme release, pH- and thermoprofiles of hydrolytic activity, effect of γ-sterilization, etc. We have shown that gelatin/sodium carboxymethyl cellulose films have adhesive force on the level of 4380 Pa. Scanning electron microscopy images shows the relative uniformity of the gelatin surface with entrapped lysozyme. Mucoadhesive films with lysozyme have 100% bactericidal effect on the test strain, Staphylococcus aureus ATCC 25923 F - 49 and thus could be considered as a perspective antimicrobial preparation.
Aim. Study of lysozyme molecules behaviour at immobilization in gelatin and carboxymethyl cellulose sodium salt solutions by matrix-assisted laser desorption/ionization (MALDI). Methods. Determination of the activity of lysozyme, both free and entrapped in gelatin and carboxymethyl cellulose sodium salt (Na-CMC) solutions, was conducted by bacteriolytic method. The enzyme interaction with polymers was confirmed by viscometry and mass-spectrometry methods. Results. The occurrence of lysozyme associates in aqueous solution in monomeric and oligomeric forms was shown. A non-valent interaction of the enzyme with solutions of polymers results in the dissociation of oligomeric associates into subunits, which depends on the support nature and mass ratio of lysozyme to polymer. The quantitative retention of immobilized lysozyme hydrolytic activity was established, which favours obtaining mucoadhesive film forms with bacteriolytic action. Conclusions. The lysozyme immobilization by non-valent interactions in gelatin solution and Na-CMC solutions causes dissociation of the enzyme oligomeric structures; a stronger lysozyme coupling with NaCMC was noted
The influence of sterilization of ocular medicinal inserts with human serum albumin (HSA), lysozyme, papain, and urea on the preservation of their physicochemical properties was studied. It was found that the physicochemical properties of inserts with HSA and lysozyme preserved totally their physicochemical properties after sterilization by g-irradiation in doses of 10, 17, and 28 kGy. The influence of g-irradiation on the proteolytic activity of ocular medicinal inserts with papain and urea was dose-dependent and reduced the activity by 85 and 93% and led to its complete loss. Adding antiseptics used in the production of ophthalmologic preparations (nipagin, sodium metabisulfite, benzyl alcohol, benzalkonium chloride, etc.) caused an activity loss of the ocular medicinal inserts with papain and urea and did not provide microbiological purity and sterility. UV irradiation of ocular medicinal inserts with papain and urea produced microbiologically pure inserts that preserved 63.8% of the proteolytic activity. Manufacturing of ocular medicinal inserts with papain and urea under aseptic conditions using sterilizing filtration (79.7% activity preservation) was recommended for sterilization.
The lysozyme immobilization in cryogel of polyvinyl alcohol and physico-chemical properties of obtained preparation was investigated. Hydrolytic activity of lysozyme was determined by bacteriolytic method, using Micrococcus lysodeikticus cells acetone powder as substrate. Protein content was determined by the Lowry-Hartree method. Immobilization of lysozyme was conducted by entrapment in polyvinyl alcohol gel with subsequent cycles of freezing-thawing. Antimicrobial activity was studied by standard disk-diffusional method. The hydrogel filmic coatings with antimicrobial action, insoluble at physiological conditions, with quantitative retaining of protein and hydrolytic activity of lysozyme were obtained. The product is characterized by the widened pH-profile of activity at acidic pH values, stability in acidic medium (pH 5.5) and at storage. Its antimicrobial action against Staphylococcus aureus ATCC 25923 F-49, Pseudomonas aeruginosa 415, Escherichia coli 055 K 59912/4 and Candida albicans ATCC 885-653 was noted. The proposed method of lysozyme immobilization allows to obtain stable, highly effective product with antimicrobial activity, prospective for usage in biomedical investigations.
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