Nisin is a peptide with antibacterial activity and represents a potential alternative to classic antibiotics, which is very important today due to the increasing resistance of bacteria to known antibiotics. Nisin is unstable, so it makes sense to incorporate it into nanoparticles to protect it from degradation.
The aim of the master's thesis was to develop a method for the production of nanoparticles based on the double emulsion method with solvent diffusion. First, we optimized the formulation of empty nanoparticles, which we checked periodically by incorporating nisin, as empty nanoparticles behaved differently than nanoparticles with nisin. We studied the effect of the sonication time of the primary and secondary emulsions, and observed the effect of the polyvinyl alcohol, which we added to the external aqueous phase and to the secondary emulsion dilution medium, but did not use in the final formulation of nanoparticles with nisin, as it was less effective than other stabilizers. Pegylated poly (lactic-co-glycolic) acid and poloxamer 188 proved to be more effective stabilizers. Both enabled the production of sufficiently small nanoparticles with a narrow size distribution. After preparing the nanoparticle dispersion, we purified the nanoparticles by centrifugation. We lyophilized the nanoparticles with nisin to increase their stability during storage. We studied how different cryoprotectants/lyoprotectants and media for dispersing the lyophilisate affect the size of the nanoparticles. The best combination was found to be sucrose in a mass ratio of 3:1 relative to the amount of poly (lactic-co-glycolic) acid and the use of poloxamer 188 in the lyophilisate dispersion medium. In the final formulation of nanoparticles with nisin, after lyophilization, where the theoretical concentration of nisin in the base nanoparticle dispersion was 0.54 μg/mL, we obtained nanoparticles with a size of 206.4 ± 5.8 nm and a polydispersity index of 0.169 ± 0.054. Where the theoretical concentration of nisin in the base nanoparticle dispersion was 3.22 μg/mL, we obtained nanoparticles with a size of 202.5 ± 9.0 nm and a polydispersity index of 0.274 ± 0.037.
Finally, in the most successful formulation of nanoparticles with 5% (w/w) pegylated copolymer of lactic and glycolic acid and poloxamer 188, we analyzed the efficiency of nisin incorporation, which was highest at a theoretical concentration of nisin in the base nanoparticle dispersion of 19.2 μg/mL and amounted to 95.9%.
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