Development of new formulations containing lysozyme and lactoferrin and determining the antibacterial effect of these formulations on mutans streptococci and lactobacilli in vitro conditions

The children under three years old are not suggested to use fluoridated toothpaste because of the risk of fluorosis and chlorhexidine for a long period resulting adverse effects and cytotoxicity. New formulations containing lysozyme and lactoferrin might be effective on preventing tooth decay in small children. Topical application of antimicrobial agents with drug delivery systems such as poloxamer and liposome is used for the treatment of oral diseases.The aim of this study was to determine the antibacterial effect of different formulations on Streptococcus sobrinus, Streptococcus mutans ve Lactobacillus acidophilus among containing lysozyme and lactoferrin and drug delivery system as poloxamer 407 with the trade name of Pluronic F-127 or/and freeze dried liposome containing DOTAP [Freeze dried Liposomal DOTAP (FLD)] in comparison with 0,2 % chlorhexidine.The formulations were (1) Sorensen?s Buffer Solution, (2) the gel formulation containing only poloxamer 407, (3) the formulation dissolving lysozyme and lactoferrin in Sorensen?s Buffer Solution, (4) the formulation combined with poloxamer 407 after dissolving lysozyme and lactoferrin in Sorensen?s Buffer Solution, (5) the formulation dissolving FLD in Sorensen?s Buffer Solution, (6) the formulation combined with FLD after dispersed into the poloxamer 407 in Sorensen?s Buffer Solution, (7) the formulation combined with FLD after dissolving lysozyme and lactoferrin in Sorensen?s Buffer Solution, (8) the formulation dissolving lysozyme and lactoferrin into Sorensen?s Buffer, then incorporting into the poloxamer 407 and combining with FLD. The control group was 0,2% chlorhexidine gel/mouthwash and positive control group consisted of empty hydroxyapatite discs. The antibacterial effect was assessed by determining the Minimal Inhibition Concentrations (MIC) of the test groups on Streptococcus sobrinus, Streptococcus mutans ve Lactobacillus acidophilus. The effects of ten groups on biofilm formation of Streptococcus sobrinus and Streptococcus mutans for 24 hours on the hydroxyapatite discs were also evaluated.In MIC experiment, the formulations which do not contain poloxamer 407 showed no inhibiton effect on these bacteria. The formulations with poloxamer 407, apart from the content of lysozyme and lactoferrin, inhibited the bacteria. The 0,2 % chlorhexidine was effective on the bacteria at very low concentrations.In biofilm experiment, the formulations with poloxamer 407 were found more effective in the inhibition of biofilm formation of Streptococcus sobrinus and Streptococcus mutans than other groups. Among these, the formulation containing Lysozyme and Lactoferrin with poloxamer 407 exhibited the highest efficiency on these bacteria (p<0,05). In the specimens with chlorhexidine, it was observed that there was no bacterial growth.The formulations which showed antibacterial effect in the present study, should be further evaluated with regard to longer periods, using kinetic methods. Additionally cytotoxicity should be determined.Key words: Lysozyme, Lactoferrin, drug delivery system, antibacterial effect