• Lysozyme and vancomycin co-loaded mesoporous silica nanoparticles; a “Trojan horse” with synergistic antimicrobial effects
  • Nasrin Namdar-Bilesuvar,1 Ayyoob Arpanaei,2,* Bahar Nayeri-Fasaei,3 Fatemeh Rahimia,4 Parvin Shariati,5
    1. National Institute of Genetic Engineering and Biotechnology (NIGEB)
    2. National Institute of Genetic Engineering and Biotechnology (NIGEB)
    4. National Institute of Genetic Engineering and Biotechnology (NIGEB)
    5. National Institute of Genetic Engineering and Biotechnology (NIGEB)


  • Introduction: Nowadays, there is a great deal of demands for the development and discovery of safe and new approaches for treatment of microbial infections. Among these methods, the use of nanoparticles in the formulation of antimicrobial agents has attracted great attention. Combination therapy which is simultaneous application of therapeutics agents is another approach to enhance the efficiency of the treatment methods. The main objective of this research is to combine these two approaches through preparation, characterization and study of antibacterial properties of mesoporous silica nanoparticles (MSNs) co-loaded with lysozyme and vancomycin against Staphylococcus aureus relative with free and separately loaded forms of these compounds.
  • Methods: At first, mesoporous silica nanoparticles were fabricated using a template removal technique. The resulting nanoparticles were studied in terms of shape and size using scanning electron microscopy and Zeta potential measurement analyses. In order to determine the optimal mass ratios of vancomycin and lysozyme, different compositions of vancomycin (0.05, 0.1, 0.2, 0.4, 0.6 μg) and lysozyme (0.25, 0.5, 1, 2, 3 mg) loaded onto nanoparticles (1 mg) were prepared and tested. Lysozyme and vancomycin co-loaded MSNs (lys-van-MSNs) were prepared and their antimicrobial activity was evaluated in terms of minimum inhibitory concentration (MIC) against Staphylococcus aureus, and then, compared to those of the free vancomycin, lysozyme, the mixture of vancomycin and lysozyme (van+lys) and their MSNs-loaded samples (van-MSNs, Lys-MSNs, van-MSNs+lys-MSNs). To investigate the antimicrobial interactions of this systems, first, the fractional inhibitory concentration (FIC) of each antibacterial agent was calculated. The calculation of the fractional inhibitory concentration index (FICI) is obtained from the sum of the FIC values of the two combined antimicrobial agents. The results were interpreted using the European Committee of Antimicrobial Sensitivity Assessment (EUCAST) guidelines. According to this guideline, for FICI ≤ 0.5, the effect is synergy; for 0.5 < FICI ≤ 1, additive, for 1< FICI ≤ 2, indifference and for FICI > 2, the interaction is antagonism.
  • Results: The average size of nanoparticles, and their surface charge were 77.22±10 and -22.8 mV, respectively. The highest loading capacities of nanoparticles obtained were found to be 53.65 μg of vancomycin and 883.3 μg of lysozyme per mg of nanoparticles. Besides, the results of antimicrobial activity assays for the free, loaded and co-loaded lysozyme and vancomycin, separately or in combination with each other, against Staphylococcus aureus showed that the minimum inhibitory concentration (MIC) for the free lysozyme and in combination with vancomycin (0.675 mg/ml and 1.562 μg/ml, respectively,) is about 75% lower than that of the free samples alone (lysozyme 2.7 mg/ml and vancomycin 6.25 μg / ml). In addition, the MIC values for the combination comprising loaded-lysozyme and loaded-vancomycin (lysozyme 0.168 mg / ml and vancomycin 0.781 μg / ml) were again about 75% lower than those of the loaded samples alone for both antimicrobial agents (0.675 mg/ml for lysozyme and 3.125 μg/ml for vancomycin). According to the results for MIC values for vancomycin, lysozyme and vancomycin + lysozyme, FICI was calculated to be 0.499. This result demonstrates the synergistic effect of these two antibacterial agents. On the other hand, considering the vancomycin-loaded and lysozyme-loaded samples, each as a distinct antibacterial agent, the interaction of the two agents in the prepared compounds was also investigated. It was found that, given the value obtained for FICI, that is 0.5, the combination of these two factors causes a synergistic effect between them. In addition, vancomycin+lysozyme co-loaded nanoparticles were also evaluated and results indicated that their antimicrobial activity is effectively retained compared to the combination separately loaded samples. It was further determined that the physical adsorption of lysozyme and vancomycin on mesoporous silica nanoparticles significantly increases the stability of the antimicrobial agents during storage for 72 hours at two different temperatures of 4 and 25 ° C. The results of analysis of the cytotoxic effects of nanoparticles in different states and different concentrations, on human fibroblast cells, human breast cancer cells and human stomach cancer cells by MTT and ROS methods and the hemolytic assay showed that the antimicrobial nanosystems generate no significant cytotoxicity effect.
  • Conclusion: All and all, these results indicate that mesoporous silica nanoparticles can be considered as a suitable carrier for both vancomycin and lysozyme molecules in the separate or dual loading modes to overcome bacterial infections
  • Keywords: Co-loading, Vancomycin, Lysozyme, Antimicrobial Effects, Mesoporous Silica Nanoparticles, Synergism