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UTILIZATION OF SELENIUM NANOPARTICLES WITH SCHIFF BASE CHITOSAN AS ANTIBACTERIAL AGENTS

PAVLINA JELINKOVA1, ZUZANA KOUDELKOVA1, VEDRAN MILOSAVLJEVIC1, PAVEL HORKY2, PAVEL KOPEL1, 3, VOJTECH ADAM 1, 3

1Department of Chemistry and Biochemistry 2Department of Animal Nutrition and Forage Production

Mendel University in Brno Zemedelska 1, 613 00 Brno

3Central European Institute of Technology Brno University of Technology,

Purkynova 123, 612 00 Brno CZECH REPUBLIC

jelinkova.pav@gmail.com

Abstract: Bacterial infections and the increasing resistance of bacteria to antibiotics are included among the major global health problems. Therefore selenium nanoparticles in complexes with chitosan and selenium nanoparticles with Schiff base chitosan were synthesized and tested as potential antibacterial agents. Selenium nanoparticles with chitosan showed the average particle size of 29.4 nm with zeta potential of -44.3 mV. The standardized disc diffusion method has been used for susceptibility testing of selenium nanoparticles with chitosan and derivates on Staphylococcus aureus, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Chitosan selenium nanoparticles show inhibitory effect on gram-negative bacteria Escherichia coli only, whereas chitosan Schiff bases inhibit growth of all bacterial strains tested. The use of selenium nanoparticles in combination with chitosan Schiff bases appears to be a good way for the reduction of bacterial infection.

Key Words: selenium nanoparticles, chitosan, nosocomial infections, antimicrobial activity, MRSA

INTRODUCTION Nosocomial infections are undesirable complication of health care undertaken in hospitals

(Valaperta et al. 2010). They extend the period of patient treatment, cause an increase in morbidity and mortality and finally cost increases for medical care (Chudobova et al. 2014). Gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli) are major and basic bacterial pathogens (Tong et al. 2015). With rapid use of antibiotics to treat infectious diseases the inhibitory effect is decreased and the effect is also manifested by bacterial resistance to antibiotics (Carvalho and Santos 2016). From this reason it is necessary to look for new antibacterial agents for inhibition of bacterial growth (Kopel et al. 2015, Nawas et al. 2016).

Nanoparticles are small molecules with diameters from 1 nm to 100 nm (Snoddy and Jayasuriya 2016). Synthesis of non-toxic and highly pure nanomaterials allows the new directions for their use as antimicrobial agents against pathogenic bacteria (Chudobova et al. 2013, Chaudhary et al. 2016).

Nowadays, polymers are increasingly used as suitable drug carriers. They are used for slow release of the active ingredient, to increase solubility and for targeted delivery (Kumar 2000). Chitosan has excellent biological properties; it is non-toxic, biocompatible and biodegradable. Due to its perfect properties, is used in fields including biomedicine, agrochemistry and cosmetics (Anitha et al. 2014). Only chitosan own antibacterial properties against gram-positive (S. aureus), but also gram-negative bacteria (E. coli). The exact mechanism is not fully elucidated. Prerequisite is to change the permeability of cell membranes, causing escape of intracellular content, which leads to cell lysis. Another mechanism can be penetration of chitosan into the cells and subsequently binding to the DNA and partial inhibition of RNA and protein synthesis (Zheng and Zhu 2003).

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MATERIAL AND METHODS

Chemicals All the chemicals were supplied by Sigma-Aldrich (St. Louis, MO, USA) in ACS purity unless

noted otherwise. The deionized water was prepared by using reverse osmosis equipment Aqual (Aqual s.r.o., Brno, Czech Republic). The water was further purified by using Milli-Q Direct QUV equipped with the UV lamp. The pH was measured by using pH meter WTW inoLab (Weilheim, Germany).

Synthesis of SeNPs with chitosan and chitosan Schiff bases Selenium nanoparticles were prepared according to published method (Chudobova et al. 2014,

Cihalova et al. 2015). Briefly, to sodium selenite solution was added 3-mercaptopropionic acid and pH was adjusted to 7 by addition of NaOH. The concentration of selenium was 160 µg/mL. Chitosan solution was prepared by dissolving chitosan (5 g) in 500 mL of water and addition of acetic acid (5 mL). C-ACP The Schiff base was prepared by mixing 2-acetylpyridine (0.59 mL) with chitosan solution (50 mL), heating at 80 °C for 1 h and neutralization with NaOH. C-PA It was prepared similarly to C-ACP, only 2-pyridinecarboxaldehyde (0.59 mL) was used instead of ACP. C-SA The same method as above, only salicylaldehyde (0.66 mL) was used. C-Se, C-ACP-Se, C-PA-Se, C-SA-Se The solutions were prepared by mixing of 5 mL of chitosan (Schiff bases) solutions with 5 mL of selenium solution. The final concentration of selenium is 80 µg/mL and the solutions were used for treatment of bacteria.

Nanoparticles characterization The SeNPs with chitosan and chitosan Schiff bases were characterized using measurement of

particle sizes and zeta potentials by Dynamic Light Scattering (DLS) (NANO-ZS, Malvern Instruments Ltd., Worcestershire, U.K.). The parameters of the measurement were as follows: temperature 25 °C, absorption coefficient 10-3 and equilibration time 120 s. In each case, the measurement duration depended on the number of runs, which varied between 20 and 40.

Cultivation of S. aureus, methicillin-resistant S. aureus (MRSA) and E. coli S. aureus (NCTC 8511), MRSA (ST239) and E. coli (NCTC 13216) were obtained from the

Czech Collection of Microorganisms, Faculty of Science, Masaryk University in Brno, Czech Republic. The bacterial strains were cultivated in Luria Bertani (LB) into 50 mL Erlenmeyer flasks for 24 h on a shaker at 130 rpm and 37 °C. Antibiotic oxacillin (3µg/mL) was added in to the MRSA for cultivation.

Testing of antibacterial activity The antimicrobial effect of selenium nanoparticles in complexes with chitosan and chitosan Schiff

bases was determined using the measurement of the inhibition zones. Agar surface in Petri dish was covered with a mixture of 100 µL of 24 h bacterial cultures in the exponential phase of growth, and 3 µL of LB medium. Excess volume of the mixture on the Petri dishes was aspirated. Discs (Ø 1 cm) were mixed with SeNPs complexes in 80 µg/mL concentration in Eppendorf tubes. Soaked discs were then laid on a Petri dish. Petri dishes were insulated against possible external contamination and placed in a thermostat (Tuttnauer 2450EL, Israel) set at 37 °C for 24 h. After 24 h of incubation, the inhibition zones were measured and photographed in each Petri dish (Richtera et al. 2015).

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RESULTS AND DISCUSSION

Figure 1 Representative samples of SeNPs with chitosan and its derivates

Legend: A) C-Se; B) C-ACP-Se; C) C-PA-Se; D) C-SA-Se

Size and zeta potential of SeNPs The stability behavior of the selenium nanoparticles were determined by using size distribution

and ζ-potential measurements (Figure 2). It was found that chitosan modified selenium nanoparticles have an average size of 29.4 nm and zeta potential was detected at -44.3 mV. The obtained results confirm that selenium nanoparticles modified with chitosan are highly stable. The results on selenium nanoparticles with chitosan Schiff bases are given in Table 1. These results demonstrated that the complexes are less stable and they probably aggregate. The high aggregations are observed in the case of all selenium nanoparticles with chitosan Schiff bases. The modification of selenium nanoparticles with chitosan Schiff bases caused the change of charge from negative to positive which allows better interaction with negatively charged bacterial membrane (Qi et al. 2004).

Figure 2 A) Size of C-Se. B) Zeta potential distribution of C-Se.

Table 1 Characterization of selenium nanoparticles and their complexes with chitosan and its derivates

Sample Size (nm) Zeta potential (mV) C-Se 29.4 ± 3.5 -44.3 ± 2.4

C-ACP-Se 586.9 ± 116 13.4 ± 1.4 C-PA-Se 144.5 ± 40.6 8.48 ± 0.2 C-SA-Se 178.1 ± 41.9 15.8 ± 0.9

Testing of antibacterial activity Antibacterial activity of selenium nanoparticles with chitosan and selenium nanoparticles with

chitosan Schiff bases was determined using disc diffusion method and expressed in terms of the size of the inhibition zone (mm). SeNPs with chitosan and SeNPs with chitosan Schiff bases were applied on different bacterial strains: G+ (S. aureus) and G- (E. coli), bacterial strain resistant to antibiotics (MRSA). Effect of selenium nanoparticles with chitosan and selenium nanoparticles with chitosan Schiff bases on bacterial strains is shown in Table 2 and Figure 3. The highest inhibitory effect after 24 hours of incubation can be seen after the addition of C-PA-Se on E. coli (Figure 3).

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Table 2 Measurement of inhibition zones (mm)

Sample Bacteria

C-Se C-ACP-Se C-PA-Se C-SA-Se

Staphylococcus aureus 0 5 4 3 MRSA 0 3 3 3

Escherichia coli 6 6 7 6

Figure 3 Characterization of resistance of bacterial strains by using standardized disc diffusion method

Legend: (a) C-Se, (b) C-ACP-Se, (c) C-PA-Se, (d) C-SA-Se, (A) S. aureus, (B) MRSA, (C) E. coli

CONCLUSION The SeNPs with chitosan and SeNPs with chitosan Schiff bases were synthesized. Almost all

compounds show good antibacterial activity. The best inhibitory effect showed on Escherichia coli (G- bacteria) after application of all selenium nanoparticles with chitosan and selenium nanoparticles with chitosan Schiff bases. These SeNPs with chitosan and SeNPs modified with chitosan Schiff bases appear to be a good kind for treatment against G- bacterial strains.

ACKNOWLEDGEMENTS The research was financially supported by the TP_02/2015.

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