Elsevier

Journal of Biotechnology

Volume 316, 10 June 2020, Pages 17-26
Journal of Biotechnology

The battle against biofilm infections: juglone loaded nanoparticles as an anticandidal agent

https://doi.org/10.1016/j.jbiotec.2020.04.009Get rights and content

Highlights

  • Juglone loaded nanoparticles and free juglone inhibit biofilm formation and pre-established biofilms of Candida albicans.

  • The use of nanoparticulate systems increases the antibiofilm activity due to the controlled release of the active substance.

  • Juglone and juglone nanoparticles are effective on the membrane structure of Candida albicans.

  • Juglone and juglone nanoparticles have fluorescence quenching effect on DiSC3(5).

ABSTRACT

In this study, juglone nanoparticles were prepared by single emulsion solvent evaporation method and their effect against Candida albicans biofilm was investigated in comparison with the free juglone and Fluconazole by performing XTT, crystal violet, standard plate count, confocal microscopy and membrane depolarization analyses. Juglone nanoparticles and free juglone were found to inhibit biofilm formation and pre-established biofilms (98-100%) at all doses tested, whereas Fluconazole did not cause a significant inhibition, even at the highest dose applied, especially against pre-established biofilms. Membrane depolarization analysis showed that free juglone and juglone loaded nanoparticles were effective on C. albicans membrane structure and have fluorescence quenching effect on DiSC3(5). It is extremely important that the antibiofilm activity of the juglone nanoparticles is similar to that of the juglone used at the same concentration, since similar effect is provided by using less active substance due to controlled release. Accordingly, it can easily be said that juglone loaded nanoparticles are much more effective in the formation and elimination of C. albicans biofilm than the free juglone and Fluconazole.

Introduction

Biofilms are highly complex structures, composed of cells clustered and attached to immobile or biological surfaces and covered with extracellular matrix of their own production, including water channels that transport nutrients and oxygen to the inner layers [1,2]. Biofilm infections are an important problem in the clinic due to more than 2 million cases per year, long-lasting and compulsive treatment and high health expenditures. The most common cause of biofilm infections in humans is a fungal pathogen, Candida albicans [2,3]. Bloodstream infections caused by C. albicans are known as candidemia and are frequently seen in patients who have decreased neutrophil count due to cancer or immunosuppressive therapies. The use of medical devices such as stents, shunts, implants, pacemakers and catheters and also surgeries that cause dissemination of C. albicans living commensally in the mouth cavity, gastrointestinal tract, vagina and skin may cause candidemia. Since the symptoms are very similar to sepsis, the diagnosis may be delayed and this can lead to the loss of patient. In order to prevent this, antifungal therapy is being started before the development of disease for patients at risk [4]. However, when it is a resistant infection, antifungal therapy fails at any stage. Candida biofilms are known to be 30-2000 times more resistant to antifungal agents than planktonic cells [5]. The mechanisms of antifungal resistance, which cause increasing rates of morbidity and mortality, have not yet been fully elucidated. Parameters such as the presence of extracellular matrix and highly resistant cells called persister cells, the expression of resistance genes and the changing growth rate are thought to have role in resistance development [[6], [7], [8], [9], [10]]. Due to antifungal resistance, the drug dose required for the treatment of biofilm infections may be well above the highest therapeutically feasible concentration [11]. For this reason, new and effective antifungal agents need to be developed.

Many of the plant secondary metabolites that are effective in growth, development and reproduction processes have a variety of biological activities and therefore take an important place in medicine and pharmacy research [12]. Juglone (5-hydroxy-1,4-naphthoquinone), a naphthoquinone obtained from walnut (Juglans sp.), is a remarkable molecule because of its properties such as antimicrobial [[13], [14], [15], [16], [17], [18]], cytotoxic and genotoxic [19], anticancer [20] and antioxidant activities [21]. However, its hydrophobic nature and toxic effect prevent the use of juglone in biological systems [22].

In recent years, the use of nanosized drug delivery systems has attracted a great deal of attention and many studies have been conducted in order to provide the use of many antimicrobial agents similar to juglone, which are not efficiently used due to their low water solubility, cytotoxicity and chemical instability [23,24]. Successful results have been obtained in studies using systems such as liposomes, polymeric nanoparticles, dendrimers and solid lipid nanoparticles. The use of these systems not only facilitates the delivery of the drug to the site of infection, but also reduces the dose and the associated toxicity needed to achieve therapeutic efficacy [25,26]. It is also preferred that nanoparticles, which are more advantageous than microparticles, will not cause aggregation and hence vascular occlusion in the circulatory system where the smallest capillaries are 5-6 μm in diameter [27].

Biodegradable polymeric nanoparticles are applicable for different molecules such as drugs, peptides or nucleic acids. They are often preferred for the delivery of antimicrobials because of their stability in blood and nonimmunogenic, noninflammatory and nonthrombogenic properties [28]. It is also a great advantage that degradation products of biodegradable polymers can be metabolized through normal pathways and thus not cause any side effects [24,29]. Besides that, the nanoparticulate systems enable the use of active agents in lesser amounts due to controlled release. Polymeric nanoparticles prepared using PLGA (poly (D,L-lactic-co-glycolic acid)), a polymer of considerable interest due to FDA approval, have been found to enhance the antimicrobial activity of active agents [30,31]. For instance, in a previous work, the nanoparticles prepared by encapsulating the juglone molecule into PLGA were effectively used against C. albicans cells and found to have 4-fold higher anticandidal activity than the free form of juglone [32]. In addition, successful results were obtained in studies investigating the effect of PLGA nanoparticulate system on biofilm structure [[33], [34], [35]]. For example, in the study conducted by Anjum et al. [36], it was found that the PLGA nanoparticles loaded with xylitol promoted the antibiofilm activity of xylitol in infected wounds. Also, Arafa et al. [37] performed an antimicrobial study against Enterococcus faecalis biofilms and noted that ciprofloxacin loaded PLGA nanoparticles caused a stronger inhibition than ciprofloxacin due to controlled release. Klodzinska et al. [38] stated that D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles improved the biofilm prevention activity of azithromycin.

The hypothesis of this study is that the encapsulation of juglone into PLGA nanoparticles can effectively increase the antibiofilm activity. Accordingly, in this research, it is aimed to investigate the effect of the juglone loaded PLGA nanoparticles on Candida albicans biofilm in comparison with the free juglone.

Section snippets

Production of Juglone Loaded Nanoparticles

The nanoparticle production was performed by the single emulsion (w/o) solvent evaporation method, due to the hydrophobic structure of juglone molecule, as described in our previous studies [32,39].

45 mg juglone was dissolved in 1.5 ml of DCM and then mixed with PLGA dissolved in 1 ml of DCM. This organic phase was added dropwise to 4 ml of 3% PVA solution in ice bath whilst sonication was performed at 80% power for 90 sec. The resulting oil-in-water (o/w) emulsion was immediately added

Production and Characterization of Juglone Nanoparticles

For the juglone loaded nanoparticles produced in the present study, the reaction yield and the encapsulation efficiency were calculated as 54.78% and 89.02%, respectively.

The mean particle size of the nanoparticles was measured as 207.60 ± 1.99 nm with 0.148 ± 0.022 polydispersity index. A narrow size distribution was detected and shown in Fig. 1A. Further, the average zeta potential was measured as -25.7 ± 1.2 mV (Fig. 1B) and the obtained zeta potential indicated that the nanoparticles are

DISCUSSION

In this study, juglone loaded PLGA nanoparticles with 207.60 ± 1.99 nm size and -25.7 ± 1.2 mV zeta potential were produced by the single emulsion solvent evaporation method.

In the release study of juglone nanoparticles; a rapid release, or burst release, occurred at 1st hour, and then the release rate gradually slowed down. At the end of the 60th day, the release rate of nanoparticles was 42.17%. In the antibiofilm studies, the activity of juglone nanoparticles was evaluated considering the

FUTURE PERSPECTIVE

In recent years, increased resistance to antibiotics and mortality rates due to infection necessiates the discovery and development of new agents against biofilm infections, which are the biggest responsible of the situation. In our previous antifungal study, it was shown that the MIC values of juglone were lower compared to the commercially available Fluconazole, and the use of juglone as an antifungal agent was promising. However, since juglone is a toxic compound, it seems to be beneficial

CRediT authorship contribution statement

Busra Gumus: Methodology, Investigation, Writing - original draft. Tayfun Acar: Investigation, Visualization. Tugba Atabey: Investigation. Serap Derman: Conceptualization, Writing - review & editing. Fikrettin Sahin: Resources, Writing - review & editing. Tulin Arasoglu: Conceptualization, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by Yildiz Technical University Scientific Research Projects Coordination Department (Project no: FBA-2018-3101).

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