Accepted Manuscript

Title: Antifungal organoselenium compound loadednanoemulsions stabilized by bifunctional cationic surfactants

Author: Magdalena Pietka-Ottlik Agnieszka Lewinska AnnaJaromin Anna Krasowska Kazimiera A. Wilk

PII: S0927-7757(16)30583-0DOI: http://dx.doi.org/doi:10.1016/j.colsurfa.2016.07.062Reference: COLSUA 20850

To appear in: Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: 29-2-2016Revised date: 7-7-2016Accepted date: 21-7-2016

Please cite this article as: Magdalena Pietka-Ottlik, Agnieszka Lewinska, AnnaJaromin, Anna Krasowska, Kazimiera A.Wilk, Antifungal organoseleniumcompound loaded nanoemulsions stabilized by bifunctional cationic surfactants,Colloids and Surfaces A: Physicochemical and Engineering Aspectshttp://dx.doi.org/10.1016/j.colsurfa.2016.07.062

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Antifungal organoselenium compound loaded nanoemulsions stabilized by bifunctional

cationic surfactants

Magdalena Piętka-Ottlika*

, Agnieszka Lewińskab, Anna Jaromin

c, Anna Krasowska

d,

Kazimiera A. Wilka

a) Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wrocław

University of Science and Technology, Wybrzeże Wyspiańskiego 29, 50-370 Wrocław,

Poland

b) Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland

c) Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wrocław,

Joliot-Curie 14a, 50-383 Wrocław, Poland

d) Department of Biotransformation, Faculty of Biotechnology, University of Wrocław, Joliot-

Curie 14a, 50-383 Wrocław, Poland

* Corresponding author

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wrocław

University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław,

Poland

phone: +48 71 320 25 34

email: magdalena.pietka@pwr.edu.pl (MPO)

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Highlights:

Organoselenium compound loaded nanoemulsions were prepared by crash dilution method.

Dicephalic cationic surfactants were used for the nanodroplet stabilization.

High stability was proven by ξ-potential and no evolution in the backscattering.

Nanoemulsions were active towards Candida albicans and its biofilm.

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Abstract

Over last few decades, organoselenium compounds have been extensively studied in organic

and medicinal chemistry. Ebselen (2-phenylbenzisoselenazol-3(2H)-one, BSe) and its

analogues have emerged as potential pharmaceuticals and promising catalysts of various

oxidation reactions and potential pharmaceuticals. For instance, their biological interest relies

on their antioxidant, anti-inflammatory and glutathione peroxidase-like activities as well as

antimicrobial, antiviral and chemopreventive effects. One of the major obstacles for their

practical use is poor water solubility. For this purpose the potential of dicephalic cationic

surfactants, N,N-bis[3,3'-(trimethylammonio)propyl]alkylamide dichlorides, to form

physically stable nanoemulsion-based drug carriers in the presence of a variety of oils (oleic

acid, Capmul MCM and PG-8) and the organoselenium-derived cargo was explored.

Nanoemulsions were characterized by visual and microscopic observations, as well as

dynamic light scattering (DLS) measurements of particle size and distributions. Results of

transmission electron microscopy (TEM) and DLS showed that nearly spherical droplets of

very similar size were obtained, with oleic acid bringing best properties. Time-depended size

(DH) and ξ -potential measurements, dispersion stability test and kinetic stability studies with

multiple light scattering (MLS) technique proved high colloid stability of the fabricated

systems. Positively charged nanoemulsions with high ξ –potential values up to 87 mV showed no

significant evolution in backscattering profiles. The nanoemulsions were evaluated for

antimicrobial efficacy towards Candida albicans and its biofilm. Strong antifungal activity

was observed, with deleterious morphological changes in cellular structures and yeasts cell

surface alterations. In conclusion, the designed oil-in-water nanoemulsion-based carriers,

stabilized by dicephalic cationic surfactants can comprise a promising delivery system for

poorly water soluble organoselenium derivatives of efficient antimicrobial activities.

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Keywords: nanoemulsion, dicephalic cationic surfactants, ebselen, organoselenium

compounds, antimicrobial activity, Candida albicans

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1. Introduction

Organoselenium compounds, particularly benzisoselenazol-3(2H)-ones, are an important class

of multifunctional synthetic products with recognized anti-inflammatory, antioxidant,

cytoprotective, chemopreventive, antinociceptive, antidepressant-like, antiviral and

antimicrobial activities. Ebselen (2-phenylbenzisoselanzol-3(2H)-one) and its analogues are

similar in action to glutathione peroxidise (GPx), thus possessing ability to protects cells from

oxidative and free radical damage. Ebselen has also been demonstrated to be a substrate for

human thioredoxin reductase [1-5]. Recently, benzisoselenazol-3(2H)-ones have started to

receive more attention as antimicrobial agents. It has been demonstrated that ebselen and its

analogues possess potent antimicrobial activity, mostly against Gram-positive bacteria and

yeasts, including multi-drug resistant strains [6-9]. It was suggested that fungicidal action of

ebselen is due, at least in part, to interference with both the proton-translocating function and

the ATPase activity of the plasma membrane H+-ATPase [9,10].

Although it is clear that organoselenium compounds should be considered as potential

pharmaceuticals due to their wide range of pharmaceutical effects, their poor water solubility

is a main challenge that may be an obstruction for further clinical trials. For this reason, the

development of appropriate nanocarriers seems to be a prominent approach to overcome this

disadvantage.

Nanoemulsions are one of the most promising nanocarriers for delivery of active substances

[11,12] as they present many advantages versus conventional emulsions, eg. lower

preparation cost, higher storage stability, good production feasibility [13]. Nanoemulsions are

heterogeneous, transparent or translucent systems consisting of fine oil-in-water dispersions

stabilized by an interfacial film of surfactant, with droplet size, in general, below 1000nm,

most frequently between 100 nm and 500 nm [14], however depending on the reference this

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range may vary, from below 100 nm [15], 20-200 nm [16], below 300 nm [17] or maximum

500 nm [14]. Nanoemulsions can be produced by high-energy emulsification methods using

high pressure-homogenisation, microfluidization or ultrasonication, where energy is provided

to the coarse emulsion system which generates intense disruptive forces and minimizes

droplet size or by low-energy emulsification methods where droplet size is reduced by

varying composition and altering the environmental factors like temperature and/or

employing the chemical properties of the system to create nanosized emulsion droplets from a

microemulsion matrix [14]. Unlike microemulsions, nanoemulsions are thermodynamically

unstable. Their high kinetic long-term stability is related to the fact that the destabilisation

phenomena such as creaming or coalescence are largely decelerated due to the small particle

size of the droplets which are not affected by gravity, but rather related to Brownian motion.

Usually, the Ostwald ripening, which is the tendency of small droplets to merge with larger

droplets due to differences in solubility is reported to be a main source of instability. In

addition, reversible destabilization phenomena such as flocculation, creaming or separation

may occur [14]. Compared to microemulsions, nanoemulsions are more robust in terms of

destabilization caused by dilution or temperature changes. Both, dilution and variation in

temperature strongly affects structure and droplet size of microemulsion whereas has limited

effect on nanoemulsion droplet size [18]. Numerous advantages of nanoemulsions as drug

delivery systems cause that the hydrophobic core of oil-in-water nanoemulsions is willingly

used as a cargo space for encapsulation of a variety of poorly water soluble therapeutic agents

and active substances [11-13]. Such encapsulation increases their bioavailability, protects

them from fast degradation upon administration, and may beneficially modify their

pharmacokinetics and biodistribution. In the recent years there has been increased interest to

use nanoemulsions to enhance drug permeation through the skin [19-21]. So far, many drugs

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have been successfully studied in nanoemulsion-based delivery systems [11,12, 21-30]

including template-assisted processes [31-33], what justifies rationale of our studies.

Herein, we report a new approach to encapsulate one of the bioactive organoselenium

compounds - hydrophobic 2-n-propylbenzisoselenazol-3(2H)-one (BSe) in oil-in-water (o/w)

nanoemulsions specifically designed as antimicrobial formulations towards Candida albicans.

The work reported here extends our recent studies on new functionalized surfactants and their

efficiency to stabilize a variety of oil-core nanocarriers, fabricated in template-mediated

processes [31-34]. Mainly, we describe potential of dicephalic cationic surfactants, N,N-

bis[3,3'-trimethylammonio)propyl]alkylamide dichlorides, which preparation methodology

and biological impact can make them suitable as stabilizing agents of oil-core nanoparticulate

drug delivery systems [35,36]. Therefore, the main objectives of this work included: (i)

development of stable nanoemulsion systems based on dicephalic cationic surfactants for BSe

within short and long-term storage (ii) the investigation of the loaded nanoemulsions in terms

of their antimicrobial efficacy against C. albicans and its biofilm.

2. Materials and methods

2.1. Chemicals

The structures of compounds used for the formation of nanoemulsions with their

abbreviations are presented in Table 1.

2-n-propylbenzisoselenazol-3(2H)-one (BSe) was prepared by tandem selenenylation-

acylation of n-propylamine with 2-(chloroseleno)benzoyl chloride following procedure

described in [7]. Dicephalic cationic surfactants, N,N-bis[3,3'-(trimethylammonio)propyl]

alkylamide dichlorides denoted as Cn-(DAPACL)2, where n = 12, 14, 16 were synthesized

according to procedure described in [37]. Oleic acid (OA, purchased from Sigma Aldrich,

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Poland; M.p. 13-14 oC), mixed medium chain mono- and diglycerides (Capmul MCM,

obtained as gift sample from Abitech Corporation, USA; liquid at RT), propylene glycol

monocaprylate (Capmul PG-8, obtained as gift sample from Abitech Corporation, USA;

liquid at RT) were used as oil phases. All reagents were of analytical grade and used as

provided. Water used for all experiments was doubly distilled and purified by means of

Millipore (Bedford, MA) Milli-Q purification system.

2.2. Preparation of nanoemulsions

Oil-in-water (o/w) nanoemulsions were prepared by crash dilution method [16] using various

oils (OA, Capmul MCM, Capmul PG-8), dicephalic cationic surfactants of different

hydrocarbon length (C12-(DAPACL)2, C14-(DAPACL)2, C16-(DAPACL)2) and water, at the

ratios presented in Table 2. Briefly, in a first step, oil (with or without BSe), surfactant and

water were mixed at a certain composition followed by sonication at 25 oC for 20 min to form

microemulsion concentrate which was subsequently rapidly diluted four times with water

resulting in nanoemulsion.

2.3. Characterization of nanoemulsions

2.3.1. Dispersibility test

The in vitro performance of the formulations was visually assessed by using the following

grading system (Fig. 1S) [38]:

Grade A: Rapidly forming (within 1 min) nanoemulsion, having a clear or bluish appearance.

Grade B: Rapidly forming, slightly less clear emulsion, having a bluish white appearance.

Grade C: Fine milky emulsion that formed within 2 min.

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Grade D: Dull, greyish white emulsion having slightly oily appearance that is slow to

emulsify (longer than 2 min).

Grade E: Formulation, exhibiting either poor or minimal emulsification with large oil

globules present on the surface.

2.3.2. Particle size and polydispersity

The droplet size of nanoemulsions, expressed as hydrodynamic diameter (DH), was

determined at 25 oC by dynamic light scattering (DLS), with Zetasizer Nano ZS (Malvern

Instruments Ltd., Malvern, UK), providing in parallel the size distribution expressed as

polydispersity index (PDI). The samples were filtered before measurements through a filter

with pore size of 0.22 μm to remove any impurities. Each value was obtained as an average of

three runs with at least 10 measurements.

2.3.3. Zeta potential (ξ-potential)

The ξ-potential of nanoemulsion droplets, which reflects the electric charge on the particle

surface, was measured by the microelectrophoretic method using Zetasizer Nano ZS (Malvern

Instruments Ltd., Malvern, UK). All the measurements were performed at 25 ◦C. Each value

was obtained as an average of three subsequent runs of the instrument with at least 20

measurements.

2.3.4. Particle shape and morphology

The shape and morphology of nanoemulsions were studied by transmission electron

microscopy (TEM) with FEI Tecnai G2 20 XTWIN electron microscope at RT. The size

distribution for each nanoemulsion sample was determined by counting the size of

approximately 250 nanoparticles from several TEM images obtained from different places of

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the grid. Energy dispersive X-ray (EDX) spectra were recorded with Thermo Scientific Ultra

Dry detector (resolution 129 eV) and analyzed by Noran System 7. Few drops of the diluted

suspension were placed on Cu-Ni grid and stained with 2% uranyl acetate before capturing

the image. The size distribution plots were fitted by using Gauss curve approximation.

2.4. Stability of nanoemulsions

In order to overcome the problem of metastable and unstable formulations, nanoemulsions

were subjected to time-depended size (DH) and ξ -potential measurements at RT as well as

dispersion stability test, including heating and cooling cycles, centrifugation and freeze–thaw

cycles [39]. The nanoemulsions which showed no phase separation, creaming, coalescence or

phase inversion upon these tests were selected for turbidity test.

2.4.1. Dispersion stability test

1. Heating/cooling cycles: six cycles between refrigerator temperature (4 oC) and 45

oC with

storage at each temperature for a minimum period of 48 h were performed. Those

formulations, which were stable at these temperatures, were subjected to centrifugation test.

2. Centrifugation: passed formulations were centrifuged at 3500 rpm for 30 min. Those

formulations that did not show any phase separation, creaming and cracking were taken for

the freeze/thaw stress test.

3. Freeze/thaw cycles: three freeze/thaw cycles between -21°C and +25°C with storage at

each temperature for a minimum period of 48 h were performed.

The formulations which showed no phase separation, creaming, coalescence or phase

inversion upon these tests were selected for the kinetic destabilization test.

2.4.2. Kinetic stability

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The kinetic stability of nanoemulsions was studied with the TurbiscanTM LAB

(Formulaction, France) which uses multiple light scattering (MLS) technique to detect and

determine destabilization phenomena suffering concentrated dispersions over time [40].

Nanoemulsion samples were placed in cylindrical glass tubes where theirs stability was

monitored at RT by measuring the transmission (T) and backscattering (BS) of a pulsed near-

infrared light source (λ= 880 nm) as a function of sample height. The profiles were analyzed

using the instrument’s software (Turbisoft version 2.0.0.33). The Turbiscan Stability Index

(TSI) is a result of kinetics computation based on raw signals and depends on the global

stability of the sample. The measurements were performed for freshly prepared

nanoemulsions and repeated on the same samples over 30 days of storage at RT.

2.5. Antimicrobial activity assessment

2.5.1. Minimum inhibitory concentration (MIC) determination

The minimum inhibitory concentration (MIC), defined as the lowest concentration of an

antimicrobial agent that completely inhibits the growth of a microorganism, was determined

by dilution method according to Clinical and Laboratory Standards Institute guidelines [41].

Briefly, C. albicans ATCC 10231 strain was incubated with various concentrations of

compounds for 48 h at 28oC in YPG medium (1% yeast extract Difco, 1% peptone Difco, 2%

glucose) in 96-well microplates. After incubation time, the optical density was measured

using a microplate reader at 600nm (ASYS UVM 340 Biogenet). Negative and growth control

wells did not contain compounds tested.

2.5.2. Effect of NE on Candida albicans’ cell shape - scanning electron microscopy (SEM).

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C. albicans ATCC 10231 strain was incubated with nanoemulsions on 96-well polystyrene

microplates for 24 h at 30˚C and then the wells were washed in PBS and prepared for SEM

analysis by fixation in 2.5% glutaraldehyde. The samples were subsequently treated with

phosphate buffer in 2.5% glutaraldehyde, then dehydrated in a series of acetone washes and

dried. The SEM measurements were made at RT with Hitachi S-3400N equipped with a

tungsten cathode (magnification 80 - 300.000x) at operation voltage of 15 keV. Micrographs

have been acquired with a secondary electron detector (SE) and a backscattered electron

detector (BSE).

2.5.3. Effect of NE on Candida albicans’ biofilm formation

C. albicans ATCC 10231 biofilms were formed in the bottom of 96-wells plate (Starstedt) by

pipetting of 0.1 mL of a standardized cells suspension in YPG medium (5×106cells/mL) and

incubation for 2 h at 37◦C on a rotary shaker (MixMate, Eppendorf, Hamburg, Germany) at

300 rpm, then wells were washed with PBS, pH 7.4 and 0.1 mL of nanoemulsions were

added. The wells containing C. albicans only were used as control. After 2h of incubation at

37˚C the wells were washed with PBS and prepared for SEM analysis by fixation with 2.5%

glutaraldehyde. The samples were subsequently treated with phosphate buffer in 2.5%

glutaraldehyde, then dehydrated in a series of acetone washes and dried and dusted with a

layer of gold sputter Cressington 108A. The SEM measurements were made with Hitachi S-

3400N equipped with a tungsten cathode (magnification 80 - 300.000x) at operation voltage

of 15 keV. Micrographs have been acquired with a secondary electron detector (SE) and a

backscattered electron detector (BSE).

Parallely, another 96-wells plate with adherent cells of C. albicans was stained with 0.1%

crystal violet for 5 min at RT and then washed three times with PBS. Next, 150 µl of 0.05M

HCl in isopropanol and 50 µl of 0.25% sodium dodecyl sulfate (SDS) were added to each

well to permeabilize the cells and resolubilize crystal violet, and the optical density was

measured at 590 nm with the Asys UVM 340 (Biogenet) microplate reader.

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2.5.4. Effect of NE on Candida albicans’ adhesion

C. albicans ATCC 10231 strain was grown on a YPG medium at 30◦C for 24 h. The cells

were then washed once with phosphate-buffered saline (PBS, pH 7.4) and diluted in fresh

YPG medium to a final concentration of 5 × 106cells/mL. BSe nanoemulsions (0.1 mL) were

added to a 96-well flat-bottom polystyrene plate (Sarstedt) and the plate was incubated for 2 h

at 37◦C on a rotary shaker (MixMate, Eppendorf, Hamburg, Germany) at 300 rpm. Then, the plate

was washed with distilled water and the tested strain was added to the final culture volume,

100 L in every well. The plate was incubated at 37◦C for 2 h to induce germination. Non-

adherent cells were removed by several washes with water. Adherent germ tube forms were

stained with 0.1% crystal violet for 5 min and washed three times with dis-tilled water. Next,

the 150 L of isopropanol 0.02 N HCl and 50 L of 0.25% SDS were added to each well to

dissolve the crystal violet. The absorbance of each well was measured using a microplate

reader at 600nm (ASYS UVM 340 Biogenet). The results were expressed as a percentage of

control (untreated C. albicans). Assays were carried out twice in three replicates.

3. Results and discussion

3.1. Preparation and characterisation of BSe-loaded nanoemulsions

Bifunctional N,N-bis[3,3'-(trimethylammonio)propyl]alkylamide dichlorides Cn-(DAPACL)2

containing two hydrophilic entities linked to one hydrophobic tail via labile amide linkage

[35, 36] were selected for the stabilization of oil-in-water (o/w) nanoemulsions, in which the

highly active antimicrobial organoselenium compound, 2-phenylbenzisoselanzol-3(2H)-one

(BSe) was successfully encapsulated. The selected dicephalics belong to a group of custom-

designed products due to their outstanding properties (e.g., behavior at the interfaces,

interactions with polyelectrolytes, biological impact, lower impact on the environment) which

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determine their usefulness in the context of biomedical and pharmaceutical applications

[31,36,42]. It should be emphasized that during the preparation of the BSe-loaded

nanoemulsions the primary stabilizing mechanism may appear in water or at the surface of

the oil droplets, depending upon the chemical components, applied to fabricate a given

nanoemulsion system. The surfactant is predominantly adsorbed at the oil/water interface,

diminishing the interfacial energy as well as giving rights to form a mechanical barrier against

coalescence or other destabilization processes. Therefore, the selection of proper and suitable

emulsifiers is critically important in controlling the long-term colloidal stability of the

nanoemuslion systems and possible interactions with biological microenvironment [43].

The final properties of nanoemulsions, as non-equilibrium systems, strongly depend not only

on composition but also on a preparation methodology. Thus, it is important to control all the

variables. In this case, BSe-free and BSe-loaded oil-in-water (o/w) nanoemulsions have been

prepared by crash dilution method using various oils (OA, Capmul MCM, Capmul PG-8),

dicephalic cationic surfactants of different hydrocarbon length (C12-(DAPACL)2, C14-

(DAPACL)2, C16-(DAPACL)2) and water, at the ratios presented in Table 2. Appropriate

formulations have been chosen for the concentrate to be diluted four times with water to

generate bluish, transparent oil-in-water (O/W) nanoemulsions following the general

approach described in [44,45]. It is important to carefully select the formulations, as

nanoemulsions have formed only if the initial concentrate was a microemulsion phase. If the

concentrate began in an emulsion-phase region then opaque emulsions were generated. The

presence of organoselenium compound in BSe-loaded nanoemulsions was confirmed by

energy-dispersive X-ray spectroscopy (EDX) used in conjunction with transmission electron

microscopy. Figure 1 shows a typical point-detection instance of BSe-loaded nanoemulsion,

in this particular case C14(DAPACL)2/OA/water. A large number of counts at voltage pulse

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(which remains proportional to the x-ray energy) characteristic for selenium indicates a high

degree of loading.

Freshly prepared nanoemulsions were examined for droplet size, its distribution and ξ-

potential by DLS technique. The mean droplet size was in a range of 76.5-461.5 nm (Table 2),

characteristic for nanoemulsions, what was also confirmed by transmission electron

microscopy, where nearly spherical droplets of very similar size were visible (Fig. 1). Most of

the freshly prepared nanoemulsions was characterized by low polydispersity indexes (below

0.4), only NE E 7-1, E 8-1, E 9-1, S 11-1, S 11-1, E 12-1 and S 12-1 showed wider size

distribution. Generally, it has been noticed that best characteristic was obtained for

nanoemulsions based on oleic acid. Those composed of Capmul MCM and PG-8 showed

higher polydispersity, with the exemption of C14-(DAPACL)2/Capmul MCM/water

formulation. Of all surfactants, C14-(DAPACL)2 forms nanoemulsions with the lowest PdI,

suggesting that this might be an optimal hydrocarbon length to stabilize the system.

Surfactants C12-(DAPACL)2 and C16-(DAPACL)2 also formed nanoemulsions with good

parameters but only if oleic acid was used as oil phase.

3.2. Physical stability of BSe-loaded nanoemulsions

As a first step, the formulations were visually assessed using the grading system from A to E

(Table 3). Formulations that pass the Grade A dispersibility test have high potential to remain

as stable nanoemulsions with the best physical features. The majority of tested formulations

passed test in Grade A which support the accurate selection of components. Some

formulations passed test in Grade B, showing minor problems during preparation, however, it

does not exclude them from further investigation. It is possible that stable nanoemulsion

systems will form in a longer period. Therefore, all formulations were selected for further

investigation.

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At this stage, all samples were divided in two sample sets. The first set was stored at RT for

30 days and evaluated via particle size (DH), its distribution and ξ-potential, the second one

was subjected to heating/cooling cycles, centrifugation and freezing/thawing cycles as well as

it has carefully been observed for phase separation, creaming, coalescence and cracking

(Tables 2 and 3). It was intended to simulate the behaviour during long-term storage and

under stress conditions and thus exclude metastable and unstable formulations.

Nanoemulsions based on oleic acid, stabilized by C12-(DAPACL)2 and C14-(DAPACL)2 had

shown good stability under stress conditions. No phase separation, creaming, coalescence,

cracking or phase inversion was observed. Furthermore, no signs of BSe precipitation were

noted. It seems that longer hydrocarbon chain in C14-(DAPACL)2 for which half-cover

isotherm occurs at a lower concentration which affecting the stability at oil/water interface has

a better potential to create stable systems with the OA oil phase. Some of C12-

(DAPACL)2/OA-based formulations with lower surfactant/oil ratio showed increase in

polydispersity index over time, whereas C14-(DAPACL)2 formed stable nanoemulsions at both

selected ratios. In this case, the parameters as size, polydispersity index and ξ-potential

remained largely unchanged during a prolonged observation period as supported by DLS

measurements (Table 2) and microscopy (Fig. 1). Moreover, it is believed that ζ-potential

higher than 30mV will ensure high stability due to high energy barrier toward coalescence of

dispersed droplets. The increase in droplet size in some cases could be a result of multiple

processes such as flocculation, coalescence and Ostwald ripening. On the other hand, the

decrease in particle diameter might be due to the movement of oil molecules from the

emulsion droplets into the surfactant micelles or the growth of some droplets due to Ostwald

ripening or coalescence. Consequently, BSe incorporated in the oily phase of nanoemulsions

had rather restricted contact with water molecules of the exterior phase. In our opinion the

developed nanoemulsion provides therefore an inert environment for BSe.

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An interesting tendency was observed for most stable nanoemulsion compositions containing

oleic acid and stabilized by C14(DAPACl)2. The addition of BSe into the oil phase resulted in

increased ζ-potential in all tested formulations in comparison to BSe-free nanoemulsions.

This effect appeared to be dose-dependent and the highest ζ-potential value of +87.4 ± 4.1

was achieved at the highest organoselenium content (0.08%). Only insignificant or minor

changes in the mean particle diameter were observed for this surfactant after one month of

storage. We therefore postulate that this finding might be a consequence of an efficient

solubilization phenomenon of the tested organoselenium molecule, which was used to

stabilize the system. Process lies at a potential stabilization of droplets present in the tested

systems, which would result from complex compensation/attraction forces occurring between

the alkyl chains of surfactants and repulsion forces directly resulting from electrostatic and

steric interactions.

Twelve most promising formulations that positively passed initial stability tests were selected

for further investigation to evaluate long-term physical stability in multiple light scattering

(MLS) measurements with Turbiscan LabExpert. This method allows detecting and

determining the destabilization phenomena, e.g. sedimentation, creaming, coalescence or

flocculation. The sedimentation phenomenon occurs when there is a transmittance decrease

over time at the bottom of the sample, whereas if this change is observed on the top of the

sample it is due to creaming process. When a transmittance decrease with time is observed

over the entire height of the sample, it indicates variation in droplet size, more likely due to

flocculation or coalescence.

The backscattering profiles of the selected oil-in-water nanoemulsions at 0 day (freshly

prepared) and after 30 days of storage in room temperature are shown in Fig. 2, where X-axis

represents the height of the tube (mm) and Y-axis shows the backscattering light percentage.

By analysing the distances between the curves, it is possible to determine the dynamics of the

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processes occurring in the sample. A large distance between the curves indicates rapid

destabilization phenomena, whereas coverage of the individual curves supports the high

stability of system and slow rate of destabilization phenomena. From the recorded profiles

(Fig. 2.) it can be noticed that there is practically no evolution in backscattering which means

that there was no significant variation in the droplet size over the 30 days period. However, it

is worth noting that the BSe-loaded nanoemulsion stabilized by the C14-(DAPACL)2

demonstrates higher stability than the one composed of C12-(DAPACL)2. The backscattering

increase in the bottom (peak at left side of the plot) and on the top (small peak at the right

side) is attributed to the meniscus that the samples form in contact with the glass or the air

entrapped in the samples [23]. Thus, the variations in backscattering profiles under the sample

height of 2 mm and over that of 10 mm were not correlated to destabilization phenomena.

Apart from that, no macroscopic changes in the formulations were observed at the last day of

the test.

Additionally to backscattering profile analysis, the nanoemulsion stability was also evaluated

based on the TSI (Turbiscan Stability Index) values. This index, depending on the global

stability of the samples, provides an easy way to characterize the tested systems and to select

the best formulation in term of stability. The smaller the TSI value, the more stable the

formulation is. The destabilization kinetics of nanoemulsions as a change of TSI value during

analysis, is given in Table 4 and Fig. 2S). All nanoemulsion systems have a relatively low TSI

values that slightly increase over a period of 30 days but again it evident that the

nanoemulsion systems stabilized by C14-(DAPACL)2 (Table 4, Fig.2S, pink lines) show

higher stability (lower TSI values) that the ones stabilized by C12-(DAPACL)2 (Table 4,

Fig.2S, blue lines). This might suggest that the longer hydrocarbon chain of 13 units is

optimal for the above mentioned systems. Generally, the stability was improved in the

presence of organoselenium cargo, whereas dilution of the BSe-free nanoemulsions increased

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destabilization process. The lowest value of the TSI for NE S4-2 indicates the most stable

formulation.

3.3. Antimicrobial efficacy

Benzisoselenazol-3(2H)-ones and dicephalic surfactants have been previously screened for

antimicrobial activity against various bacterial and fungal strains, showing some positive

results [7,36]. This was motivating to select 2-n-propylbezisosenazol-3(2H)-one as the cargo

and dicephalic cationic surfactants of different alkyl chain length as nanoemulsion

components for further studies. The minimum inhibitory concentrations (MICs) against C.

albicans 10231 assessed for both, the BSe and dicephalic cationic surfactants were as follows:

BSe – 0.2 mM, C12-(DAPACL)2 – 6.0 mM, C14-(DAPACL)2 – 4.8 mM and C16-(DAPACL)2 –

0.5 mM.

C. albicans is a dimorphic organism that forms either a yeast or a mycelium depending on the

growth environment. As a first step, the selected nanoemulsions which showed the best

physicochemical characteristics were incubated with C. albicans in a planktonic form to

evaluate their antifungal activity and to study the effect on yeast’s cell shape. Both, reduction

of C. albicans viability with BSe-loaded nanoemulsion exposure (Fig. 3B) and morphological

alteration in the treated vs. untreated C. albicans cells (Fig. 3E-G) were observed by the

scanning electron microscopy (SEM). Figure 3 shows representative images of untreated and

BSe-loaded nanoemulsion treated C. albicans cells. It is clearly visible the cells treated with

the nanoemulsions’ samples at MIC (Fig. 3G) and even sub-MIC levels (Fig. 3F) underwent

considerable morphological alterations in comparison to the control (Fig. 3E). The untreated

cells appeared round-shape, with normal smooth surfaces (Fig. 3E) while nanoemulsion

treated cells lost their original shapes, appeared to be shrunken, wrinkled and deformed (Fig.

3F,G). As expected, much more dramatic changes in morphology were observed at the MIC

22

levels (Fig. 3G), where cells appeared to be ruptured and partially degraded. Although the

sub-MIC concentrations did not cause such a profound damage in the planktonic cells, it is

evident that they were still capable of modifying their outermost surface (Fig. 3F). These

morphological changes might have an impact on microbial virulence. It has been reported that

antimicrobial agents at sub-MIC concentrations although do not active on microbial growth,

they still have a numerous effects on microbes by altering their biochemistry, morphology,

physico-chemical characteristics, and interfering with some important cell functions such as

adhesiveness, surface hydrophobicity, motility and host–microbe interactions, thus reducing

microbial virulence factors [46,47].

As most Candida spp. infections are associated with biofilm formation on host surfaces that

leads to resistance to antimicrobials and host defenses, the second part of the study was

focused on the evaluation of the effect of BSe-loaded nanoemulsions on the adherence

properties and biofilm formation capacity of C. albicans [48,49]. The SEM analysis revealed

the reduction in the preformed C. albicans biofilm after exposure to the BSe-loaded

nanoemulsions (Fig. 3D). The biofilm architecture of the C. albicans control (untreated) was

heterogeneous, composed of a dense layer of yeasts, pseudohyphae, and hyphal forms

(Fig. 3C) whereas after the nanoemulsions treatment the number of hyphae and pseudohyphae

significantly decreased (Fig. 3D). Further analysis with crystal violet staining confirmed the

biofilm reduction to the level of 46.6 to 86.2 %, depending on the nanoemulsion system

applied (Table 5). It is possible that in this case the BSe-loaded nanoemulsions may interfere

with the adhesion properties of C. albicans. A significant reduction of adhesion to polystyrene

surface was observed, when C. albicans cultures were treated with the BSe-loaded

nanoemulsions (Table 5). Depending on the type of nanoemulsion system, adhesion was at

the level of 35.7-57.4 %, as compared to 100% of untreated C. albicans cells. The ability to

23

adhere to various materials constitutes important virulence factor of C. albicans being the first

step in its biofilm formation [50,51].

4. Conclusions

We have successfully developed novel nanoemulsion-based nanoparticulate delivery systems,

stabilized by bifunctional cationic surfactants, N,N-bis[3,3'-(trimethylammonio)propyl]

alkylamide dichlorides, for the delivery of poorly water soluble organoselenium compound,

2-n-propylbenzisoselenazol-3(2H)-one (BSe), having a broad spectrum of outstanding

antimicrobial activity. The BSe-loaded nanocarriers fabricated by the crash dilution method

constituted nanodispersions of nearly monodisperse size, efficient BSe encapsulation and very

good kinetic stability proven by high positive ξ -potential values and no evolution in

backscattering profiles. The treatment of C. albicans and its biofilm with the BSe-loaded

nanoemulsions resulted in strong antifungal effect, by leading to deleterious morphological

changes and cell surface alterations, as well as ultimately loss in viability.

This work presents a new general approach and provides guidelines for the design of

convenient nanocarriers for a group of water-insoluble/sparingly soluble bioactive

organoselenium compounds. The results reported in this work demonstrate the suitability and

potential of developed BSe-loaded nanoemulsions for a local antimicrobial effect upon

possible topical administration. Additionally, such physically stable and unimodal

nanoemulsions of low surfactant content seem to provide very promising vehicles, with a

further perspective to use them in a variety of template-mediated processes, including

multilayering approaches.

Acknowledgements

24

The authors wish to acknowledge the financial support of the research by the National

Science Centre (NCN) under grant no. UMO-2013/09/D/ST5/03814 and the kind help of Dr.

Urszula Bazylińska from the Department of Organic and Pharmaceutical Technology, Faculty

of Chemistry, Wrocław University of Technology, Wrocław, Poland for her valuable advices

related to nanoemulsions preparation.

Contributions

Author MPO generated the concept of developing nanocarriers to enhance the solubility of

antimicrobial organoselenium compounds and designed general studies presented in this

manuscript. Author MPO carried out the literature searches and summaries of previous work.

Author MPO synthesized organoselenium cargo. Author KAW provided cationic surfactants

and guidelines for nanoemulsion composition. Author AL prepared nanoemulsions and run

TEM, SEM and MLS measurements. Authors AJ and AL run DLS measurements. Author AJ

run dispersion stability test. Author AK run antimicrobial tests. Author MPO collected the

data and performed the analyses of the collected data. Authors MPO and AL prepared the

figures and tables. Author MPO wrote the first draft of the manuscript. Author AL supported

a part of discussion related to nanoemulsion stability. All authors contributed to correcting the

manuscript. All authors contributed to and have approved the final manuscript.

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Figure Captions

Fig. 1. TEM images of NE S4-2: A – freshly prepared nanoemulsion system, B –

nanoemulsion system 30 days after preparation, and point-detection EDX analysis (C), with

the analyzed particle circled.

Fig. 2. Backscattering profiles of nanoemulsions as a function of sample height (mm)

analyzed over 30 days of storage at RT: A – NE E1-2, B – NE E4-2, C – NE S1-2, D – NE

S4-2; blue line represents measurement at 0 day (freshly prepared nanoemulsions) while pink

line was recorded after 30 days of storage. ΔBS is defined as a difference in mean BS value

between the first scan and nth

scan.

Fig. 3. SEM images of C. albicans: A – untreated population (control), B – population treated

with BSe-loaded nanoemulsion, C – untreated biofilm (control), D – biofilm treated with

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cell treated at concentration above the MIC. Images represent typical fields of view.

32

Table 1

Molecular structures and abbreviations of dicephalic cationic surfactants and organoselenium

cargo.

Structure R Abbreviation MW [g/mol]

C

O

R N

N

N

H

H

2Cl-

n-C11H23

n-C13H27

n-C15H31

C12-(DAPACL)2

C14-(DAPACL)2

C16-(DAPACL)2

442.55

470.60

498.66

Se

N

O

R

n-C3H7 BSe 240.16

33

Table 2

Composition, droplet size, polydispersity index and ξ-potential of nanoemulsion systems.

Nanoemulsion system

Weight fraction DHa [nm] PdI

b ξ

c [mV]

Surf.

[%]

Oil

[%]

BSe

[%] 0 day 30 days 0 day 30 days 0 day 30 days

C12(DAPACl)2

/OA/water

NE E1-1 0.6 0.2 0 138.3±1.9 139.1±0.7 0.20±0.02 0.14±0.01 +68.3±2.2 +23.5±9.2

NE E1-2 0.15 0.05 0 129.9±0.7 179.2±41.5 0.29±0.02 0.57±0.05 +27.1±1.5 +28.0±3.0

NE S1-1 0.6 0.16 0.04 129.0±2.2 231.5±5.9 0.18±0.01 0.26±0.01 +46.1±3.4 +41.0±3.0

NE S1-2 0.15 0.04 0.01 119.8±2.7 287.6±0.2 0.30±0.01 0. 27±0.16 +15.4±2.3 +12.2±0.4

NE E2-1 0.8 0.4 0 201.5±0.9 246.9±17.9 0.12±0.01 0.42±0.05 +76.8±3.9 +12.6±5.9

NE E2-2 0.2 0.1 0 292.7±9.7 252.5±2.1 0.29±0.04 0.45±0.08 +39.3±2.4 +30.9±3.7

NE S2-1 0.8 0.32 0.08 194.9±1.9 84.1±65.0 0.14±0.01 0.53±0.26 +67.5±3.1 +20.9±3.3

NE S2-2 0.2 0.08 0.02 290.8±4.4 262.1±47.9 0.29±0.01 0.46±0.05 +33.6±1.6 +25.4±1.6

C14(DAPACl)2

/OA/water

NE E3-1 0.6 0.2 0 198.9±2.7 197.4±2.1 0.05±0.01 0.15±0.01 +61.6±3.2 +67.2±2.0

NE E3-2 0.15 0.05 0 159.7±0.5 158.5±1.5 0.09±0.02 0.28±0.00 +48.2±1.5 +64.2±1.0

NE S3-1 0.6 0.16 0.04 136.6±1.8 162.4±5.6 0.04±0.01 0.08±0.03 +72.0±1.8 +67.2±3.1

NE S3-2 0.15 0.04 0.01 162.4±1.6 138.7±3.3 0.06±0.01 0.38±0.02 +54.7±0.7 +36.8±1.6

NE E4-1 0.8 0.4 0 118.5±4.9 106.2±0.9 0.28±0.01 0.22±0.01 +80.7±5.1 +77.4±4.4

NE E4-2 0.2 0.1 0 120.9±1.3 140.7±1.2 0.13±0.01 0.18±0.01 +60.5±1.8 +53.0±2.6

NE S4-1 0.8 0.32 0.08 170.4±6.3 142.7±1.9 0.40±0.06 0.10±0.02 +87.4±4.1 +76.1±3.5

NE S4-2 0.2 0.08 0.02 146.1±0.3 141.3±0.9 0.12±0.01 0.21±0.02 +67.8±1.1 +35.1±1.0

C16(DAPACl)2

/OA/water

NE E5-1 0.6 0.2 0 151.0±1.8 85.8±0.5 0.26±0.01 0.20±0.004 +84.7±3.3 +70.8±3.1

NE S5-1 0.6 0.16 0.04 78.4±0.22 73.8±0.3 0.24±0.01 0.22±0.004 +39.2±1.2 +74.6±2.2

NE E6-1 0.8 0.4 0 102.0±6.9 102.6±2.7 0.32±0.03 0.28±0.01 +81.6±3.7 +85.2±1.6

NE S6-1 0.8 0.32 0.08 461.5±31 126.9±1.2 0.51±0.03 0.27±0.01 +93.5±5.7 +87.3±2.9

C12(DAPACl)2

/MCM/water

NE E7-1 0.6 0.2 0 124.0±11.5 129.9±3.1 0.46±0.02 0.22±0.003 +52.7±2.3 +42.4±2.3

NE S7-1 0.6 0.16 0.04 151.6±3.3 79.4±1.3 0.36±0.01 0.27±0.004 +39.5±3.0 +65.2±2.3

C14(DAPACl)2

/MCM/water

NE E8-1 0.6 0.2 0 112.6±3.1 91.3±3.3 0.43±0.05 0.56±0.03 +62.4±3.4 +59.2±6.1

NE S8-1 0.6 0.16 0.04 149.2±1.5 79.7±25.5 0.18±0.01 0.55±0.09 +68.4±3.5 +79.9±5.4

C16(DAPACl)2

/MCM/water

NE E9-1 0.6 0.2 0 224.8±80.6 34.1±6.4 0.63±0.34 0.93±0.08 +89.5±2.2 +52.7±13.0

NE S9-1 0.6 0.16 0.04 297.5±66.3 97.5±58.6 0.39±0.03 0.68±0.32 +53.2±4.4 +67.9±8.6

C12(DAPACl)2

/PG-8/water

NE E10-1 0.6 0.2 0 186.3±18.1 109.8±3.3 0.40±0.16 0.51±0.09 +63.7±3.8 +67.0±5.5

NE S10-1 0.6 0.16 0.04 355.7±8.9 151.9±1.6 0.40±0.03 0.35±0.02 +38.1±3.5 +35.2±3.6

C14(DAPACl)2

/PG-8/water

NE E11-1 0.6 0.2 0 308.1±17.6 227.7±2.4 0.36±0.04 0.24±0.01 +64.1±2.2 +74.4±7.3

NE S11-1 0.6 0.16 0.04 76.5±35.3 12.6±1.3 0.59±0.28 0.58±0.01 +44.2±4.7 +31.6±10.7

C16(DAPACl)2

/PG-8/water

NE E12-1 0.6 0.2 0 241.4±73.1 80.4±52.8 0.76±0.21 0.35±0.15 +4.5±3.7 +55.7±15.0

NE S12-1 0.6 0.16 0.04 205.2±30.1 122.0±58.5 0.67±0.22 0.62±0.14 +46.7±2.2 +56.2±0.6

a DH: hydrodynamic diameter b PdI: polydispersity index c ξ: zeta potential

34

Table 3

Stability and dispersibility studies of nanoemulsion systems.

Nanoemulsion system H/Ca

Cent.b

F/Tc

D.T.d

Inference

NE E1-1 + + + Grade A Passed

NE E1-2 + + + Grade A Passed

NE S1-1 + + + Grade A Passed

NE S1-2 + + + Grade A Passed

NE E2-1 + + - Grade A Failed

NE E2-2 + + - Grade A Failed

NE S2-1 + + - Grade A Failed

NE S2-2 + + - Grade A Failed

NE E3-1 + + + Grade A Passed

NE E3-2 + + + Grade A Passed

NE S3-1 + + + Grade A Passed

NE S3-2 + + + Grade A Passed

NE E4-1 + + + Grade A Passed

NE E4-2 + + + Grade A Passed

NE S4-1 + + + Grade A Passed

NE S4-2 + + + Grade A Passed

NE E5-1 + + - Grade A Failed

NE S5-1 + + - Grade A Failed

NE E6-1 + - - Grade B Failed

NE S6-1 + - - Grade B Failed

NE E7-1 - - - Grade B Failed

NE S7-1 - - - Grade B Failed

NE E8-1 + + - Grade B Failed

NE S8-1 + + - Grade B Failed

NE E9-1 - - - Grade B Failed

NE S9-1 - - - Grade B Failed

NE E10-1 - - - Grade B Failed

NE S10-1 - - - Grade B Failed

NE E11-1 + + - Grade B Failed

NE S11-1 + + - Grade B Failed

NE E12-1 - - - Grade B Failed

NE S12-1 - - - Grade B Failed a H/C: heating/cooling cycles b Cent.: centrifugation, c F/T: freeze/thaw cycles d D.T.: dispersibility test

35

Table 4

Turbiscan Stability Index of nanoemulsions as a function of time.

Nanoemulsion system TSI

a

5 days 10 days 15 days 20 days 25 days 30 days

NE E1-1 0.7 1.4 2 2.4 2.7 3.1

NE E1-2 0.7 1.3 1.9 2 2.1 2.2

NE S1-1 0.5 1.0 1.5 1.8 2.1 2.3

NE S1-2 0.5 1.0 1.4 1.5 1.7 1.9

NE E3-1 0.3 0.7 0.9 1.1 1.3 1.5

NE E3-2 0.3 0.5 0.8 1.0 1.1 1.3

NE S3-1 0.3 0.5 0.8 1.0 1.2 1.5

NE S3-2 0.2 0.5 0.7 1.0 1.2 1.5

NE E4-1 0.2 0.4 0.6 0.9 1.1 1.4

NE E4-2 0.2 0.3 0.5 0.6 0.8 1.0

NE S4-1 0.1 0.2 0.4 0.5 0.6 0.7

NE S4-2 0 0 0.1 0.1 0.1 0.1 a TSI: Turbiscan Stability Index; depends on the global stability of the sample

36

Table 5

The influence of BSe-loaded nanoemulsions on the C. albicans ATCC 10231 biofilm

formation and adhesion to the polystyrene surface.

Nanoemulsion system Biofilm

a [%] Adhesion

b [%]

NE S1-1 83.0 55.7

NE S3-1 84.8 51.0

NE S4-1 86.2 40.0

NE S1-2 63.2 42.6

NE-S3-2 46.6 57.4

NE S4-2 78.4 35.7 a,b The values, expressed as a percentage of control (untreated C. albicans), are means of three experiments

1

Fig. 1

Figure(s)

2

Fig. 2

3

Fig. 3

A B

G

F

E

C D