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Antifungal activity against Candida

albicans of starch Pickering emulsion
with thymol or amphotericin B in
suspe...
Andrea Cossu

International journal of pharmaceutics

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 International Journal of Pharmaceutics 493 (2015) 233–242

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Antifungal activity against Candida albicans of starch Pickering

emulsion with thymol or amphotericin B in suspension and calcium
alginate films
Andrea Cossua,1, Min S. Wanga,1, Amol Chaudharia , Nitin Nitina,b,*
a
Food Science and Technology Department, University of California Davis, Davis, CA 95616, USA
b
Department of Biological and Agricultural Engineering, University of California Davis, Davis, CA 95616, USA

A R T I C L E I N F O A B S T R A C T

Article history: Conventional antifungal treatments against Candida albicans in the oral cavity often result in increased
Received 10 February 2015 cytotoxicity. The goal of this study was to determine the potential of starch Pickering emulsion as a
Received in revised form 18 July 2015 delivery vehicle for an antifungal natural phenolic compound such as thymol in simulated saliva fluid
Accepted 26 July 2015
(SSF) compared to amphotericin B. An oil-in-water (o/w) emulsion was stabilized using starch particles.
Available online 29 July 2015
Physical stability of the emulsion and disruption induced by a-amylase activity in SSF was evaluated.
Encapsulated thymol in o/w emulsion was compared to encapsulated amphotericin B for antifungal
Keywords:
activity against C. albicans in suspension using emulsions or zone inhibition assay on agar plates using
Candida albicans
Pickering emulsion
emulsions dispersed in alginate films. Results showed that the emulsions were stable for at least three
Alginate film weeks. Digestion of the emulsion by a-amylase led to coalescence of emulsion droplets. The antifungal
Controlled-release activity of thymol and amphotericin B in emulsion formulation was enhanced upon incubation with
Antifungal compound a-amylase. Results from the zone inhibition assay demonstrated efficacy of the emulsions dispersed in
alginate films. Interestingly, addition of a-amylase to the alginate films resulted in a decreased inhibitory
effect. Overall, this study showed that starch Pickering emulsions have a potential to deliver hydrophobic
antifungal compounds to treat oral candidiasis.
ã 2015 Elsevier B.V. All rights reserved.

1. Introduction Therefore, to improve the efficacy of localized delivery and

reduce systemic side effects observed with the current antifungal
Oral candidiasis is a common fungal infection affecting infants treatments, antifungal compounds may be formulated into
and patients with immunocompromised pathologies (Ramos-E- mucosal adhesive films (Lalla and Bensadoun, 2011; Vazquez
Silva et al., 2010) or those following antitumoral therapies (Bulacio and Sobel, 2012) such as poly(acrylic acid) (PAA) or other
et al., 2012). polymeric coatings (Quirós-Sauceda et al., 2014) to achieve a
This fungal infection can be treated with either systemic or controlled and localized delivery of bioactives in the oral cavity.
topical antifungal therapy. Systemic antifungal therapy is com- To address the toxicity issues with synthetic antifungal drugs,
monly used for the treatment of immunocompromised patients prior studies have evaluated the antifungal efficacies of natural
but can cause significant side effects due to their toxicity to the essential oils, which are natural phenolic and isoprenic molecules
host. In contrast, topical treatment of oral candidiasis possesses commonly found in food and are generally recognized as safe
fewer side effects but does not provide sufficient contact of the (GRAS) compounds. Among the many essential oils, carvacrol,
drug with the oral mucosa and require multiple dosages to thymol, eugenol and isoeugenol have been shown to have effective
effectively treat the infection (Lalla and Bensadoun, 2011; Vazquez antimicrobial activity against a broad-spectrum of microorganisms
and Sobel, 2012). (Gallucci et al., 2014). Despite the potential for medicinal uses,
these antifungal compounds have poor bioavailability and bio-
absorptivity due to their low water solubility (Celia et al., 2013). In
addition, essential oils are susceptible to oxidation as well as light
* Corresponding author at: Food Science and Technology Department, University and heat degradation, thus reducing their stability and shelf-life
of California Davis, RMI South, 392 Old Davis Road, CA 95616, USA. Fax: +1 530 752
(Turek and Stintzing, 2013). As such, encapsulation and controlled
4759.
E-mail address: [email protected] (N. Nitin). delivery of these natural essential oils could be a promising
1
Both authors contributed equally to this work. approach to overcome these drawbacks.

http://dx.doi.org/10.1016/j.ijpharm.2015.07.065
0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
234 A. Cossu et al. / International Journal of Pharmaceutics 493 (2015) 233–242

Oil-in-water (o/w) emulsions are commonly used for dispersion wicked away using a filter paper and 8 mL of 1% ammonium
of hydrophobic compounds in aqueous environments or films. molybdate was added to negatively stain the sample. The excess
Among diverse approaches for formulating emulsions, particle stain solution was removed by wicking with a filter paper and the
stabilized emulsions, also known as Pickering emulsion, have TEM grid was allowed to dry in air for 10 min. The TEM sample was
shown significant potential in reducing coalescence and improving viewed using a Philips CM120 BioTWIN TEM (FEI Company,
stability of emulsions (Marku et al., 2012). Hillsboro, OR, USA) operated at 80 kV and the image was acquired
This study evaluated the potential of starch stabilized Pickering using the Gatan TEM Imaging software.
emulsions as delivery vehicles for controlled release of antifungal
compounds from emulsions in suspension and film formulation. 2.4. Starch Pickering emulsion preparation
Starch was chosen as the stabilizer because it is a biodegradable
and GRAS material (Tan et al., 2012) and it can be broken down by An o/w starch Pickering emulsion was prepared as follows:
a-amylase, an active enzyme in the oral cavity. 15 wt% of extra virgin oil was added to 15 wt% of starch and 70 wt%
An o/w emulsion was prepared using extra virgin olive oil as the water. A stock of thymol-oil was made by adding thymol directly to
oil phase and a modified food grade starch (CAPSUL1TA) derived the olive oil (500 mg/mL in oil) and stirred at 90  C for 1 h until the
from cassava (Manihot esculenta) as the stabilizer/emulsifier. thymol was completely dissolved. In a different emulsion
Stability of the starch Pickering emulsion was evaluated using formulation, amphotericin B was added to the oil phase (final
dynamic light scattering (DLS) and optical microscopy. Thymol (2- concentration in the emulsion of 1 mg/mL). The water and oil
isopropyl-5-methylphenol), a natural essential oil, was used as a phases were dispersed at 24,000 rpm for 2 min with a hand
potential antifungal compound in inhibition assays against disperser (Ultra-Turrax model T25, IKA Works, Wilmington, NC,
Candida albicans in the Pickering emulsion. The antifungal efficacy USA) and the ultrafine o/w emulsion was generated by sonication
of thymol was compared to amphotericin B, a well-established using a tip sonicator (Q55 QSonica, Newtown, CT, USA) for 30 s at
antifungal compound using the same emulsion based formulations 50% power. The o/w starch Pickering emulsions of thymol (75 mg/
(Chavanet et al., 1997). Antifungal efficacy was evaluated using mL final concentration) and amphotericin B (1 mg/mL final
both suspension and film formulation. The influence of a-amylase concentration) were stored at 4  C until use.
activity in a simulated salivary conditions on antifungal efficacy
was also assessed using both suspension and film formulations. 2.5. Size and zeta-potential measurements
This study aims to evaluate the potential of starch Pickering
emulsions for a controlled oral delivery of hydrophobic antifungal The stability of the prepared emulsions was evaluated by
compounds both in suspension and in film formulation for the measuring changes in particles size and zeta potential of the
treatment of oral candidiasis. emulsion during a period of 3 weeks. The hydrodynamic diameter
and zeta potential of the starch Pickering emulsion were measured
2. Materials and methods using a particle size analyzer (Malvern Nano Series, Malvern
Instruments, Inc., Westborough, MA, USA) using a 1:1000 dilution
2.1. Reagents and media of the emulsion. Refractive indices of 1.45 and 1.33 were used for
the emulsion and disperse water phase, respectively. Size and zeta
Modified food starch CAPSUL1TA was purchased from National potential measurements were performed in triplicate at 25  C.
Starch Food Innovation (Bridgewater, NJ, USA). Sodium phosphate
monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), 2.6. Starch Pickering emulsion digestion
sodium chloride (NaCl), potassium chloride (KCl), magnesium
chloride (MgCl2) and agar were obtained from Fisher Scientific A starch Pickering fine emulsion with Nile red as encapsulant at
(Hampton, NH, USA). Tween 20, calcium chloride, alginic acid a final concentration of 50 mM in the oil phase was prepared using
sodium salt, glycerol, Nile red, amphotericin B and a-amylase were the same method as mentioned above. The emulsion was digested
purchased from Sigma–Aldrich (St. Louis, MO, USA). Extra virgin oil using 10–150 U/mL of a-amylase in a simulated salivary fluid (SSF)
was obtained from a local retail store. Thymol was purchased from (5 mM NaH2PO4, 5 mM Na2HPO4, 16 mM urea, 2 mM CaCl2, 4 mM
Acros Organics (Fair Lawn, NJ, USA). Yeast mold (YM) broth was MgCl2, 10 mM NaCl, 10 mM KCl, pH 6.5). The a-amylase concen-
purchased from Amresco (Solon, OH, USA). tration range was chosen based on the reported peak diurnal
salivary a-amylase concentration in human subjects (Nater et al.,
2.2. Atomic force microscopy 2007). The digestion was carried out by mixing a 1:1 (v/v) of
emulsion to SSF with a-amylase and incubating the mixture under
Surface morphology of the starch particle was analyzed using shaking conditions at 37  C for 1 h. The disruption of the starch
atomic force microscopy (AFM). A 10 mL drop of aqueous starch Pickering emulsion was monitored by measuring the fluorescence
dispersion (0.001 wt%) was deposited on a freshly cleaved mica and intensity of the encapsulated Nile red at 2 min intervals for up to
incubated at room temperature for 10 min. The surface was rinsed 1 h using a Molecular Devices M5 platereader (Carlsbad, CA, USA).
3 times with DI water and dried under a gentle stream of N2 gas. The fluorescence intensity of Nile red was measured from the
The sample was imaged using a tapping mode AFM (Asylum bottom of each well. The excitation and emission wavelengths
Research, MFP-3D, Santa Barbara, CA, USA) with NSC15 silicon were 530 nm and 640 nm, respectively.
probes (MikroMasch 325 kHz, 46 N/m). The topographic image of
the sample was acquired at 1 Hz and 512  512 pixels resolution 2.7. Visualization of starch Pickering emulsion after digestion using
and processed using the IGOR Pro 6.21 software. optical microscopy

2.3. Transmission electron microscopy A coarse emulsion (dispersed without sonication) with Nile red
at a final concentration of 50 mM in the oil phase was prepared as
An individual starch particle was observed by TEM. A drop of mentioned above. The structures of the coarse starch Pickering
aqueous starch dispersion (8 mL, 0.0001 wt%) was placed on a emulsion before and after digestion with 100 U/mL of a-amylase
carbon coated TEM grid (400 mesh, Ted Pella, Redding, CA) and were visualized using an inverted optical microscope (Olympus IX-
incubated for 10 min. The excess aqueous starch dispersion was 7). The fluorescence images were acquired after 0, 10, 30 and
 A. Cossu et al. / International Journal of Pharmaceutics 493 (2015) 233–242 235

60 min of digestion time using a 63X objective and the fluorescence completely dissolved. Solutions were then poured in a polystyrene
excitation and emission were 541 nm and 572 nm, respectively. petri dish and air dried at 30  C for 24 h. Squares of 1 cm  1 cm
were cut from the dried films and placed on YMA plates previously
2.8. Fungal cultures and suspension inhibition assay (SIA) seeded with 1.0  107 CFU of C. albicans. Then, 20 mL of SSF with or
without a-amylase (100 U final) were spotted over the film
C. albicans ATCC 90028 was cultured in YM or YM plus 1.5% agar squares. YMA plates were incubated for 24 h at 37  C to detect an
(YMA) media at 30  C. An optical density of 1.0 at 530 nm (8.0  107 inhibition halo. Inhibition was measured as distance (mm) from
CFU/mL, confirmed by plate count) was reached after incubation at one side of the film square. Experiments were conducted in
30  C at 250 rpm agitation in tubes containing 10 mL of prewarmed triplicate for all the conditions.
YM broth. Cultures of approximately 2.0  106 CFU/mL were
prepared in YM broth for further experiments. 2.10. Confocal microscopy visualization of starch Pickering emulsion
For the controlled release experiments, aliquots of 50 mL of SSF digestion in calcium alginate film after digestion with a-amylase
containing 20 or 200 U/mL of a-amylase were added to 50 mL of a
2.0  106 CFU/mL culture of C. albicans in wells of a microtest u- To demonstrate the a-amylase digestion of the starch Pickering
bottom transparent 96 wells plate (BD, Franklin Lakes, NJ, USA). emulsion cast within the alginate film, 1 mL of Nile red emulsion
Then, thymol or amphotericin B emulsions were added at different (50 mM) was added to 10 mL of calcium alginate gel and a thin film
concentration ranges (10–1000 mg/mL thymol and 1–20 mg/mL was prepared as mentioned above. The final concentration of Nile
amphotericin B) to a final volume of 110 mL and incubated red in the poured solution in a petri plate was 5 mM. A 1 cm  1 cm
overnight at 37  C in a spectrophotometer spectramax 340 plater- of the alginate film was cut and placed on a microscope slide. Then,
eader (Molecular Devices, Sunnyvale, CA). Optical density (OD) of a drop of 20 mL of SSF with a-amylase (100 U) was spotted over the
each sample was measured at 530 nm with a time interval of 4 h film and a cover slip was placed over the hydrated film. The
between successive readings for a period of 24 h. The sample was alginate film was immediately visualized using a Zeiss LSM
stirred with a 10 s shaking every 5 min. Control samples with SSF 510 Meta confocal microscope. The fluorescence images were
without a-amylase or emulsions without thymol and amphoteri- acquired after 0, and 30 min of digestion using a 63X objective with
cin B were evaluated. All experiments were performed in a laser excitation at 543 nm and emission at 560 nm.
triplicates. The OD530 measurement of each sample was subtracted
from the initial OD530, and the data was plotted as the mean OD530 2.11. Release assay of thymol or amphotericin B from starch Pickering
against time. A negative subtracted OD530 after 24 h was emulsion in suspension after digestion with a-amylase
considered as a fungicidal concentration.
A starch Pickering emulsion containing thymol in the oil phase
2.9. Alginate film preparation and agar inhibition assay (AIA) was diluted in SSF with 2% Tween 20 until a final concentration of
the encapsulant was 10,000 mg/mL. An emulsion containing a final
Calcium alginate gels with varying amounts of starch Pickering concentration of amphotericin B at 100 mg/mL was also prepared
emulsions were prepared as follows: starch Pickering emulsions as above. One hundred units of a-amylase per millilliter were
with thymol (1000–20,000 mg/mL) or amphotericin B (1–500 mg/ added to the emulsions and the samples were incubated at 37  C at
mL) were mixed with calcium chloride (5 mM) and glycerol (1.5 wt 170 rpm agitation. Five hundred microliters of the digested sample
%) in water in a 100 mL beaker. Alginic acid sodium salt (1.5 wt% were taken at 30 min and 60 min of incubation time as well as
final concentration) was added last and water was added to a final before the incubation period (t0). The sample volume was pipetted
volume of 10 mL. The solution was continuously agitated at room into a Nanosep centrifugal device 30 K Omega (Pall Life Sciences,
temperature with a magnetic stirrer until the alginate was Ann Arbor, MI, USA) and centrifuged at 16,000  g for 5 min. The

Fig. 1. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) images of starch particles. (A) AFM image of the starch particles. Inset shows the height
profile of one starch particle. (B) A negatively stained TEM image shows a close up view of one individual starch particle. Scale bar = 100 nm.
236 A. Cossu et al. / International Journal of Pharmaceutics 493 (2015) 233–242

flow through was recovered and diluted with 1.5 volumes of 3. Results
absolute ethanol before spectrophotometric measurement. Detec-
tion of thymol or amphotericin B from the disrupted emulsions 3.1. AFM and TEM analysis of starch particles
was evaluated by measurement of the absorbance of the diluted
sample at 240 nm or 276 nm, respectively using a GENESYS 10S The starch particles were evaluated using AFM and TEM
UV–vis spectrophotometer (Thermo Scientific, Waltham, MA, USA) imaging to obtain the size and morphological characteristics of the
in UV–cuvettes (BRAND, Wertheim, Germany). The blank for this starch. A representative AFM (Fig. 1A) and TEM images (Fig. 1B) of
experiment consisted of a solution of 1.5 volumes of ethanol and 1 the starch particle are shown. The results of image analysis from
volume of SSF with 2% Tween 20. The absorbance measurements both the AFM and TEM images demonstrate that individual starch
were normalized by subtracting the background absorbance particles are approximately 100 nm in diameter and have an
measured using the control emulsion without the encapsulants. irregular shape (Fig. 1). The height profile analysis from the AFM
These control emulsions were treated using the same experimen- images further revealed that the thickness of the starch particles
tal procedures as the emulsions with encapsulants (thymol or was only 5–6 nm thick (Fig. 1A, inset). Together these results
amphotericin B). suggest that the starch particles were flat discs rather than spheres
(Fig. 1).
2.12. Statistical analyses
3.2. Stability of the starch Pickering emulsion
Mean and standard deviation of each data were calculated and
one-way ANOVA was used for the statistical analysis of the data Results from the DLS measurements showed that the starch
obtained from droplet size and zeta potential of the emulsion. The Pickering emulsion was stable for up to three weeks of storage in
two-tailed t-student’s test was used for the statistics of data from the dark at room temperature (Fig. 2). The mean droplet diameter
the means of the time points of the release and permeation assay of the emulsions increased slightly from 311 15 nm to
from emulsion or emulsion in calcium alginate film. A p- 328  11 nm in three weeks, while a slight increase in the net
value  0.05 was considered as statistically significant. negative zeta potential of the emulsion was observed from
44.5  0.5 mV to 46.7  0.9 mV. During storage of the Pickering
emulsion for three weeks (Fig. 2), no significant change in the
mean diameter (F = 1.318; Dfn = 3; Dfd = 8; p = 0.334) of the
emulsion was observed while a statistically significant change in

Fig. 2. Stability measurement of the starch Pickering emulsion. DLS measurement of (A) the mean droplet diameter and (B) zeta potential of the emulsions from 0 to 21 days.
Data represents the mean  SD from 3 independent samples. An asterisk indicates statistical significance (p < 0.05). Images of the Pickering emulsion (C) at day 0 (left) and
21 days (right) are also shown.
 A. Cossu et al. / International Journal of Pharmaceutics 493 (2015) 233–242 237

zeta potential (F = 11.840; Dfn = 3; Dfd = 8; p = 0.003) was detected 150 U/mL of a-amylase were added, the half-lives (t1/2) of Nile red
after three weeks. It is important to note that a change of 2–3 mV in fluorescence were 18, 14 and 14 min, respectively (Fig. 3A). On the
the zeta potential of colloidal systems has limited practical other hand, when 10 U/mL a-amylase was added, a slower
implications. In addition to particle size and zeta potential disruption rate (t1/2 = 38 min) was observed (Fig. 3A). When no
analysis, stability of the emulsion samples was also evaluated a-amylase was added, 80% of the initial fluorescence was retained
based on visual analysis. Representative visual images of the after 1 h of incubation with SSF (Fig. 3A).
emulsion formulation at t = 0 and t = 21 days are shown in Fig. 2B. The release trend of thymol or amphotericin B during the
The results indicate lack of any creaming in the emulsion sample digestion of the emulsions with a-amylase is shown in Fig. 3B and
during storage. C. The results (Fig. 3B) show significant increase (p < 0.05) in the
release (both at 30 min and 60 min sampling times) of thymol from
3.3. Digestion of the emulsion with a-amylase starch stabilized emulsion following incubation with a-amylase as
compared to the control emulsions with thymol not incubated
To characterize changes in the starch Pickering emulsion upon with a-amylase. It is important to note that a decrease in the
incubation with a-amylase, Nile red was selected as a model concentration of released thymol in the aqueous phase after
hydrophobic encapsulant. The effect of varying concentrations of 60 min of simulated oral digestion compared to its concentration
a-amylase ranging from 10 U/mL to 150 U/mL on digestion of after 30 min of incubation may result due to relatively high
starch Pickering emulsion is summarized in Fig. 3A. These results volatility of thymol (Nieddu et al., 2014).
indicate that the starch Pickering emulsion was disrupted in the Similarly in the case of emulsions encapsulating amphotericin
presence of a-amylase, and the phase separation of the oil and B, the release of amphotericin B was also significantly higher
aqueous layers was clearly visible by eye after 1 h of incubation (p < 0.05) after 60 min of incubation with a-amylase compared to
with 100 U/mL of a-amylase. Based on fluorescence spectroscopy the control emulsion with amphotericin B not digested (Fig. 3C).
measurements, a rapid disruption of the emulsion and decrease in However in the case of amphotericin B encapsulation emulsions,
fluorescence intensity of Nile red in the colloidal suspension was the amount of amphotericin B released from emulsions incubated
observed, indicating coalescence of oil droplets and phase with and without a-amylase at 30 min incubation time was not
separation between the oil and water phase. When 50, 100 and significantly (p > 0.05) different.

Fig. 3. Measurement of encapsulated Nile red dye in starch Pickering emulsion and release assay of thymol and amphotericin B during a-amylase treatment. (A) Graph of the
decay of fluorescence intensity over time of the encapsulated Nile red after addition of different amount (0, 10, 50, 100 and 150 U/mL) of a-amylase. A control emulsion was
incubated for the same period in SSF without enzyme. Relative fluorescence was normalized to the fluorescence intensity at 0 min. (B) Thymol or (C) amphotericin B release
assays from the respectively starch Pickering emulsions after a-amylase treatment. Absorbance at 240 nm and 276 nm for thymol and amphotericin B respectively were
recorded at 0, 30 and 60 min of digestion. Data were subtracted from readings derived by the control emulsion without encapsulant in the oil phase prepared in the same
conditions. All the data of the figure represent the mean  SD from 3 independent samples for each set.
238 A. Cossu et al. / International Journal of Pharmaceutics 493 (2015) 233–242

Fig. 4. Fluorescence microscopy imaging of starch Pickering emulsion during a-amylase digestion. (Top) Fluorescence microscopy images of the starch Pickering emulsion
after 0, 10, 30 and 60 min of digestion with 100 U/mL a-amylase at 37  C in the dark. The control Pickering emulsion sample was incubated in SSF under the same conditions
without a-amylase addition. Scale bar = 20 mm. (Bottom) Photographs showing the Nile red encapsulated starch Pickering emulsion with and without 100 U/mL a-amylase
digestion at the same incubation conditions and time intervals as above.

To visualize the changes in emulsion structure with a-amylase to 1 h of incubation (Fig. 4). At 0 and 10 min after digestion, no
digestion, 100 U/mL of a-amylase was added to the coarse changes in emulsion structure were observed between the SSF
emulsion sample with encapsulated Nile red and the digestion control and a-amylase samples. However, the emulsions started to
process was monitored by optical imaging at several time points up degrade after 30 min of incubation with a-amylase, as evident by

Fig. 5. Suspension inhibition assay (SIA) of Candida albicans with encapsulated antifungal compounds in starch Pickering emulsion. Inhibitory effects of thymol (10–1000 mg/
mL) (D–F) and amphotericin B (1–20 mg/mL) (G–I) encapsulated starch Pickering emulsions against C. albicans in SSF and SSF with 10 U/mL and 100 U/mL a-amylase.
Incubatation with varying amounts of control starch Pickering emulsions (1–20 mL) without encapsulant was also carried out in parallel (A–C) without any added antifungal
encapsulant in the emulsion. Data were subtracted to the initial absorbance (t = 0 min) of the culture at 530 nm. Growth curves show the averages from biological
triplicates  SD from three biological replicates.
 A. Cossu et al. / International Journal of Pharmaceutics 493 (2015) 233–242 239

the formation of bigger oil droplets in the fluorescence image 3.5. Growth inhibition of C. albicans using thymol or amphotericin B
(Fig. 4). At 1 h, the size and fluorescence intensity of the Nile red oil encapsulated Pickering emulsion in alginate films
droplets were shown to have increased in the a-amylase treated
sample. This was in contrast to the SSF control sample at 1 h that Calcium alginate films with starch Pickering emulsion con-
showed no change in the size of Nile red oil droplets (Fig. 4). taining thymol or amphotericin B were tested for the inhibition of
C. albicans growth on YMA plates inoculated with approximately
3.4. Enhancement of the antifungal activities of thymol and 1.0  107 cells of C. albicans. The inhibition zones after the addition
amphotericin B emulsions after digestion with a-amylase of 1 1 cm2 squares of the testing films were measured (Fig. 6).
Alginate films containing the thymol emulsions at final
In the suspension inhibition assays, the antifungal activity of concentrations between 1000 and 8000 mg/mL did not reveal an
the emulsions containing thymol or amphotericin B was tested in a inhibition effect. However, alginate films with thymol concen-
liquid culture of C. albicans. A minimum fungicidal concentration trations at 9000 mg/mL or higher showed concentration-depen-
(MFC—as defined in the Section 2) of 200 mg/mL and 5 mg/mL was dent inhibition of the C. albicans fungal lawn. The addition of 100 U
determined for thymol and amphotericin B respectively, upon of a-amylase to the alginate film with the thymol emulsions did
incubation of emulsions with C. albicans for 24 h without the not change the effective inhibitory concentrations but reduced the
addition of a-amylase (Fig. 5D and G). Upon incubation of radius of the inhibition zones compared to the control without
emulsion samples with thymol or amphotericin B with 10 U/mL a-amylase (Fig. 6).
of a-amylase, no difference in MFC was observed in either the For the alginate films containing amphotericin B emulsions, no
thymol or amphotericin B samples compared to the control growth inhibition of C. albicans was observed at concentrations
without a-amylase (Fig. 5E and H). However, the addition of 100 U/ lower than 5 mg/mL (Fig. 6). However, an inhibition halo on the
mL of a-amylase showed a decrease in the MFC to 100 mg/mL and fungal growth was observed when 10 mg/mL or higher concentra-
1 mg/mL for thymol and amphotericin B, respectively (Fig. 5F and I). tion of amphotericin B was used (Fig. 6). Interestingly, the addition
This corresponded to a 50% and 80% reduction in the MFC for of 100 U of a-amylase to the alginate films resulted in a decrease in
thymol and amphotericin B, respectively compared to the control. size of the inhibition zone of amphotericin B, but the minimum

Fig. 6. Agar inhibition assay (AIA) of Candida albicans with encapsulated antifungal compounds in starch Pickering emulsion in calcium alginate film. Calcium alginate films
(1 1 cm2) with encapsulated thymol (8000–20,000 mg/mL) (A and B) and amphotericin B (5–500 mg/mL) (C and D) were treated with either SSF (A and C) or 20 mL of
SSF + 5 U/mL of a-amylase (B and D). Distances of inhibition (mm) from a side of each alginate film square on a microbial lawn derived from 1.0  107 seeded CFU are shown.
Data show the averages  SD from biological triplicates.
240 A. Cossu et al. / International Journal of Pharmaceutics 493 (2015) 233–242

inhibitory concentration was the same (10 mg/mL) as in the case of biopharmaceutical formulation approach for the controlled-
the control without a-amylase (Fig. 6). Similar to the thymol release of the API to the oral cavity is needed.
emulsion alginate films, the amphotericin B film after digestion by Several studies have reported the use of natural essential oils as
a-amylase also resulted in a smaller inhibition zones around the antifungal compounds in yeast inhibition assays (Aznar et al.,
films (Fig. 6). 2015) either as antifungal agents (Gallucci et al., 2014) or as
enhancers in combination with already well known antifungal
3.6. a-amylase digestion of starch Pickering emulsion in calcium drugs (Pemmaraju et al., 2013; Faria et al., 2011). Thymol, carvacrol,
alginate film cymene, eugenol and isoeugenol are a few of the natural essential
oils that have been tested and recognized as antifungal compounds
Changes to the starch Pickering emulsion cast within the (Aznar et al., 2015; Gallucci et al., 2014). Among these essential oils,
alginate film due to digestion by a-amylase was observed by thymol has been shown to be a potent antibacterial and antifungal
fluorescence imaging. The emulsion droplets appeared evenly agent, and was used previously to control fungal (Abbaszadeh
distributed within the film matrix at t = 0 (Fig. 7) either with or et al., 2014) and listerial growth in food using a nanoemulsion
without a-amylase. However, after 30 min of incubation with preparation (Xue et al., 2013; Pan et al., 2014). Furthermore, it has
a-amylase, bigger oil droplets induced by coalescence of individual been demonstrated by atomic force microscopy (AFM) imaging
were observed, indicating disruption of the interface of oil droplets that thymol affects the envelope of C. albicans and increases cell
within the alginate film (Fig. 7). It should be noted that a few larger permeability by entering the lipid bilayers and modifying the
oil droplets were also observed in the sample without a-amylase; membrane fluidity (Braga and Ricci, 2011).
however no coalescence was observed. In this study, we designed a controlled delivery system based on
a formulation of a starch Pickering o/w emulsion for the release of
4. Discussion both natural and synthetic hydrophobic antifungal compounds in
suspension and edible film formulations.
Current antifungal therapies for the treatment of oral candidi- The Pickering emulsion prepared in this study showed good
asis involve the administration of antifungal compounds in physical stability for up to at least three weeks when stored at
concentrations which are toxic to the host and can cause severe room temperature under dark conditions (Fig. 2). The selected time
side effects (Gallucci et al., 2014). In addition, the increased period was based on previous studies that have evaluated the
number of fungal strains with drug resistance properties against stability of food grade emulsions (Anton et al., 2013; Surh et al.,
azole based antifungal drugs (Cannon et al., 2009) requires the 2007) and emulsion based fungicide formulations (Narayanan
development of new pharmaceutical agents and their formulations et al., 2000). Data from particle size measurment and zeta potential
to improve activity and bioavailability while reducing side effects. analysis together with the visual inspection and absence of
Rapid dilution of the active pharmaceutical ingredient (API) in the creaming from the emulsion during the chosen stability period
oral cavity and the possibility of drug ingestion by the patient confirmed the stability of the Pickering emulsion. This result was
makes it difficult to design controlled-release formulations consistent with the observed stability of similar Pickering
(Steinberg and Friedman, 1999). As such, the actual bioavailability emulsions made using starch nanocrystals (Li et al., 2014) and
of the API in the oral cavity could be significantly less than the quinoa starch granules (Rayner et al., 2012).
required concentration for complete fungal eradication (Samar- Image analyses based on AFM and TEM measurements showed
anayake and Ferguson, 1994). For this reason, an alternate that the structure of the starch particles were flat and irregular in
shape, with an aspect ratio of approximately 1:20 (height:diameter)
(Fig. 1). Similar starch nanocrystals structures derived from the acid
hydrolysis of waxy maize starch have also been previously reported
(Lin et al., 2011). This unique flat disc-like structure of the modified
starch particle could contribute to its exceptional stabilizer/
emulsifier property. Compared to other Pickering emulsions that
use spherical particles like silica nanoparticles (He and Yu, 2007), the
low aspect ratio of the starch particles may allow them to be stacked
and packed tightly around the oil droplet. The tight packing of starch
particles around the oil droplet may prevent the o/w emulsion
droplets from coalescing, thus improving stability of the the
emulsions and encapsulated compounds.
Coalescence of emulsion droplets and phase separation
between the oil and water phase induced by a-amylase treatment
of emulsion was monitored using Nile red as a model encapsulant.
In this study, the digestion of starch molecules with a-amylase at
50 U/mL or higher concentration resulted in a reduction of
fluorescence intensity of Nile red in a suspension (Fig. 3A) over
a period of 1 h, indicating a phase separation of the oil and water
phase. This observation was further supported by fluorescence
imaging that showed the formation of larger oil droplets after 1 h of
incubation with 100 U/mL a-amylase (Fig. 4).
The controlled-release of hydrophobic encapsulants due to
Fig. 7. Confocal fluorescence microscopy visualization of starch Pickering emulsion starch digestion by a-amylase was directly observed in the
in calcium alginate film during a-amylase digestion. Confocal fluorescence spectrophotometric assays for the detection of the two antifungal
microscopy images of the calcium alginate film with the starch Pickering emulsion compounds from the emulsions and indirectly observed in the
containing Nile red (5 mM final concentration in the film) after 0 and 30 min of
digestion with 100 U of a-amylase at room temperature in the dark. Images are
antimicrobial assay of the emulsions suspended in microbial
shown as black and white negative pictures. Scale bar = 10 mm. Arrows indicate cultures of C. albicans.
coalescence phenomena.
 A. Cossu et al. / International Journal of Pharmaceutics 493 (2015) 233–242 241

Thymol and amphotericin B were added to the oil phase of the the YM agar. Accordingly, the addition of a-amylase would cause a
emulsion and tested for their antifungal activity. The inhibitory rapid disruption of the emulsion in the film, which would result in
effects of thymol and amphotericin B in the suspension assay emulsion coalescence and phase separation of the oil and water
showed that the addition of 100 U/mL a-amylase resulted in an phases, as demonstrated in the imaging measurements of
enhanced effect against C. albicans due to the increased bioavail- emulsion droplet coalescence within the alginate films (Fig. 7).
ability of the antifungal compounds that were released from the Therefore, the antifungal compounds would co-migrate with the
emulsions (Fig. 5). The MFC for thymol and amphotericin B were oil phase away from the lawn of C. albicans on the plate surface. In
50% and 80% lower, respectively in the 100 U/mL a-amylase addition, increase in size of oil droplets upon addition of a-amylase
samples compared to the control samples without a-amylase may also inhibit the release of oil droplets from the hydrated
samples or with the 10 U/mL a-amylase samples (Fig. 5). This alginate gel as compared to the control formulation without
result demonstrated that the release of encapsulant using starch a-amylase. This inhibition in release of coalesced emulsion
Pickering emulsions could be controlled by varying the amount of droplets is different from the enhanced binding of the hydrophilic
a-amylase in the system. drug molecules with the alginate film as reported in the prior study
Data from the release assay of the antifungal compounds from (Labovitiadi et al., 2013). Together, these processes could reduce
emulsions subjected to digestion with a-amylase show that the the inhibitory effect of the alginate in the presence of a-amylase.
release of both thymol and amphotericin B was greater than the Nevertheless, the results of this study demonstrated the potential
release from the same emulsion which was not incubated with of hydrophobic bioactive delivery to a semi-solid agar surface
a-amylase (Fig. 3A and B). These data are consistent with the result using this starch Pickering emulsion in an alginate film formula-
from the SIA where the addition of a-amylase to the colloid system tion, which may have significant applications for delivery in the
had increased the antifungal activity of the emulsions added to the oral cavity. Since the oral cavity niche contains the most
experimental system. The two merged data confirm that a-amy- hydrophobic soft tissues in a human body (Van der Mei et al.,
lase increases the release of the antifungal compounds from the 2004), a different absorption/diffusion pharmacokinetics is
emulsion and this suggests that the inhibitory effect against expected in an in vivo assay compared to the agar inhibition assay
Candida can be increased and controlled. results reported in this study.
Biodegradable alginate films and microbeads formulated antimi-
crobial and antifungal compounds have been shown previously to be 5. Conclusions
potential materials for wound dressing and controlled drug delivery
(Liakos et al., 2013; Sikareepaisan et al., 2011). Furthermore, bucco- The present study demonstrated that starch particles can form
adhesive films made from alginate have also been developed Pickering emulsions that were stable at room temperature for at least
previously for the controlled release of the antifungal drugs into the three weeks. Incubation of starch Pickering emulsionwith increasing
buccal cavity (Juliano et al., 2008). In the current approach, the starch concentration levels of a-amylase led to rapid coalescence of oil
Pickering emulsion was incorporated into alginate films, and the droplets and phase separation of the oil phase. Antifungal activity of
antifungal activity of thymol and amphotericin B was characterized thymol or amphotericin B in a simulated saliva was enhanced upon
using static inhibition zone assays both with and without a-amylase incubation with a-amylase. Results from the zone inhibition assay
in simulated salivary fluid. demonstrated efficacy of the emulsions dispersed in alginate films.
The static inhibition assay also served as a comparative assay to Interestingly, addition of a-amylase to the alginate films resulted in a
evaluate the inhibitory activity of the antifungal alginate films on a decreased inhibitory effect. Together, the results showed that starch
fungal lawn system versus a suspension inhibition assay. Pickering emulsions has potential to deliver hydrophobic antifungal
In the static inhibition assay, the addition of a-amylase did not compounds in oral cavity. Further studies are required to assess the in
decrease the minimum inhibitory concentration of thymol or vivo efficacy of these Pickering emulsions for the treatment of fungal
amphotericin B encapsulated in the drug formulation compared diseases.
with the control without a-amylase (Fig. 6). This is in contrast to the
result obtained in Fig. 5 with emulsion samples in aqueous phase. Authors’ contributions
Interestingly, a decrease in inhibitory effects of thymol and
amphotericin B was observed when a-amylase was added, as the ACo and MSW designed and performed the experiments,
inhibition zones were also noticeably smaller compared to the analyzed the data and wrote the paper. ACh performed the
control (Fig. 6). experiments. NN conceived and design the study, analysed the data
The decrease in inhibitory effect observed in the static and contributed to reagents, materials, instrument tools.
inhibition assay could be attributed to the differences in the fate
of emulsions in static and suspension assays that may influence the Conflict of interest
interaction of antifungal compounds with C. albicans. In the
suspension inhibition assay, addition of a-amylase resulted in The authors declare that the research was conducted in the
digestion of starch at the emulsion interface but due to mechanical absence of any potential conflict of interest. The manuscript has
shaking during incubation phase, separation between the oil and been read and approved by all the authors.
water phase was suppressed. The destabilized emulsion droplets
remained dispersed during incubation, which significantly in- Acknowledgments
creased the bioavailability and diffusion rates of thymol and
amphotericin B. As a result, enhanced inhibitory effects against C. This work was supported by NSF/CAPPS grant # 201224478 and
albicans were observed when a-amylase was added to the ACS/PRF grant # 51459/DNI5.
emulsion compared to the control without a-amylase.
On the other hand, in the static inhibition assay, the emulsion- References
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