Astaxanthin-alpha tocopherol nanoemulsion formulation by emulsification methods: Investigation on anticancer, wound healing, and antibacterial effects

Accepted Manuscript Title: Astaxanthin-alpha tocopherol nanoemulsion formulation by emulsification methods: Investigation on anticancer, wound healing, and antibacterial effects Authors: Shanmugapriya Karuppusamy, Hyejin Kim, Periaswamy Sivagnanam Saravana, Byung-Soo Chun, Hyun Wook Kang PII: DOI: Reference:

S0927-7765(18)30573-3 https://doi.org/10.1016/j.colsurfb.2018.08.042 COLSUB 9574

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

14-6-2018 11-8-2018 19-8-2018

Please cite this article as: Karuppusamy S, Kim H, Saravana PS, Chun B-Soo, Kang HW, Astaxanthin-alpha tocopherol nanoemulsion formulation by emulsification methods: Investigation on anticancer, wound healing, and antibacterial effects, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.08.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Astaxanthin-alpha tocopherol nanoemulsion formulation by emulsification methods: Investigation on anticancer, wound healing, and antibacterial effects

Shanmugapriya Karuppusamya, Hyejin Kimb, Periaswamy Sivagnanam Saravanac, Byung-Soo Chunc and Hyun Wook Kanga,b*

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Department of Biomedical Engineering and Center for Marine-Integrated Biomedical Technology (BK 21 Plus), Pukyong National University, Busan, South Korea b Interdisciplinary program of Biomedical Mechanical & Electrical Engineering, Pukyong National University, Busan, South Korea c Department of Food Science and Technology, Pukyong National University, Busan, South Korea

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*Corresponding author

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Hyun Wook Kang, Ph.D. Associate Professor, Department of Biomedical Engineering, Pukyong National University, Busan, South Korea. Tel: +82-51-629-5774 E-mail: [email protected] & [email protected]

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Graphical abstract

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HIGHLIGHTS

ATNE nanoemulsion showed small droplet size.

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It has maintained a kinetically high stability for three months.

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ATNEs at lower concentrations reduced cancer cell viability and induced apoptosis.

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It also increased fibroblast cell migration and disrupted bacterial cell membrane.

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The remarkable findings are good wound healing and antibacterial activities.

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ABSTRACT

Emulsion-based delivery systems have been fabricated and developed to increase the bioavailability of astaxanthin and alpha-tocopherol as active compounds for various biomedical applications.

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Astaxanthin-alpha tocopherol nanoemulsion (ATNE) is well known for its potential 6.to 6.30 effect.

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The current study investigated ATNE by spontaneous (SENE) and ultrasonication emulsification (USNE) methods to optimally fabricate oil/water nanoemulsion characterized for biomedical applications. The

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two methods were compared by using a response surface method of 3-level Box-Behnken design

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(BBD) with significant factors. Transmission electron microscopy (TEM) confirmed spherical-shaped nanoemulsion from SENE and USNE methods and dynamic light scattering (DLS) proved the good

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stability of the fabricated nanoemulsion. Cytotoxicity studies on three different cancer cells confirmed that the nanoemulsion at higher concentrations was more toxic than one at lower concentrations by accompanying a significant decrease in the cellular viability after 24 and 48 h of exposure. The wound-

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healing potential using scratch assay evidenced faster healing effect of the nanoemulsion. Both minimal inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) methods

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confirmed significant antibacterial activity to disrupt the integrity of the bacterial cell membrane. The current results suggested that ATNE act as effectively targeted drug delivery vehicles in the future for cancer treatment applications due to its significant results of anticancer, wound healing, and

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antimicrobial effects.

Keywords:

astaxanthin; nanoemulsion; spontaneous emulsification; ultrasonication; toxicity; wound healing; antimicrobial.

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Short statistical summary: Total number of words: 5881 (except title page, abstract, highlights)

1. Introduction

Nanotechnology-based applications in the medical field have gained popularity in recent years,

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especially in drug delivery, biomedical imaging, and therapeutics. Currently, most attention is

focused on the study of nanoemulsions with a mean droplet size of 100–600 nm [1] due to their

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bioavailability, and biocompatibility efficacy. The unique characteristics of nanoemulsions

include clear, thermodynamically stable, isotropic liquid mixtures of oil, water, surfactant, and co-surfactant systems, much larger surface-area-to-volume ratio [2], transparency or translucency to the naked eye, good stability against droplet aggregation [3], and a decrease in

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the particle size [4]. Thus, nanoemulsions play an important role in various biomedical

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applications: drug delivery, cosmetics, and cell culture technology [5, 6]. High-energy emulsification method and low energy emulsification were highly applicable in various

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dosage forms and can be administered by equally varying routes [7]. It can be optimized for various

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parameters with its different conditions [8, 9]. Low-energy nanoemulsion methods require large amounts of surfactants for stabilization of droplets. Ultrasound-assisted emulsification is an easy, cost-

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efficient, and safer method for preparing nanoemulsions for development of novel drug delivery carriers and also suitable for efficient delivery of active ingredients through rough skin surface [10, 11]. different

types

of

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Recently,

nanotechnology-based

methods,

including

nanoemulsion,

nanoencapsulation, nanoliposomes, carbon nanotubes, and nanomicelles have been used for

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delivering drugs to the targeted site without any adverse effects [10, 12]. The development of alternative drugs and delivery systems has a vital role in the treatment of emerging bacterial infections [13]. The application of nanoemulsion as an antimicrobial agent is a new and promising innovation due to nanoemulsion having a broad-spectrum activity against bacteria, enveloped viruses, fungi, and

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spores [5, 14]. The recent studies reported that use of nanoemulsion as an antimicrobial agent leads to research and development of new antimicrobial agents targeting specific pathogens [15]. Astaxanthin is a natural pigment carotenoid that belongs to xanthophylls or oxygenated carotenoids. It can protect the human body against neurodegenerative conditions, ultraviolet light effects, and cancer. The previous toxicity studies have demonstrated excellent biocompatibility, biodegradability, and stability for cancer treatment [1]. Another advantage is the FDA-approved biosafety for human

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consumption [16]. A natural nutritional component, astaxanthin is marketed as a dietary supplement for its potential health benefits around the world. Several in vivo and in vitro studies have reported that it possesses a wide variety of biological activities and nutraceutical functions [17]. Alpha-tocopherol (α-tocopherol) is an important form of vitamin E that may prevent various health issues. Ethylenediaminetetraacetic acid (EDTA) and α-tocopherol improved the chemical stability of astaxanthin that was loaded into nanostructured lipid carriers. Astaxanthin stability increased during nanoemulsion production with an increase of α-tocopherol from 50 to 100 ppm, indicating further

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protection of astaxanthin against free radicals. The fact that α-tocopherol is effective at protecting astaxanthin suggested that free radicals also have an important role in astaxanthin degradation [18].

Previous studies have shown that α-tocopherol was effective at retarding the oxidation of carotenoids

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in emulsion forms [19]. Thus, α-tocopherol can be combined with astaxanthin for the formulation of

nanoemulsion in aspects of various clinical applications. With this concept in mind, the main aim of the current study was to investigate the anticancer potential of astaxanthin-α-tocopherol

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nanoemulsion by optimizing the fabrication conditions for toxicity tests with better resistance against

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microbial infections.

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2. Methods and Materials 2.1 Materials Astaxanthin (>97%), α-tocopherol, polyethylene glycol, Span® 80, Tween® 80, sodium caseinate, sodium azide, medium chain triglyceride oil, and phosphate bovine saline were purchased from SigmaAldrich Co. (St. Louis, MO, USA). The test bacterial strains were obtained from a Korean collection type cultures, Korea. Selective media were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and used

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for culturing bacteria. Absolute ethanol was purchased from Samchun Pure Chemical Co., Ltd. (Korea). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO)

and Annexin V (AV)-FITC apoptosis detection kit was obtained from Sigma-Aldrich Co. (St. Louis, MO,

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USA). Double-distilled water was used for all aqueous solutions in the experiments. All chemicals were of analytical grade and were used directly as received without further purification. 2.2. Formulation of nanoemulsion 2.2.1 Spontaneous emulsification method

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The homogeneous organic phase composed of oil (medium chain triglyceride oil), bioactive compound

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(α-tocopherol, 200 mg), and a lipophilic surfactant (Span® 80), co-surfactant (PEG) in a water-miscible solvent (ethanol, 40 mL). The homogeneous aqueous phase was formed with hydrophilic surfactant

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(Tween® 20), bioactive compound (astaxanthin, 200 mg), emulsifier (sodium caseinate in sodium

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azide) in water (80 mL). The organic phase was injected in aqueous phase under magnetic stirring, then o/w nanoemulsion was formed instantaneously, leading to the formation of nanodroplets [20],

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then it was stored in a brown bottle or covered with aluminium foil and kept in the dark for further studies.

2.2.2 Ultrasonication method

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Nanoemulsions formulations were prepared by the ultrasonication method according to Li et al. [21] and Ma et al. [22]. The organic phase and aqueous phase were prepared as mentioned above in

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Section 2.2.1. Then it was coarsely homogenized by a high-speed blender at 18000 rpm/min for 5 min. Then, nanoemulsion formed was sonicated by ultrasonic homogenizer in an ultrasonic sonicator VCX750 (SONICS Vibracell, Newtown, USA), with aid of 13 mm probe (threaded end type) at the frequency of 20 kHz, intensity of 90% for 5-15 min [23]. The reactor was closed, it was purged with nitrogen gas

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through a valve, and was maintained at its required temperature, pressure, agitation speed, S/L ratio, and reaction time. Finally, nanoemulsion samples were prepared by spontaneous emulsification and ultrasonication emulsification method and represented as SENE and USNE samples. 2.3. Characterization of nanoemulsion 2.3.1. Morphology of nanoemulsion droplets

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Nanoemulsion droplet morphology and structure were visualized by transmission electron microscopy (TEM). The sample was prepared by placing one drop of the nanoemulsion (negatively stained with phosphotungstic acid) onto a copper grid. Images were captured by a TEM supplied with a W-source and a working voltage of 80 kV [1]. 2.3.2. Physicochemical characterization Physicochemical parameters of nanoemulsion were measured using dynamic light scattering. The nanoemulsion formed was diluted in the ratio 1:30 with water to minimize the multiple scattering

2.3.3. Stability

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effects prior to each experiment. Each measurement was carried out in triplicate [3].

The stability analysis was performed by centrifuging the nanoemulsion at 3500 rpm for 30 min [4, 24],

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then checked at both refrigerator temperature (4°C) and room temperature (25°C). 2.3.4 Quantification of astaxanthin

Astaxanthin in nanoemulsion samples was extracted using solvent: 80 μL of nanoemulsion samples

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were diluted in 10 mL organic solvent (dichloromethane: methanol = 2:1(v/v)) and then centrifuged at 10,000 rpm for 20 min. It was quantified in the supernatant using an ultraviolet-visible

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spectrophotometer (Multiskan GO, Thermo Fisher Scientific., Ltd, Seoul, Korea) at 480 nm. The

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measurements were conducted in triplicates [4]. The pure methanol and dichloromethane solution

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was used as a blank. The astaxanthin concentration was calculated and expressed in μg mL−1. 2.4. In vitro cytotoxicity

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2.4.1 Cell culture

Four cell lines (CT26, HeLa, Panc1, and T24) were purchased from the Korea Cell Line Bank, Seoul, Korea and used in this study. For cytotoxicity test, T24 cells were cultured in RPMI 1640 with HEPES

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(Corning, New York, NY, USA) with 10% fetal bovine serum (FBS) (Corning, New York, NY, USA) and 1% antibiotic (Gibco, Thermo Scientific, Waltham, MA, USA); CT26, HeLa, and Panc1 cells were cultured

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in Dulbecco’s modified eagle’s medium (DMEM), trypsin–ethylenediaminetetraacetic acid (trypsinEDTA), antibiotics, FBS, and phosphate-buffered saline (PBS) (Corning, New York, NY, USA) with 10% bovine calf serum (Welgene, Gyeongsan, Korea) and 1% antibiotic (Gibco, Thermo Scientific, Waltham, MA, USA), 50 IU mL−1 penicillin, and 50 µg mL−1 streptomycin. The cultured cells were maintained at

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37°C in a humidified atmosphere of 5% CO2 and 95% air. CT26, HeLa, Panc1, and T24 cells were seeded into 96-well plates at a seeding density of 1 ×104 cells/well and incubated overnight. 2.4.2 MTT assay A cytotoxicity assay was performed on the basis of cell viability using the MTT viability assay [25]. It was evaluated on CT26, HeLa, Panc1, and T24 cells. Then, 100 µL of the media from each sample was taken and transferred into each well. The cells were then incubated for 24 and 48 h (37ºC, 99% RH,

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5% CO2). After 24 h incubation, the extract solutions were removed, and 100 μL MTT solution (0.5 mg mL-1 in PBS) was added to each well and incubated for 4 h in the dark. Finally, the MTT solutions were sucked, and 100 mL DMSO was added to each well, followed by incubation for 20 min to dissolve purple crystal. Absorbance was measured at 570 nm with an ELISA reader, and cell viability (%) was expressed as the percentage relative treated to control cells and was determined using the following equation [7]: 𝑇𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑋100 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

(1)

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Cell Viability (%) =

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2.5. Apoptosis analysis

The AV-FITC apoptosis detection kit was used to measure the apoptotic cells [26]. HeLa, CT26, and T24 cells were cultured in 12-well plates at 1×105 cells/well for 24 h, then it was treated with each sample

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for 24 h at different concentrations from 25–125 mg/mL. Afterward, the cells were trypsinized and washed twice with cold PBS. Then it was suspended in 1X binding buffer at a concentration of 1X10 6

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cells/mL and transferred 100 µL of the solution to culture tube that contained 5 µL of FITC AV and 5

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µL propidium iodide (PI). The mixed solution gently vortexed the cells and incubated for 15 min at 25

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°C in the dark. Then 400 µL of 1X binding buffer was added to each tube. The values were expressed as the percentage of cells positive for a given marker relative to the total number of cells: FITC labeled cells (viable cells), PI-labeled cells (apoptotic cells), and cells with the ruptured cell membrane

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(necrotic cells will be double labeled). Green AV and red PI fluorescence intensities of apoptosis analysis were analyzed on a flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA) within 1 h.

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Cells with AV-positive and PI-negative fluorescence were defined as apoptotic. Cells with AV-negative and PI-positive fluorescence (PI-permeable) cells were defined as necrotic. AV-/PI-, AV+/PI-, AV+/PI+

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and AV-/PI+ populations are corresponding to viable, early apoptotic, late apoptotic and necrotic cells, respectively. Data were analyzed through a dot plot using the BD Accuri C6 software.

2.6. Scratch wound healing assay CT26, HeLa, Panc1, and T24 cells were seeded in 24-well plates with each well containing 3×105 cells

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and kept for incubation [27]. After cell adhesion, a wound was formed by scratching with a 10 μL tip, and residual cells were washed twice with PBS. Then, a photograph was taken by an optical microscope at 0,12 and 24 h, which was subjected to ImageJ software analysis. The cell migration area of both sides could be calculated based on the difference between the whole area and middle wound area. The migration rate over 24 h period was calculated by dividing the cell migration area after 24 h of treatment by that at 0 h.

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2.7. Antimicrobial activity Antibacterial activity was determined by using minimal inhibitory concentration (MIC). Minimum bactericidal concentrations (MBCs) against Gram-positive (Bacillus subtilis (KCTC 1028), Staphylococcus aureus (KCTC 1916) and Streptococcus mutans (KCTC 5244)) and Gram-negative ((Pseudomonas aeruginosa (KCTC 1750) and Escherichia coli (KCTC 42934)) bacteria were used as test microorganisms

2.7.1. Antibacterial activity by agar well diffusion method

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The sterile cotton swab containing bacterial culture was swabbed in Mueller–Hinton agar [28, 29]. Using a sterile well cutter, 3 wells of 8 mm diameter were punched in the agar. To each well, both samples were added, and to other wells, sterile distilled water was added as a control. The

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experiments were carried out in triplicate manner. At 37°C, both plates were incubated for 24 h. Antibacterial activity was evaluated by measuring the zone of inhibition (mm).

2.7.2 Determination of Minimal inhibitory concentration (MIC)/ Minimum bactericidal

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concentrations (MBC)

The antimicrobial activity was also evaluated against Gram-positive and Gram-negative bacteria by

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determining MIC and MBC. The turbidity of microorganism was detected and examined MIC by

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subculturing in various dilutions onto the agar plates, and at minimal concentration, which inhibited

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the growth of microbes. The lowest concentration of sample killed all the microorganisms was noted as MBC values [28]. Nanoemulsion samples were serially diluted with sterile nutrient broth in a 96-

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well plate, and each well, containing 180 µL of diluted nanoemulsion in nutrient broth, was inoculated with 20 µL of standardized bacterial culture with an O.D from 0.2 to 0.4 at 490 nm. The cell density in wells was 2 x 107/mL. Plates were incubated at 37°C overnight, and MIC was determined as the highest

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dilution that resulted in a 99.9% reduction in the bacterial cell number showing no bacterial growth. To determine MBC, 100 µL of culture broth from wells containing no growth was plated onto nutrient

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agar and incubated at 37°C overnight. The purity of cultures was confirmed by plating growth from wells. Due to different compositions of nanoemulsions, MIC and MBC were expressed as the dilution of nanoemulsions.

2.8. Experimental design

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The experiment was designed by three-level Box–Behnken design (BBD) with 3 factors and 15 runs were made in total, with 5 repeats of the central point. In the experiment, the normal and coded values of independent variables were A: ultrasonic intensity (30,60 and 90%), B: sonication time (5, 10 and 15 min), and C: temperature (20, 40 and 60 °C) (Table 1S). Based on initial trials, independent variables were used. The succeeding equation for individual variables is given as follows:

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𝑋=

(𝑥𝑖 − 𝑥𝑜 ) ∆𝑥

(2)

where Y is the response, xi and xj are independent variables (i and j range from 1 to k) affecting response, β0 is constant, βi, βii, and βij are regression coefficients for linear, quadratic, and interactive terms, respectively, and k is the number of parameters. The data obtained from design were fitted to second-order polynomial equations (Eq. 2). 3

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𝑌 = 𝛽0 + ∑ 𝛽𝑖 𝑋𝑖 + ∑ 𝛽𝑖𝑖 𝑋𝑖 + ∑ ∑ 𝛽𝑖𝑖 𝑋𝑖 𝑋𝑗 𝑖=1

𝑖=1

𝑖
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(3)

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The RSM was performed using Design-Expert v.10 (Stat-Ease, Minneapolis, Minnesota, USA). 2.9 Statistical analysis

Triplicate experiments were carried out and data were subjected to statistical analysis by using SPSS

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software. All data were expressed as a mean ± standard deviation, and statistical difference (P<0.05)

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of mean comparison among various treatments was performed by using Student’s t-test.

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3. Results and Discussion 3.1. Statistical analysis of USNE results 3.1.1. Effect of operational variables Experimental design is an excellent tool to analyze and optimize the preparation of astaxanthin-alphatocopherol loaded nanoemulsion with desirable properties using the right amount of materials [30]. Nevertheless, selection of design in optimizing nanoemulsion can be another challenge, as the different design will yield different results [8]. BBD of experiment has an advantage over other designs

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in that it does not contain combinations for which all factors are simultaneously at their highest or lowest levels [9].

Nanoemulsion obtained from trials using 3 factors and three-level BBD (Table 1) and F-values for all

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variables (Table 2) were presented, while the p-value was found to be p < 0.0001. In addition, lack of

fit was insignificant for droplet size, PI, zeta potential and astaxanthin content of nanoemulsion (p > 0.05). On the other hand, F-values suggested that pure error was not significant. The most important

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value for BBD was obtained for R2, which showed around 0.9 for all variables. The analysis of variance (ANOVA) of experiments (Table 2) was summarized and suggested that the obtained model exactly

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showed an actual correlation between independent response and variables [31]. A second-order

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polynomial equation was used to maximize nanoemulsion formulation with its chemical composition.

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Response surface 3D graphs were plotted for all responses according to the model equations established (Table 2S). The response surface plots of droplet size were: +227.55 -17.21A -7.20B 38.19C + 64.08AB + 43.60AC - 8.57BC + 27.75A2 + 11.58B2 + 0.65C2 (Fig. 1A (a-c)), PI: +0.17 + 0.010A +

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0.003B + 0.001C + 0.035AB - 0.028AC + 0.001BC + 0.010A2 -0.026B2 -0.008C2 (Fig. 1B (a-c)), zeta potential: -22.74 - 2.46A - 0.67B - 0.26C - 1.71AB - 0.36AC - 0.81BC + 0.75A2 + 2.75B2 + 2.20C2 (Fig. 1C

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(a-c)), and astaxanthin content: +1.63 + 0.054A - 0.030B - 0.030C - 0.063AB + 0.023AC - 0.025BC 0.27A2 - 0.27B2 - 0.43C2 (Fig. 1D (a-c)) in USNE sample. Such behavior can be described by fact that

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stabilization of oil droplets was a result of localization of surfactant molecules at oil-water interface [9, 24, 32]. Nanoemulsion showed an area where responses can be maximized, which takes place during ultrasonic intensity (90%), sonication time (10 min) and temperature (20 ⁰C). In contrast, chemical composition showed different combinations of response surface in 3D graphs for each [33].

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Determination of importance of 3 factors in BBD model by standardized coefficient demonstrated that interaction among 3 factors has a positive contribution to droplet size, PDI, zeta potential, and astaxanthin content. Therefore, the existence of interaction terms between the main factors under conditions of our experiments emphasizes the necessity to carry out active multifactor experiments for determining an optimal condition of nanoemulsions [34]. 3.1.2. Selection of the optimum conditions

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Optimization was carried out by identifying the optimum composition in the formulation of nanoemulsions. The optimal condition for nanoemulsion was determined to obtain a high amount using Derringer’s desirability function analysis. Derringer’s function will search the grouping of various factor levels that improve a range of responses by sustaining its necessities for each response [35]. The cause of the thermodynamic instability of nanoemulsions is mainly dependent on their physical properties and preparation method. A product must have a shelf life of at least 6 months/a year to be marketed to ensure equilibrium system maintained its best quality. Hence, extreme conditions were

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applied to the samples for fast prediction of resistance level against phase separation [8].

The optimal conditions determined from the ultrasonic intensity, sonication time, and temperature

were kept in a range [11, 23]. Using the method of the desired function, optimized levels of different

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factors, i.e., ultrasonic intensity (90%), sonication time (10 min), and temperature (20 ⁰C) were

obtained. The optimal conditions exhibited a predicted value of droplet size: 217.81 nm, PI: 0.11, zeta potential: -15.76 mV, and astaxanthin content: 1.07 mg/mL-1 in triplicates to confirmed similarity with

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predicted values [21]. The results obtained from validated trials exhibited a reputable covenant with predicted values, demonstrating the adequacy of developed quadratic models. Thus, BBD integrated

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with desirability functions could be successfully used to improve extraction efficiency of nanoemulsion

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by maintaining optimum range of droplet size, PDI, zeta potential, and astaxanthin content.

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3.2. Characterization of nanoemulsion 3.2.1. Morphology of nanoemulsion droplets

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A TEM micrograph confirmed as spherical in shape (Fig. 2 (a–d, e–h)) with droplet size ranging from 189–216.6 nm (SENE) and 106-213.7 nm (USNE). Most of the nanoemulsion droplets were aggregated that might be due to being a fusion and combination of nanoemulsion droplets with each other [1]. It

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showed that nanodroplets should be nanosized and was correlated with particle size measurement determined by DLS (Fig. 1S & 2S). This is mainly because particle size determined by DLS was a

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hydrodynamic diameter, whereas nanoemulsion droplets were suspended in solutions that enhanced aggregation.

3.2.2. Physicochemical characterization Physicochemical parameters were analyzed by DLS and their measurements were conducted in the

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triplicate manner (Table 3S). The droplet size of SENE and USNE possessed an average diameter of 216.6 nm and 213.7 nm, whereas average diameter ranged from 189–216.6 nm and 106–213.7 nm. The droplet size of nanoemulsion was much reduced after sonication process was confirmed in TEM images [3, 8]. Zeta potential studies were performed to show an impact of encapsulation on surface charge and stability of prepared nanoemulsion. It was a measure of the surface charge carried by particles

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suspended in a liquid [2]. SENE and USNE showed a zeta potential value of -20.57 Mv and -22.59 Mv that confirmed the highest stability occurred. The increasing astaxanthin content by α-tocopherol significantly maintained zeta potential values and found to be in agreement with recent findings [3, 10, 15, 23] due to a shielding effect and coating of protonated NH2 group by α-tocopherol on astaxanthin. PI was found to be 0.153 and 0.200 for SENE and USNE, and PDI remained below 0.2, which reflects their relative homogeneity [3]. The PI was found in the range of 0.2–0.4, indicated high monodispersity

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of oil droplets and a narrow size distribution [2].

The refractive index of SENE and USNE was similar and found as 1.3328, whereas the dielectric

constant was 78.3. The viscosity was found to be 0.8898 and 0.8878 for SENE and USNE and confirmed

6 months, with no significant change in particle size distribution.

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the physical stability of nanoemulsion system. All nanoemulsion size evaluations were repeated after

Every nanoemulsion formulation was required to have an acceptable thermodynamic stability. In

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order to assess physical stability of a nanoemulsion, use of accelerated stability tests may be a useful approach. The size of nanoemulsion samples was found to be physically stable up to 6 months at room

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and refrigerator temperature. On further checking, no phase separation was observed during storage

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and centrifugation and showed that formulated nanoemulsion survived stability tests [3, 24].

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Nanoemulsions exhibit enhanced shelf stability against gravitationally driven creaming over microscale emulsions at same φ [20]. The results showed that when the concentration of surfactant

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was low, stability was maximum, regardless of the concentration of oil/co-surfactant. The only exception was at low oil concentrations when a small decrease in the stability was observed. Nevertheless, when surfactant concentration was medium or high, stability profile became

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complicated [36].

Most of the nanoemulsions have good stability against droplet growth, but different storage

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conditions and environmental factors may affect stability [4]. Nanoemulsion was able to maintain its particle size, PDI, zeta potential under refrigerated conditions. Under room temperature, nanoemulsion was also able to maintain its properties until day 14, when particle size and PDI significantly increased, which may be owing to the Ostwald ripening effect whereas small particles

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clumped together to formed bigger ones [5]. 3.3. in vitro cytotoxicity by MTT assay HeLa, CT26, and T24 cells were performed cytotoxicity assay on basis of cell viability using MTT viability assay [25, 37]. The cytotoxic effect of SENE sample was evaluated and indicated that cell viability was found to be 97% after 24 h and 94% after 48 h on HeLa cells (Fig. 3a); for CT26, it was 98% after 24 h and 92% after 48 h (Fig. 3c); for T24, it was 98% after 24 h and 94% after 48 h (Fig. 3e), whereas

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cytotoxic effect of USNE samples was evaluated on HeLa, CT26, and T24 cells and indicated that cell viability was found to be 100% after 24 h and 97% after 48 h on HeLa cells (Fig. 3b); for CT26, it was 100% after 24 h and 98% after 48 h (Fig. 3d); for T24, it was 100% after 24 h and 97% after 48 h (Fig. 3f). Cell viability data revealed that nanoemulsion does not exhibit any toxicity after 24 and 48 h in contact with HeLa, CT26, and T24 cells. No significant toxicity (P < 0.05) was observed for both samples and found to be statistically significant, which displayed cell viability values in the range of 66–100% after 24 h of incubation, and these slightly decreased to 92% after 48 h of incubation. Cell viability may

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be adversely affected by exposure to bioactive molecules as well as the delivery vehicle or carrier,

thereby influencing the wound-healing process. The cytotoxicity study shows that nanoemulsion at

lower concentrations did not showed any toxicity, while at higher concentrations, it was found to

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induced toxicity [24, 29, 37]. 3.4. Apoptosis analysis

Based on the percentage of live cells (Fig. 4) at the higher concentration in all three cell lines, the USNE

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sample presented higher cytotoxic potential, whereas SENE sample showed slightly lower cytotoxic potential in relation to control so we carried out apoptosis analysis for nanoemulsion samples were

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less toxic or non-toxic in nature [37]. These data suggested that cytotoxicity observed in cells treated

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with nanoemulsion samples could be associated with surfactant employed as the diluent of

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nanoemulsion samples. Apoptosis and necrosis were different types of cell death. It can be differentiated by flow cytometry using distinct dyes. Annexin V staining give better information about

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early/late apoptotic and necrotic cells death. AV was a marker of apoptosis, is a Ca (2+)-dependent phospholipid-binding protein with a high affinity for phosphatidylserine. PI was a fluorescent dye necrosis indicator and a cell-impermeant dye that intercalates DNA and RNA of cells with the damaged

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plasma membrane [7]. Annexin-V is used as a probe to detect cells that have expressed phosphatidylserine on the cell surface, an event found in apoptosis as well as other forms of cell death.

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PI is membrane impermeant and excepted from viable cells, it does not pass through intact cell membranes. Therefore, AV-/PI-, AV+/PI-, AV+/PI+ and AV-/PI+ populations are corresponding to viable, early apoptotic, late apoptotic and necrotic cells, respectively. An early increase in Annexin+ and PI- followed by an increase in Annexin+/PI+ are represent as apoptosis. In contrast, a direct

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increase in Annexin+ and PI+ without Annexin+ and PI- are necrosis. Both possibilities will occur.

3.5. in vitro wound-healing assay (scratch assay) The potential wound-closure properties of nanoemulsion samples were assessed using a monolayer scratch model. Wound healing assay was used in a range of disciplines to study the coordinated movement of a cell population. Developing a standard approach for the wound-healing assay promoted comparability between studies and could lead to a user database containing collective

13

migration data for a variety of cell lines [6, 37]. The wound-healing process showed significant cell

migration on HeLa, CT26, and T24 cells. A photograph by an optical microscope at 0 h, 12 h, and 24 h (Labelled as (a-c)) was subjected to ImageJ software analysis and labeled as A: HeLa, B: CT26, and C: T24 cells (Fig. 5 A-C (a-c)). After cell adhesion, a wound size formed by scratching was reduced from 6 h itself. A long-term wound healing assay (>24 h) cannot be distinguished cell proliferation and changes in cell survival from cell motility [38]. Cells can either migrated as single cells, as loosely connected population or collectively as sheets of cells (epithelial cells). A wide variety of cells had been

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analyzed for migration with this assay. [39]

Nanoemulsions are broadly antimicrobial oil-in-water emulsions containing nanometer sized

droplets stabilized with surfactants. It represents a novel potential antimicrobial and anti-

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inflammatory treatment for reduces bacterial wound infection and dermal inflammation after burn

wounds. This are on the U.S. Food and Drug Administration (FDA) Generally Recognized as Safe (GRAS) list. Cell migration can be monitored with the help of microscope, as that the cells traveled from intact

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zones into the scratched region. Cell movement can be calculated by measuring the decrease of an

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uncovered region at different time points until ‘‘wound’’ was closed [38]. The cell migration area of both samples against three cells was measured (Fig. 5D). The improvement in “scratch closure”

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produced by nanoemulsions compared with untreated and treated, which showed a gradual

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repopulation of the scratch area [12]. Furthermore, concentrations of 25 mg/mL achieved using nanoemulsions resulted in significantly higher closure activity at 12 h. The wound closure was higher

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rate at 25mg/ml in 12h when compared to other concentrations due to viable cells in the wounded tissue area. There is high cell variability without toxicity that was confirmed by MTT assay. MTT data also have high cell viability at 25mg/ml concentration So higher wound closure rate is depending on

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cell viability. This finding strongly supported MTT-cell viability data [37]. 3.6. Antimicrobial activity

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The antibacterial activity showed a significant inhibitory effect at the minimum concentration of 0.5 mg/mL (Table 3). MIC was determined using various concentrations of nanoemulsion samples (0.25 to128 mg/mL). Nanostructure materials, including nanoparticles, nanoemulsions, or nanotubules, can be used to effectively manipulate or deliver immunologically active components to target sites in the

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setting of infectious or noninfectious diseases [35]. MIC and MBC values ranged from 0.5 to 32 mg/mL for various bacteria. The results showed better activity against S. aureus by USNE than SENE. This results were supported by previous studies [40-43]. A nanoemulsion acts as a novel microbicidal agent [41] and have been reported against Gram-postive than Gram-negative species [42]. Among this, antibacterial property was effective against Staphylococcus aureus infection, because this common organism that is found in skin abrasions and open wounds can threat to public health with limited

14

therapeutic effect. Nanoemulsion study reported that Staphylococcus aureus infected wounds significantly decreased bacterial load and had no toxic effects on healthy skin tissues and can greatly reduce inflammation characteristic of infected wounds [43]. Nanoemulsion formulations were proven to exhibit broad antimicrobial properties and were ideally suited for treatment of burn wounds. Nanoemulsion played a significant role in causing physical damage to bacteria, facilitating its uses as an effective antibacterial agent as well as preventing them from developing resistance [29].

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Antimicrobial nanoemulsions were surfactant-containing oil in water emulsions (particle size, 100 to 800 nm) which were very effective against many bacteria, enveloped virus, fungi, and spores at

concentrations that were non-irritating to skin or mucous membranes of animals. Antimicrobial

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activity and mechanism of nanoemulsions were believed to function as a result of the ability of

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nanoemulsions to fuse with outer membranes of microorganisms [44].

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4. Conclusion In conclusion, we developed ATNE to detect toxicity level, wound healing potential with antimicrobial effect. This study proved significant anticancer potential against different cancer cells and promoted wound healing with antimicrobial resistance against bacteria. Astaxanthin-alpha-tocopherol nanoemulsion by spontaneous and ultrasonication emulsification methods was characterized and optimized its stability with specific physical properties for in vitro biomedical applications. Fabricated nanoemulsion was non-toxic and safe to exhibit broad antimicrobial properties and was ideally suited

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for treatment of wounds and enhanced wound repair as demonstrated by in vitro cytotoxicity and wound healing, apoptosis, and antimicrobial studies. This results can be a starting point for further studies aiming at cancer treatment applications. In the future, this fundamental mechanism of

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nanoemulsions is needed for proposing novel experimental approaches in cancer treatment and

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diagnosis, and antimicrobial resistance.

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Acknowledgment

This research was supported by a grant from the Marine Biotechnology Program (20150220) funded by the Ministry of Oceans and Fisheries, Korea.

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References

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[1] K. Bouchemal, S. Briançon, E. Perrier and H. Fessi, Nano-emulsion formulation using spontaneous emulsification: solvent, oil and surfactant optimisation, Int.J.Pharm. 280 (2004) 241-251. [2] S.Y. Teo, M.Y. Yew, S.Y. Lee, M.J. Rathbone, S.N. Gan and A.G. Coombes, In vitro evaluation of novel phenytoin-loaded alkyd nanoemulsions designed for application in topical wound healing, J. Pharm.Sci., 106 (2017) 377-384. [3] A. Kaur, Y. Saxena, R. Bansal, S. Gupta, A. Tyagi, R.K. Sharma, J. Ali, A.K. Panda, R. Gabrani and S. Dang, Intravaginal Delivery of Polyphenon 60 and Curcumin Nanoemulsion Gel, AAPS Pharm.Sci.Tech., 18 (2017) 2188-2202. [4] N. Khalid, G. Shu, B.J. Holland, I. Kobayashi, M. Nakajima and C.J. Barrow, Formulation and characterization of O/W nanoemulsions encapsulating high concentration of astaxanthin, Food Res.Int., 102 (2017) 364-371. [5] Q. Hu, H. Gerhard, I. Upadhyaya, K. Venkitanarayanan and Y. Luo, Antimicrobial eugenol nanoemulsion prepared by gum arabic and lecithin and evaluation of drying technologies, Int.J.Biol. Macromol., 87 (2016) 130-140. [6] J.E. Jonkman, J.A. Cathcart, F. Xu, M.E. Bartolini, J.E. Amon, K.M. Stevens and P. Colarusso, An introduction to the wound healing assay using live-cell microscopy, Cell Adh. Migr., 8 (2014) 440-451. [7] A.B. Vermelho, V. da Silva Cardoso, E. Ricci Junior, E.P. dos Santos and C.T. Supuran, Nanoemulsions of sulfonamide carbonic anhydrase inhibitors strongly inhibit the growth of Trypanosoma cruzi, J. Enzyme Inhib. Med. Chem., 33 (2018) 139-146. [8] C.L. Ngan, M. Basri, F.F. Lye, H.R.F. Masoumi, M. Tripathy, R.A. Karjiban and E. Abdul-Malek, Comparison of Box–Behnken and central composite designs in optimization of fullerene loaded palm-based nano-emulsions for cosmeceutical application, Ind.Crops Prod., 59 (2014) 309-317. [9] J. Wadhwa, A. Asthana, G. Shilakari, A.K. Chopra and R. Singh, Development and evaluation of nanoemulsifying preconcentrate of curcumin for colon delivery, The Sci. World J., 2015 (2015) 510-541. [10] M. Kaci, A. Belhaffef, S. Meziane, G. Dostert, P. Menu, E. Velot, S. Desobry and E. Arab-Tehrany, Nanoemulsions and topical creams for the safe and effective delivery of lipophilic antioxidant coenzyme Q10, Colloids Surf., B Biointerf., 167 (2018) 165-175. [11] T. Leong, T. Wooster, S. Kentish and M. Ashokkumar, Minimising oil droplet size using ultrasonic emulsification, Ultrason. Sonochem., 16 (2009) 721-727. [12] Y.C. Chen and B.H. Chen, Preparation of curcuminoid microemulsions from Curcuma longa L. to enhance inhibition effects on growth of colon cancer cells HT-29, RSC Adv., 8 (2018) 2323-2337. 17

A

CC E

PT

ED

M

A

N

U

SC R

IP T

[13] T. Mason, J. Wilking, K. Meleson, C. Chang and S. Graves, Nanoemulsions: formation, structure, and physical properties, J.Phys.: Condens. Matter, 18 (2006) 635666. [14] V.A. Dolgachev, S.M. Ciotti, R. Eisma, S. Gracon, J.E. Wilkinson, J.R. Baker Jr and M.R. Hemmila, Nanoemulsion therapy for burn wounds is effective as a topical antimicrobial against Gram-negative and Gram-positive bacteria, J.Burn Care Res., 37 (2016) 104-114. [15] D.J. McClements and J. Rao, Food-grade nanoemulsions: formulation, fabrication, properties, performance, biological fate, and potential toxicity, Crit.Rev.Food Sci. Nutr., 51 (2011) 285-330. [16] V.P. Nguyen, S. Park, J. Oh and H. Wook Kang, Biocompatible astaxanthin as novel contrast agent for biomedical imaging, J. Biophotonics, 10 (2017) 1053-1061. [17] J.S. Stewart, A. Lignell, A. Pettersson, E. Elfving and M. Soni, Safety assessment of astaxanthin-rich microalgae biomass: Acute and subchronic toxicity studies in rats, Food Chem. Toxicol., 46 (2008) 3030-3036. [18] F. Tamjidi, M. Shahedi, J. Varshosaz and A. Nasirpour, EDTA and α‐ tocopherol improve the chemical stability of astaxanthin loaded into nanostructured lipid carriers, Eur. J.Lipid Sci.Technol., 116 (2014) 968-977. [19] Y. Liu, Z. Hou, J. Yang and Y. Gao, Effects of antioxidants on the stability of βCarotene in O/W emulsions stabilized by Gum Arabic, J. Food Sci. Technol., 52 (2015) 3300-3311. [20] T. Tadros, P. Izquierdo, J. Esquena and C. Solans, Formation and stability of nanoemulsions, Adv. Colloid Interface Sci., 108 (2004) 303-318. [21] C. Li, Y. Zhang, C. Zhao, Y. Ni, K. Wang, J. Zhang and W. Zhao, Ultrasonic AssistedReflux Synergistic Extraction of Camptothecin and Betulinic Acid from Camptotheca acuminata Decne. Fruits, Molecules, 22 (2017) 1076-1087. [22] P. Ma, Q. Zeng, K. Tai, X. He, Y. Yao, X. Hong and F. Yuan, Preparation of curcumin-loaded emulsion using high pressure homogenization: Impact of oil phase and concentration on physicochemical stability, LWT-Food Sci. Technol., 84 (2017) 34-46. [23] S. Kentish, T. Wooster, M. Ashokkumar, S. Balachandran, R. Mawson and L. Simons, The use of ultrasonics for nanoemulsion preparation, Innovative Food Sci. Emerg. Technol., 9 (2008) 170-175. [24] K. Cinar, A review on nanoemulsions: preparation methods and stability, Trakya Univ. J. Eng. Sci., 18 (2017) 73-83. [25] I. Khan, A. Bahuguna, P. Kumar, V.K. Bajpai and S.C. Kang, In vitro and in vivo antitumor potential of carvacrol nanoemulsion against human lung adenocarcinoma A549 cells via mitochondrial mediated apoptosis, Sci. Rep., 8 (2018) 144-158. [26] Q. Xie, W. Deng, X. Yuan, H. Wang, Z. Ma, B. Wu and X. Zhang, Seleniumfunctionalized liposomes for systemic delivery of doxorubicin with enhanced pharmacokinetics and anticancer effect, Eur.J. Pharm. Biopharm., 122 (2018) 87-95. [27] T. Chen, T. Gong, T. Zhao, Y. Fu, Z. Zhang and T. Gong, A comparison study between lycobetaine-loaded nanoemulsion and liposome using nRGD as therapeutic adjuvant for lung cancer therapy, Eur. J. Pharm. Sci., 111 (2018) 293-302. [28] P.S. Saravana, Y.J. Cho, Y.B. Park, H.C. Woo and B.S. Chun, Structural, antioxidant, and emulsifying activities of fucoidan from Saccharina japonica using pressurized liquid extraction, Carbohydr. Polym., 153 (2016) 518-525.

18

PT

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M

A

N

U

SC R

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[29] J. Jerobin, P. Makwana, R.S. Kumar, R. Sundaramoorthy, A. Mukherjee and N. Chandrasekaran, Antibacterial activity of neem nanoemulsion and its toxicity assessment on human lymphocytes in vitro, Int. J. Nanomed., 10 (2015) 77-86. [30] Y.H. Lin, M.J. Tsai, Y.P. Fang, Y.S. Fu, Y.B. Huang and P.C. Wu, Microemulsion formulation design and evaluation for hydrophobic compound: Catechin topical application, Colloids Surf., B, 161 (2018) 121-128. [31] H.P. Thakkar, J.L. Desai and M.P. Parmar, Application of Box-Behnken design for optimization of formulation parameters for nanostructured lipid carriers of candesartan cilexetil, Asian J. Pharm, 8 (2014) 81-89. [32] S. Sood, K. Jain and K. Gowthamarajan, Optimization of curcumin nanoemulsion for intranasal delivery using design of experiment and its toxicity assessment, Colloids Surf., B, 113 (2014) 330-337. [33] E. Gonzalez-Mira, M. Egea, M. Garcia and E. Souto, Design and ocular tolerance of flurbiprofen loaded ultrasound-engineered NLC, Colloids Surf., B, 81 (2010) 412-421. [34] M. Nikbakht, S. Honary and P. Ebrahimi, Optimization of finasteride nano-emulsion preparation by using chemometrics approaches (and/or Box-Behnken design and regresion model), Res.Pharm. Sci., 7 (2012) 225-237. [35] P.S. Saravana, A.T. Getachew, R. Ahmed, Y.J. Cho, Y.B. Lee and B.S. Chun, Optimization of phytochemicals production from the ginseng by-products using pressurized hot water: Experimental and dynamic modelling, Biochem. Eng. J., 113 (2016) 141-151. [36] N. Seyedhassantehrani, R. Karimi, G. Tavoosidana and A. Amani, Concurrent study of stability and cytotoxicity of a novel nanoemulsion system–an artificial neural networks approach, Pharm. Develop. Tech., 22 (2017) 383-389. [37] Z.P. Gumus, E. Guler, B. Demir, F.B. Barlas, M. Yavuz, D. Colpankan, A.M. Senisik, S. Teksoz, P. Unak and H. Coskunol, Herbal infusions of black seed and wheat germ oil: Their chemical profiles, in vitro bio-investigations and effective formulations as phyto-nanoemulsions, Colloids Surf., B, 133 (2015) 73-80. [38] N. Kramer, A. Walzl, C. Unger, M. Rosner, G. Krupitza, M. Hengstschläger and H. Dolznig, In vitro cell migration and invasion assays, Mutat. Res./Rev. Mutat.Res., 752 (2013) 10-24. [39] C.C. Liang, A.Y. Park and J.L. Guan, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro, Nat. Protoc., 2 (2007) 329333.

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[40] S. Sugumar, V. Ghosh, M. J. Nirmala, A. Mukherjee and N. Chandrasekaran, Ultrasonic emulsification of eucalyptus oil nanoemulsion: Antibacterial activity against Staphylococcus aureus and wound healing activity in Wistar rats, Ultrason. Sonochem., 21 (2014) 1044–1049. [41] Z. Cao, T. Spilker, Y. Fan, L.M. Kalikin, S. Ciotti, J.J. LiPuma, P.E. Makidon, J.E. Wilkinson, J.R. Baker Jr and S.H.Wang, Nanoemulsion is an effective antimicrobial for methicillin-resistant Staphylococcus aureus in infected wounds, Nanomedicine.,12(10) (2017)1177-1185. [42] M.H.Alkhatib, M.M.Aly and S.Bagabas,Antibacterail activity and maechanism of action of lipid nanoemulsions against Staphylococcus aureus, J. Pure.Appl.Microbiol.,7(20132) 259-267. [43] K.Thakur, G.Sharma, B. Singh, A. Jain, R.Tyagi, S. Chhibber and O. P. Katare, Cationic-bilayered nanoemulsion of fusidic acid: an investigation on eradication of methicillin-resistant Staphylococcus aureus 33591 infection in burn wound, Nanomedicine, 13(8)(2018)825-847.

[44] M. Almada, B. Leal-Martínez, N. Hassan, M. Kogan, M. Burboa, A. Topete, M. Valdez and J. Juarez, Photothermal conversion efficiency and cytotoxic effect of gold nanorods stabilized with chitosan, alginate and poly (vinyl alcohol), Mater. Sci. Engi., C, 77 (2017) 583-593. 19

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Fig. 1. Response surface plots showing combined effects of ultrasonic intensity, sonication time, and temperature on A(a-c): droplet size, B(a-c): polydispersity index, C(a-c): zeta potential, and D(a-c): astaxanthin content in nanoemulsion samples prepared by ultrasonication emulsification method

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Fig. 2. Transmission Electron Microscopy images of astaxanthin-α-tocopherol nanoemulsion samples prepared by spontaneous (a-d) and ultrasonication emulsification (e-h) methods at various magnifications.

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Fig. 3. Cytotoxicity profile by MTT assay of nanoemulsion sample prepared by spontaneous emulsification method (SENE) and ultrasonication emulsification method (USNE) at 24 and 48h on HeLa (a & b), CT26 (c & d), T24 (e &f) cells from 25-125 mg/mL against control of 100 % cell viability. Each value is the mean of three replicates with standard deviation (±SD) and analyzed by Student’s t-test (n = 3; *p<0.05 vs. Control).

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Fig.4. Annexin V Apoptosis analysis of nanoemulsions samples. Induction of cell apoptosis in HeLa, CT26, and T24 cells cell treated with spontaneous (SENE) and ultrasonication emulsification nanoemulsion (USNE) samples at 100 mg/ml. The apoptotic cells and necrotic cells were analyzed by flow cytometry.

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Fig. 5. Wound healing process of nanoemulsions samples by scratch assay. Black solid lines represent wound size (µm in length) of (A) HeLa, (B) CT26, and (C) T24 cells by spontaneous (a-c) and ultrasonication emulsification (d-f) nanoemulsion methods. (D) Cell migration area in wound healing process of nanoemulsions samples by scratch assay at 0, 12, and 24h in HeLa, CT26, and T24 cells. Each value is the mean of three replicates with standard deviation (±SD) and analysed by Student’s t-test. (n = 3; *p<0.05 vs control of each cell line).

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Table 1. Box–Behnken design with actual and coded USNE conditions and experimentally obtained values for selected parameters.

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40

4

90

15

40

5

30

10

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6

90

10

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7

30

10

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8

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10

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9

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5

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10

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15

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5

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12

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Astaxanthin content (mg/g)

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Zeta potential

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Polydispersity Index

(nm) (mV) Predict Actual Predicte Actu Predicte Actual Predicte Actual ed d al d d 357.86 355.36 0.17 0.17 -17.83 0.96 1.00 18.88 187.79 192.79 0.12 0.12 -19.34 1.30 1.23 19.02 217.81 212.81 0.11 0.11 -15.76 1.00 1.07 16.07 304.04 306.54 0.21 0.20 -24.09 1.09 1.05 23.04 353.70 354.95 0.13 0.13 -17.45 0.96 0.93 17.03 239.57 233.32 0.20 0.21 -21.65 0.91 0.99 22.59 185.13 191.38 0.20 0.19 -17.24 0.90 0.82 16.29 245.40 244.15 0.16 0.15 -22.88 0.95 0.98 23.30 275.34 276.59 0.14 0.13 -17.69 0.99 0.97 17.06 275.59 279.34 0.13 0.13 -17.41 1.00 0.96 17.51 221.11 217.36 0.13 0.13 -16.58 0.92 0.96 16.47 187.06 185.81 0.13 0.13 -19.54 0.83 0.85 20.17 229.73 227.55 0.16 0.17 -22.74 1.65 1.63 23.09 228.51 227.55 0.17 0.17 -22.74 1.64 1.63 23.03 224.40 227.55 0.17 0.17 -22.74 1.60 1.63 22.11

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Droplet size

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A: B: C: Std. Ultrasonic Sonicatio Temperat Run intensity n time ure (°C) (%) (min) 1 30 5 40

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Table 2. Analysis of variance (ANOVA) of fitted second-order polynomial model for selected parameters of nanoemulsion.

110.38 5.96 5.36 0.60 116.34

Model Residual Lack of fit Pure error Total

1.13 0.030 0.029 0.001265 1.16

122.30

<0.0001

7.47

0.1203

14.47

0.0045

5.50

0.1577

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Model Residual Lack of fit Pure error Total

p-Value

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0.012 0.0004625 0.0004125 0.00005 0.013

F-value

10.29

0.0097

5.92

0.1479

21.01

0.0019

15.09

0.0628

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Model Residual Lack of fit Pure error Total

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41963.61 190.63 175.02 15.61 42154.24

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Model Residual Lack of fit Pure error Total

DF Mean square Droplet size a 9 4662.62 5 38.13 3 58.34 2 7.80 14 Polydispersity Index b 9 0.001339 5 0.0000925 3 0.0001375 2 0.000025 14 Zeta potential c 9 12.26 5 1.19 3 1.79 2 0.30 14 Astaxanthin content d 9 0.13 5 0.00598 3 0.009544 2 0.0006323 14

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Sum of squares

6.17; R2: 0.9955; mean: 248.87; adjusted R2: 0.9873; C.V.%: 2.48; predicted R2: 0.9327; adequate precision: 33.631,

b S.D.:

0.009618; R2: 0.9630; mean: 0.15; adjusted R2: 0.8965; C.V.%: 6.23; predicted R2: 0.4635; adequate precision: 12.448,

c

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a S.D.:

S.D.: 1.09; R2: 0.0.9488; mean: -19.71; adjusted R2: 0.8565; C.V.%: 5.54; predicted R2: 0.2515; adequate precision: 9.349, 0.077; R2: 0.9742; mean: 1.11; adjusted R2: 0.9279; C.V.%: 6.94; predicted R2: 0.0.6027; adequate precision: 12.826.

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d S.D.:

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Table 3. Antimicrobial study by minimal inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) methods of nanoemulsion samples.

8±0.02

2

8

10±0.04

7±0.01

4

16

9±0.02

6±0.04

2

16

8±0.03

5±0.05 6±0.02

4 4

16 32

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Staphylococcus mutans Pseudomonas aeruginosa Staphylococcus aureus Bacillus subtilis Escherichia coli

0.5

2

0.5

4

1

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0.5 0.5

1 2

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Microorganisms

Nanoemulsion samples Spontaneous emulsification Ultrasonication emulsification method method Zone of Zone of MIC MBC MIC MBC inhibition inhibition (µg/mL) (µg/mL) (µg/mL) (µg/mL) (mm) (mm)

6±0.02 7±0.04

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Minimal inhibitory concentration (MIC); Minimum bactericidal concentrations (MBC); B.sub: Bacillus subtilis, S.aur: Staphylococcus aureus, S.mut: Streptococcus mutans, and P.aer: Pseudomonas aeruginosa. Each value is the mean of three replicates with standard deviation (±SD). (n = 3; *p<0.05 vs. control).

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