Characterisation of ionic liquids nanoemulsion loaded with piroxicam for drug delivery system

Accepted Manuscript Characterisation of ionic liquids nanoemulsion loaded with piroxicam for drug delivery system

Siti Balkis Mahamat Nor, Pei Meng Woi, Sook Han Ng PII: DOI: Reference:

S0167-7322(16)33599-1 doi: 10.1016/j.molliq.2017.03.042 MOLLIQ 7077

To appear in:

Journal of Molecular Liquids

Received date: Accepted date:

16 November 2016 9 March 2017

Please cite this article as: Siti Balkis Mahamat Nor, Pei Meng Woi, Sook Han Ng , Characterisation of ionic liquids nanoemulsion loaded with piroxicam for drug delivery system. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Molliq(2017), doi: 10.1016/j.molliq.2017.03.042

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Characterisation of Ionic Liquids Nanoemulsion Loaded with Piroxicam for Drug Delivery System. Siti Balkis Mahamat Nor1,2 a , Pei Meng Woi1,2 b Sook Han Ng3, c Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2 3

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Center of Ionic Liquids (UMCiL), University of Malaya, 50603 Kuala Lumpur, Malaysia.

School of Pharmacy, International Medical University, 57000 IMU Bukit Jalil, Kuala Lumpur, Malaysia.

[email protected], [email protected], [email protected]

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Corresponding author: Department of Chemistry, Faculty of Science, University Malaya, 50603

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Kuala Lumpur, Malaysia. Email: [email protected]. Tel.: +60379674271. School of Pharmacy, International Medical University, 57000 IMU Bukit Jalil, Kuala Lumpur,

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Malaysia. Email: [email protected]. Tel.: +60327317507.

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Abstract In this study, ionic liquid-in-oil nanoemulsions (IL/o NEs) system were formulated by using two types of ionic liquids, 1-hexyl-3-methylimidazolium chloride [Hmim][Cl] and 1-butyl-3methylimidazolium hexafluorophosphate [Bmim][PF6] in differences mass ratio with Tween80/Span-20 1:1, 1:2, 2:1 and 2:3. They were tested for stability study before undergo characterisation, rheology behaviour and released study in order to get the best result of NEs system. The high concentration of Tween-80 in the formulation of NEs show high stability from separation, creaming, sedimentation and flocculation. The droplet sizes, zeta potential, drug encapsulation efficiency (%) and pH value for all formulations were considered in the range of 100 to 500 nm, -37.3 to -55.3 mV, 60.02% to 98.76% and 4.72 to 5.50 respectively. Spherical droplets were seen in the transmission electron microscopy (TEM) images of the nanoemulsions. Rheological studies showed non-Newtonian shear thinning behaviour at low shear rate up to 14 S -1 of NE for both ionic liquids. Nanoemulsions insertion of Piroxicam was used to investigate the in vitro drug releases via dialysis bag method. The permeation of drug demonstrated the optimised surfactant ratio is 2:1 and ionic liquid is [Hmim][Cl] with 93% of drug released. It is concluded that the NEs prepared from ionic liquids offered a good potential as a carrier for drug delivery of Piroxicam. Keywords

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Nanoemulsions, 1-hexyl-3-methylimidazolium hexafluorophosphate, Piroxicam.

chloride,

1-butyl-3-methylimidazolium

1. Introduction

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In recent years, the use of ionic liquids (ILs) as a potential environmentally benign solvent in pharmaceutical industries are become interest. ILs are salts comprise of cations and anions and can be categorised into; hydrophobic and hydrophilic. They can be synthesised with differences properties by changing anions/cation combination [1] and have been used as a “green” replacement for toxic, hazardous, flammable and highly volatile organic solvents [2]. Since they are organic salts which are liquids at ambient room temperature [3] and have low melting points, they can act as solvents for various reactions [4]. Numerous reports also demonstrated ILs as solvent and were used as enhancer, active pharmaceutical ingredients (APIs) [5], and preservative [6]. Regarding to the previous study [7, 8], ILs show an excellent solvent in solubilising hydrophobic and hydrophilic drug. They were used as a component of microemulsion carrier system whether ionic liquid in oil (IL/o) [9] or ionic liquid in water (IL/w) [10] system which depends on the polarity of ILs.

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In pharmaceutical processing, the used of IL as a solvent is still debatable even it is known as “green solvent” especially in medicine and biomedicine, and in biology-related area. Most researchers concerns about ILs toxicity as the main challenge in drug delivery application, due to lack of scientific knowledge on it. However, nontoxic ILs for pharmaceutical can be produced by altering their physical chemical properties by changing anions/cations combination [2]. Fortunately, there are some literature demonstrated synthesizing of nontoxic ILs by selecting biocompatible organic cations and inorganic anions [11, 12]. Recent study had proven that the incorporation of ether groups into the ester side chain of the ILs lower the toxicity comparable to alkyl derivatives [13]. Moreover, some researchers demonstrated low/negligible toxicity of ILs towards HaCaT cells [6] and ICP-81 leukaemia as well as the glioma cells [14]. As ILs are debatable about their toxicity, it should be noted there are a lot of pharmaceutical excipients demonstrated similar toxicities to ILs for example dimethylsulfoxide and non-ionic surfactant. Thus, ILs with low toxicity and exhibit good biodegradability should be explored its potential as pharmaceutical ingredients.

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Topical drug delivery systems served as a tool to deliver the drug and to control or sustain the active moieties. Therefore, they were used as an alternative routes to overcome the side effects and widely used in several disease treatment like osteoarthritis [15]. However, the major problem administration of Piroxicam via topical drug delivery system is barrier properties of stratum corneum, which is considered as one of the most impermeable epithelia of the human body to exogenous substances [16, 17]. The problems were minimised by using several techniques such as ion pairing and permeation enhancer for skin treatment [18]. But the use of chemical enhancers cause an adverse effect especially in chronic application, since many of them are usually irritants [19]. Herein this study, we adopt the development of nanoemulsion system as the solution to overcome this issue. Nanoemulsion is an emulsion with a droplet size in nanometre scale which defined as an emulsion where oil or water droplets are finely dispersed in the opposite phase with the help of a suitable surfactant to stabilize the system. Nanoemulsion has small and uniform droplet size of less than 200 nm [20]. An emulsion also can be considered as a NE even the size is less than 500 nm provided that the emulsion has low surfactant content and is kinetically stable [21, 22]. NEs have a lot of interesting properties which are large surface area, small droplet size, kinetically stable [2325] with a possible long-term physical stability against from sedimentation or creaming [26]. Due to their excellent properties, they were used as a promising tool to deliver drug especially in dermatology [27, 28] and protecting the bioactive compound [24].

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Piroxicam is a class of nonsteroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic effects which belongs to a new class of compounds known as Oxicams [29, 30]. Piroxicam broadly used for the medication of musculoskeletal and joint disorders such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, in soft-tissue disorder, in acute gout and in post-operative pain [17]. It is easily absorbed and given oral administration. However, it will contribute adverse effects to human if it is given oral route, especially on the stomach, kidneys and also gastric mucosal damage. Therefore, to achieve better therapeutic effects, the Piroxicam was administered or delivered through the skin or topical drug delivery system as a promising method [15, 17].

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Various methods were used in designing a new carrier for delivering Piroxicam. They were developed in order to obtain maximum therapeutic efficacy and reduces side effects on the patients. Microsphere, organogel, alginate beads, solid lipid nanoparticles, microemulsion and nanoemulsions are carrier system used for skin delivery Piroxicam. Nevertheless, some of the methods are inapplicable for delivering Piroxicam due to their limitation. As compared to the others method, microemulsions and nanoemulsions show excellent results and have advantageous with improved solubility, absorption and diffusion [18, 31]. However, new study demonstrated combined formulation nanoemulsion and gel matrix produced better carrier system which showed higher permeation rate and relatively less drug retained [17, 18].

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Basically, most of drugs are hydrophobic in nature, same goes with Piroxicam, Acyclovir and Etomidate. Thus, they are difficult to soluble or sparingly soluble in water and in most of pharmaceutical grade organic liquids. In consequence, the drugs which are sparingly soluble never enter a formulation stage as a result from the poor solubility and difficulties in delivery. Hence, researchers developed some methods to overcome the problem in order to increase the solubility of the drug by using excipients for example ethanol and dimethyl sulfoxide (DMSO). These types of excipients will lead to the toxicity and others undesirable side effect if used in high concentration [32]. Therefore, in this study ILs that act as green solvent were used in developing ILs based nanoemulsion which help in solubilising and as drug reservoirs [8].

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Nanoemulsion as carrier system has been exploited for transdermal delivery of various drugs [16]. In one of the in vitro release study, they were investigated Piroxicam released in oil-in-water NE and it shown 95.21% release of drug. In this study, ionic liquids (1-hexyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium hexafluorophosphate) were used as nonaqueous phase to substitute the water phase. We developed ionic liquid-in-oil nanoemulsion (ILs/o NE) by using ionic liquids as a solvent and loaded with Piroxicam. Next, the IL/o NEs system were characterised by varying the weight ratio of surfactant (Tween-80/Span-20) 1:1, 1:2, 2:1 and 2:3. Permeation released of Piroxicam was compared by using in vitro released study which analysed by UV visible technique. 2. Experimental 2.1 Materials Disodium hydrogen phosphate, sodium dihydrogen phosphate, and xanthan gum were purchased Friendemann Schmidt Chemicals. The dialysis tubing cellulose membrane was obtained from Sigma Aldrich, St. Louis, MO, USA. Polyoxyethylene sorbitan monooleate (Tween 80) and sorbitan laurate (Span 20) were purchased from Fluka Chemie GmbH, USA. Labrafac Lipophile WL 1349 was purchased from Gattefossé, France. Piroxicam was purchased from Nutrisoul, Xi’an

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Yiyang Bio Tech Co., China. 1-Butyl-3-methylimidazolium hexafluorophosphate was obtained from Merck and 1-hexyl-methylimidazolium chloride was obtained from Fluka, Water was doubly distilled and deionized. All analytical grade of reagents were used in this experiment. 2.2 Methods 2.2.1 Preparation of IL-in Oil Nanoemulsion

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IL-in-oil nanoemulsion (IL/o NE) was prepared according to the method describes by previous researchers [2] with minor modification. The surfactant mixture of Tween-80/Span-20 was prepared by blended Tween-80 and Span-20 at fixed ratios (w/w); 1:1 at 25°C. The composition of surfactant and Labrafac Lipophile WL 1349 (oil) was evaluated by mixing 10% of surfactant mixtures (Tween-80/Span-20) with Labrafac Lipophile WL 1349 (84.5%). After that, the mixtures were mixed thoroughly until clear and optically transparent solution was obtained. On the other hand, 5% of ionic liquids (1-hexyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium hexafluorophosphate) were mixed with 0.8% thickeners (xanthan gum). Then, the mixture of surfactant and ionic liquids were heated at temperature 70°C. The mixtures of ionic liquids with thickeners were mixed with a transparent solution (a mixture of oil and surfactant). Next, the mixtures of that components were subjected to high shield homogeniser at 15000 rpm for 15 min. in order to ensure the homogeneity of components. After that, coarse emulsion were optimized for NE by subject to ultrasonic cavitation at room temperature, 60% energy intensity for 15 min. and undergoing high pressure homogenizer for five cycles. Lastly, the drug was inserted into NE and stirred for 4 hrs. 2.2.2 Stability Study of Nanoemulsions

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The physical stability of the NE loaded with Piroxicam during the prolong storage is determined by size measurements and by visual inspection at regular time intervals. In addition, the centrifuge test is carried out to assess the physical stability of NE. To this approach, NEs are centrifuged for 30 min. at 4500 rpm. On the other hand, NEs were introduced into freeze thaw cycle test for storage stability determination. In the case freeze thaw cycle, test tubes were filled with NEs were sealed and vertically stored for 16 hrs in a freezer at -5°C and then for 8 hrs at room temperature (25°C) and repeated for three cycles [16]. Other than that, NEs also were stored for 90 days in the storage at room temperature. 2.2.3 Characterization of Nanoemulsions 2.2.3.1 Droplet Size Analysis and Zeta Potential Measurement The droplet size and zeta potential of NEs were measured using a Zeta sizer ZEN 3600 (Malvern, Worcestershire, UK) at room temperature (25 ± 1°C). 1:100 of factor dilution with deionised water was used to dilute the formulated NEs prior subjected to analysis. The instrument was calibrated using zeta potential standard and polystyrene latex standard. Zeta potential was determined by using electrophoretic light scattering (ELS) and droplet size measurement was

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determined by using dynamic light scattering (DLS). This measurement was performed in triplicate [16].

2.2.3.2 Determination of Drug Encapsulation Efficiency

Encapsulation (1)

efficiency

(%)

=

(Winitial

drug

Wfinal

drug

)/

Winitial

drug

x

100

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2.2.3.3 pH Determination

–

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The encapsulation efficiency of Piroxicam was determined spectrophotometrically at 353 nm. After centrifugation of the NEs suspension, amount of the free drug was detected in the supernatant and the amount of incorporated drug was determined as the result of the initial drug minus free drug. [33]. The encapsulation efficiency (EE %) could be achieved by the following equation [34]:

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pH of the pure and drug encapsulated NEs were measured with Mettler Toledo pH meter (Mettler Toledo Inc., Columbus, Ohio) standardised using standard buffer of pH 4.0 and 7.0. pH measurement was essential to ensure non-irritating nature of the formulation on the skin and also for stability of NEs over a time. All the measurements were repeated three times [35].

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2.2.3.4 Transmission Electron Microscopy (TEM)

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The morphology and size of loaded drug NEs were done using TEM with negative staining of phosphor tungstic acid (PTA). Nanoemulsions were diluted with deionised water for 20 times and were applied on the 300 mesh copper grid for 1 min. Then, the grid was kept in an inverted state and a drop of PTA was applied to the grid for 10 sec. Filter paper was used to remove excess of PTA [36]. The NEs were left for two days to ensure they are dry. The grid was analysed by using transmission electron microscopy LEO LIBRA-120.

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2.2.3.5 Rheology Behaviour and Viscosity Measurement A rotational/ oscillatory viscometer, Anton Paar Rheometer (Anton Paar MCR 301, US) was used for determining the viscosity of NE. All measurements were performed with a stainless steel cone-plate sensor at 25.0 ± 0.1°C with 4°/40 mm. Sample of NEs were tested at a controlled temperature 25.0 ± 0.1°C under shear rate control condition within the range 1 s-1 to 100 s-1. Rheograms of apparent viscosity against shear rate and shear stress against shear rate were plotted and the data obtained were fitted to the Power Law model according to the following equation: τ = Kγn

(2)

where τ is the shear stress, γ is the shear rate, K is the consistency index (Pa.sn), and n is the flow behaviour index [37].

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2.2.3.6 In Vitro Release Studies

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The in vitro release of Piroxicam from the formulation was studied through a dialysis tubing cellulose membrane (molecular cut-off 14 kDa) using modified apparatus. The dissolution medium used was freshly prepared 0.01 M phosphate buffer saline solution (pH 7.4). Dialysis tubing cellulose membrane, previously soaked overnight in the dissolution medium was tied to one end of a specifically designed glass cylinder (open at both ends). 4 mL of formulation was accurately placed into this assembly. The cylinder was attached to a stand and suspended in 40 mL of dissolution medium maintained at 35 ± 1°C so that the membrane just touched the receptor medium surface. The dissolution medium was stirred at low speed using magnetic stirrer. Aliquots, each 2 mL volume were withdrawn at hourly intervals and replaced by an equal volume of the receptor medium [1]. Control sample with the same composition of oil, ionic liquids, and surfactant were treated as before in order to eliminate the effect of NE components on the UV absorption of Piroxicam. The amount released of Piroxicam was determined spectrophotometrically at 353 nm from their formulation by measuring the test samples against blank samples. The measurements were carry out three times and mean of the results were reported [24]. The drug content was quantified by Beer-Lambert law:

(3)

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A = εbc

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where A is absorbance, ε is the molar absorptivity with unit of L mol-1 cm-1, b is the path length of sample and c is the concentration of the compound in solution, mol L-1.

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3. Results and Discussion

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3.1 Stability Study

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3.1.1 Physical Stability

For physical stability, centrifugation method was applied in order to evaluate the physical instabilities for example phase separation, phase inversion, and cracking of the NEs formulation [38]. Based on the Table 1, it was showed that all the surfactant ratio (1:1, 1:2, 2:1 and 2:3) for [Hmim][Cl] have good physical stability which remained homogenous with a white colour and macroscopic homogeneity. However, for [Bmim][PF6] separation was occurred for surfactant ratio 1:2 and 2:3, whereas for ratio 1:1 and 2:1, white colour and macroscopic homogeneity were observed which indicates the NE was good in stability. The instabilities of [Bmim][PF6] can be suggested due to the types of ILs and mass ratio of Tween-80/Span-20. [Bmim][PF6] is a hydrophobic IL that containing non coordinating anion that poorly soluble as compared to [Hmim][Cl] which is categorised as hydrophilic IL that having coordinating anion which highly soluble and possess a strong hydrogen bond acceptors. The anions of ILs can form hydrogen bonds with hydroxyl group of Span-20 and Tween-80 [39] as the head groups of surfactant have a lot of hydrogen bonding sites. Moreover, hydrophilic ethylene oxide groups of Tween-80 which has hydrogen bonding interaction between imidazolium cation [40] causes strong affinity to

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imidazolium cation appended in ILs. Thus, it can be suggested that the present of hydrogen-bond interaction in the system of [Bmim][PF6]/Tween-80/Span-20 NE is the main factor to stabilise the system of NE. Hence, the higher Tween-80 content in the surfactant mixture stimulate the higher hydrogen bond interaction, thus increased the stability of NE.

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After a freeze thaw cycle (three times cycles), all formulations of NEs were homogeneous and the aqueous phase did not change the macroscopic aspect of NEs. All of them were found stable. During the freezing process, ILs were segregated from emulsion by crystallised oil particle. Lipid film that surrounded emulsion droplet were disrupted. Meanwhile, the thaw cycle, the droplets were melted and instantly coalesced between approaching droplets which is determined by phase separation. However, all formulations for NEs were maintained homogeneity. Xanthan gum reduced the re-association of ILs droplets and that phenomena in the damaged network led to less syneresis [41]. In addition, it reduced the formation of ice crystals. Thus, NEs with xanthan gum formulations are suitable to be stored at room temperature and freezing temperature.

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In addition, physical stability of NEs system were evaluated through droplet size determination after centrifugation and freeze thaw cycle. This evaluation gave a confirmation result for the stability of NEs system. It was observed that droplet sizes of NEs were slightly increased for all ratio [HMIM][Cl]-NEs system which is from 176.50 ± 0.66 nm to 179.13 ± 0.83 nm (ratio 1:1), 200.87 ± 1.54 nm to 202.87 ± 0.75 nm (ratio 1:2), 174.57 ± 0.68 nm to 175.93 ± 0.21 nm (ratio 2:1) and 189.10 ± 0.92 to 199.80 ± 1.25 nm (ratio 2:3) (shown in Table 1) after centrifugation testing respectively. Results for ratio 1:1 and 2:1 for [BMIM][PF6]-NEs also indicated significant changes on the droplets from 245.00 ± 1.01 nm to 258.10 ± 0.62 nm (ratio 1:1) and 211.57 ± 0.59 nm to 222.43 ± 0.70 nm (ratio 2:1). The results pointed out the systems were stable enough to prevent from occurring of instability. However the other two ratios; 1:2 and 2:3 displayed high increases in the droplets size after testing; from 347.83 ± 0.81 nm to 477.60 ± 6.54 nm for ratio 1:2 and 344.13 ± 0.86 nm to 478.60 ± 6.78 nm for ratio 2:3 respectively. These results can be suggested through the interaction of surfactant with ILs. These systems contained higher amount of Span-20 compared to Tween-80 and as a consequences the concentration of Span-20 at the interfacial region increased. Thus by reducing the PEO groups interaction with the electropositive imidazolium ring and anions of [BMIM][PF6] the droplets becomes bigger. Therefore, they can lead to instability of NEs system. All surfactant ratio [HMIM][Cl]-NEs system (1:1, 1:2, 2:1 and 2:3 ratios) and 1:1 and 2:1 ratios for [BMIM][PF6]-NEs system were considered stable and could be a potential for carrying drug. On the other hand, the stability of IL/o NE was observed for an extended time by examining the changes of droplet sizes along with direct visual observation. For this investigation, the IL/Tween-80/Span-20/Labrafac Lipophile NEs that contained Piroxicam at the different ratio of Tween-80/Span-20 (1:1, 1:2, 2:1 and 2:3) and different ILs were prepared and stored at 25°C. [Hmim][Cl]-NEs shown all the formulations have no precipitation over 90 days. However, there are small amount linear increasing of size for all surfactant ratio; 1:1 (21.7 nm), 2:1 (10.3 nm) and 2:3 (18 nm) except for 1:2 ratio (122.1 nm) as shown in Figure 1(a). Despite of the size increases, they are still in the range of nanoemulsion scale, thus, confirming the physical stability of the system. On the other hand, for [Bmim][PF6]-NEs the surfactant ratio of 1:1 and 2:1 were considered as stable because no precipitation was occurred over 90 days and only increased about 17.1 nm for ratio 1:1 and 5.4 nm for 2:1. For the others two surfactant ratios; 1:2 and 2:3, no precipitation was occurred

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over 90 days. Nevertheless, after 60 days, some presence of bacteria or fungi was detected in the NEs as indicated by the black deposition on the surface of NEs and the droplet sizes also increased about 1100 nm (within 90 days) for ratio 1:2 and 135.3 nm (within 90 days) for 2:3 (Figure 1(b)).

3.1.2 Chemical Stability

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Nanoemulsions stability were analysed by checking their behaviour in various pH values over the storage period for 90 days at room temperature. IL/o NEs showed small significant changes within 90 days for both systems (Figure 2) for ratio 1:1 from pH 4.75 to 4.78; ratio 2:1 from pH 4.83 to 4.85 for [Hmim][Cl] and for ratio 1:1 from pH 5.11 to 5.09 and ratio 2:1 from pH 5.50 to 5.57 for [Bmim][PF6]. The acidic values shown that NEs are resisted from hydrolytic degradation. However, the pH value for ratio 1:2 and 2:3 (Hmim][Cl] and [Bmim][PF6] systems) showed relatively decreased in pH value after 60 days, whereas others were maintained. The decrease in pH values of NEs from pH 4.77 to 3.87 for 1:2 and pH 5.41 to 4.53 for [Hmim][Cl] and from pH 5.02 to 3.93 and pH 5.23 to 4.53 for [Bmim][PF6] may come from the changes in the chemical structure of ingredients in emulsion. These results can be suggested due to the hydrolysis of fatty acid esters in Labrafac Liphophile into free fatty acid degradation products [42], thus decreased the pH value of 1:2 and 2:3 ratio.

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Based on the stability study, it is shown that [Hmim][Cl]-NEs are more stable compared to [Bmim][PF6]-NEs. This is presumably due to the coordinating anion bringing the NEs much easier to solubilise in the core of Tween-80/Span-20/Labrafac Lipophile micelles as they are strong hydrogen bond acceptor [2]. Thus, their droplet sizes are smaller rather than [Bmim][PF6]-NEs. The smaller droplet sizes of micelles were attributed to the physical stability that against the gravitational separation. On the other hand, the addition of xanthan gum to both NEs also increased the stability by retarding the droplet from the thinning process which slow down droplets movement and the number of collisions [43]. Due to the stability study for both types of NEs, it was shown that the surfactant ratio (Tween-80: Span-20) 2:1 is the best result. This can be suggested that the present of higher Tween-80 than Span-20 for both ILs/o NEs system which it has hydrophilic PEO groups that gave strong affinity to imidazolium cations in ILs and hydroxyl groups that can form hydrogen bonds with anions ILs [2]. Due to this behaviour, surfactant ratio 2:1 was chosen for further characterisation and release study.

Types of nanoemulsions

[Hmim][Cl]-NE

[Bmim][PF6]-NE

Ratio Tween80:Span-20

Table 1: Stability study of ILs/o NEs (mean±SD, n=3)*. Storage room temperature (25°C)

Stability

Droplet size (nm)* Before

After centrifuge

After freeze thaw cycle

Centrifugation

Freeze thaw cycle

30 days

60 days

90 days

1:1

176.50 ± 0.66

179.13 ± 0.85

177.50 ± 0.60

√

√

√

√

√

1:2

200.87 ± 1.54

202 87 ± 0.75

201.83 ± 0.60

√

√

√

√

√

2:1

174.57 ± 0.68

175.93 ± 0.21

174.80 ± 0.50

√

√

√

√

√

2:3

189.10 ± 0.92

199.80 ± 1.25

190.27 ± 1.07

√

√

√

√

√

1:1

245.00 ± 1.01

258.10 ± 0.62

245.77 ± 1.15

√

√

√

√

√

1:2

347.83 ± 0.81

477.60 ± 6.54

371.40 ± 1.44

x

√

√

√

X, ■

2:1

211.57 ± 0.59

222.43 ±0.70

219.70 ± 0.75

√

√

√

√

√

2:3

344.13 ± 0.86

478.60 ± 6.78

374.67 ± 1.34

x

√

√

√

X, ■

√ = Stable nanoemulsion system, X = Unstable nanoemulsion system,

■ = Present of fungus or bacteria

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Droplet size (nm)

a 400 300 200 100 0

Ratio 1:1 Ratio 1:2 Ratio 2:1 1 Day

14 Days 30 Days 60 Days 90 Days

2000

Ratio 2:3

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Droplet size (nm)

Time (days)

b

Ratio 1:1

1000

Ratio 1:2

0 1 Day

14 Days 30 Days 60 Days 90 Days

Ratio 2:3

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Ratio 2:1

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Figure 1: (a) Droplet size of [Hmim][Cl]-NEs and (b) Droplet size of [Bmim][PF6]-NEs.

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pH Value

6

a

14 Days

Ratio 1:2 Ratio 2:1

30 Days

60 Days

90 Days

Ratio 2:3

Time (days)

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Ratio 1:1

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b

pH Value

6 4

Ratio 1:1

2

Ratio 1:2 Ratio 2:1

0 1 Day

14 Days

30 Days

60 Days

90 Days

Ratio 2:3

Time (days)

Figure 2: (a) pH value of [Hmim][Cl] and (b) pH value of [Bmim][PF6].

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3.2 Physicochemical Evaluation of Nanoemulsion 3.2.1 Droplet Size

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Particle size analysis is an important parameter in order to identify the stability and determines the rate and permeability of NEs through the skin. In terms of stability, the Brownian motion produced by the small droplet size of NEs were contributed to the stability against sedimentation or creaming. As a result, the diffusion rate is higher than the sedimentation rate induced by the gravitational force [44]. On the other hand, the small droplet size of emulsion is important for permeability or penetration of carrier. Smallest droplet size will affect the absorption of drug which has the larger interfacial surface area for drug absorption [45]. The result droplet size of NEs were shown in Table 2. Based on the result, the droplet sizes were considered in the range of NEs diameter which are below 500 nm.

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Formulation of [Hmim][Cl]-NEs loaded Piroxicam shows the droplets size for surfactant ratio 2:1, 174.57 ± 0.68 nm (Table 2) is the smallest droplet size compared to the other ratios which were 176.5 ± 0.66 nm, 200.9 ± 1.54 nm, 189.1 ± 0.92 nm for 1:1, 1:2 and 2:3 surfactant ratio respectively. Besides that, for the formulation of [Bmim][PF6]-NEs the smallest size of droplet attributed to the surfactant ratio is 2:1, 211.6 ± 0.59 nm. Other ratios, which belong to 1:1, 1:2, and 2:3 gave droplet sizes of 245.0 ± 1.01 nm, 347.8 ± 0.81 nm and 344.1 ± 0.86 nm respectively. These patterns may be affected by the HLB value and the molar ratio of surfactant [32, 46]. By using the differences ratio of Tween-80 and Span-20, it was found that the droplet sizes of ILs aggregates increased as the decreased of Span-20. It can be explained that at higher concentration of Tween-80 at the interface, larger interfacial area was imposed and the POE (polyoxyethylene) chains on the head group experienced steric interaction [47]. In addition, its contain hydroxyl group that can form hydrogen bonds with anions of ILs.

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Furthermore, [Hmim][Cl]-NEs have fine droplet sizes compared to the [Bmim][PF6]-NEs. The formation of small ILs droplets in formulation NEs may be due to the differences in polarity and lipophilicity between ionic liquids [6]. As mention earlier, [Hmim][Cl] is a hydrophilic ILs that having coordinating anions which is easier for it to dissolve in the Tween-80/Span-20 rather than [Bmim][PF6] which is a hydrophobic IL. Thus, the size of the [Hmim][Cl]-NEs droplets decreased and become smaller compared to [Bmim][PF6]-NEs. The addition of imidazolium cations to the formulation also can lead to the formation of smaller droplet sizes due to the changes arrangement of the emulsifiers on the surface of the ILs droplets [48]. On the other hand, the addition of Piroxicam into the micelles only contributed to slight increases in droplet size. This indicated the emulsions were stable enough to load the drug into the micelles. The polydispersity index (PDI) is a parameter used to indicate the nanocapsules size distribution. A lower PDI value describes a monodisperse droplet population in the particle size distribution and a unimodal system, while the value closer to 1 indicates a wide range of droplet size [49]. All the formulations of [Hmim][Cl]-NEs were found PDI value below 0.3, whereas [Bmim][PF6]-NEs showed above 0.3 for ratio 1:2 and 2:3 and for others are below 0.3. Polydispersity index until 0.3 is considered suitable and indicates that the suspension is monodisperse presenting a narrow sizes range [50].

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Table 2: Content of the nanoemulsion formulation (mean ± SD, n=3)*. Tween80:Span-20

[Hmim][Cl]-NE

1:1

Drug entrapment efficiency (DEE %)

11.80

1:2

10.78

2:1

[BMIM][PF6]-NE

HLB value

12.82

2:3

11.16

1:1

11.80

1:2

10.78

2:1

12.82 11.16

PDI value*

Zeta (mV)*

potential

95.50 %

175.17 ± 0.57

0.094 ± 0.011

-38.60 ± 1.28

4.72 ± 0.006

176.50 ± 0.66

0.061 ± 0.011

-37.33 ± 0.60

4.75 ± 0.006

-

199.73 ± 0.42

0.179 ± 0.011

-44.80 ± 0.85

4.74 ± 0.035

93.75 %

200.87 ± 1.54

0.066 ± 0.011

-38.70 ± 0.98

4.77 ± 0.015

-

173.90 ± 0.85

0.053 ± 0.006

-42.00 ± 0.56

4.79 ± 0.006

98.76 %

174.57 ± 0.68

0.071 ± 0.012

-43.80 ± 0.66

4.83 ± 0.006

93.42 %

187.53 ± 0.61 189.10 ± 0.92

0.180 ± 0.010 0.087 ± 0.010

-41.13 ± 0.96 -38.56 ± 1.01

5.20 ± 0.090 5.41 ± 0.015

-

244.00 ± 0.92

0.132 ± 0.072

-44.80 ± 0.61

5.01 ± 0.025

70.65 %

245.00 ± 1.01

0.156 ± 0.051

-50.20 ± 1.18

5.11 ± 0.015

-

311.33 ± 1.04

0.221 ± 0.020

-55.30 ± 0.66

5.13 ± 0.046

60.02 %

347.83 ± 0.81

0.340 ± 0.043

-46.03 ± 0.76

5.02 ± 0.040

-

210.53 ± 0.91

0.068 ± 0.020

-40.00 ± 0.82

5.46 ± 0.050

80.04 %

211.57 ± 0.59

0.089 ± 0.027

-39.03 ± 1.04

5.50 ± 0.058

63.98 %

336.00 ± 0.75 344.13 ± 0.86

0.212 ± 0.036 0.356 ± 0.027

-41.33 ± 0.93 -39.60 ± 0.61

5.38 ± 0.010 5.23 ± 0.095

pH value*

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2:3

Droplet size mean ± SD (nm)*

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Types of nanoemulsion

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3.2.2 Zeta Potential

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Zeta potential is a potential exists between the particle surface and the dispersing liquid at the slipping plane. The stronger the interfacial film will lead to lesser zeta potential decay rate and increased the stability of emulsions. This reveals the strength of interfacial film is governed by the stability of emulsions. [51]. The interfacial film of NEs were determined by measuring electrophoretic mobility of particle by using zetasizer. The zeta potential value for the NE is tabulated in Table 2. According to the result, zeta potential value for [Hmim][Cl]-NEs and [Bmim][PF6]-NEs showed high negative value. This indicates the electrostatic forces on the dispersed NEs interfacial film were great enough to prevent coalescence. Thus, the phase of the emulsion will be separated if the repulsive force decreased [45]. Zeta potential value for formulation of [Hmim][Cl]-NEs and Bmim][PF6]-NEs were between -37.3 mV to -55.3 mV. These values are sufficient enough to produce an energy barrier between the droplet that can preclude from the formation of coelescence. The non-ionic surfactant (Tween-80/Span-20) shields the ionic liquids and drugs to form micelles from the oil and created high negative charge of interfacial film and thus prevent from coalescence and coagulation. As a result, the stability of the NEs was increased. As shown in Table 2, the surface charge of micelles were negative although the emulsions were stabilised with non-ionic surfactant. This phenomenon is presumably comes from hydrogenbond interactions arise from the hydroxyl group at the head of surfactants (Tween-80 and Span-20) with anions and hydrogen-bond interactions between the C2-H of the [Bmim]+ and [Hmim]+ cation of ILs and the EO units in Tween-80 [40]. This in turn contributed to high negative charge of zeta potential for both of the ILs/o NEs. Regarding to these interaction, it was also likely that they were effected by high concentration of Tween-80 rather than Span-20 in formulation of NEs, and lead to the small droplet sizes of micelles. Maruno and Rocha-Filho also found significant negative zeta potentials using non-ionic surfactant, and associated this phenomenon with some chemical properties of the polyoxyethylene chains in the surfactant used [52]. In general, if the zeta potential of NE is above +30 mV or below -30mV, the charge stabilization is considered to be effective [37]. Thus the formulation of NEs were considered stable enough for the accelerated stability tests.

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3.2.3 Drug Encapsulation Efficiency The encapsulation efficiency (EE %) of Piroxicam into IL/o NEs systems were studied spectrophotometrically. Encapsulation efficiency can be assigned as the ratio of quantity encapsulated/adsorbed drug in relation to the total (theoretical) amount of drug used for production of NE [53]. Table 2 shows the EE % results of IL/o NEs and they were proven to be successful in encapsulated drug in carriers for both types of ILs. It was observed that EE % for [Hmim][Cl]-NEs (98.76 %) are higher compared to [Bmim][PF6]-NEs (80.04 %) for ratio 2:1. The encapsulation of drug was also supported by the increases droplet sizes of NEs (Table 2).

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Scheme 1 shows the proposed mechanism for the encapsulation of Piroxicam into NEs. As illustrated in Scheme 1, ILs were located inside the core of micelle which were stabilised by the surfactant (mixture of Tween-80 and Span-20) by creating an interfacial film between Labrafac Lipophile and ILs. The interaction of hydrogen binding between ILs ([Hmim][Cl] and [Bmim][PF6]) and Tween-80 is the driving force for solubilising ILs into the core of during the aggregation of Tween-80 [40]. The addition of Piroxicam into the NEs was soluble in ILs. Since, the solubility of Piroxicam in the Labrafac Lipophile are low [54], it can be assumed Piroxicam was solubilised and encapsulated in the core of ILs. Based on the previous study, Piroxicam molecules were successful dissolution in the ILs due to the formation of hydrogen bonds between the IL anions and the polar groups of Piroxicam molecules acting just like cellulose compounds [39]. Anions of ILs (Cl- and PF6-) acted as the hydrogen bond acceptor has an interaction with the hydroxyl group of Piroxicam. Thus, Piroxicams are more easily to dissolve in ILs.

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According to Moniruzzaman and co-workers, the solvency of drug molecules were affected by hydrophilic ILs which having coordinating anions, while hydrophobic ILs possessing noncoordinating anions could not perform as such [5]. Generally, the longer the alkyl chain of imidazolium cation causes greater hydrophobicity which in turn increases the solvency for the hydrophobic drug. Similar phenomenon was reported for the effect of anions hydrophobicity which also able to dissolve high value of hydrophobic drug molecules [3]. Interestingly in this study both types of the ILs, i.e. hydrophilic and hydrophobic could dissolve the Piroxicam, thus encapsulated into NEs. [Hmim][Cl] possessed high encapsulation of drug rather than [Bmim][Pf6]. This is presumably due to the Cl- anion has a stronger hydrogen bond and longer alkyl chain in imidazolium salt compared to [Bmim][PF6] which has weaker hydrogen bond properties than Cl-.

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13

Span-20

PT

Labrafac Lipophile Wl 1349

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Tween-80

Xanthan gum Ionic liquid Drug

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Scheme 1: Proposed mechanism for the formation ionic liquids-in-oil nanoemulsion,(a) Hydrogen-bond interaction between Tween-80 and [Bmim][PF6], (b) Hydrogen-bond interaction between Tween-80 and [Bmim][Cl] and value x+y+z = 20. 3.2.4 pH Value The pH value is important for the topical formulation which should be close to the pH of human skin. Generally, the pH of skin is between 4 to 6 and depends on the age of individuals. NEs formulation in alkaline will contribute to the skin irritation and render the skin susceptible to bacterial infection. Thus, in order to avoid the risk of irritation or alteration of the cutaneous tegmentum the pH value should be between 4 and 6.5 [37]. In this investigation, all the formulation NEs were exhibited a pH value in the range of 4.72 to 5.5 considering that they would not irritate the skin (Table 2).

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3.2.5 Morphology

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Transmission electron microscopy (TEM) is used for determination of droplet size of NEs. In this study, only the selected NEs were measured which is the surfactant with ratio 2:1 for [Hmim][Cl]-NE and [Bmim][PF6]-NE that contained Piroxicam inside the micelles. Through these images (Figure 3), NEs were appeared dark with a spherical shape of NEs droplets with a particle size in the range of 100 to 300 nm. The gel network of xanthan gum has no influence on the structure of NEs, even though it increased the viscosity of NEs. These results have confirmed that the droplet size for NEs were in nanosize range (less than 500 nm) and the shape of droplet are sperical, thus emulsion formulated was NE.

b

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a

Figure 3: TEM image of [Hmim][Cl]-NE (a) and [Bmim][PF6]-NE (b).

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3.2.6 Rheology Study

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The rheological evaluation of pharmaceutical NEs are a useful and important parameter for development of NEs. This method gives the information about the behaviour of emulsion. The rheological behaviour of the NEs formulation are influenced by the spreadability of topical formulation their retention on the skin surface [37] and the rate of drug released [55]. It can be observed in Figure 3 the changes in viscosity of ILs/o NEs acted as a function of shear rate. Based on the figure, the viscosity of all NEs were decreased sharply with increasing shear rate and then plateaued. It is shown that the flow behaviour is non-Newtonian behaviour, shear thinning behaviour at low shear rates up 14 S- 1 for both types of ionic liquids. The rheological behaviour of these NEs were confirmed by applying the Power Law model [37]. Power Law shown in equation (2) was used to evaluate the flow index of each formulation NEs. The flow index indicates the deviation of the system from Newtonian behaviour. A value of n = 1 indicates Newtonian behaviour, n < 1 indicates shear thinning or pseudoplastic flow and n > 1 represents shear thickening or dilatant flow. Table 3 indicates the flow index, n for both NEs, where [Hmim][Cl]-NE is 0.0689 and [Bmim][PF6]-NE is 0.0795. Other than that, the presence of the xanthan gum in the formulation of NEs also affecting the flow index. The elastic gel-like of xanthan gum offers a resistance to flow initially under low shear forces, which decreases under increasing shear forces, dispute leading to the unrestricted flow of droplets in the direction of the shearing force [37]. The present of xanthan gum, which was composed of large molecules formed a polymer network structure surrounding the ionic liquid droplets through hydrogen bonding between xanthan gum molecules, which resulted in high shear viscosity when low shear rate was applied [56]. Figure 4 indicated the viscosity of the NEs were

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decreased as the shear rate increased based on the breakdown of colloid structure [57]. The high correlation coefficient, r2 data ranging between 0.998 and 0.999 for both NEs were acceptable and considered as a good data [37]. Therefore, this suggesting that the dependence of the viscosity on the shear rate indicated that the NEs were non-Newtonian behaviour.

20 15 10 5 0 0

5

10

15

20

[Hmim][Cl]-Ne [Bmim][PF6]-Ne

25

NU

Shear rate [1/s]

SC RI PT

Viscosity [Pa.s]

25

Figure 4: Rheological behaviour for ratio 2:1 of [Hmim][Cl]-NE and [Bmim][PF6]-NE.

[Hmim][Cl]-NE [Bmim][PF6]-NE

1.4004 1.4836

n

Maximum viscosity (Pa.s)

Yield stress (Pa)

0.0689 0.0795

21.2 20.5

2.12 2.05

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K

PT

Type of emulsion ratio 2:1

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Table 3: Rheological properties of nanoemulsions.

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Figure 5 indicates the functional relationship between shear stress and shear rate were affected by increasing energy input. The data obtained were fitted to the Herschel-Bulkley model according to the following equation [58]:

σ= σo + Kγn

(4)

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where σ is the shear stress (Pa), σo is the apparent yield stress K is the consistency index, γ is the shear rate (s-1) and n is the flow behaviour index. This equation reveals the fact that the initial application of shear for any system that possess a network structure resulted in distortion of the network. Then non-Newtonian flow will occur when the applied stress is greater than the yield stress. Yield stress of the NEs were shown in the Table 3. The increased yield stress of emulsion were resulted from the attractive force which one of the colloidal interaction. This is due to the magnitude of yield stress relies on the strength of the attractive force between the droplets [59]. The decreased droplet size of emulsion will increase in the total droplet surface area that causes from the increase in the dispersed phase volume fraction. The growth strength of the attractive force resulting from the upsurges of total surface area. Therefore, when a high attractive force is holding the droplets, then the greater stress needed to initiate flow which resulted in high yield stress [59]. Therefore, NEs may have tendency to separate when the yield stress is low.

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Shear stress (Pa)

10

[Hmim][Cl]-NE [Bmim][PF6]-NE

1 1

Shear rate (s-1)

10

100

SC RI PT

0.1

Figure 5: A plot of shear stress as a function of shear rate for ratio 2:1 of [Hmim][Cl]-NE and [Bmim][PF6]-NE.

3.3 In Vitro Released Study

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Penetration and partitioning of the drug through the skin are very important for drug delivery in order to release a drug to the target area. In many cases, the different forms of solubilised drug is formed when drug loaded NEs come in contact with aqueous medium which formed, including free molecular state, drug in NEs and drug micellar solution. Because of the situation is mimicked in vivo dissolution condition, the separation of the free drug molecules from those entrapped in the NE droplets or micelles becomes important things [45]. To overcome this problem, dialysis bag method was used compared to the conventional release testing due to the deficient to this system [45]. For this purpose the membrane used in the dialysis bag method must be porous and inert in order to allow penetration of drugs [18].

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The calibration curve of Piroxicam powder in phosphate buffer pH 7.4 was constructed and was found to be linear. From the plotted graph of absorbance (A) vs. concentration of drug released (µg/mL) the equation of the resulting line was “Absorbance = 0.0065 + 1004x concentration”, r2 = 0.9996. From the linear region of the graph (Figure 6), it was confirmed that the penetration of drug molecules through membrane has been reached. The slope was calculated and its value was used for comparison and other calculation for other parameters.

Absorbance, A

AC

1 0.8 0.6 0.4 0.2 0 0

50

100

150

200

250

300

350

Concentration, ug/mL

Figure 6: Calibration curve of Piroxicam powder in phosphate buffer pH 7.4. Figure 7 showed the release profile of Piroxicam in [Hmim][Cl]-NE and [Bmim][PF6]-NE formulation for surfactant ratio 2:1. The percentage of cumulative drug released that penetrates the dialysis membrane was plotted as a function of time (hours). In this study, the pH for buffer

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solution used is 7.4 to suits the acidic properties of Piroxicam. This attributed to the partitioning of Piroxicam that is higher from the oil phase to the buffer solution, hence the drug transfer rate is higher as well [18]. To calculate the percentage cumulative drug released, Equation 5 was used [60]. Cumulative percentage released (%) = Sample volume/Bath volume x P(t-1) + Pt

(5)

where, Pt is percentage release at time t and P(t-1) is percentage release previous to t.

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As shown in Figure 7, after 48 hrs about 93% of Piroxicams were released from formulation of [Hmim][Cl]-NE and 66% for [Bmim][PF6]-NE. The high amount of releasing Piroxicam for [Hmim][Cl]-NE can be suggested due to the smaller particle sizes of the micelles of the NEs formulation as mentioned in Table 2. The solubility pressure of the droplets was increased due to the higher surface area in the NEs and thus led to higher releasing of Piroxicam [61]. Besides that, the released of the Piroxicam also depends on the component formulation of NE. The difference in types of ionic liquids used resulted in the differences percentage of drug released. This can be suggested due to the hydrophilic and hydrophobic differences in both ILs. Therefore, [Hmim][Cl]NE is easier to pass across the dialysis membrane to buffer solution which contain water rather than [Bmim][PF6]-NE due to its hydrophilicity behaviour.

100

AC

Cumulative release drug (%)

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Apart from that, the in vitro release for both formulations of NEs showed an interesting permeation release of Piroxicam (Figure 7). In the first hour, Piroxicams were released very fast compared to the second and the next hour. At the first minute of measurement, the present of Piroxicam in the phosphate buffer was detected. After the first hour, the drug release followed a steady pattern. The released of the Piroxicam in the first hour can be ascribed to the Piroxicam loaded on the surface of nanoparticles [34] where they clearly shown that the penetration rates of Piroxicams relied on the viscosity of the system. This suggests the viscosity of the NE was higher compared to the phosphate buffer saline. This findings is in agreement with the data reported by previous study which revealed the rate of drug release from emulsion formulations is dependent on the vehicle used and the viscosity of the system as well as the existence of surfactant micelles [62]. Thus, the viscosity of the vehicle used was found to be inversely proportional to the amount of penetration of Piroxicam. On the other hand, the percentage release of Piroxicam is not 100%. This results is presumably due to the restricted movement of micelles to across the dialysis membrane [45] and trapped inside the NEs system. However, these NEs can be considered as promising drug delivery systems for topical administration of the drug due to the fast response of penetration drug is required.

80 [Hmim][Cl]-NE [Bmim][PF6]-NE

60 40 20 0 0

10

20

30

40

50

Time (hour)

Figure 7: Drug released profile for ratio 2:1 of [Hmim][Cl]-NE and [Bmim][PF6]-NE.

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4. Conclusion

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The result of the present study showed that IL/o NEs can be used as drug delivery of Piroxicam which is sparingly soluble in water. The replacement of water into nonaqueous emulsions were successful achieved by using hydrophilic IL, [Hmim][Cl] and hydrophobic IL,[Bmim][PF6]. Based on the formulation of NEs, both ionic liquids [Hmim][Cl] and [Bmim][PF6] show the 2:1 is the best ratio of mixture surfactant Tween-80/Span-20. Moreover, stability studies show excellent physical and chemical stability of IL/o NEs. The encapsulation efficiency results indicated that both ILs can dissolve the drug and encapsulated into the micelles of NEs. Other than that, both of the ILs shows an excellent result for physicochemical properties which led to the best result for penetration of Piroxicam through in vitro release study. For rheology study, IL/o NEs demonstrated the shear thinning for both ILs. However, the formulation of [Hmim][Cl]-NEs gave the best result for stability, physicochemical properties and in vitro release study. Therefore, IL/o NEs are considered to having optimistic potential for drug delivery of Piroxicam.

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Acknowledgments

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This research is supported by UMRG Sub-Program RP012B-14 SUS and PPP Research Grant PG010-21015A from University of Malaya and supported by International Medical University (IMU). References

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Alam, M.S., et al., Stability testing of beclomethasone dipropionate nanoemulsion. Tropical Journal of Pharmaceutical Research, 2015. 14(1): p. 15-20. Kaur, K., R. Kumar, and S.K. Mehta, Formulation of saponin stabilized nanoemulsion by ultrasonic method and its role to protect the degradation of quercitin from UV light. Ultrason Sonochem, 2016. 31: p. 29-38. Teo, S.Y., et al., Evaluation of biosourced alkyd nanoemulsions as drug carriers. Journal of Nanomaterials, 2015. 2015: p. 1-8. Chen, H., et al., A study of microemulsion systems for transdermal delivery of triptolide. J Control Release, 2004. 98(3): p. 427-36. Fukaya, Y., et al., Cellulose dissolution with polar ionic liquids under mild conditions: required factors for anions. Green Chemistry, 2008. 10(1): p. 44-46. Zheng, Y., W. Eli, and G. Li, FTIR study of Tween80/1-butyl-3-methylimidazolium hexafluorophosphate/toluene microemulsions. Colloid and Polymer Science, 2009. 287(7): p. 871876. Pongsawatmanit, R. and S. Srijunthongsiri, Influence of xanthan gum on rheological properties and freeze–thaw stability of tapioca starch. Journal of Food Engineering, 2008. 88(1): p. 137-143. Bernadi, D., et al., Formation and stability of oil-in-water nanoemulsion containing rice bran oil: in vitro and in vivo assessment. Journal of Nanobiotechnology, 2011. 9(44): p. 9-44. Krstonošid, V., et al., Influence of xanthan gum on oil-in-water emulsion characteristics stabilized by OSA starch. Food Hydrocolloids, 2015. 45: p. 9-17. Gadhave, A.D., Nanoemulsions: formation, stability and application. International Journal for Research in Science and Advanced Technologies 2014. 2(3): p. 038-043. Avachat, A.M. and V.G. Patel, Self nanoemulsifying drug delivery system of stabilized ellagic acidphospholipid complex with improved dissolution and permeability. Saudi Pharm J, 2015. 23(3): p. 276-89. Matsaridou, I., et al., The influence of surfactant HLB and oil/surfactant ratio on the formation and properties of self-emulsifying pellets and microemulsion reconstitution. AAPS PharmSciTech, 2012. 13(4): p. 1319-30. Lu, D. and D.G. Rhodes, Mixed composition films of Spans and Tween 80 at the air−water Interface. Langmuir, 2000. 16(21): p. 8107-8112. Bataller, H., et al., Cutting fluid emulsions produced by dilution of a cutting fluid concentrate containing a cationic/nonionic surfactant mixture. Journal of Materials Processing Technology, 2004. 152(2): p. 215-220. Tang, S.Y., et al., Formulation development and optimization of a novel Cremophore EL-based nanoemulsion using ultrasound cavitation. Ultrason Sonochem, 2012. 19(2): p. 330-45. Jia, F., et al., Nanostructured lipid carriers with liquid crystal structure encapsulating phenylethyl resorcinol: Characterization and in vitro study. Molecular Crystals and Liquid Crystals, 2016. 633(1): p. 1-13. Bhatt, N., et al., Stability study of O/W emulsion using Zeta potential. Journal of Chemical and Pharmaceutical Research, 2010. 2(1): p. 512-527. Maruno, M. and P.A.d. Rocha-Filho, O/W nanoemulsion after 15 years of preparation: A suitable vehicle for pharmaceutical and cosmetic applications. Journal of Dispersion Science and Technology, 2009. 31(1): p. 17-22. Silva, R., et al., Protein microspheres as suitable devices for piroxicam release. Colloids and Surfaces B: Biointerfaces, 2012. 92: p. 277-285. Park, E.-S., et al., Transdermal delivery of piroxicam using microemulsions. Archives of pharmacal research, 2005. 28(2): p. 243-248. Hamed, R., et al., Nanoemulsion-based gel formulation of diclofenac diethylamine: design, optimization, rheological behavior and in vitro diffusion studies. Pharm Dev Technol, 2015: p. 1-10. Ngan, C.L., et al., Skin intervention of fullerene-integrated nanoemulsion in structural and collagen regeneration against skin aging. Eur J Pharm Sci, 2015. 70: p. 22-8. Pal, R., Yield stress and viscoelastic properties of high internal phase ratio emulsions. Colloid & Polymer Science, 1999. 277(6): p. 583-588.

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Antonio, G.C., et al., Rheological behavior of blueberry. Food Science and Technology (Campinas), 2009. 29(4): p. 732-737. Pal, R., Rheology of Emulsions Containing Polymeric Liquids: Encyclopedia of Emulsion Technology. Vol. 4. 1996, New York: Taylor & Francis. 94-225. Chandrasekaran, A.R., et al., Invitro studies and evaluation of metformin marketed tabletsMalaysia. Journal of Applied Pharmaceutical Science, 2011. 1(5): p. 214. Başpınar, Y., et al., Pitavastatin-containing nanoemulsions: Preparation, characterization and in vitro cytotoxicity. Journal of Drug Delivery Science and Technology, 2015. 29: p. 117-124. Abd-Allah, F.I., H.M. Dawaba, and A.M. Ahmed, Development of a microemulsion-based formulation to improve the availability of poorly water-soluble drug. Drug Discoveries & Therapeutics, 2010. 4(4): p. 257-66.

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Hydrophilic and hydrophobic ILs promote the formation of IL/o NEs. Hydrogen-bond interaction helps in solubilise IL into core of surfactant. Nano particle size of prepared IL/o NE offers excellent stability. Drug released study via dialysis bag showed IL/o NEs as an excellent carrier.

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