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Journal of Nanomaterials
Volume 2015, Article ID 537598, 8 pages
http://dx.doi.org/10.1155/2015/537598
Research Article

Evaluation of Biosourced Alkyd Nanoemulsions as Drug Carriers

1School of Postgraduate Studies and Research, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia
2School of Pharmacy, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia
3Department of Chemistry, Faculty of Science, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia
4ULTI Pharmaceuticals, 19 Pembroke Street, Hamilton Lake, Hamilton 3204, New Zealand

Received 27 August 2015; Accepted 15 October 2015

Academic Editor: Michele Laus

Copyright © 2015 Siew Yong Teo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Novel oil-in-water (O/W) nanoemulsions were formulated using short, medium, and long oil length alkyds synthesized from palm kernel oil by a two-stage alcoholysis-polyesterification reaction. Alkyd/surfactant/water ternary phase diagrams identified a composition of 1% alkyd, 9% Tween 80, and 90% water where spontaneous production of nanoemulsions occurred. The pH, droplet size, and zeta potential of all formulations were in the range of 6.4–6.6, 11–14 nm, and −6 mV to −8 mV, respectively. Rheological studies showed that the nanoemulsions displayed non-Newtonian shear thinning behavior at low shear rates up to 20 s−1 with conversion to Newtonian behavior above this shear rate. All nanoemulsions were found to be stable against phase separation on storage at 4°C and 25°C for three months. Short oil length alkyd nanoemulsions exhibited significantly higher stability compared with medium and long oil length alkyd nanoemulsions, as demonstrated by an absence of phase separation and only minor changes of droplet size on storage at an elevated temperature of 45°C for 3 months. The drug carrying capacity and storage stability of the nanoemulsions were assessed using phenytoin. The entrapment efficiency of alkyd nanoemulsions was in excess of 90% and loss of phenytoin content was restricted to less than 4% during storage of the nanoemulsions for three months at 4°C, 25°C, and 45°C. Taken together, these findings indicate that nanoemulsions prepared from palm kernel oil-based alkyds offer potential as nanocarriers for drug delivery applications.

1. Introduction

Alkyds (vegetable oil modified polyesters) are typically synthesized from a polybasic acid, a polyhydric alcohol, and a triglyceride oil or its fatty acids. Alkyds are conventionally applied in the manufacture of paints, adhesives, inks, and various coatings and are generally classified as short (<45% oil content in formulation), medium (45–55%), or long (>55%) oil length alkyds. Research on alkyds has generally focused on the different types of vegetable oils used in their preparation, such as linseed, soybean, African locust bean seed, and rubber seed oil [13]. Alkyds synthesized from palm oil have been investigated extensively by Gan et al. for various industrial applications [47]. Previous studies have also demonstrated the potential advantages of palm oil-based formulations for wound repair, for treatment of skin conditions including skin infections, and for regulation of the body temperature of patients in the control of high fever and convulsion [810]. However, there is a marked absence of information on the use of palm oil-based alkyds for pharmaceutical applications.

The lipid component of alkyds suggests their utility as micro- and nanoemulsions for drug delivery. In particular, oil-in-water nanoemulsions are attracting major interest for oral administration and topical applications [1113]. Borhade et al. found that nanoemulsions greatly enhanced the solubility of clotrimazole to a level of 25 mg/mL and showed 100% drug release within 15 minutes irrespective of pH of medium [11]. Jain et al. demonstrated that nanoemulsions significantly enhanced the oral bioavailability of atorvastatin by more than 2.8- and 2.4-fold compared with conventional tablet and suspension dosage forms, respectively [12]. Alam and coworkers reported that nanoemulsion formulations of clobetasol propionate were advantageous for topical delivery to the skin since they were not irritant and reduced inflammation by 84% in the rat model compared with commercial cream formulations (41%) [13].

Nanoemulsions are typically transparent, kinetically stable disperse systems, comprising oil droplets of size less than 100 nm dispersed in an aqueous continuous phase. They are frequently formulated using high-pressure homogenization methods. However, the extreme processing conditions of high pressure and high shear stress coupled with relatively high temperatures may lead to structural and thermal degradation of the drug carrier (excipient) and the incorporated drug [14]. As a result, emulsion phase inversion (EPI) methods have attracted increasing interest for the production of drug-loaded nanoemulsions. EPI requires low energy input and relies on the spontaneous formation of droplets when certain combinations of oil, water, and surfactant are combined under controlled conditions. EPI has been shown to form monodispersed emulsions of very fine droplets with good storage stability [1517]. For example, Sun et al. reported that bovine serum albumin- (BSA-) loaded nanoemulsions formulated by EPI exhibited a droplet size of approximately 22 nm and the BSA content was retained for 180 days at room temperature [16].

Nanoemulsions generally incorporate a surfactant to produce an interfacial film around the dispersed oil droplets causing steric repulsion, preventing coalescence, and thus producing stable nanoemulsions [18]. The stability of nanoemulsions is a key determinant of their utility for pharmaceutical manufacture and is generally dictated by the integrity and density of the stabilizing interfacial film at the droplet surface. Nanoemulsion instability may result from a number of interrelated factors including the chemical composition of the stabilizing film, pH of the continuous phase, and storage temperature [19]. Several studies have highlighted the tendency of nanoemulsions to destabilize under conditions involving steric or electrostatic mechanisms. Nikiforidis et al. found that both steric and electrostatic repulsive forces contributed to the stability of nanoemulsions, by counteracting van der Waals attractive forces [20]. de Morais et al. also concluded that that the stability of canola oil/water nanoemulsions involved electrostatic and steric mechanisms, but the steric contribution was dominant [21].

The aim of this study was to evaluate the potential of novel nanoemulsions based on palm kernel oil-derived alkyds as drug delivery systems for oral and topical applications and to determine the storage stability of drug-loaded formulations. Optimum EPI conditions were established using ternary phase diagrams and the physicochemical characteristics of the resultant nanoemulsions were determined to assess their suitability as drug delivery vehicles. The drug loading capacity, encapsulation efficiency, and long-term storage stability of drug-loaded alkyd nanoemulsions were investigated using the hydrophobic drug, phenytoin.

2. Materials and Methods

2.1. Materials

Glycerol, toluene, potassium hydrogen phthalate, phenolphthalein, and Tween 80 were purchased from Fisher Scientific Sdn. Bhd., Selangor, Malaysia. Sebacic acid, ethanol, and potassium hydroxide were purchased from Merck Sdn. Bhd., Selangor, Malaysia. Palm kernel oil (PKO) was received as a gift from IFFCO (S.E.A) Sdn. Bhd., Kuala Lumpur, Malaysia. Phenytoin was purchased from Chemolab Sdn. Bhd., Kuala Lumpur, Malaysia. The chemicals were of reagent grade and used as received.

2.2. Synthesis and Characterization of Alkyds

Short, medium, and long oil length alkyds (designated as SOA, MOA, and LOA, resp.) were synthesized from palm kernel oil, glycerol, and sebacic acid using the two-stage alcoholysis-polyesterification method. The reaction was carried out in a reaction flask equipped with a mechanical agitator, thermometer, nitrogen gas inlet, and Dean-Stark decanter. In brief, PKO and glycerol were reacted to form monoglyceride during alcoholysis with the presence of sodium hydroxide (NaOH, 0.1% w/w) as a catalyst at a temperature of °C. Subsequently, sebacic acid was added to the mixture to initiate the second polyesterification stage, which was carried out at °C to form alkyd.

The acid number and hydroxyl number of the alkyd were determined according to ASTM D4274-05 and ASTM D1980-87(1998) standard test methods, respectively. Both tests were carried out in triplicate and the average results were reported.

2.3. Formulation of Nanoemulsions

Alkyd nanoemulsions were formulated from the palm kernel oil-derived alkyds, the nonionic surfactant (Tween 80), and ultrapure water at ambient temperature using the EPI method. The oil phase (SOA, MOA, and LOA, resp.) and Tween 80 were weighed separately and mixed thoroughly by vortexing at 1400 rpm for 2 minutes in specific alkyd/surfactant ratios (range of 1 : 9 to 9 : 1), to yield mixtures of a constant weight of 1 g. Subsequently, the mixture was slowly titrated with 100 μL aliquots (0.1 g) of ultrapure water. Following each water addition, the mixture was vortexed at 1400 rpm for 2 minutes and visually inspected for changes in appearance and phase separation. Ternary phase diagrams were constructed using the Chemix School v3.50 software (Arne Standnes, Norway).

Phenytoin-loaded alkyd nanoemulsions were formulated by first mixing phenytoin into the mixture of alkyd and surfactant at room temperature. The mixture was vortexed thoroughly at 1400 rpm, until a clear dispersion was formed, indicating that drug solubilization was complete. The mixture was slowly titrated with ultrapure water using the EPI method as described above until a nanoemulsion was obtained.

2.4. Characterization of Nanoemulsions
2.4.1. Physicochemical Evaluation

The alkyd nanoemulsions were subjected to visual inspection, pH, zeta potential, and droplet size determinations. The zeta potential and droplet size of nanoemulsions were measured using a Zetasizer ZEN 3600 (Malvern, Worcestershire, UK) at room temperature (°C). The instrument was calibrated using zeta potential standard and polystyrene latex standard, respectively. Zeta potential measurements were determined using electrophoretic light scattering (ELS). Droplet size measurements were determined using dynamic light scattering (DLS). All measurements were performed in triplicate.

2.4.2. Rheology

The rheological behavior of nanoemulsions was investigated using a MCR 301 rheometer (Anton Paar, Österreich, Austria) with a concentric cylinder system operating in rotational mode. Nanoemulsion samples (10 mL) were tested at a controlled temperature of °C under shear rate control conditions within the range 1 s−1 to 50 s−1. Rheograms of apparent viscosity against shear rate were plotted and the data obtained were fitted to the power law model according to the following equation:where is the shear stress, is the shear rate, is the consistency index (), and is the flow behavior index.

2.4.3. Emulsion Stability

Nanoemulsion samples were stored in an incubator (Heraeus, Hanau, Germany) at temperatures of °C, °C, and °C for three months. The physical appearance, pH, droplet size, and zeta potential were recorded for triplicate samples at monthly intervals.

2.4.4. Drug Loading, Entrapment Efficiency, and Stability

The drug loading and entrapment efficiency of alkyd nanoemulsions were determined using a high performance liquid chromatography (HPLC) method [22]. The test facility employed a reverse phase HPLC system (1200 series, Agilent Technologies) equipped with a pump, injector valve with 20 μL sample loop, ZORBAX Eclipse Plus C-18 analytical column (250 mm × 4.6 mm, 5 μm particles), and UV detector with data processor (Chem Station Software). The mobile phase of acetonitrile and ultrapure water (50 : 50) was delivered at a flow rate of 1.0 mL/min. Aliquots of samples (phenytoin in alkyd nanoemulsion) were dispersed in the mobile phase and the injected sample volume was 20 μL. A standard curve of peak area against concentration of phenytoin was plotted and the drug content of each sample was determined by comparison with the standard curve. The following equation was used to calculate the percentage drug loading and encapsulation efficiency of the nanoemulsion:The stability of phenytoin in the nanoemulsions was determined using the stability-indicating HPLC assay described above. Measurements were carried out using freshly prepared samples and samples stored for three months at 5°C, 25°C, and 45°C. Significant differences in drug content between the two groups were assessed using one-way analysis of variance.

3. Results and Discussion

3.1. Synthesis and Characterization of Alkyds

SOA, MOA, and LOA were synthesized using the two-stage alcoholysis-polyesterification method yielding a family of materials of low molecular weight (<2000 Da) with the chemical composition shown in Figure 1 and properties shown in Table 1. The low acid number (in the range of 5–7) indicates low content of free acid in the samples and suggests that they are suitable for application on the skin as topical drug delivery systems. In addition, the high hydroxyl numbers listed in Table 1 suggest that the PKO-based alkyds would exhibit good adhesive properties for increasing retention on the skin surface. Furthermore, the presence of pendant hydroxyl groups in the alkyd structure (Figure 1) presents opportunities for crosslinking with other materials (e.g., surfactants) to form three-dimensional structures, which may permit further control of drug release rate.

Table 1: Properties of alkyds prepared by two-stage alcoholysis-polyesterification method.
Figure 1: Plausible chemical structure of a section of the alkyd chain, where R is alkyl group.
3.2. Formulation of Alkyd Nanoemulsions

Ternary phase diagrams were constructed for mixtures of each type of alkyd (SOA, MOA, and LOA, resp.) with Tween 80 surfactant and water and found to be similar (Figure 2), indicating that the oil length (or content) of the alkyds investigated had no significant effect on the formation of O/W nanoemulsions. Three regions of significance were identified: an isotropic phase region (I), homogenous phase region (H), and two-phase region (T). The two-phase (T) region identified those alkyd/Tween 80/water mixtures (typically <50% water, >50% surfactant) where phase equilibria were not established and constituted the largest part of the phase diagram. This region was a result of the inability of the surfactant to successfully stabilize the alkyd phase. The homogenous phase (H) (typically >50% water, <50% Tween 80) was characterized by a uniform creamy, fluid system. This phase was generally less stable compared with the isotropic region due its high polydispersity. The isotropic phase region (I) (typically 1% alkyd, 9–29% Tween 80, and 70–90% water) appeared as a transparent, colorless fluid suggesting that the alkyd is solubilized efficiently within the aqueous surfactant phase. Hence, nanoemulsions were formulated from each type of alkyd (SOA, MOA, and LOA) using a composition in the transparent, isotropic region (I) comprising 1% w/w alkyd, 9% w/w Tween 80, and 90% ultrapure water (Table 1). Incorporation of surfactant is necessary to form an interfacial film around the dispersed alkyd (oil phase) droplets causing steric repulsion, preventing coalescence, and thus producing physical stability. However, nanoemulsion compositions having less than 10% w/w of Tween 80 were selected for further investigation in this study in order to minimize the content of surfactants in formulations that are intended ultimately for clinical application in patients.

Figure 2: Ternary phase diagram formed by Tween 80 (HLB 15.0), short oil length alkyd, and water mixtures. T, H, and I: two-phase region, homogeneous region, and isotropic region.

3.3. Characterization of Alkyd Nanoemulsions
3.3.1. Physicochemical Evaluation

The pH of topical formulations for the skin should be close to the pH of skin. The pH of the skin is generally in the range of 4 to 6, depending mainly on the skin area and the age of the individual; elderly individuals have a skin pH close to neutral. Alkaline vehicles are known to cause skin irritation and render the skin susceptible to bacterial infection [23]. Lucero et al. recommended that the pH values of skin formulations should be between 4 and 6.5 to avoid the risk of irritations or alterations of the cutaneous tegmentum [24]. SOA, MOA, and LOA nanoemulsions exhibited a pH value in the range of 6.4–6.6, indicating that they would not result in irritation, on the basis of a response to acidity/alkalinity, if applied topically on the skin. The alkyd formulations satisfied the size criterion of nanoemulsions (droplets < 100 nm) and exhibited a very narrow size range between 11 nm and 13 nm (Table 2). Zeta potential measurements carried out on the nanoemulsions (Table 2) provided a measure of the electrical charge carried by the droplets suspended in the aqueous phase. Zeta potential values of all alkyd nanoemulsions ranged between −6 mV and −8 mV. The small negative charge may be attributed to the hydroxyl groups in the alkyd structure (Figure 1) or the use of the nonionic surfactant (Tween 80) in the formulation and the water dipoles which influence the negative surface charge of the droplets. Marinova et al. reported that the adsorption of hydroxyl ions at an oil-water interface is the most likely mechanism for negative interfacial charging [25]. Overall, the alkyd nanoemulsions may be considered to be almost neutral in terms of electrical charge since the zeta potential results were in the range between −10 mV and +10 mV [26].

Table 2: Properties of alkyd nanoemulsions.
3.3.2. Rheological Behavior

The viscosity of nanoemulsions affects the ease of distribution and film formation on the skin surface. The change in viscosity of alkyd nanoemulsions as a function of shear rate is shown in Figure 3. The viscosity of all nanoemulsions decreased sharply with increasing shear rate and then plateaued. The nanoemulsions thus displayed two distinct types of flow behavior, non-Newtonian shear-thinning behavior at low shear rates up to 20 s−1 and a transition to Newtonian behavior (identified by constant nanoemulsion viscosity) at higher shear rates up to 50 s−1. The rheological behavior of the nanoemulsions revealed by the rheograms was confirmed by applying the power law model (1). Good data fits were obtained with high correlation coefficients (). The values of and are shown in Table 3. The flow index of all nanoemulsions was less than 1, which indicated shear-thinning (pseudoplastic) behavior and is indicative of the presence of weak interactions (attractive forces) between the droplets and surrounding aqueous medium. The weak, elastic gel-like network offers a resistance to flow initially under low shear forces, which decreases under increasing shear forces, eventually leading to unrestricted flow of droplets in the direction of the shearing force. The finding of non-Newtonian rheological behavior of the alkyd nanomeulsions at low shear rates below 20 s−1 is comparable with the pseudoplastic behavior of several nanoemusions reported by Jaworska et al. [27].

Table 3: Rheological properties of alkyd nanoemulsions.
Figure 3: Rheological behavior of alkyd nanoemulsions.
3.3.3. Nanoemulsion Stability

Nanoemulsions prepared from SOA, MOA, and LOA generally exhibited no visible sign of phase separation over 3 months at storage temperatures of 4°C and 25°C. However, evidence of phase separation (as indicated by precipitate formation) was apparent after 1 month storage at 45°C in the case of MOA and LOA nanoemulsions and distinct phase separation occurred between two and three months. It is likely that the elevated temperature imparts higher thermal energy to the droplets, causing an increase in collision frequency, which consequently leads to coalescence and phase separation. Interestingly, nanoemulsions prepared from short oil length alkyds remained stable at 45°C for 3 months, suggesting that the interfacial surfactant film provides greater resistance to droplet coalescence in this system, possibly due to an increased packing density of surfactant chain molecules at the droplet surface.

All nanoemulsions (SOA, MOA, and LOA) maintained a pH value between 6.0 and 6.6 during storage for three months at 4°C and 25°C and there was negligible effect of alkyd oil length (Figure 4). This behavior indicates that the nanoemulsions exhibit high resistance to hydrolytic degradation and are advantageous for the preparation of pharmaceutical formulations. However, there was a significant decrease in pH of the nanoemulsions from around 6.5 to 3.5–4.2 on storage at 45°C for three months, indicating an increased rate of hydrolysis. Dissolution of degradation products (organic acid monomers) in the water phase at high storage temperature leads to increasing acidity of the nanoemulsion over time.

Figure 4: Effect of storage time on the pH of alkyd nanoemulsions stored at 4°C, 25°C, and 45°C.

Zeta potential is widely used to predict the stability of colloidal systems since it indicates the magnitude of the surface charge on particles and thus the degree of repulsion which prevents coalescence. The low zeta potential values (−6 mV to −8 mV) of the nanoemulsions may arise from spontaneous adsorption of hydroxyl ions (OH) from the water phase to the hydrophilic head of surfactants as a result of hydrogen bonding or desorption of hydrogen ions (H+) from the droplet surface into the water phase to form hydronium ions (H3O+). Therefore, the decrease of absolute zeta potential of all alkyd nanoemulsions with increasing storage time and storage temperature (Figure 5) may indicate a charge neutralization effect between hydroxyl ions (OH) and hydronium ions (H3O+). Generally, charge stabilization of nanoemulsions is considered to be effective if the zeta potential is above +30 mV or below −30 mV. Stabilization of nanoemulsions prepared using nonionic surfactants including Tween 80 occurs mainly via steric mechanisms [21, 28] and the low values of zeta potential measured in the present study to support this principle.

Figure 5: Effect of storage time on zeta potential of nanoemulsions stored at 4°C, 25°C, and 45°C.

The change in droplet size of nanoemulsions prepared from SOA, MOA, and LOA over 3 months at storage temperatures of 4°C, 25°C, and 45°C is presented in Figure 6. The droplet size of all nanoemulsions remained fairly constant at around 12 nm on storage at 4°C for three months, indicating that 4°C is a suitable, long-term storage temperature for alkyd nanoemulsions.

Figure 6: Effect of storage time on the droplet size of nanoemulsions stored at 4°C, 25°C, and 45°C.

A small increase in droplet size from around 12 nm to 17 nm was recorded for all alkyd nanoemulsions after 3 months of storage at 25°C. These minimal changes may be explained by the high surfactant concentration in the nanoemulsions that improves stability by forming a strong interfacial film around the dispersed droplets to impede coalescence. The high surfactant concentration is also expected to lower the rate of droplets ripening by reducing the rate of dissolution of the alkyd in water due to the formation of surfactant micelles that shield the oil droplets from the continuous aqueous phase.

When nanoemulsions were stored at 45°C for 3 months, the droplet size of MOA and LOA nanoemulsions increased significantly by a factor of 10 (from approximately 10 nm to 115–130 nm), indicating a decrease in nanoemulsion stability. This behavior may be explained by increases in thermal energy of the droplets in the nanoemulsions and increased collision frequency leading to droplet coalescence. The decrease in stability may also be connected with a decrease in density (or viscosity) of the stabilizing interfacial film with increasing temperature, which results in film-drainage effects and subsequent drop coalescence and instability of the system. Interestingly, the droplet size of SOA nanoemulsion increased by only 17% when retained at 45°C for 3 months, demonstrating improved storage stability compared with MOA and LOA nanoemulsions, in line with visual observations. An increased packing density of surfactant chain molecules at the droplet surface provides a possible explanation for this behavior, as noted previously.

3.3.4. Drug Loading and Entrapment Efficiency of Nanoemulsions

In the present study, phenytoin was incorporated in the three types of alkyd nanoemulsion using the EPI method. Phenytoin is an anticonvulsant drug and rated as a Class II compound under the Biopharmaceutics Classification System (BCS). Such compounds permeate efficiently across biological membranes but have limited aqueous solubility and thus require precise formulation to achieve acceptable levels of oral bioavailability and therapeutic activity. A high drug loading of 10 mg/mL was measured for alkyd nanoemulsions irrespective of oil length. The encapsulation efficiency of SOA, MOA, and LOA nanoemulsions was calculated as %, %, and %, respectively, indicating extremely efficient drug encapsulation within the alkyd nanoemulsions.

3.3.5. Measurement of Drug Stability of Nanoemulsions

No significant change () in the drug content of nanoemulsions was measured following storage at 4°C, 25°C, and 45°C for three months when compared with the freshly prepared alkyd nanoemulsions (Table 4). This finding indicates absence of drug-alkyd interactions over an extended time period and the absence of drug degradation on storage of nanoemulsions at an elevated temperature of 45°C.

Table 4: Study of drug stability in alkyd nanoemulsions.

4. Conclusions

Nanoemulsions having a droplet size around 12 nm were successfully prepared from mixtures of palm kernel oil-derived alkyds and aqueous Tween 80 surfactant using the emulsion phase inversion technique. The nanoemulsions exhibited favorable pharmaceutical characteristics, namely, pH values around 6.5, pseudoplastic flow behavior, and impressive physical stability on storage at ambient temperature for three months. Furthermore, the alkyd nanoemulsions were capable of efficiently encapsulating a hydrophobic drug, phenytoin, with no significant drug loss on storage at 4°C, 25°C, and 45°C for three months. Alkyd-based nanoemulsions thus show significant potential as drug carriers for topical and oral administration.

Conflict of Interests

The authors declare no conflict of interests.

Acknowledgments

This project was funded by International Medical University under Project no. IM130272-95000, Malaysia Toray Science Foundation (MTSF) Science and Technology Grant 2014 (IMUR160/2014), and University of Malaya under Project no. PG050-2014A. The authors thank IFFCO (S.E.A) Sdn. Bhd. for providing PKO used in this study.

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