Fullerene nanoemulsions were formulated in palm kernel oil esters stabilized by low amount of mixed nonionic surfactants. Pseudoternary phase diagrams were established in the colloidal system of PKOEs/Tween 80 : Span 80/water incorporated with fullerene as antioxidant. Preformulation was subjected to combination of high and low energy emulsification methods and the physicochemical characteristics of fullerene nanoemulsions were analyzed using electroacoustic spectrometer. Oil-in-water (O/W) nanoemulsions with particle sizes in the range of 70–160 nm were formed. The rheological characteristics of colloidal systems exhibited shear thinning behavior which fitted well into the power law model. The effect of xanthan gum (0.2–1.0%, w/w) and beeswax (1–3%, w/w) in the estimation of thermodynamics was further studied. From the energetic parameters calculated for the viscous flow, a moderate energy barrier for transport process was observed. Thermodynamic study showed that the enthalpy was positive in all xanthan gum and beeswax concentrations indicating that the formation of nanoemulsions could be endothermic in nature. Fullerene nanoemulsions with 0.6% or higher xanthan gum content were found to be stable against creaming and flocculation when exposed to extreme environmental conditions.
Nanoemulsions are non-equilibrium colloidal systems formed by forcing two immiscible liquids into homogeneous state which is kinetically stable with particle size ranging from 20 to 200 nm [
In drug delivery system, toxicity of the final product depends on the ingredients which constitute the emulsion system. Microemulsions have a presence of noxious cosurfactant and higher concentration of surfactant that leads to unwanted toxicity, whereas nanoemulsions are devoid of such disadvantage. Nonionic surfactants have prominent advantages as compared to anionic or cationic surfactants in regard to lesser toxicity and biodegradability [
The present study intends to develop a novel nanoemulsion for transdermal application using palm kernel oil esters as biocompatible carrier for fullerene. The core of this study is based on characterization of physicochemical behavior of the developed system in emphasizing the involved thermodynamic. Fullerene is a hydrophobic molecule composed entirely of carbon atoms which are interconnected to each other via single and double bonds forming a spherical shape [
There have been a number of reports indicating the utility of fullerene and its derivatives in oxidative stress-related diseases, such as HIV, Parkinson’s disease, and allergy [
In this study, the physical and structural aspects of fullerene nanoemulsions comprised of mixed nonionic surfactants and palm kernel oil esters which are presented here. Different formulations of fullerene stabilized by xanthan gum were prepared by using combination of high and low energy emulsification techniques. The physicochemical properties of the novel formulations were characterized with an emphasis on the thermodynamic and flow behavior.
Powdered fullerene (C60) with 99.5% purity, xanthan gum from
The percentage of fatty acid composition in palm kernel oil esters.
Palm kernel oil esters composition | (number of carbon atoms : number of double bonds) | Percentage (%) |
---|---|---|
Oleyl caproate | C24 : 1 | 0.5 |
Oleyl caprate | C26 : 1 | 5.6 |
Oleyl caprylate | C28 : 1 | 5.9 |
Oleyl laurate | C30 : 1 | 54.1 |
Oleyl myristate | C32 : 1 | 13.9 |
Oleyl palmitate | C34 : 1 | 6.2 |
Oleyl stearate | C36 : 1 | 1.2 |
Oleyl oleate | C36 : 2 | 6.4 |
Oleyl linoleate | C36 : 3 | 1.7 |
Fullerenes were assayed at 160
The phase behavior study for mixed surfactant system (Tween 80 : Span 80) was conducted. The pseudoternary phase diagram was constructed at room temperature (298.0 ± 0.5 K) and was plotted using SigmaPlot version 12 by Systat Software Inc. (San Jose, USA). Classification of phase state was made according to the physical appearance observed which was categorized into isotropic, homogeneous, and two or multiphase regions.
An approach using HLB concept, an empirical parameter, was applied to determine the hydrophilic and hydrophobic content of surfactants and was used to study the phase behavior of the mixtures. This method aims to achieve the required HLB value for a given oil to obtain stable emulsion system. High HLB value indicates high hydrophilicity of the surfactant system or vice versa. The HLB values of mixed binary surfactant systems were calculated by Griffin’s formula which was applicable to nonionic surfactants as shown in [
Preformulation (Pre-F) was selected from the pseudoternary phase diagrams based on the following: low percentage of surfactant to avoid potential adverse toxicology and dermatology effects [ appreciable percentage of dispersed phase (oil phase) to favor the formation of O/W emulsion system which gained the consumer acceptance for most skin care products.
Selected Pre-F was prepared using high energy followed by low energy emulsification technique. Formulations with xanthan gum at varying concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0% (w/w) and beeswax concentrations of 1, 2, and 3% (w/w) were prepared. 0.7% (w/w) of phenonip was added into the formulations as antimicrobial agent. Nanoemulsions were prepared by stirring oil and aqueous phase separately at 400 rpm at 373 K to fully dissolve all the ingredients. The oil phase was added slowly into the aqueous phase and homogenized at 4,000 rpm for 15 min using a high shear homogenizer (PT3100, Kinematica AG, Switzerland). The emulsions formed were then cooled down to room temperature (298 ± 2 K) while stirring at 200 rpm using an overhead stirrer (RW20 digital, IKA-Werke, Germany).
The FNEs mean droplet size was measured using acoustic and electroacoustic spectrometer (DT-1200, Dispersion Technology, USA) equipped with electroacoustic probes and a stirrer. It allows the determination of the distribution and the droplet size down to the nanometer range. During the measurement process, parameters such as ultrasonic attenuation, sound velocity, and acoustic impedance could be determined at different frequencies. The mathematical modeling of these measurements results in the calculation of the droplet size. With electroacoustic spectroscopy, it is unnecessary to carry out sample dilution which allows the characterization of the real concentrated dispersions. Sample dilution will alter the overall system properties of the emulsion due to the changes in actual percentage composition of each component. 100 mL of FNEs was loaded into the spectrometer chamber. The parameters of Debye length and dynamic mobility of the FNEs droplets were also determined with the software provided by feeding the respective conductance data.
The viscosity of colloidal systems was measured using a dynamic shear rheometer (Kinexus rotational rheometer, Malvern Instrument, UK) at a fixed shear rate of 0.1
The rheological behavior of fullerene-based colloidal systems was examined at varying controlled shear rate mode from 0.1 to 100
Densities of FNEs were measured at four different temperatures (298.15, 303.15, 308.15, and 313.15 K) using a densitometer (DM2911, Rudolph Analytical Research, USA). Sample was injected slowly into the valve where the sample moves through the glass tube to ensure that no air pocket was trapped along the tube. The densitometer was calibrated by using pure water and dry air. The densities have precision <±10−5 kgm−3. The pH values of FNEs were measured using a pH meter (S20-SevenEasy, Mettler Toledo, Switzerland) and the conductivities of FNEs were measured using a conductivity meter (SG3-SevenGo, Mettler Toledo, Switzerland). Calibration was carried out using different pH buffer solutions (pH 4, 7, and 10) and conductivity standards (1413
An accelerated storage testing was carried out to predict the long-term physical stability of the nanoemulsions. Samples were subjected to centrifugal force of 4000 rpm for 15 min. In the thermal stress test, FNEs were kept at storage temperatures of 298 K and 318 K for 90 days. FNEs were also tested for freeze-thaw cycles by adding 5 mL of samples into thin-walled glass tubes which were allowed to freeze at 269 K for 24 h and thawed at room temperature for another 24 h. The above step was repeated for three cycles. Visual assessment under cross polarized plates was done to evaluate the physical appearance of the samples.
The pseudoternary phase diagram was constructed on the basis of initial experiments to show the relationship between composition involving PKOEs : fullerene, single surfactant (Tween 80), and water using minimum energy through vortexing. The phase-forming behavior of the PKOEs : fullerene/Tween 80/water system at 298 K is presented in Figure
Pseudoternary phase diagram of PKOEs : fullerene (Flln)/Tween 80 : Span 80/Water in the MSRs of (a) 10 : 0, (b) 9 : 1, (c) 8 : 2, and (d) 7 : 3 at 298 K. ■ = Isotropic; ● = Homogeneous; ▼ = Two-phase.
Mixture of surfactants was used instead of a single surfactant. This combination of surfactants exhibited better performance in many industrial applications [
The HLB values of single and binary surfactant systems of Tween 80 and Span 80 relating to the formation of monophasic region in the pseudoternary phase diagram are shown in Table
Calculated hydrophilic-lipophilic balance (HLB) values of the nonionic surfactants in the single and binary surfactant systems.
Surfactant/mixed surfactant | HLB number |
---|---|
Tween 80 | 15.00 |
Span 80 | 4.30 |
Tween 80 : Span 80 (9 : 1) | 13.93 |
Tween 80 : Span 80 (8 : 2) | 12.86 |
Tween 80 : Span 80 (7 : 3) | 11.79 |
As seen in Figure
The mean droplet size of FNEs with different components concentration ranged from 70 to 160 nm with fitting error less than 5% as tabulated in Table
Composition and physicochemical characteristics of FNE formulations.
Formulation | Components concentration (%, w/w) |
Diameter |
pH | ||||||
---|---|---|---|---|---|---|---|---|---|
PKOEs | Tween 80 | Span 80 | Phenonip | Xanthan gum | Beeswax | Water | |||
Pre-F | 20 | 4 | 1 | 0 | 0 | 0 | 75.0 | 89.7 ± 0.10 | 5.70 |
FNE1 | 20 | 4 | 1 | 0.7 | 0 | 0 | 74.3 | 72.8 ± 0.05 | 5.60 |
FNE2 | |||||||||
(a) | 20 | 4 | 1 | 0.7 | 0.2 | 0 | 74.1 | 112.7 ± 0.12 | 5.66 |
(b) | 20 | 4 | 1 | 0.7 | 0.4 | 0 | 73.9 | 133.5 ± 0.12 | 5.64 |
(c) | 20 | 4 | 1 | 0.7 | 0.6 | 0 | 73.7 | 104.5 ± 0.13 | 5.53 |
(d) | 20 | 4 | 1 | 0.7 | 0.8 | 0 | 73.5 | 90.9 ± 0.10 | 5.47 |
(e) | 20 | 4 | 1 | 0.7 | 1.0 | 0 | 73.3 | 113.6 ± 0.11 | 5.36 |
FNE3 | |||||||||
(a) | 20 | 4 | 1 | 0.7 | 0.2 | 1 | 73.1 | 115.2 ± 0.13 | 5.65 |
(b) | 20 | 4 | 1 | 0.7 | 0.4 | 1 | 72.9 | 144.8 ± 0.08 | 5.61 |
(c) | 20 | 4 | 1 | 0.7 | 0.6 | 1 | 72.7 | 94.9 ± 0.11 | 5.55 |
(d) | 20 | 4 | 1 | 0.7 | 0.8 | 1 | 72.5 | 98.6 ± 0.10 | 5.54 |
(e) | 20 | 4 | 1 | 0.7 | 1.0 | 1 | 72.3 | 99.8 ± 0.15 | 5.40 |
FNE4 | |||||||||
(a) | 20 | 4 | 1 | 0.7 | 0.2 | 2 | 72.1 | 103.5 ± 0.14 | 5.73 |
(b) | 20 | 4 | 1 | 0.7 | 0.4 | 2 | 71.9 | 144.3 ± 0.09 | 5.70 |
(c) | 20 | 4 | 1 | 0.7 | 0.6 | 2 | 71.7 | 114.7 ± 0.15 | 5.66 |
(d) | 20 | 4 | 1 | 0.7 | 0.8 | 2 | 71.5 | 91.6 ± 0.08 | 5.68 |
(e) | 20 | 4 | 1 | 0.7 | 1.0 | 2 | 71.3 | 99.0 ± 0.13 | 5.34 |
FNE5 | |||||||||
(a) | 20 | 4 | 1 | 0.7 | 0.2 | 3 | 71.1 | 110.8 ± 0.11 | 5.68 |
(b) | 20 | 4 | 1 | 0.7 | 0.4 | 3 | 70.9 | 155.0 ± 0.12 | 5.64 |
(c) | 20 | 4 | 1 | 0.7 | 0.6 | 3 | 70.7 | 124.6 ± 0.14 | 5.58 |
(d) | 20 | 4 | 1 | 0.7 | 0.8 | 3 | 70.5 | 103.7 ± 0.13 | 5.45 |
(e) | 20 | 4 | 1 | 0.7 | 1.0 | 3 | 70.3 | 100.7 ± 0.13 | 5.40 |
The pH of a healthy human skin on average is 5.5 [
Effects of beeswax and xanthan gum concentration on (a) electrical conductivity, (b) surface charge, (c) dynamic mobility, and (d) Debye length of FNEs.
The electrical conductivity of the FNEs is also related to the ability of emulsion droplets to conduct an electric current which correlates to their mobility. The dynamic mobility of the colloidal systems is presented in Figure
The shear viscosities,
Viscosities of Pre-F and FNEs at different temperatures with varying concentration of xanthan gum containing 0% (●), 1% (○), 2% (▼), and 3% (Δ) of beeswax.
Steady shear flow properties of FNEs with different compositions of xanthan gum and beeswax concentrations were measured over a range of shear rates. The flow behaviors of FNEs are particularly important to predict the end-product’s texture attribute and to ease the industrial handling processes such as flow pump and agitation tank [
Power law model parameters for selected FNE formulations.
Formulation | Components concentration (%, w/w) | Consistency coefficients |
Flow behavior indices, |
Regression |
|
---|---|---|---|---|---|
Xanthan gum | Beeswax | ||||
FNE3 | |||||
(b) | 0.4 | 1 | 3.173 | 0.202 | 0.983 |
(c) | 0.6 | 1 | 7.305 | 0.144 | 0.976 |
(d) | 0.8 | 1 | 9.438 | 0.137 | 0.988 |
(e) | 1 | 1 | 12.49 | 0.147 | 0.99 |
FNE4 | |||||
(b) | 0.4 | 2 | 4.318 | 0.217 | 0.981 |
(c) | 0.6 | 2 | 8.742 | 0.104 | 0.98 |
(d) | 0.8 | 2 | 10.32 | 0.137 | 0.971 |
(e) | 1 | 2 | 15.78 | 0.132 | 0.984 |
FNE5 | |||||
(b) | 0.4 | 3 | 5.247 | 0.19 | 0.986 |
(c) | 0.6 | 3 | 8.886 | 0.143 | 0.981 |
(d) | 0.8 | 3 | 11.02 | 0.141 | 0.98 |
(e) | 1 | 3 | 21.32 | 0.095 | 0.974 |
Shear rate dependence of steady shear viscosity for FNEs with 1% (w/w) of beeswax with different xanthan gum concentrations.
The activation enthalpy,
The values of molar volume were kept constant as the experimental densities of the nanocolloidal systems showed very insignificant dependence over temperature. Furthermore, the values of
The activation parameters for FNEs with different compositions are tabulated in Table
Temperature-dependent activation parameter for PKOEs/Tween 80 : Span 80/water systems.
Formulation | Δ |
Δ |
Δ |
|||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Temperature, K | Temperature, K | Temperature, K | ||||||||||
298 | 303 | 308 | 313 | 298 | 303 | 308 | 313 | 298 | 303 | 308 | 313 | |
Pre-F | 23.8 | 16.2 | 8.0 | −0.7 | 38.8 | 38.4 | 38.1 | 37.9 | −50.5 | −75.3 | −101.6 | −129.1 |
FNE1 | 96.1 | 67.3 | 36.4 | 3.3 | 39.9 | 38.7 | 38.4 | 38.3 | 188.6 | 92.0 | −10.7 | −117.8 |
FNE2 | ||||||||||||
(a) | 47.6 | 29.3 | 9.8 | −11.1 | 57.6 | 56.5 | 56.0 | 55.5 | −33.8 | −92.9 | −156.1 | −221.6 |
(b) | 98.9 | 80.2 | 59.9 | 38.3 | 60.9 | 59.8 | 58.5 | 58.1 | 127.5 | 63.7 | −1.7 | −72.7 |
(c) | 107.5 | 82.1 | 54.9 | 25.7 | 62.9 | 61.7 | 60.9 | 60.6 | 149.6 | 64.1 | −26.3 | −121.3 |
(d) | 108.2 | 69.2 | 27.3 | −17.4 | 64.0 | 62.5 | 62.2 | 62.0 | 148.3 | 18.5 | −120.0 | −263.8 |
(e) | 81.7 | 60.8 | 38.3 | 14.3 | 64.7 | 63.9 | 63.3 | 63.3 | 57.1 | −13.7 | −88.0 | −166.6 |
FNE3 | ||||||||||||
(a) | 52.8 | 59.2 | 65.9 | 72.9 | 57.8 | 57.1 | 56.3 | 55.4 | −16.8 | 3.7 | 25.0 | 46.9 |
(b) | 91.7 | 71.9 | 50.6 | 27.9 | 61.3 | 60.0 | 59.5 | 59.0 | 102.0 | 36.0 | −35.4 | −109.0 |
(c) | 96.5 | 72.3 | 46.3 | 18.5 | 63.2 | 61.8 | 61.2 | 60.7 | 111.8 | 31.1 | −55.0 | −144.7 |
(d) | 107.0 | 77.0 | 44.9 | 10.5 | 64.1 | 62.8 | 62.2 | 62.1 | 144.0 | 43.3 | −63.1 | −174.8 |
(e) | 91.0 | 65.8 | 38.8 | 9.9 | 65.0 | 64.1 | 63.5 | 63.5 | 87.0 | 2.2 | −87.1 | −181.2 |
FNE4 | ||||||||||||
(a) | 63.3 | 64.7 | 66.0 | 67.4 | 57.9 | 57.2 | 56.4 | 55.7 | 18.0 | 21.5 | 25.2 | 28.4 |
(b) | 69.0 | 52.2 | 34.2 | 15.0 | 61.4 | 60.4 | 59.7 | 59.3 | 25.3 | −30.4 | −89.1 | −151.0 |
(c) | 81.2 | 58.8 | 34.7 | 9.0 | 63.5 | 62.2 | 61.6 | 61.1 | 59.5 | −14.7 | −94.1 | −176.4 |
(d) | 98.1 | 73.9 | 48.0 | 20.2 | 64.3 | 63.1 | 62.6 | 62.3 | 113.4 | 32.0 | −54.3 | −144.5 |
(e) | 78.3 | 60.4 | 41.1 | 20.4 | 65.2 | 64.3 | 63.8 | 63.6 | 44.2 | −16.6 | −80.7 | −148.2 |
FNE5 | ||||||||||||
(a) | 68.0 | 61.1 | 53.7 | 45.7 | 58.2 | 57.5 | 56.7 | 56.2 | 32.7 | 8.8 | −15.8 | −42.7 |
(b) | 83.7 | 62.1 | 38.9 | 14.1 | 61.9 | 60.6 | 60.0 | 59.5 | 73.1 | 1.6 | −75.2 | −154.7 |
(c) | 95.2 | 68.7 | 40.2 | 9.8 | 63.9 | 62.6 | 62.1 | 61.8 | 105.3 | 16.6 | −77.6 | −176.1 |
(d) | 81.7 | 60.0 | 36.8 | 11.9 | 64.4 | 63.5 | 63.3 | 63.1 | 57.9 | −14.9 | −93.0 | −173.8 |
(e) | 73.5 | 56.1 | 37.5 | 17.5 | 65.2 | 64.4 | 63.9 | 63.7 | 27.6 | −31.0 | −92.7 | −157.8 |
Assessment of long-term stability of FNEs shelf life under environmental storage conditions can be very tedious and time consuming which is considered uneconomical. The development of products ought to be fast-paced yet reliable to meet the demand of the consumers. Thus, FNEs were let to experience a variation of extreme storage conditions to predict the samples’ ability to withstand over a period of time. Centrifugation can accelerate the rate of creaming or sedimentation which demonstrates that the breakdown of an emulsion can be related to the action of gravitational force. O/W emulsion system often exhibits creaming rather than sedimentation due to the lower density of the oil droplet compared to the aqueous medium. The result of the accelerated stability study of FNEs is shown in Table
Accelerated stability assessment of the Pre-F and FNEs.
Formulation | Centrifugation test at 4000 rpm for 15 min | 298 K | 318 K | Freeze-thaw cycles |
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Pre-F |
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FNE1 |
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FNE2 | ||||
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FNE3 | ||||
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FNE4 | ||||
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FNE5 | ||||
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Note:
Sample storage at elevated temperature contributed to the higher kinetic energy in the Brownian motion of the oil droplets. This is possibly to speed up the movement and more collision between the oil droplets at higher temperature. Only FNEs which were stable against centrifugation were tested on storage at elevated temperature. Our results showed that FNEs with more than 0.6% xanthan gum (w/w) were able to maintain the homogeneity of the emulsion only up to 90 days. The phase separation decreases with the increase in xanthan gum concentration possibly due to the formation of more ordered polymer structure formed within the emulsion system.
In the freeze-thaw cycle, FNEs, except Pre-F and FNE1, were found to be stable by maintaining their homogenous state. On freezing the samples, oil droplets segregated themselves from the emulsion by the crystallized ice particle resulting in the disruption of lipid film surrounding the droplets. When the samples were thawed, the droplets melted and immediately coalesced between approaching droplets, resulting in oiling off from the emulsions which was indicated by phase separation. However, FNEs with xanthan gum maintained their homogeneity whereas Pre-F and FNE1 (without xanthan gum) exhibited phase separation. Xanthan gum being dissolved in the water reduced the reassociation of oil droplets and that phenomenon in the damaged network led to less syneresis [
Pseudoternary phase diagrams served as template to obtain emulsion-based preformulation and were further developed into nanoemulsions with small particle size (<200 nm) by high-low energy emulsification method. Mixed surfactant systems demonstrated better performance in obtaining larger monophasic zones compared to single surfactant system. The relationship between viscosity and temperature of the fullerene nanoemulsions agreed well with Arrhenius equation at all tested temperatures of 298–313 K. In the presence of xanthan gum, fullerene nanoemulsions exhibited pseudoplastic behavior. The activation enthalpy for viscous flow (
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to acknowledge financial support from Ministry of Higher Education (MOHE), Malaysia, under the Fundamental Research Grant Scheme (FRGS) (02-01-13-1235FR) and My Brain15.