Int. J. Mol. Sci. 2012, 13, 13049-13064; doi:10.

3390/ijms131013049
OPEN ACCESS
International Journal of
Molecular Sciences
ISSN 1422-0067
[Link]/journal/ijms
Article

Formulation Optimization of a Palm-Based Nanoemulsion

System Containing Levodopa
Syafinaz Zainol 1,*, Mahiran Basri 1,2,*, Hamidon Bin Basri 3, Ahmad Fuad Shamsuddin 4,5,
Siti Salwa Abdul-Gani 1, Roghayeh Abedi Karjiban 1 and Emilia Abdul-Malek 1
1
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia; E-Mails: salwa@[Link] (S.S.A.-G.);
[Link]@[Link] (R.A.K.); emilia@[Link] (E.A.-M.)
2
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
3
Department of Medicine, Faculty of Medicine and Health Science, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia; E-Mail: hamidon@[Link]
4
Centre for Drug Delivery Research, Faculty of Pharmacy, Universiti Kebangsaan Malaysia,
Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia; E-Mail: afsna@[Link]
5
Quality Use of Medicines Research Group, Faculty of Pharmacy, Universiti Kebangsaan Malaysia,
Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

* Authors to whom correspondence should be addressed; E-Mails: syafinazzainol@[Link] (S.Z.);

mahiran@[Link] (M.B.); Tel.: +603-8946-7266 (M.B.).

Received: 2 August 2012; in revised form: 26 August 2012 / Accepted: 9 October 2012 /
Published: 11 October 2012

Abstract: Response surface methodology (RSM) was utilized to investigate the influence
of the main emulsion composition; mixture of palm and medium-chain triglyceride (MCT)
oil (6%12% w/w), lecithin (1%3% w/w), and Cremophor EL (0.5%1.5% w/w) as well
as the preparation method; addition rate (220 mL/min), on the physicochemical properties
of palm-based nanoemulsions. The response variables were the three main emulsion
properties; particle size, zeta potential and polydispersity index. Optimization of the
four independent variables was carried out to obtain an optimum level palm-based
nanoemulsion with desirable characteristics. The response surface analysis showed that the
variation in the three responses could be depicted as a quadratic function of the main
composition of the emulsion and the preparation method. The experimental data could be
fitted sufficiently well into a second-order polynomial model. The optimized formulation
was stable for six months at 4 C.
Int. J. Mol. Sci. 2012, 13 13050

Keywords: palm oil; nanoemulsions; levodopa; Response Surface Methodology; central

composite design

1. Introduction

Parkinsons disease (PD) is one of the central nervous system (CNS) diseases which is expected to
rise with increasing lifespan and population demographics in the future. Various types of drugs are
used to treat and relieve the symptoms of the disease including levodopa, ropinirole, apomorphine and
selegiline. However, levodopa is the gold standard for anti-parkinsonian therapy and consequently,
nearly every PD patient eventually receives this drug [1].
At present, 95% of all new potential therapeutics has poor pharmacokinetics and biopharmaceutical
properties [2]. Therefore, it is necessary to produce efficient drug delivery systems in which the drug
molecule reaches only the target site of action, without influencing other organs and tissues.
Nanotechnology plays an important role in therapies of the future as nanomedicines could lower doses
required for efficacy as well as increasing the therapeutic indices and safety profiles of new
therapeutics [3]. The drug delivery systems include liposomes, micelles, nanoemulsion, nanoparticulate
systems and dendrimers.
Nanoemulsion systems are potential carriers for efficient delivery of drugs across the blood-brain
barrier. In general, they are biocompatible, biodegradable, physically stable (particularly nanoemulsion
and microemulsions) and relatively easy to produce on a large scale using proven technology [4]. Their
non toxic and non irritant nature makes them ideal therapeutic agents as they do not damage human
and animal cells [5]. Their long term physical stability confers an additional advantage. Nanoemulsion
in parenteral delivery has been adopted for supplying nutritional requirements, controlled drug
release, vaccine delivery and for drug targeting to specific sites [6]. Intravenous administration of
nanoemulsions is shown to be very advantageous, particularly due to their droplet size of less than
1 m [7]. In addition, the emulsions can minimize the pain associated with intravenously administrated
drugs by exposing the tissue to lower concentrations of the compounds [4].
Palm oil is yielded from the fruit of the Elaeis guineensis tree. As a vegetable oil, palm oil is one of
the promising resources of industrial fats and oils due to its various beneficial properties such as high
thermal, high productivity and oxidative stability. Palm oil has been widely used in the food industry.
It has not been explored in pharmaceutical applications. Palm oil for pharmaceutical application should
be explored and expanded due to its favorable properties such as a long chain triglyceride, nontoxicity
and low cost. In order to meet some application requirements such as pharmaceutical and cosmetic
formulations, there is a need to convert the oil into an emulsion. In the preparation of palm oil
emulsion, organic solvents are not needed. However, until now, there does not seem to have been any
report on the use of palm oil in an emulsion formulation for drug delivery.
Response Surface Methodology (RSM) is a tool which consists of mathematical and statistical
techniques which are derived from the fit of empirical models to the obtained data from experiments.
In order to explain the studied system, linear or square polynomial functions are utilized. Hence, the
experimental conditions can be investigated for the optimization study. RSM has been used broadly to
Int. J. Mol. Sci. 2012, 13 13051

develop and optimize new formulations as it can evaluate all potential factors simultaneously. From
the experimental design, the influence of the formulation variables on responses can be determined and
the effects of factor interaction can be investigated. The advantages of using RSM are reported to be
the reduction in the number of experimental runs needed to evaluate multiple variables and the ability
of the statistical tool to identify interactions [8]. Therefore, it is less laborious and time consuming
compared to studying one-variable at a time [9]. Additionally, this experimental methodology
generates a mathematical model which is presented in graphical form [10].
In this work, levodopa was selected as the targeted drug to be loaded in the nanoemulsion as it is
widely used to treat and relieve the symptoms of PD. To date, levodopa is only available for the patient
in tablet form. Therefore, the aims of this work by using RSM were to formulate an optimal novel
palm-based nanoemulsion containing levodopa and to evaluate simultaneously the main effects and
interaction effects between the factors including composition of oil, lecithin and Cremophor EL as well
as the addition rate on the responses; particle size, zeta potential and polydispersity index.

2. Results and Discussion

2.1. Screening of Variables

A preliminary study was carried out to evaluate the levels of independent variables. Based on the
resultant data, the lower, middle and upper levels of the four independent variables were determined.
Levodopa nanoemulsions showed particle size below 200 nm, narrow size distribution and zeta
potential of more or less 25 mV by restraining the range of oil, lecithin, Cremophor EL composition
and addition rate at levels of 6%12%, 1%3%, 0.5%1.5% and 220 mL/min, respectively.

2.2. Fitting the Response Surface Models

The variation in the particle size, polydispersity index and zeta potential were predicted by
employing response surface methodology as the responses were the function of the emulsion
composition and preparation variables of levodopa loaded nanoemulsions. Table 1 shows the
experimental data obtained for the three response variables based on central composite design (CCD)
matrix. The experimental data was statistically analyzed. The statistic analysis was used to determine
the best fitted model for the four independent variables. The estimated regression coefficients, R2,
adjusted R2, regression (p-value), lack of fit (p-value) and probability values related to the effect of the
four independent variables are shown in Table 2. A positive value in the regression equation represents
an effect that favors optimization due to a synergistic effect, while a negative value indicates an
inverse relationship or antagonistic effect between the factor and the response [11]. It should be
mentioned that non-significant (p < 0.005) linear terms were included in the final reduced model if
quadratic or interaction terms containing these variables were found to be significant (p < 0.05) [12].
In this work, the response surface analysis demonstrated that the second-order polynomial used for
particle size has a higher coefficient of determination (R2 = 0.9757) compared to the polydispersity
index (R2 = 0.9632) and the zeta potential (R2 = 0.9093). The obtained coefficient of determination
showed that more than 90% of the response variation of the particle size, zeta potential and
polydispersity index could be described by RSM models as the function of the main nanoemulsion and
Int. J. Mol. Sci. 2012, 13 13052

preparation variables. It was observed that the lack of fit had no indication of significant (p < 0.05) for
the final reduced model, therefore proving the satisfactory fitness of the response surface model to the
significant (p < 0.05) factors effect (Table 2).

Table 1. The experimental data obtained for the three responses.

Experiment Number R1 R2 R3
1 89.49 23.10 0.14
2 101.87 25.40 0.12
3 100.90 24.10 0.12
4 145.40 28.93 0.23
5 101.85 30.42 0.14
6 101.00 23.10 0.13
7 93.54 36.80 0.16
8 104.70 1.19 0.18
9 107.23 2.76 0.15
10 133.25 26.70 0.13
11 103.47 7.67 0.20
12 96.88 33.80 0.19
13 119.70 35.45 0.22
14 106.70 28.70 0.16
15 186.30 25.45 0.13
16 91.39 36.45 0.15
17 100.48 26.35 0.14
18 107.93 29.65 0.15
19 145.50 18.30 0.16
20 99.87 37.00 0.10
21 108.95 23.05 0.16
22 117.50 30.90 0.32
23 113.80 32.00 0.16
24 84.68 36.10 0.17
25 122.80 26.33 0.19
26 105.10 23.37 0.14
27 88.68 35.88 0.16
28 99.50 20.23 0.13
29 99.47 29.43 0.11
30 87.89 26.10 0.19
R1: Particle Size; R2: potential; R3: Polydispersity index.
Int. J. Mol. Sci. 2012, 13 13053

Table 2. Regression coefficients, adjusted and probability values for the final reduced models.
Regression Coefficient R1 R2 R3
A0 102.958 24.802 0.140
A 3.83 1.938 0.004
B 10.623 1.2 0.0013
C 0.896 5.727 0.009
D 7.206 1.245 0.039
A2 0.155 2.36 0.0017
B2 5.896 0.609 0.0008
C2 0.783 2.622 0.00051
D2 0.855 2.564 0.024
AB 10.3 0.643 0.0069
AC 2.081 1.436 0.0011
AD 6.749 3.825 0.008
BC 2.559 1.628 0.0039
BD 2.674 2.052 0.0067
CD 1.429 0.885 0.0157
R2 0.976 0.909 0.963
R2 (Adjusted) 0.922 0.671 0.882
Regression (p-value) <0.0001 0.0284 0.0003
Lack of Fit (p-value) 0.0514 0.0711 0.2241
A0 is constant, A, B, C and D are the linear, quadratic and interaction coefficients of the quadratic polynomial
coefficient. R1: Particle Size; R2: potential; R3: Polydispersity index.

From Table 2, it was observed that only two independent variables (C and D) exhibited a positive
effect on the response of particle size (R1). For zeta potential (R2), three independent variables (A, B, C)
presented a positive effect while for polydispersity index (R3) all independent variables presented
negative effects except for one variable (D). Coefficients with more than one factor, or higher order
terms in the regression equation, represent the interaction between terms or the quadratic relationship,
respectively which suggest a non-linear relationship between factors and responses [13]. In this
condition, factors can produce a different degree of response than is predicted by the regression
equation if they are varied at different levels or more than one factor is changed simultaneously [11].
All the responses R1, R2 and R3 were affected by the interaction of independent variables, presenting a
quadratic relationship. The interaction effects between A and B and between A and D were favorable
only for response R2. A favorable effect was also noticed for all responses, R1, R2 and R3 for the
interaction between B and C. The interaction between B and D was favorable only for R1. However, it
was observed that the interaction between A and C and between C and D had an inverse effect for all
responses. Quadratic effects of all four independent variables were noticed for all the responses. The
highest and positive quadratic effect for all variables was noticed for R1 while the highest and negative
quadratic effect was noticed for R2.
The coefficient significance of the quadratic polynomial models was evaluated by using Analysis of
Variance (ANOVA). For any of the terms in the models, a large F-value and a small p-value indicated
a more significant effect on the respective response variables [14]. Table 3 shows the effect of
independent variables on the variation of the physicochemical properties of levodopa-loaded
Int. J. Mol. Sci. 2012, 13 13054

nanoemulsions. The independent variables that most affect the particle size of the nanoemulsion for the
linear term were lecithin composition, followed by the linear term of addition rate; the other two linear
terms (oil and Cremophor EL composition) did not indicate any significant effect (p > 0.05). The
quadratic term of lecithin composition also had a significant effect (p < 0.05) on the particle size of
nanoemulsions. Conversely, the effect of the other three quadratic terms was insignificant (p > 0.05).
Furthermore, the interaction between oil and lecithin composition and between oil composition and
addition rate showed a significant effect (p < 0.05) on the particle size of nanoemulsions.

Table 3. Analysis of variance (ANOVA) of regression coefficient of the fitted quadratic equation.
R1 R2 R3
Variables
F Value p Value F Value p Value F Value p Value
Main effects A 3.393 0.0986 1.087 0.3277 0.606 0.4564
B 78.41 <0.0001 0.417 0.5366 0.201 0.6648
C 0.558 0.474 9.492 0.0151 9.242 0.014
D 12.028 0.0071 0.449 0.5218 58.543 <0.0001
Quadratic effects A2 0.019 0.8929 5.527 0.0466 0.404 0.541
B2 27.606 0.0005 0.368 0.561 0.102 0.7567
C2 0.487 0.5027 6.823 0.031 0.034 0.8573
D2 0.581 0.4655 6.522 0.034 75.862 <0.0001
Interaction effects AB 49.143 <0.0001 0.24 0.6377 3.611 0.0898
AC 2.006 0.1904 1.193 0.3065 0.091 0.7703
AD 21.101 0.0013 8.467 0.0196 5.114 0.0501
BC 3.034 0.1155 1.535 0.2505 1.155 0.3104
BD 3.034 0.1021 2.438 0.1571 3.418 0.0975
CD 3.034 0.3562 0.454 0.5195 18.856 0.0019
A: Composition of oil; B: Composition of Lecithin, C: Composition of Cremophor EL; D: Addition rate.
R1: Particle Size; R2: Zeta potential; R3: Polydispersity index.

The variable which exhibited the largest effect on the zeta potential of the nanoemulsion for the
linear term was Cremophor EL composition. The other three variables (oil composition, lecithin
composition and addition rate) showed insignificant effects. The quadratic terms of Cremophor EL
composition, addition rate and oil composition exhibited significant effects on the zeta potential as
well. The interaction between oil composition and addition rate showed a significant effect on the zeta
potential compared to the other interactions.
For the polydispersity index, the linear and quadratic term of the addition rate had the most
significant effect (p < 0.001), followed by the linear term of Cremophor EL composition and
interaction between Cremophor EL composition and addition rate. Thus, it was indicated that in
evaluating the response variation of the polydispersity index, it was important to consider the
Cremophor EL composition and the addition rate.

2.3. Response Surface Analysis

In general, there is a high demand in the pharmaceutical industry for the production of
nanoemulsions with a smaller droplet size (<1 m). Due to the nano-sized and kinetically stable
Int. J. Mol. Sci. 2012, 13 13055

characteristics, nanoemulsions are very efficient in encapsulating and/or solubilising the drugs and can
successfully deliver them to the targeted part of the body. Direct contact of the drug with the body
fluids and tissues can be avoided and the drug is released slowly over a prolonged period of time,
which may lead to minimization of side effects [1517].
For the optimization of levodopa-loaded nanoemulsions, response surface analyses were plotted in
three dimensional model graphs. The response surface plots for particle size, zeta potential and
polydispersity index which are used to interpret the interaction effect of the variables are presented in
Figures 13 respectively. The third and forth factors were kept at constant level. Figure 1A,B
demonstrated that the particle size increases with increasing oil composition. The oil phase
composition influences the physicochemical properties and the stability of parenteral lipid
emulsions [18]. A few explanations need to be considered to describe the observed results. First, with
the rise in the oil content, the droplet disruption process becomes more difficult which is due to an
increase in the flow resistance and hence the droplet break-up rate becomes severely restricted [19,20].
Second, part of the effect can be attributed to the increased rates of collision frequency, particularly at
lower concentration, between the emulsion droplets followed by an ultimate increase of coalescence
frequency which subsequently lead to a higher probability of coalescence of the droplets [21].

Figure 1. Response surface plots showing the interaction effects of (A) lecithin
composition and oil composition as well as (B) oil composition and addition rate on
response. R1, particle size.

(A) (B)

As shown in Figure 1A, the increasing in lecithin content led to an increase in particle size.
However, additional increase in the lecithin composition resulted in a decrease of the particle size. The
increase in particle size may result from an impoverishment of the surfactant at the interface with
increasing surface of the dispersed oil phase [20]. In addition, the increase in particle size may also be
due to an insufficient amount of lecithin to emulsify the oil and the aqueous phase. Decrease of particle
size by further increase in lecithin is due to the fact that the emulsifier plays a vital role in the
formation of emulsion as it lowers the interfacial tension, thereby the Laplace pressure, p is reduced
and the stress required for droplet deformation is reduced [21]. Figure 1B demonstrates increasing
particle size when the oil composition is increased (9%12% w/w) and the addition rate is reduced.
Generally, particle size was reduced when the addition rate was decreased. However, the phenomenon
Int. J. Mol. Sci. 2012, 13 13056

did not occur in this case. The possible reason for this observation might be due to the relatively higher
oil content. The addition rate factor did not help significantly in the emulsification process.
The zeta potential is a stability indicative parameter in colloidal systems like submicron
emulsions [2224] due to electrostatic repulsion. Figure 2 demonstrated that by decreasing the addition
rate and the composition of oil, the zeta potential increased. This observation could be due to the
stabilization mechanisms; electrostatic and steric mechanism. At low addition rate and composition of
oil, the effect of steric stabilization is caused by the incorporation of surfactant and co-surfactant.
However, by increasing the addition rate and oil composition, the surfactant and co-surfactant were
unable to increase the electrostatic repulsion between emulsion droplets, as the amount was
insufficient. In addition, decreasing the addition rate at high range (10.520 mL/min) increased the
zeta potential with increasing oil composition. The reason for this behavior could be attributed to the
strong repulsive Coulomb force between charged particles which counterbalances the Van der Waals
attraction force and this phenomenon is not only contributed by the surfactant role but is also due to
the decreasing addition rate.

Figure 2. Response surface plot showing the interaction effects of oil composition and
addition rate on response R2, zeta potential.

Polydispersity index (PI) characterizes the disperse systems with respect to deviation from the
average size, and values up to 0.250 are acceptable for parenteral emulsions [25]. The polydispersity
index varied from 0 to 1. The composition of materials and the preparation method used play an
important role in the emulsion formulation as both may affect each other and thus influence the
physicochemical properties of the emulsions. Figure 3 demonstrated that the polydispersity index
increased with increasing addition rate and Cremophor EL composition at low range (0.5%0.8% w/w).
The increasing polydispersity index could be due to the high addition rate of the oil phase over the
mixture of the aqueous phase, thus limiting the breaking down of the oil droplet during the
emulsification process. This promoted the formation of a large particle size. The presence of a high
concentration of emulsifier ultimately led to an increase in the flow resistance in the batch
emulsification process which in turn resulted in the larger magnitude of apparent viscosity of the
prepared emulsions [19,26]. The higher concentration of co-emulsifier produced an emulsion with high
viscosity, which can considerably affect the emulsification efficiency. Consequently, this condition
increased the coalescence rate resulting in a larger particle size. The large particles tend to coalesce
Int. J. Mol. Sci. 2012, 13 13057

faster than small particles. This phenomenon contributed to the high polydispersity index as the
distribution of the particle size became broad. In addition, the low amount of Cremophor EL used, was
insufficient to emulsify the emulsion system.
By increasing the addition rate (1120mL/min) and composition of Cremophor
EL (0.85%1.5% w/w) the polydispersity index decreased. The decrease in polydispersity index could
be due to the co-surfactant, Cremophor EL which possesses polyethylene glycols and ethoxylated
glycerol polar covalent parts. Both structures have excellent capability to solubilize many types of
essential oil. Because of these two highly hydrophilic groups in the aqueous phase, the difference in
viscosity between the two immiscible phases is reduced, thereby leading to lowering of the critical
Weber number followed by an increased droplet break-up efficiency [21]. Cremophor EL, which is a
non-ionic surfactant, was chosen as co-surfactant not only due to its great emulsifying properties but
also due to its characteristically low toxicity.

Figure 3. Response surface plot showing the interaction effects of Cremophor EL

composition and addition rate on response R3, polydispersity index.

2.4. Optimization of Responses for Formulating Levodopa Nanoemulsions

By using Design-Expert software, the desirability function was probed to acquire an optimized
formulation. An optimum levodopa nanoemulsion is that with smallest particle size, lowest
polydispersity index and highest zeta potential. The response surface and contour plot were used to
visualize the interaction between the independent variables. By investigating the interaction effect
between the independent variables and evaluating the optimization constraints, the optimum levodopa
nanoemulsion was prepared with a composition of 7.14% oil, 2.2% lecithin, 1.24% Cremophor EL,
and an addition rate of 5.5 mL/min. Based on the optimum formulation, the predicted values of particle
size, zeta potential and polydispersity index are 104.04 nm, 29.18 mV and 0.136, respectively.

2.5. Verification of the Reduced Models

Experimental and predicted values of the responses were compared to check the adequacy of the
response surface equations. The optimized formulation of levodopa-loaded nanoemulsion has a
particle size of 109.63 nm, zeta potential of 31.06 and polydispersity index of 0.174. As displayed in
Table 4, no significant (p > 0.05) difference was noted between the experimental and theoretical
Int. J. Mol. Sci. 2012, 13 13058

predicted value. The sufficiency of the corresponding response surface models was verified based on
the observations.

Table 4. The predicted and observed response values for the optimized nanoemulsion.
Response Predicted Observed
R1: Particle size 104.04 109.63
R2: Zeta potential 29.18 31.06
R3: Polydispersity index 0.136 0.174

2.6. Stability Study

For parenteral emulsions, the droplet size and polydispersity index (PI) are important
physicochemical parameters since large particle sizes are clinically unacceptable due to emboli
formation [22,27]. Figure 4 depicts the particle size and polydispersity index of optimized formulation
over time. All levodopa-containing nanoemulsions prepared based on the recommended optimum
condition were stable at 4 C 1 C during the tested period (6 months). No significant changes of
particle size and polydispersity index were observed over a period of six months. The excellent
stability could be due to the steric stabilizing effect of the non-ionic emulsifier (lecithin) in which a
bulk steric barrier is formed against particle collision. Thus, this phenomenon prevents the occurrence
of flocculation and coalescence. Improved emulsion stabilization could be explained by the presence
of some free emulsifier in micellar form as it plays a vital role in preventing the coalescence after
emulsification and storage [20]. Furthermore, it has been reported that MCT can destabilize the
emulsion with respect to droplet coalescence, while LCT can increase the viscosity of MCT and the
particle size distribution of emulsions, which would increase the stability of emulsions during
storage [23]. In essence, it is hypothesized that the whole nano-system is in a stable state and this
might be due to the rapid absorption of the non-ionic surfactant, lecithin onto the droplet interface

Figure 4. Stability of the optimized levodopa nanoemulsion upon storage at 4 C as a

function of particle size and polydispersity index over storage of 6 months.
Int. J. Mol. Sci. 2012, 13 13059

3. Experimental Section

3.1. Material

Palm oil was purchased from Sime Darby Jomalina Sdn Bhd, Malaysia. The composition of palm
oil is 44.3% palmitic acid, 38.7% oleic acid, 10.5% linoleic acid, 4.6% stearic acid, 1.0% myristic acid,
and 0.9% other material which can be considered as impurities. Medium-chain Triglyceride oil was
purchased from Pharm-D Sendirian Berhad, Malaysia. Pure soy bean lecithin with 70%
phosphatidylcholine (Lipoid S 75) was purchased from Lipoid GmbH, Ludwigshafen-Germany.
Polyethylene glycol 400 (PEG 400) was purchased from Merck, USA. Cremophor EL, a non-ionic
surfactant with pH of 6.0 to 8.0 was purchased from Sigma-Aldrich, France. Glycerol was purchased
from JT Baker, USA. Levodopa was purchased from Noveltek Lifescienceco, Limited, Hong Kong,
China. Water was deionised by Milli-Q filtration system.

3.2. Formation of Nanoemulsions Containing Levodopa

Nanoemulsions were formulated using a mixture of palm and MCT oil containing levodopa as
dispersed oil phase and Mili-Q water as the continuous aqueous phase. Lecithin was dissolved in the
oil phase containing a mixture of palm oil with MCT oil (1:1) at 55 C for 30 min. Levodopa was
added to the oil phase and stirred. PEG 400, Cremophor EL and glycerol were dissolved in the
deionised water. The preparation was continued by adding the oil phase dropwise to the aqueous
solution with continuous stirring using the overhead stirrer (IKA RW 20 Digital, Nara, Japan) at
300 rpm. The mixture was pre-emulsified with a high shear homogenizer (Kinematica, Lucerne,
Switzerland) at 4000 rpm. The pre-emulsification step was performed for 5 min and repeated three
times. The resultant coarse emulsion was subjected to a high pressure homogenizer (Gea Niro Soavi
S.p.a) for 10 cycles at 800 bars. Table 5 depicts the composition of formulated nanoemulsion.

Table 5. Composition of oil and aqueous phase formulated nanoemulsion.

Materials Amount (w/w, %)
Oil phase
Palm Oil 5
MCT Oil 5
Lecithin 3
Levodopa 0.9
Aqueous phase
Polyethylene glycol 400 0.45
Cremophor EL 0.4
Glycerol 2
Deionised water q.s 100

3.3. Experimental Design

A four-factor CCD was utilized to study the effect of oil composition (6%12% w/w, A), lecithin
composition (1%3% w/w, B), Cremophor EL composition (0.5%1.5% w/w, C) and addition rate
Int. J. Mol. Sci. 2012, 13 13060

(220 mL/min, D) on the three response variables; particle size (R1), zeta potential (R2) and
polydispersity index (R3). Hence, based on the CCD, a total of 30 experiments were run using Design
Expert software (version 6.0.6, Stat ease Inc, Minneapolis, USA). The experimental runs involved
16 factorial points, 8 axial points and 6 replicates of centre points at 3 levels. The choice of CCD as the
experimental design is for the following reasons: it is more precise for estimating factor effects, the
interaction effect between factors can be evaluated and permits optimization in the full factor space.
The experimental data was analyzed by response surface regression procedure and the results were
statistically analyzed by corresponding analysis of variances. An appropriate polynomial model was
chosen based on the significant terms (p < 0.005), the least significant lack of fit, coefficient of
variance, the multiple correlation coefficient, and adjusted multiple correlation coefficient provided by
Design-Expert software. The experiments were carried out in randomized order in order to minimize
the effect of unexplained variability on the actual response due to extraneous factors [28]. To
determine the repeatability of the method, the center point was repeated six times. The quadratic model
was obtained in the design of the experimental space and the upper and lower levels are exhibited in
Table 6. The CCD matrix is shown in Table 7.

Table 6. Levels of independent variables in central composite design (CCD).

Coded Levels
Independent variables
Axial () Low Centre High Axial (+)
Palm oil:MCT oil (1:1) (%, w/w) 3 6 9 12 15
Lecithin (%, w/w) 0 1 2 3 4
Cremophor EL (%, w/w) 0 0.5 1 1.5 2
Addition rate (mL/min) 7 2 11 20 29

Table 7. The matrix of central composite design (CCD).

Experiment Number Blocks A B C D
1* Block 1 9 2 1 11
2 Block 1 12 3 0.5 20
3 Block 1 12 3 1.5 2
4 Block 1 6 3 0.5 2
5 Block 1 6 1 0.5 20
6 Block 1 12 1 1.5 20
7* Block 1 9 2 1 11
8 Block 1 6 3 1.5 20
9 Block 1 12 1 0.5 2
10 Block 1 6 1 1.5 2
11 Block 2 6 3 1.5 2
12 * Block 2 9 2 1 11
13 Block 2 12 3 1.5 20
14 Block 2 6 1 1.5 20
15 Block 2 12 1 1.5 2
16 Block 2 12 3 0.5 2
17 * Block 2 9 2 1 11
18 Block 2 6 3 0.5 20
Int. J. Mol. Sci. 2012, 13 13061

Table 7. Cont.
Experiment Number Blocks A B C D
19 Block 2 12 1 0.5 20
20 Block 2 6 1 0.5 2
21 * Block 3 9 2 1 11
22 Block 3 9 2 0 11
23 Block 3 15 2 1 11
24 Block 3 9 2 1 7
25 * Block 3 9 2 1 11
26 Block 3 9 4 1 11
27 Block 3 9 2 2 11
28 Block 3 9 0 1 11
29 Block 3 3 2 1 11
30 Block 3 9 2 1 29
A: Composition of oil; B: Composition of Lecithin, C: Composition of Cremophor EL; D: Addition rate.
* Center point.

3.4. Statistical Analysis

The optimum condition of the independent variables was ascertained by conducting Response
Surface Methodology to predict the variation of material compositions as well as preparation
conditions. The optimal composition and conditions of preparation of levodopa-loaded nanoemulsions
were chosen based on the condition of attaining minimum particle size (R1), maximum zeta potential
(R2) and minimum polydispersity index (R3). By using the polynomial regression equation, the
response surface behavior was explored for the response function (Yi). The generalized response
surface model is as shown below:
Yi = 0 + 1x1 + 2x2 + 3x3 + 4x4 11x12 + 22x22 + 33x32 + 44x42 + 12x1x2 + 13x1x3 + 14x1x4
+ 23x2x3 + 24x2x4 + 34x3x4
Where Yi is the predicted response; 0 is constant; i, ii are the linear, quadratic and interaction
coefficients, respectively [21]. The significant differences between the independent variables were
determined by utilizing Analysis of Variance (ANOVA). Only the significant (p < 0.05) independent
variables effects were involved in the reduced model. The non-significant (p > 0.005) independent
variables were eliminated. Then, the experimental data was refitted to the significant regression
coefficient (p > 0.05). Consequently, the final reduced model was obtained. Two dimensional contour
plots and three dimensional response surface plots were constructed to see the interaction effect of the
variables on the responses. It was suggested that for a good fit of a model, R2 should be at
least 0.80 [21].

3.5. Verification of Models

Quantitative comparison between the theoretical prediction and obtained experimental values was
made to validate the models. In addition, the percentage of the calculated value was also determined.
Int. J. Mol. Sci. 2012, 13 13062

The predicted error is the difference between the experimental value and the predicted value per
predicted value [29].

3.6. Particle Size and Polydispersity Index

Dynamic light scattering was used to analyze the particle size and polydispersity index of the
nanoemulsion by using Malvern Nano ZS90, Malvern, UK. The measurement was performed at a
scattering angle of 173 at 25 C. The nanoemulsions were diluted with deionised water to the required
concentration. Then the diluted emulsions were placed in the cuvette. The count rate was maintained
between 100 and 300 kcps.

3.7. Zeta Potential

Dynamic light scattering was used to measure the zeta potential of the nanoemulsion by using
Malvern Nano ZS90, Malvern, UK. The measurement was carried out at a scattering angle of 173 at
25 C. The nanoemulsions were diluted with deionised water to the required concentration. A folded
capillary electrophoresis cell was used to measure the zeta potential. The count rate was maintained
between 100 and 300 kcps.

3.8. Stability Study

After preparation of the Levodopa-containing nanoemulsion based on the optimized formulation,

the nanoemulsion was observed over a period of 6 months at 4 C or until instability was observed at
room temperature, 25 C. The particle size, polydispersity index and zeta potential were evaluated.

4. Conclusions

The current study showed that Response Surface Methodology is a beneficial tool for carrying out
the optimization study of levodopa nanoemulsion formulations. The variation of the average particle
size, zeta potential and polydispersity index were predicted by employing second order polynomial
regression. Generally, the linear effect of Cremophor EL had a significant effect (p < 0.05) on the zeta
potential and polydispersity index while the addition rate had a significant effect on the particle size
and polydispersity index. Conversely, all the responses were significantly (p < 0.05) affected by the
interaction effect between oil composition and lecithin composition, oil composition and addition rate
and between Cremophor EL and addition rate. The quadratics of all four independent variables had
significant (p < 0.05) effect on the the three response variables studied. The high stability of the
Levodopa-loaded nanoemulsion was due to the stabilizing effect of lecithin and Cremophor EL.

Acknowledgments

The authors gratefully acknowledge the financial support from the Ministry of Science, Technology
and Innovation of Malaysia (MOSTI) under the Nanotechnology Top Down (NND) project number
5489103 and National Science Fellowship (NSF) grant for the scholar, Syafinaz Zainol.
Int. J. Mol. Sci. 2012, 13 13063

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