Bull. Pharm. Sci., Assiut University, Vol. 36, Part 1, 2013, pp. 31-47.

Bulletin of Pharmaceutical Sciences

Assiut University

FORMULATION AND EVALUATION OF ACYCLOVIR

MICROEMULSIONS
E. Ramadan, Th. Borg*, G. M. Abdelghani and N. M. Saleh
Department of Pharmaceutics, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
Certain oils and surfactants were screened to select the most suitable ones for the
preparation of microemulsions. Labrafil M 1944 CS, Labrafil M 2125 CS and Capryol 90
were selected as oils while Tween 80 was selected as a surfactant. Tween 80 was mixed with nbutanol (n-Bu) at different w/w ratios (Km) 1:2, 1:1, 2:1 and 3:1 as surfactant to cosurfactant
(S/CoS) respectively. Pseudoternary phase diagrams were constructed using the selected oils
(Labrafil M 1944 CS, Labrafil M 2125 CS and Capryol 90) with the different Km ratios.
Microemulsion formulations were prepared using Labrafil M 1944 CS, Tween 80 and n-Bu
with Km 2:1. The physicochemical characteristics involving viscosity, refractive index (RI),
conductivity and pH were determined for these systems. In addition, the solubility of acyclovir
in the prepared microemulsion systems (MEs) was measured. Acyclovir was incorporated to
three MEs (ME10, ME50 and ME80) representing three different regions in the phase diagram;
water in oil (w/o), bicontinuous (Bc) and oil in water (o/w) respectively. All of the prepared
formulations were subjected to thermodynamic stability studies. The droplets size,
polydispersity index (PDI) and zetapotential (ZP) of both before and after drug incorporation
were determined. Acyclovir release from drug loaded MEs was determined and the kinetic of
the release data was calculated.
The conductivity and viscosity results proved the presence of three regions in the phase
diagram (w/o, Bc and o/w). The refractive index showed that the prepared MEs were
transparent and isotropic. All the formulations were thermodynamically stable. The droplet size
of drug loaded MEs was higher than that of corresponding unloaded ones but all have droplet
size in nano range. PDI was found to be less than 0.5 and ZP in the range of -0.101 to 2.5 mV.
Release of acyclovir from ME80 (0.1, 1.99, 17.98 and 79.92 w/w for acyclovir, oil, S/CoS and
water respectively) was the highest among the other ones. Model of non-Fickian "anomalous"
transport release was the mechanism of drug release from the selected MEs.
In conclusion, stable isotropic microemulsion systems using Labrafil M 1944 CS, Tween
80 and n-butanol as oil, surfactant and cosurfactant respectively with Km 2:1 could be
formulated. The viscosity and electric conductivity results proved the presence of three areas;
w/o, Bc and o/w in phase diagram. Acyclovir could be loaded into different microemulsion
systems. The percent drug release increased by increasing the water content of microemulsion.

INTRODUCTION
Microemulsions are small sized droplets
with high surface to volume ratios and
modifiable
surfaces1.
They
are
thermodynamically stable transparent, single
optically isotropic liquid system2. These
properties allow infinite shelf-life3. In addition,
the rate of penetration of drug is much faster
from microemulsion systems than from other

Received in 27/1/2013 & Accepted in 30/6/2013

*Corresponding author: Th. Borg, E-mail: mborgun@yahoo.com

drug delivery vehicles and having controlled

drug release rates, slow degradation and target
specificity4.
MEs
can
enhance
the
bioavailability of drugs via topical and
systemic routes5.
Acyclovir is a synthetic purine nucleoside
antiviral drug6. Acyclovir absorption in the
gastrointestinal tract is slow, variable and
incomplete7. Its bioavailability after oral
administration ranges from 10% to 20% of the

E. Ramadan, et al.

administered dose8. Acyclovir is one of those

agents that belong to class III according to the
Biopharmaceutical
Classification
System
(BCS) because of its high solubility and low
permeability9. Acyclovir is one of the most
effective and selective drug for treatment of
herpes viruses infections10. Herpes simplex
virus (HSV) causes a variety of infections
including oro-facial and cutaneous herpes,
genital herpes, chicken pox, varicella zoster
infections and herpes keratitis.
Hence study aim was to formulate and
characterize MEs containing acyclovir. The
prepared systems have to be investigated by
measuring conductivity, viscosity, droplet size,
PDI and ZP. In addition, the acyclovir release
and its mechanism were also determined.
EXPERIMENTAL
Materials
Acyclovir (ACV) was kindly supplied by
Memphis pharmaceutical Co., Cairo, Egypt.
Transcutol P (Highly purified diethylene
glycol
monoethyl
ether),
Labrasol
(Caprylocapryol
macrogol-8-glycerides),
Capryol 90 (Propylene glycol monocaprylate
Type II), Lauroglycol 90 (Propylene glycol
monolaurate Type II), Labrafac Lipophile WL
1349 (Medium chain triglycerides), Labrafil
M 1944 CS (Oleoyl macrogol-6-glycerides)
and Labrafil M 2125 CS (Linoleoyl macrogol6-glycerides) were kindly donated by
Gattefoss, France. Isopropyl myristate (IPM)
was purchased from Fluka, Sigma Aldrich
Chemie GmbH, Belgium. Cellophan membrane
(semipermeble membranes spectrapor) was
obtained from Fisher Scientific Co., standard
cellulose dialysis tubing 100 ft per roll, m.w.
Cutoff: 12,000-14,000. All other chemicals
were of analytical grade.
Methodology
Selection of oils and surfactants for MEs
The solubility of ACV in Capryol 90,
Lauroglycol 90, Labrafac Lipophile WL
1349, Labrafil M 2125 CS, Labrafil M 1944
CS and IPM as oil phase and Labrasol,
Tween 40, Tween 60, Tween 80 and
Transcutol P as nonionic surfactants and n-Bu
as cosurfactant was determined.
The experiment was conducted according
to the method reported by Brajesh et al.11.

32

Briefly, an excess amount of ACV was added

to 5 ml of the vehicle in stoppered vials
separately and shaken continuously at 25C for
72 hrs to get equilibrium. The equilibrated
samples were removed and centrifuged at 5000
rpm for 30 min. The supernatant was separated,
filtered through a membrane filter (0.45 m)
and after appropriate dilution with methanol;
solubility was determined spectrophotometrically
by
UV
Spectorophotometer
(Shimadzu, UV-150-02, Sersakusho, Ltd,
Kyoto, Japan) at max 252 nm. The oil
preparations which were highly solubilized
ACV were selected for further study.
Construction of phase diagrams
Tween 80 and n-Bu were selected as
surfactant (S) and cosurfactant (CoS)
respectively. To prepare S/CoS mixtures, they
are weighed at different w/w ratios which
called Km (1:2, 1:1, 2:1 and 3:1 w/w)
respectively in separate tubes. The prepared
mixtures were vortexed vigorously for 30
second (Tube vortex mixer, Gemmy Industrial
Corporation, Taiwan). On the other hand
Capryol 90, Labrafil M 1944 CS and
Labrafil M 2125 CS were selected as oil
phases. The oil and S/CoS were mixed in ratios
of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1
respectively. Distilled water was added in daily
increments of 10%-90% to the prepared
mixtures of oil and S/CoS, then mixed by
vortex mixer for 5 min and left to equilibrate
for 24 hrs at ambient temperature. Following
the addition of each aliquot of water phase, the
mixture was visually examined for their
transparency and clarity. Points from clear to
turbid and turbid to clear were designated as
emulsion and microemulsion, respectively.
Pseudoternary phase diagrams were
constructed to obtain the components and the
concentration ranges that produced the greatest
area of microemulsion. Based on these
findings, appropriate oil and S/CoS with
selected percentage ratios were used in the
preparation of MEs containing ACV.
Preparation of MEs
Table 1 shows the components of the
formulated
MEs.
Surfactant/Cosurfactant
mixture (S/CoS) was prepared in 2:1 w/w ratio
(Km) using Tween 80 and n-Bu respectively.
Then, a stock mixture of the selected oil

Table 1: The components of the prepared microemulsions.

Oilb

(S/CoS)c

Water

ME1

81

10

ME2

72

20

ME3

63

30

ME4

54

40

ME5

45

50

ME6

36

60

ME7

27

70

ME8

18

80

ME9

90

ME10

0.05

8.99

80.96

9.995

ME50

0.1

4.99

44.96

49.95

ME80

0.1

1.998

17.98

79.92

Plain MEs
ACVa
loaded
MEs
a

acyclovir
Tween 80 : n-Bu (2:1 w/w respectively)

(Labrafil M 1944 CS) and S/CoS mixture in a

ratio of 1:9 (w/w%) respectively was prepared.
To prepare a microemulsion, the calculated
amounts of oily phase (stock mixture) were
weighed and the calculated amount of aqueous
phase was added and mixed gently till
homogenity.
For drug loaded microemulsion, ACV was
added to the previously prepared MEs and
dissolved under ultrasonication (Ultrasonic
homogenizer, Chicago, USA) at output control
of 5 and pulsed duty at 90% duty cycle for 3
min or until complete solubilization of ACV.
Determination of ACV solubility in MEs
MEs prepared using Labrafil M 1944 CS
were tested for their ability to dissolve ACV in
order to determine the maximum drug loading
capacity of them. Excess amounts of ACV (50
mg) were added to the prepared plain MEs and
water in separated stoppered tubes. The tubes
were continuously shaken at 25C for 24 hrs.
Excess undissolved ACV was removed by
centrifugation at 5000 rpm for 30 min. The
supernatant was separated, filtered through a
membrane filter (0.45m).
After appropriate dilution with methanol,
solubility of ACV in MEs was determined
spectrophotometrically by UV at max 252 nm

Total Weightd

100

ACVa

MEs

Labrafil M 1944 CS
(% w/w)

versus blank. The blank was prepared by the

same manner but without ACV.
Physicochemical properties evaluation of the
prepared MEs
The pH values of the MEs were measured
by a pH meter (pH-meter, Beckman
Instrumentals fullerton, CA 92634, USA) at
ambient conditions. However, refractive
indexes of different MEs were measured using
Abbe refractometer (NAR- 4T, Atago, Japan)
in presence of a visible light source. The
experiment included placing one drop of
microemulsion on the refractometer prism and
RI value was determined. The percent
transmittance (T%) of MEs were measured at
650 nm using UV spectrophotometer
(Shimadzu, UV-150-02, Sersakusho, Ltd,
Kyoto, Japan) with distilled water as a blank.
While, the electroconductivity of the MEs were
measured using the method reported by Ghosh
et al.7. In this method for the conductivity
(mS/m) measurement, the tested MEs were
prepared with a 0.01 N aqueous solution of
sodium chloride instead of distilled water.
Electrical conductivity measurements were
performed at ambient temperature on points
along the S/CoS: Lab 1944 9:1 dilution line of
the diagram obtained with Km 2:1 using

33

E. Ramadan, et al.

conductivity/total dissolved solids meter

(Mannix, Model DCM 22CT, New York,
USA). The electrode was dipped in the
microemulsion samples until equilibrium was
reached. The viscosities of MEs were measured
with rotational viscometer (Rotaional digital
viscometer, Myr Version, Spain) which was
equipped with spindle L2 & L1. The
measurement was done at ambient temperature.
Size analysis, homogeneity and morphology
of MEs droplets
The mean droplet size, Zeta potential and
polydispersity index of MEs droplets before
and after ACV incorporation were measured by
photon correlation spectroscopy instrument
(Marlven, ZEN 3600, UK) at 25C. The
morphology
before
and
after
ACV
incorporation were checked by Transmission
electron microscope (TEM) (JOEL 1010
Tokyo, Japan) using method reported by Cho et
al.12. Microemulsion were placed on copper
grids with films and dried in air for 10 min.
They were then observed using TEM. The MEs
were photographed without dilution because it
can produce a phase transition or a molecular
reorganization13.
Accelerated physical stability tests of MEs
The method reported by Frantzen et al.14
was used to evaluate the physical stability of
MEs. MEs were centrifuged at 5000 rpm for 30
minutes. The formulations that did not show
any phase separations were taken for the
heating and cooling cycle. Six cycles between
refrigerator (4C) and oven (45C) (Gering
model SPA-GELMAN, Instrument No. 16414,
Germany) with storage at each temperature for
48 hrs were done. The stable systems were
subjected to a freeze-thaw cycle test. Three
freeze-thaw cycles were done for the
formulation between 21C and 25C. The
formulations
were inspected regarding
creaming, coalescence and phase separation or
cracking
and
those
that
survived
thermodynamic stability tests were selected for
further study.
Release of ACV from medicated MEs
Release experiments were conducted in
modified Franz-type diffusion cells with
diffusional surface area of 7.07 cm2 using
equilibrated
semipermable
cellophane

34

membrane in phosphate buffer (pH 7.4). The

release experiments were conducted for 8 hrs
with microemulsion amount equivalent to 5 mg
ACV in the donor chamber. The donor
chambers were covered using aluminum foil to
prevent evaporation of n-Bu content. The
solution in the receptor compartment was
stirred at 100 rpm and temperature was
maintained at 370.2C throughout the
experiment.
One milliliter sample was withdrawn from
the receiver compartment at predetermined
time intervals and replaced with equal volume
of fresh phosphate buffer pH 7.4 equilibrated at
370.2C. ACV concentration was determined
spectrophotometrically at ACV max (252 nm).
Three replicates of each experiment were
performed and the average was calculated.
Analysis of ACV release data
In order to determine the release model
which describes the pattern of drug release
across cellulose membrane, the release data
were analyzed with the following mathematical
models: zero order kinetic; first order kinetic15,
Higuchi equation16 and Korsmeyer-Peppas
equation17.
Zero-order kinetics:
F= Kot
First-order kinetics:
ln(1F) = K1t
Higuchi model:
F= KHt1/2
Korsmeyer-Peppas model: F= Kptn
Where F represents the fraction of drug
released in time t, Ko is the zero order release
constant, K1 is the first-order release constant,
KH is the Higuchi dissolution constant, Kp is
the Korsmeyer-Peppas release rate constant and
n is the diffusion exponent.
RESULTS AND DISCUSSION
Selection of oils and surfactants
The results of ACV solubility in oils,
surfactants and n-Bu revealed that the
maximum solubility of ACV in surfactants was
achieved by Tween 80 (3.3140.224 mg/ml)
and Tween 40 (3.2430.177 mg/ml), with
nonsignificant difference (P> 0.05). However,
a significant decrease (P< 0.05) of drug
solubility was noticed in Transcutol P
(0.8310.104 mg/ml), Tween 60 (0.780.014
mg/ml) and Labrasol (0.620.031 mg/ml)
(Table 2).

Table 2: Solubility of acyclovir in oils, surfactant and cosurfactant.

Surfactants

Screened components

Oils

Cosurfactant

Labrasol
Tween 40
Tween 60
Tween 80
Transcutol P
n- Butanol
Capryol 90
Labrafil M 1944 CS
Labrafil M 2125 CS
LabrafacLipophile WL
1349
Isopropyl Myristate
Lauroglycol 90

The higher solubility of ACV in Tween 80

and Tween 40 more than Tween 60 could be
attributed to the effect of hydrophilic-lipophilic
balance (HLB) of these polysorbates on
solubilization of the drug. Tween 60, being the
most hydrophobic surfactant (HLB 14.9),
solubilized smaller amount of ACV. Replacing
Tween 60 with more hydrophilic polysorbates
Tween 40 and Tween 80 with higher HLB 15.6
and 15 respectively, increased solubilization of
ACV. This is in agreement with Kogan et al.18.
Tween 80 (polyoxyethylene 20 sorbitan
monooleate, HLB 15) has suitable hydrophilic
surfactant properties19. Moreover, it was
reported that Tween 80 which is an ester of the
unsaturated C18 fatty acid, oleic acid, is more
effective in forming a microemulsion than
other polysorbate of the saturated fatty acid
such as Tween 20, Tween 40 and Tween 6020.
Tween 80 has been reported to accelerate
permeation of drugs such as hydrocortisone
and lidocaine and it has minimal toxicity13&21.
So that Tween 80 was selected as a surfactant
for the preparation of MEs.
Short chain amphiphilic nature alcohol
enables formulation of microemulsion with a
variety of oily phase and surfactant22. It was
reported that alkanols with higher carbon
numbers facilitate microemulsion formation
and it was more satisfactory with addition of nBu than methanol or ethanol as CoS5. n-Bu has
been reported to be an enhancer for
levonorgesterol23. So that, n-Bu was selected as
a CoS for the preparation of MEs.

Solubility (mg/ml)
mean S.D.
0.620 0.031
3.243 0.177
0.780 0.014
3.314 0.224
0.831 0.104
0.112 0.004
0.450 0.030
0.390 0.014
0.330 0.042
0.215 0.019
0.071 0.001
0.022 0.000

As shown in table 2, each of Capryol 90,

Labrafil M 1944 CS and Labrafil M 2125 CS
exhibit good solubilization properties of ACV
(0.450.03, 0.390.014, 0.330.042 mg/mL
respectively). Hence, Capryol 90, Labrafil M
1944 CS and Labrafil M 2125 CS were
selected for further studies due to their abilities
to solubilize ACV more than other oils. The
higher solubility of ACV in these three oils
than other oils may be attributed to the polarity
of the poorly soluble drugs that favor their
solubilization in small or medium molar
volume oils, such as medium chain
triglycerides or mono- or diglycerides of fatty
acids13. These oils have low HLB (4) and can
be used in selfemulsifying lipidic formulations.
These findings are extensively explained by
Friend et al.23, Yin et al.24 and Balakrishnan
et al.25.
To allow presentation of the drug in MEs
consisted of oil, nonionic surfactants,
cosurfactants and drug, MEs should be clear
and isotropic liquid at ambient temperature and
should have good solvent properties26.
Therefore, Capryol 90, Labrafil M 1944 CS
and Labrafil M 2125 CS, Tween 80 and n-Bu
were selected as oils, surfactant and
cosurfactant respectively for subsequent
construction of phase diagram.
Construction of phase diagram
Pseudoternary phase diagrams were
constructed to obtain appropriate components
and their concentration ranges for the MEs. So

35

E. Ramadan, et al.

that, phase diagrams using different Km 1:2,

1:1, 2:1 and 3:1 were constructed and the effect
of surfactant and cosurfactant mass ratio on
microemulsion formation was evaluated for
further optimization of the system.
Effect of Km on the microemulsion region
Figure 1 shows pseudoternary phase
diagrams consisted of Labrafil M 1944 CS (A,
D, G, J), Labrafil M 2125 CS (B, E, H, K) and
Capryol 90 (C, F, I, L) as oils, Tween 80 as
surfactant and n-Bu as cosurfactant with Km
1:2, 1:1, 2:1 and 3:1 respectively. The
transparent microemulsion region (ME) which
composed of constituents including Capryol
90 or Labrafil M 1944 CS or Labrafil M
2125 CS, Tween 80, n-Bu and water are
presented in phase diagrams. The rest of the
phase diagram represents the turbid and
conventional emulsions based on visual
observation. No distinct conversion from w/o
to o/w microemulsion was observed. The effect
of different Km values (1:2, 1:1, 2:1 and 3:1) on
the ME area of the system consisted of Labrafil
M 1944 CS, Tween 80 and n-Bu as oil,
surfactant and cosurfactant respectively is
shown in figure 2.
It was found that addition of cosurfactant
with surfactant in equal amounts (Km 1:1
respectively) produces ME area which is larger
than that of Km 1:2 and smaller than the
corresponding one of Km 2:1 (2:1 > 1:1 > 1:2).
When the surfactant concentration is further
increased to Km 3:1 (Fig. 1), a decrease in ME
area was observed to be less than that of Km
2:1. This increase in ME area from Km 1:2 to
2:1 was from water rich side (water: S/CoS mix
miceller phase) of phase diagram toward the oil
rich region (oil-water axis), indicating that by
increasing the surfactant concentration, the
maximum amount of water and drug that could
be solubilized into the microemulsion
increased.
The expansion of ME accompanying Km
increase may be due to that at higher
concentration of surfactant (larger Km value),
the amount of micelles will be reduced which
consequently decreased the solubilization
capacity
of
microemulsion.
Large
microemulsion regions indicate the flexibility
of the surfactant/cosurfactant film, which
allows the existence of continuous structural

36

transitions with an increasing water phase

volume fraction in the oil and S/CoS mixture.
This finding is in agreement with Zhang
et al.27 (nimodipine in microemulsion system of
Labrafil M 1944 CS, Cremophor RH 40,
ethanol and water), Djordjevic et al.28
(diclofenac diethylamine in a microemulsion of
Isopropyl myristate, Labrasol, Plurol Oleique
and water) and Yuan et al.9 (microemulsion
system of meloxican in Isopropyl myristate,
Tween 85, ethanol and water).
It was concluded that, when surfactant
concentration was increased in comparison to
cosurfactant, ME area increased up to Km 2:1,
however in Km 3:1, it was decreased. This
indicating that the optimum emulsification has
been achieved up to Km 2:1. The small ME area
at Km 3:1 could be attributed to the relatively
lower content of cosurfactant (n-Bu) which is
polar with its high tendency to incorporate into
water in the microemulsion systems. The
decrease of cosurfactant content may lead to
decrease of the overall hydrophilicity of the
S/CoS mixture, so the area of o/w
microemulsion was small. Similar results were
observed by Azeem et al.29 (ropinirole in a
microemulsion system of Capryol 90, Tween
20, Carbitol and water) and Yuan et al.9.
Finally, S/CoS mixture at Km 2:1 was
selected as optimum Km because it allowed the
formation of the largest single phase region
than other Km. It has been reported that at the
optimum Km, the cosurfactant is inserted into
the cavities between the surfactant molecules
exactly and the formed MEs has a maximum
solubilizing
capacity30.
Unlike
oral
administration, there is no worry from the high
surfactant content (Km (2:1)) in the transdermal
delivery. As enhanced skin permeation is the
aim in transdermal delivery, it is not purposeful
to select the lowest surfactant concentration.
The surfactant concentration should be chosen
so that it gives the maximum flux, which is an
important criterion for drug permeation. This is
in agreement with Shinoda & lindman31 and
Azeem et al.29.
Regarding phase diagrams of Labrafil M
1944 CS, Labrafil M 2125 CS and Capryol
90 at the optimum Km (2:1), it was found that
ME area of Capryol 90 is smaller than
corresponding ones of the two Labrafils (Fig.
1). This may be due to emulsion forming
ability the two labrafils have more hydrophilic

(A)

(B)

100

Wa
ter

Labrafil M 1944 CS

70

40

% Labrafil M 2125 CS
60

60
50
60

40

80

ME
50

60

70

80

90

100

100% S/CoS
1:2

100 % Water

20
10

10

20

30

40

50

60

70

80

90

% S/CoS

(D)

Wa
ter

90

100

30

% Labrafil M 2125 CS

70

100

10

70

80

90

100

0
10

20

30

40

50

60

70

20

90

10

30

80

20

80

90

100

100%S/CoS
1:1

100 % Water

30

100

0
60

40

70

90

ME

90

50

60

40

80

20

% Caproyl 90
60

50

T
ME

80

70

40
50

70

30

80

30

60

50

90

20

80

60

40

50

80

100

10

40

50

40

70

100% Caproyl 90

90

60

30

60

100% S/CoS
1:2

% Labrafil M 1944 CS

Wa
ter
%

70

50

20

50

(F)

20

80

40

10

40

100

10

90

60

30

(E)

30

70

20

100% Labrafil M 2125 CS

100

20

0
10

% S/CoS

100% Labrafil M 1944 CS

0

10

0
100 % Water

% S/CoS

10

20

ME

100

100

100% S/CoS
1:2

100 % Water

30

90
0

40

80

100

100% S/CoS
1:1

100 % Water

10

ME

40

50

70

100

0
30

% Caproyl 90
60

60
30

90

10

100
20

80

20

90

40

70

30

70

40
50

ME

60

80

30

50

50

70

10

80

Wa
ter

70

40

90

20

30

W
ate
r

80

30

100

10

90

20

50

100

10

90

10

100% Caproyl 90

Wa
ter

20

(C)

100% Labrafil M 2125 CS

100% Labrafil M 1944 CS

10

20

30

40

50

60

70

80

90

100

100% S/CoS
1:1

100 % Water

% S/CoS

% S/CoS

S/CoS

(G)

(H)

100% Labrafil M 1944 CS

100

60

50

100

0
30

40

50

60

70

80

90

100

100% S/CoS
2:1

100 % Water

30

40

50

Wa
ter

70

% Labrafil M 1944 CS
60

50

10

20

30

40

50

60

70

80

90

100 % Water

100

100% S/CoS
2:1

% S/CoS

60

80

30

70

50

100 % Water

80

90

100
100% S/CoS
3:1

% S/CoS

20

30

40

50

60

70

10
80

40

90

30
20

90

ME

10

100

0
10

50

80

20

100
100 % Water

% Caproyl 90
60

70

30

0
70

70

60

40

90

80

50

80
10

90

30
40

60

ME

ME

20

100
60

% Labrafil M 2125 CS

70

100

20

60
30

90

0
10

90

50
40

80

100% Caproyl 90

100

40

50

50

(L)

20

80

Wa
ter
%

0
10

40

40

10

100

100% Labrafil M 2125 CS

90

30

30

90

100% S/CoS
2 :1

100

10

20

80

(K)

20

10

100

% S/CoS

(J)

70

100 % Water

100% Labrafil M 1944 CS

70

60

20

90

0
20

% S/CoS

20

100
10

30

80

10

40

70

30

Wa
ter

20

50

10

50

90

10

% Caproyl 90
60

60

40

ME

90

70

40

50

80

20

80

60

70

30

ME

80

%Labrafil M 2125 CS

50
60

40

70

40

50

70

30

30

ME

% Labrafil M 1944 CS

60

80

40

Wa
te

70

30

90

20

20

80

100

10

90

Wa
ter

20

100

10

90

Wa
ter

0
10

(I)
100% Caproyl 90

100% Labrafil M 2125 CS

100
100% S/CoS
3:1

0
10

20

30

40

50

100 % Water

60

70

80

90

100
100% S/CoS
3:1

% S/CoS

%S/CoS

Fig. 1: Pseudoternary phase diagrams consisted of Labrafil M 1944 CS (A, D, G, J), Labrafil M 2125
CS (B, E, H, K) and Capryol 90 (C, F, I, L) as oils, Tween 80 as surfactant and n-Bu as
cosurfactant with Km 1:2, 1:1, 2:1 and 3:1 respectively. ME is the microemulsion transparent
area and T is the turbid emulsion area.

37

E. Ramadan, et al.

100% Labrafil M 1944 CS

0

100

10

90

20

80

30
40

wa
te
r
%

70
% Labrafil M 1944 CS

60

50

50

60

40

70

30

80

20

90

10

100
0

0
10

20

30

40

100% water
Km 1:2
Km 1:1

50

60

70

80

90

100

100% S/Cos

% S/Cos

Km 2:1
Km 3:1

Fig. 2: Pseudoternary phase diagrams consisted of Labrafil M 1944 CS as oil, Tween 80 as surfactant
and n-Bu as cosurfactant with Km 1:2, 1:1, 2:1 and 3:1 respectively.
nature than Capryol 90, emulsion forming
ability and still have lipophilic surfactant
nature which may reduce the water-oil
interfacial tensions. Labrafil M 1944 CS was
selected for further study because the ME of it
larger than that of Capryol 90. Furthermore, it
was reported that the hydrophilic nature of
Labrafil M 1944 CS (Oleoyl macrogol
glyceride) is more than that of Labrafil M 2125
CS (Linoleoyl macrogol glyceride)24.
ACV solubility in microemulsions
Solubility of ACV in MEs was given in
table 3. It was found that the amounts of ACV
entrapped in MEs (ME2 - ME9) were
significantly higher (P< 0.05) than that
entrapped in water. However, ACV entrapped
in
ME1
(0.5580.238
mg/ml)
was
insignificantly different than that entrapped in
water (0.78970.018 mg/ml). The higher drug
solubility in MEs could be attributed to drug
solubilization in the interfacial film between

38

the oil and water phase which may lead to

additional solubilization sites for the drug.
Table 3: Solubility of acyclovir in microemulsions and water.
MEs
ME1
ME2
ME3
ME4
ME5
ME6
ME7
ME8
ME9
Water

Solubility of ACV (mg/ml)

mean S.D.
0.5580 0.238
1.0709 0.117
1.4796 0.145
1.9851 0.139
2.0274 0.126
1.9511 0.068
2.1691 0.190
2.1064 0.085
2.2331 0.021
0.7897 0.018

Table 3 shows that increase of water content

(H2O) from 10 to 40% (ME2 to ME4) was
accompanied with sharp and highly significant

increase of drug entrapment (One-way

ANOVA, P< 0.0001). Further increase of H2O
from 80 to 90% was accompanied with a
significant increase of ACV solubility from
2.10.085 to 2.230.021 mg/ml respectively
(Student t-test, P= 0.0279). However, increase
of percentage water content (H2O) from 40 to
70% (ME4 to ME7), resulted in insignificant
change of ACV entrapment (One-way
ANOVA, P= 0.178). The observed change of
ACV solubility in different MEs could be
related to the difference in microstructures of
the MEs (w/o, Bc, o/w).
The increase of ACV entrapment
accompanying H2O increase in ME2 - ME4
may be due to the polar nature of ACV. Each
ACV molecule has two ionizable groups; basic
amino group (pKa of 2.4) and acidic hydroxyl
group (pKa of 9.2). This polarity may make
ACV dissolved in the gradually growing
aqueous internal phase (w/o).
While the insignificant change which was
accompanying increase of H2O from 40% to
70%, could be attributed to Bc phase which
consist of interwoven water and oil channels
separated by a surfactant monolayer. When
H2O increased to 80% and 90%, o/w MEs
were formed (ME8 and ME9 respectively) with
the highest chance of drug incorporation to
both the interfacial surfactant film and the
external aqueous phase.
Physicochemical properties
Measurement of pH
The pH values of the prepared MEs were
listed in table 4. The measured pH of the oily
mixture (H2O = zero) was 6.88. It was
observed that the pH values decreased
gradually from 6.615 to 5.955 as H2O
progressively increased from 10% to 90%
(from ME1 to ME9 respectively). This may be
due to that when H2O increased, the ionization
of the organic acids present increased,
releasing more protons into the solution and
reducing the pH. These results were similar to
observed data by Hathout et al.32 for a system
of testosterone in oleic acid, Tween 20,
Transcutol and water and Spernath et al.33 for
microemulsion made of ethyl oleate, propionic
acid, phosphatidylcholine, propylene glycol
and water.
Finally, the pH values of the formulated
MEs are physiologically acceptable and

suitable for topical as well as transdermal

application because it was reported that values
of the pH of skin are in the range of 5.5 to
7.011.
Refractive index of the prepared MEs
The RI values of the MEs were listed in
table 4. It was observed that the RI values
decreased gradually from 1.44 to 1.35 as H2O
increased from 10% to 90% (from ME1 to ME9
respectively). The gradual decrease of RI may
be due to the corresponding increase of H2O
and the low refractive index of water (1.33).
This result was similar to that obtained by
Azeem et al.29.
Percentage transmittance
Percentage transmittance (%T) of MEs
was found in the range of 94.351.23 to
98.350.21 (Table 4). Both refractive index
and percent transmittance data prove the
transparency of the systems.
Electric conductivity of MEs
It was reported that in order to study
electric
conductivity
of
nonionic
microemulsion, a small amount of aqueous
electrolyte must be added to provide the
charges necessary for the charge transport34.
Electric conductivity of MEs at a constant
S/CoS : oil ratio of 9:1 as a function of H2O in
the systems obtained with Km 2:1 are shown in
table 5 and figure 3.
It was found that the electric conductivity
values of MEs were zero mS/m and not
affected by subsequent increase of aqueous
phase up to H2O = 20% (w/w). However,
when H2O increased from 30 to 90% (w/w),
the electric conductivity increased from 0.016
to reach 0.879 mS/m.
It is possible to correlate the electric
conductivity behaviour with the three possible
microstructures of MEs (w/o, Bc and o/w). The
initially observed increase in conductivity is a
phenomenon known as Percolation. The
critical H2O at which percolation occurs is
known as percolation threshold (P). After
percolation threshold, gradual increase in the
electric conductivity was observed. This
finding is in agreement with Bennett et al.35
and Bumajdad & Eastoe36. Moreover,
Djordjevic et al.28 obtained a very similar
electric conductivity profile for MEs.

39

E. Ramadan, et al.

Table 4: pH, % transmittance and refractive index of MEs at a constant S/CoS:oil ratio(9:1) as a
function of water content (H2O)in the systemwith Km 2:1.
MEs and
water
ME1
ME2
ME3
ME4
ME5
ME6
ME7
ME8
ME9
Water

H2O
(% w/w)
10
20
30
40
50
60
70
80
90
100

pH*
6.615
6.535
6.470
6.365
6.295
6.220
6.095
6.020
5.955
6.924

% Transmittance*
0.021
0.049
0.000
0.007
0.049
0.028
0.063
0.085
0.091
0.167

96.68
95.18
95.74
96.15
94.35
94.92
95.14
98.35
97.29
99.99

0.83
1.19
1.00
0.84
1.23
1.32
1.31
0.21
2.33
0.00

Refractive
Index*
1.44 0.001
1.43 0.001
1.42 0.000
1.41 0.000
1.39 0.000
1.38 0.000
1.37 0.000
1.36 0.002
1.35 0.000
1.33 0.000

*mean S.D.
Table 5: Apparent viscosityand electric conductivity of MEs at a constant S/CoS : oil ratio of 9:1 as a
function of H2O in the systemsobtained with Km 2:1.
H2O
(% w/w)
Zero
10
20
30
40
50
60
70
80
90

MEs
S/Cos : oil
ME1
ME2
ME3
ME4
ME5
ME6
ME7
ME8
ME9

Viscosity*
(mPa.S)
36.5 0.70
52.5 2.12
57.5 2.12
54.5 2.12
47.0 4.24
43.0 1.41
41.0 2.82
29.5 2.12
6.0 0.00
4.5 0.71

Electric conductivity*
(mS/m)
0.000 0.000
0.000 0.000
0.000 0.002
0.016 0.001
0.079 0.002
0.185 0.001
0.317 0.007
0.476 0.011
0.659 0.023
0.879 0.000

*mean S.D.

1.0
0.9

Electric Conductivity (mS/m)

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0

10

20

30

40

50

60

70

80

90

100

water content (%w/w)

Fig. 3: Electric conductivity of MEs at a

constant S/CoS: oil ratio of 9:1 as a
function of water content (% w/w) in the
systems obtained with Km 2:1.

40

In the region of low water content, w/o

microemulsion is formed. Beyond p, a
network of conductive channels exists, which
corresponds to the formation of water cylinders
or channels in an oil phase due to the attractive
interaction between the spherical microdroplets
of water phase in the w/o microemulsion
(formation of Bc). In the region of high water
content (H2O > 50%), an o/w microemulsion is
formed. Therefore, three structural regions w/o,
Bc and o/w were found in the analysis of the
electric conductivity behavior. For further
verification of the formation of three types of
MEs, their viscosity measurements were
performed.
Apparent viscosities of MEs
Figure 4 and table 5 show the apparent
viscosities of MEs at a constant S/CoS : oil
ratio of 9:1 as a function of H2O in the systems

obtained with Km 2:1. It was found that

increase of H2O from 10 to 20% was
accompanied with initial increase of viscosity
from 52.5 to 57.5 mPa.s. Further increase of
H2O to 60% led to a decrease of viscosity to
reach 41 mPa.s although these viscosity values
are greater than that of S/Cos : oil mixture,
which confirm the formation of microemulsion.
The viscosity continued to decrease gradually
with increasing H2O from 40 to 60% (47 and
41 mPa.s. respectively).
65
60
55

Viscosity (mPa.s)

50
45
40
35
30
25
20
15
10
5
0
0

10

20

30

40

50

60

70

80

90

100

water content (%w/w)

Fig. 4: Apparent viscosities of MEs at a constant

S/CoS : oil ratio of 9:1 as a function of
water content (%w/w) in the systems
obtained with Km 2:1.

A sudden and sharp decrease in viscosity

was observed when H2O > 60% (w/w) but
viscosity values were still larger than water
viscosity (1.002 mPa.s).
The initial increase of viscosity with
increase of H2O is probably due to the
consequence of attractive interaction and
aggregation of droplets of water phase
including molecular reorganization on the
interface. This initial increase in viscosity with
increasing H2O represents P. Beyond P, the
following slow decrease in viscosity suggests
transformation of system structure from w/o
system to Bc microstructure. The subsequent
sharp decrease of viscosity could be attributed
to formation of o/w system. Furthermore,
increasing the water content can lower the
viscosity of a microemulsion. Similar results

were obtained by Djordjevic et al.28, Hathout et

al.32 and Arajo et al.37. It was found that
viscosity changes can reflect a transformation
of system microstructure in the function of
H2O. The viscosity of a microemulsion can be
affected by the component ratio and
concentration of oil, water and surfactant. This
result is in agreement with Yuan et al.38 and
Djordjevic et al.28.
It could be concluded from rheological
and conductivity measurements of investigated
MEs that they exhibited the percolation theory
which
proves
the
transformation
of
microemulsion systems at fixed temperature.
Moreover, the electrical conductivity data and
viscosity measurements could allow us to
quantitatively identify Bc structure from
droplet microemulsion structures.
So that, based on the conductivity and
viscosity results, the microemulsion systems
containing H2O = 10 and 20% (w/w) are w/o
type, the microemulsion systems containing
H2O = 30, 40, 50 and 60% (w/w) are Bc type
with nonspherical isolated droplet structure
(possibly locally cylindrical) and H2O = 70, 80
and 90% (w/w) are o/w type. From these
measurements, three MEs representing
different regions in the pseudo-ternary phase
diagram as w/o (H2O = 10%), Bc (H2O =
50%) and o/w (H2O = 80%), were selected for
further studies.
Size analysis, homogeneity and morphology
of MEs droplets
Table 6 shows mean droplet size, PDI and
ZP of MEs determined using photon
correlation spectroscopy technique. It was
found that the mean droplet size was in the
range of 0.655 to 47.9 nm, so that all the
droplets sizes are in the nano range (47.9 nm <
100 nm). PDI of MEs were found to be in the
range of 0.11 to 0.47 (< 0.5) and ZP values
were small and ranged from -0.101 to 2.5 mV.
TEM images of MEs before ACV
incorporation (ME1, ME5, ME8) and the
corresponding ones after its incorporation
(ME10, ME50, ME80) revealed that droplets
appear dark and they were well dispersed
without aggregation or cluster formation,
spherical in shape. Representive TEM images
of ME1 and ME10 before and after ACV
incorporation are shown in figure 5.

41

E. Ramadan, et al.

Table 6: The droplets size, polydispersity index and zeta potential of MEs.
MEs
ME1
ME5
ME8
ME10
ME50
ME80

Droplet size*
(nm)
0.655 0.28
47.900 2.55
8.580 0.04
5.050 2.47
40.110 2.88
45.040 12.5

(A)

Polydispersity
index*
0.11 0.10
0.33 0.04
0.48 0.01
0.47 0.13
0.34 0.03
0.28 0.02

Zeta potential
(mV)
0.0063
0.0454
2.5
-0.101
0.011
1.62

(B)

Fig. 5: TEM images of ME1 (A) and ME10 (B).

The mean particle diameter and the PDI of

microemulsion are important parameters
predicting the physical stability39 and the invivo fate of colloidal drug carriers such as
microemulsion40. The obtained small droplet
size was preferred in term of skin penetration.
It was observed that the size of the droplets of
drug loaded ME10 and ME80 were significantly
(Paired t-test, P< 0.05) higher than those of the
corresponding plain ones (ME1 and ME8
respectively). This significant increase of
droplet size may be due to insertion of ACV
molecules between surfactant molecules in the
interfacial film to expand the droplets
diameters. This is in agreement with Yuan et
al.9, Arajo et al.37 and Zachar41. Furthermore, it
was reported that incorporated drug can take
part in the microstructure of the microemulsion
system, thus influencing the microemulsion
arrangement and properties via molecular
interaction42. This increase of droplets size was
not observed in case of ME50 when compared
with the corresponding plain one (ME5) but
instead there is a decrease in droplet size. This
could be attributed to the bicontinuous

42

microstructure of these MEs containing

interwoven channels of water and oils which
represent a transient step in between w/o MEs
(ME1 and ME10) and o/w MEs (ME8 and
ME80).
It was reported that PDI describes the
homogeneity of the samples and PDI values
larger
than
0.5
indicate
a
higher
heterogeneity43. The low PDI values of the
prepared MEs indicate homogenous vesicles.
Generally, an increase of electrostatic
repulsive forces between microemulsion
droplets prevents the coalescence of
microemulsion droplets. On the contrary, a
decrease of electrostatic repulsive forces will
cause phase separation44. For microemulsions
with nononic surfactants the zeta potential can
be used to analyse the charge of the system43. It
can be seen that MEs droplets were almost
positive. It was found that when the water
content of MEs increased, the ZP increased.
This may be due to that increase of water
content leads to more ionization of functional
groups responsible for droplet surface charge.
For the same water content, it was found that

Accelerated physical stability

A) Centrifugation
MEs (plain and drug loaded) showed
absence of phase separation or drug
precipitation (for drug loaded Mes) after
centrifugation at 5,000 rpm for 30 min which
verify the stability of these tested Mes.
The stability of Mes could be attributed to
the Brownian movement associated with their
small droplets (less than 15 nm). The
microparticles in this size range are small
enough to absorb kinetic energy from
bombardment by the molecules of dispersion
medium. This keeps the dispersed droplets in a
state of violent motion preventing their settling
or creaming under gravitational field. This
explanation is in agreement with Jain et al.45.
B) Heat-cool cycling
The developed MEs showed no turbidity,
creaming, phase separation or cracking at the
end of heat-cool cycling.

portions are simultaneously dehydrated due

to the freezing action of water. As MEs were
thawed, water was released and travels rapidly
through the microemulsion. This finding is in
agreement with Attwood46.
Moreover, the MEs stability against
sedimentation, flocculation and coalescence
could be related to their small droplets sizes
(Table 6). The MEs did not show any evidence
of instability, maintain the physical integrity
and confers longer shelf life when compared
with ordinary emulsions. Similar findings were
obtained by Narang et al.47 and Cho et al.48. In
conclusion, all the tested MEs were found to be
stable.
Release profiles of ACV
Percentage released of
MEs and from phosphate
dissolved ACV (PBACV) as
semipermeable cellophane
shown in figure 6.

ACV from tested

buffer containing
a control through
membrane were

100
90
80
Cumulative ACV % Released

ZP decreased significantly by incorporation of

ACV for medicated MEs. This could be
attributed to the change in the microstructure of
the droplets produced due to insertion of ACV
molecules in the interfacial film. Each ACV
molecule has two ionizable groups (basic and
acidic pka of 2.27 and 9.25 respectively) which
could be ionized in a manner decreased ZP.
Plain MEs (ME1, ME5 and ME8) and the
corresponding drug loaded ones (ME10, ME50
and ME80) were subjected to accelerated
physical stability tests.

70
60

M E50 (Bc)

50

M E80 (O/W)
PBACV

40

M E10(W/O)

30
20
10
0

C) Freeze-thaw cycling
During freezing, it was observed that the
appearance of MEs turned from transparent to
white color which increased by increasing
H2O. When MEs were thawed, their
appearance turned to transparent again. No
turbidity, creaming, phase separation or
cracking was observed at the end of freezethaw cycling. This result indicates that the MEs
can heal themselves before coalescence
occurs, so the MEs survive the test.
These results can be explained on the
basis that at freezing temperature the formation
of ice crystals in MEs may cause oil particles
to elongate and flatten. In addition, the
lipophilic portion of the emulsifier molecule
will lose their mobility while the hydrophilic

60

120

180

240

300

360

420

480

540

600

Tim e (m in)

Fig. 6: Release of ACV from microemulsion

formulations and phosphate buffer pH
7.4 through cellophane membrane.

It was revealed that ACV release profile

of ME80 > ME50 > ME10 (Fig. 6). This indicates
that ACV release increased with increasing
water content of microemulsion. The
percentage of drug released after eight hours
were 68.23, 61.36, 23.5 and 69.74% from
ME80, ME50, ME10 and PBACV respectively. So
that, there is a strong correlation between the
water content and the drug release. This release
behavior of ACV (polar drug) may be due to

43

E. Ramadan, et al.

Table 7: Acyclovir release kinetics from the tested microemulsions.

Microemulsions
ME 10
ME 50
ME 80

Zero-order
ko
r2
0.0448
0.939
0.1236
0.896
0.1371
0.829

First-order
k1
r2
0.0005
0.9507
0.0020
0.9455
0.0025
0.8867

the presence of the drug in the aqueous phase.

Therefore in case of o/w microemulsion
(ME80), ACV is present in direct contact with
the release barrier. However in case of w/o
microemulsion (ME10), ACV needs to travel
longer distance from internal phase (water) to
external one (oil) and then from the last one out
to the release media through cellophane
membrane. Moreover, the viscosity values of
the tested MEs were markedly affected by the
water content H2O (Table 5) which in turn
may influence on the ACV release.
This is in agreement of Djordjevic et al.49
and Parsaee et al.50. Those authors found that,
the maximum flux value of diclofenac
diethylamine through cellophane membrane
was obtained from microemulsion containing
the highest percentage of water.
Drug release mechanisms
ACV release data from microemulsion
formulations were analyzed using different
models; Zero-order, First-order, Higuchi
diffusion and Korsmeyer-Peppas models. The
results were illustrated in table 7.
Table 7 illustrates that all the tested
formulations followed the Korsmeyer-Peppas
model as indicated by the highest r2 (r2 from
0.972 to 0.985). To find out the drug release
mechanism from Korsmeyer-Peppas model,
moreover the exponent n values of the tested
MEs were 0.55, 0.53 and 0.54 for ME10, ME50
and ME80 respectively. So, non-Fickian
"anomalous" transport release is expected to be
the drug release mechanism from MEs as 0.5 <
n < 1. This finding indicates that the ratecontrolling stage in the release process was
diffusion of the dissolved drug through the
microemulsion to the external medium.
Conclusion
Acyclovir could be incorporated in
different stable isotropic microemulsion
systems in w/o, bicontinous and o/w regions
using Labrafil M 1944 CS, Tween 80 and n-

44

Higuchi
kH
r2
1.063 0.977
3.017 0.984
3.427 0.955

Korsmeyer-Peppas
kP
N
r2
0.75 0.5511 0.983
2.54 0.5306 0.985
2.91 0.5364 0.972

butanol as oil, surfactant and cosurfactant

respectively with Km 2:1. The release pattern of
the drug was affected by the water content in
the system. Kinetic analysis of release data
revealed that non-Fickian transport is the
release mechanism.
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