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Formulation and Pharmacokinetics of Diclofenac Lipid

Nanoemulsions for Parenteral Application
Srividya Ramreddy, Prabhakar Kandadi and Kishan Veerabrahma

PDA J Pharm Sci and Tech 2012, 66 28-37

Access the most recent version at doi:10.5731/pdajpst.2012.00735
 Downloaded from journal.pda.org on February 19, 2015

Formulation and Pharmacokinetics of Diclofenac Lipid

Nanoemulsions for Parenteral Application
SRIVIDYA RAMREDDY, PRABHAKAR KANDADI, and KISHAN VEERABRAHMA*

Nanotechnology Research Lab, Department of Pharmaceutics, University College of Pharmaceutical Sciences,

Kakatiya University, Warangal, Andhra Pradesh, India—506009 ©PDA, Inc. 2012

ABSTRACT: The objective of the present study was to formulate and determine the pharmacokinetics of stable o/w
parenteral lipid nanoemulsions (LNEs) of diclofenac acid used to treat arthritic conditions. The LNEs of diclofenac
acid with a mean size ranging from 200 to 240 nm and a zeta potential of ⫺29.4 ⫾ 1.04 mV (negatively charged
LNEs) and 62.1 ⫾ 3.5 (positively charged LNEs) emulsions were prepared by hot homogenization and ultrasonication
process. The influence of formulation variables, such as the change in proportion of cholesterol, was studied, and
optimized formulations were developed. The optimized formulations were relatively stable during centrifugal stress,
dilution stress, and storage. The drug content and entrapment efficiency were determined using high-performance
liquid chromatography. The in vitro drug release was carried out in phosphate-buffered saline pH 7.4 and cumulative
amount of drug released was estimated using a UV-visible spectro-photometer. During in vivo pharmacokinetic
studies in male Wistar rats, diclofenac serum concentration from LNEs was higher than that of Voveran injection and
was detectable up to 12 h. Diclofenac in LNEs showed improved pharmacokinetic profile with increase in area under
the curve, elimination half-life and mean residence time in comparison to Voveran.

KEYWORDS: Diclofenac lipid nanoemulsions, Pharmacokinetics of diclofenac, Voveran injection

LAY ABSTRACT: Our aim was to prepare and determine the pharmacokinetics of injectable lipid nanoemulsions of
diclofenac acid for treating arthritic conditions by reducing the frequency of dosing and pain at site of injection. The
nanoemulsions of diclofenac acid were prepared by homogenization and ultrasonication process. The sizes and
charges of oil globules were determined. The effect of cholesterol on stability of emulsion was studied, and an
optimized preparation was developed. The optimized formulations were stable during centrifugation, dilution, and
storage. The total amount of drug in emulsion and percentage amount of drug present in emulsion globules were
determined using high-performance liquid chromatography. The drug release from preparation was carried out in
phosphate-buffered saline pH 7.4. The cumulative amount of drug released was estimated using a spectrophotometer.
The time course of the released drug in rat serum was determined. Diclofenac concentrations from lipid nanoemul-
sions were higher than that of Voveran injection (solution form) in serum.

1. Introduction erties, diclofenac sodium is used to reduce pain in

Diclofenac sodium, a water-soluble, non-steroidal an-
ti-inflammatory drug (NSAID) available in the form of
tablets and injection, possesses potent cyclooxyge-
nase-inhibiting activity (1). Due to its analgesic prop-
* Corresponding Author: Professor Veerabrahma Kis-
han, Department of Pharmaceutics, University College
of Pharmaceutical Sciences, Kakatiya University,
Warangal—506009, Andhra Pradesh, India. Tele-
phone: 91-87-02446259. Fax: 91-87-02438844.
Email: vbkishan@yahoo.com.
doi: 10.5731/pdajpst.2012.00735
patients with arthritis, kidney stones, gallstones, men-
strual pain, dysmenorrhoea, or cancer. It is also widely
used in long-term therapy of various arthritic condi-
tions such as rheumatoid arthritis (2), osteoarthritis
(3), spondylarthritis, and ankylosing spondylitis. The
biological half-life of diclofenac is short and varies
from 1 to 2 h (4 – 6). Therefore, multiple dosing is
required to maintain a therapeutic concentration of
drug for an extended period. The long-term oral ad-
ministration of diclofenac sodium results in gastroin-
testinal bleeding, gastrointestinal disturbances, peptic
ulceration, blood dyscrasias, and anaphylaxis (7). Ad-
ministration by the parenteral route produces irritation
and pain at the site of injection, a great discomfort to
the patient without any sustained effect. Hence, a

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sustained release parenteral delivery system is desir-
able.
Novel drug delivery systems (liposomes, nanopar-
ticles, and lipid emulsions) are proposed to overcome
the limitations of conventional dosage forms and for
sustained delivery of drugs. Commercial parenteral
lipid (fat) emulsions are available in market for nutri-
tional (Lipofundin, Intralipid, Elolipid, Neutralipid,
etc.) and medical (Diagemuls, Diprivan, Liple, etc.)
applications. Lipid nanoemulsions (LNEs) have been
studied as drug carriers for sustained release and tissue
targeting. LNEs are excellent carriers for lipophilic
and amphiphilic drugs, possess many favorable prop-
erties such as biocompatibility, biodegradability, sta-
bility, and ease of preparation and handling (8, 9). The
structure of LNE consists of a neutral lipid core (i.e.,
triglyceride) stabilized by a monolayer of amphiphilic
lipid (phospholipids). LNEs can be scaled up for in-
dustrial purposes. By using LNEs, direct contact of the
drug with body fluid and tissues can also be avoided,
thereby minimizing any possible side effects (10, 11).
Therefore, to reduce the frequency of administration
and pain associated with the injection and to improve
patient compliance, diclofenac, being a lipophilic drug
(log P 4.058) was formulated into parenteral o/w lipid
nanoemulsion. In our previous work, diclofenac LNEs
were studied for the pharmacodynamic effects in rats
with carragenan-induced inflammation and found to
have a sustained effect when compared to parenteral
injection (12). In this work, formulation development,
characterization, and in vivo pharmacokinetics of di-
clofenac LNEs for parenteral application are de-
scribed.
2. Experimental
Materials
The diclofenac sodium was a kind gift from Dr. Red-
dy’s laboratories (Hyderabad, India). The soybean oil
was purchased from Sigma Chemicals Ltd. (Mumbai,
India). The egg lecithin (PC-80) was a kind gift from
Lipoid (Ludwigshafen, Germany). Cholesterol, meth-
anol, chloroform, and glycerol (98%) were purchased
from Merck Ltd. (Mumbai, India). The alpha-tocoph-
erol acetate was purchased from Hi-Media (Mumbai,
India). Oleic acid and ammonium acetate were pur-
chased from SD Fine Chemicals Ltd. (Mumbai, India).
All other chemicals used were of reagent grade.
Vol. 66, No. 1, January–February 2012
Methods
Conversion of Diclofenac Sodium Salt to Diclofenac
Acid: An aqueous solution of diclofenac sodium was
acidified with 0.1 N HCl solution; a white precipitate
formed and was separated by filtration. The precipitate
was washed with water and then extracted into chlo-
roform. The chloroform was removed by a rotavaccum
evaporator (Laborota 4000, Heidolph, Kelheim, Ger-
many) to obtain a thin film of solid residue. The purity
of diclofenac acid was determined by differential
scanning calorimetry (DSC).
Preparation of Lipid Nanoemulsion (LNE) Con-
taining Diclofenac Acid: Diclofenac acid, egg leci-
thin, ␣-tocopherol acetate and oleic acid were added to
soya bean oil (oil phase); glycerol was dissolved in
sufficient amount of double-distilled water (aqueous
phase); and both phases were heated to 70 °C. The
aqueous phase was added to the oil phase to produce
a coarse emulsion (10 mL). The formed emulsion was
homogenized (Heidolph DIAX 900, Kelheim, Ger-
many) for 3 min at 15000 rpm and then sonicated
(Vibra Cell, 750 watt, Sonics and Materials Inc., New-
town, CT, USA) at 30% amplitude using a 12 T probe
for about 20 min. The formulations were prepared
using different proportions of cholesterol.
Determination of Particle Size and Zeta Potential
(Zp): The LNEs were characterized for globule size,
polydispersity index (PDI), and Zp before and after
sterilization (autoclaving at 15 lbs, 121 °C for 15 min).
The emulsions were diluted (1:100) with double-dis-
tilled water and taken in a cuvette. The cuvette was
placed inside the Zeta Sizer (Nano ZS 90, Malvern
Instruments, Malvern, UK). The observations for
globule size were recorded at a 90° light-scattering
angle and the temperature was maintained at 25 °C.
The average particle count rate was maintained be-
tween 50 and 500 kcps during the measurement pro-
cess (13). The hydrodynamic diameter of the globules
via Brownian motion was determined using the prin-
ciple of photon correlation spectroscopy. The Zp mea-
surement was based on the electrophoretic mobility of
globules, using in-built software, which uses Helm-
holtz-Smoluchowski equation (13, 14).
Effect of Varying Concentration of Cholesterol:
LNEs were prepared as described above using differ-
ent concentrations of cholesterol, 0%, 0.1%, 0.2%, and
0.3% w/v, while keeping the other ingredients con-
stant. The prepared formulations were compared with
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that of the control LNE (without cholesterol) for glob-
ule size, PDI, and Zp before and after sterilization to
get an optimized formulation.
Preparation of Positively Charged LNEs: Positively
charged emulsions were prepared using formulation
LNE-3, in which the oleic acid was substituted with
stearyl amine (0.3%) while keeping all other ingredi-
ents constant. The prepared emulsion was character-
ized for globule size, PDI, and Zp before and after
sterilization.
Determination of the Effects of Centrifugal Stress,
Desorption Stress, and Storage on Stability of
LNEs: The formulations LNE-0 (without cholesterol),
LNE-3 (0.3% cholesterol), and LNE-4 (0.3% stearyl
amine) were studied to determine the effect of centrif-
ugal stress, desorption stress, and stability on storage.
Effect of Centrifugal Stress on Stability: Three
LNEs (LNE-0, LNE-3, and LNE-4) were studied. One
milliliter of each formulation was placed in 1.5 mL
centrifuge tubes, and the emulsions were centrifuged
(Biofuge primo, Heraeus, Germany) at 15,000 m/s2
(1529 ⫻ g) for 10 min, to assess the stability of
emulsions under centrifugation. The creaming volume
percentage for each emulsion was calculated by using
the formula (15) in eq 1 and then compared.
Vt ⫺ Vs
C ⫽ 100 (1)
Vt
where C ⫽ creaming volume percentage, Vt ⫽ total
volume of sample, and Vs ⫽ volume of the lower
phase layer.
Effect of Dilution (Desorption Stress) on Stability:
The LNEs were diluted with double-distilled water 50
to 5000 times (1:50, 1:100, 1:200, 1:500, 1:1000, and
1:5000), and the effect of dilution on size and Zp was
studied using a zeta sizer.
Stability of LNEs during Storage: Three sterile for-
mulations (LNE-0, LNE-3, and LNE-4) were tested
for stability on storage. LNE-0 and LNE-3 were ster-
ilized by autoclaving and LNE-4 by membrane filtra-
tion. One milliliter of each emulsion was placed in 1.5
mL eppendorf tubes, and these were stored at room
temperature (RT). They were tested for globule size,
PDI, and Zp at 0, 15, 30, 45 and 60 days.
30
Determination of Total Drug Content and Entrap-
ment Efficiency (EE): A high-performance liquid
chromatography (HPLC) system consisting of a LC-
10AT vp solvent delivery system containing double
reciprocating plunger pump (Shimadzu, Kyoto, Ja-
pan), a SPD-10A vp UV–visible variable wavelength
detector with deuterium lamp (Shimadzu), and a
250 ⫻ 4.6 mm Luna 5␮ C-18 reverse-phase analytical
column (Phenomenex, Utrecht, Netherlands) was used
to determine total drug content and EE of the formu-
lations. The mobile phase consisted of methanol: 0.1M
ammonium acetate buffer: acetonitrile (60:30:10). The
flow rate was 1 mL/min and detection was performed
at 280 nm (16).
Total Drug Content: Drug-loaded formulations (0.1
mL) were diluted to 2 mL with a chloroform/methanol
mixture (1:1) and further sufficiently diluted with mo-
bile phase, and 20 ␮L was injected into HPLC. The
total drug content was obtained by using the standard
calibration curve obtained with standard concentration
of diclofenac acid.
Entrapment Efficiency (EE): Entrapment efficiency
was determined by measuring the concentration of free
drug (unentrapped) in an aqueous medium as reported
previously (12). The aqueous medium was separated
by ultra-filtration using Centrisort tubes (Sartorius,
Goettingen, Germany), which consisted of filter mem-
brane (molecular weight cutoff 20,000 Da) at the base
of the sample recovery chamber. About 4 mL of the
formulation was placed in the donor chamber and
centrifuged at 3500 rpm for 5 min (Biofuge Primo R,
Heraeus, Hanau, Germany). The LNE and the encap-
sulated drug remained in the donor chamber, and the
aqueous phase moved into the receiver chamber through
filter membrane. The amount of drug in the aqueous
phase was estimated using HPLC method, and the %EE
was calculated using the following equation.
EE 共%兲
⫻ weight of drug used in the formulation
⫽ 共Weight of drug used in the formulation
⫺ Weight of drug in aqueous phase兲 ⫻ 100.
(2)
In Vitro Drug Release Studies of Formulations: The
drug release from different formulations was studied
using the dialysis bag method. Initially, the dialysis
tubing (the cellulose membrane) (DM 60, molecular
PDA Journal of Pharmaceutical Science and Technology
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weight cutoff 12,000 –14,000, Himedia, Mumbai, In-
dia) was hydrated by soaking in phosphate buffer pH
7.4 overnight at RT. The LNEs (1 mL) were placed in
dialysis tubing, tied at both ends, and then suspended
in 100 mL of phosphate buffer pH 7.4 in 250 mL
beakers as dialysis medium. The dialysis medium was
stirred continuously at 100 rpm by a magnetic stirrer
(Remi Equipments, Mumbai, India). At different time
intervals (0.25, 0.5, 1, 2, 3, 4, 6, 8, 10 and 12 h), 1 mL
of samples were withdrawn from the beaker and re-
placed with an equal volume of fresh medium. The
drug concentration in collected samples was estimated
using standard calibration curve obtained with stan-
dard concentrations of diclofenac acid in phosphate
buffer (pH 7.4) measuring the absorbance at 276 nm
(17) on a UV-visible spectrophotometer (ELICO SL
159, Hyderabad, India). The cumulative percentage of
drug released versus time was plotted to determine the
release pattern of the drug from the formulations.
Pharmacokinetic Studies: Healthy male Wistar rats
(280 –300 g) were used for the pharmacokinetic study.
The animals were given a standard diet and had free
access to water ad libitum. The studies were conducted
with prior approval of institutional animal ethical
committee.
Study Protocol: The rats were fasted overnight with
free access to water ad libitum. The animals were
divided into four groups (n ⫽ 6) and were randomly
administered with different formulations. One group
of animals was administered with Voveran injection (5
mg/kg body weight) by intravenous (IV) bolus injec-
tion via the tail vein. The other three groups received
the LNE-0, LNE-3 and LNE-4 formulations. After
injection at predetermined time intervals, 0.25, 0.5, 1,
2, 4, 6, 8 and 12 h, the blood samples were collected
from the retro-orbital venous plexus. The blood was
allowed to clot, was centrifuged, and serum was col-
lected. The serum samples were stored at ⫺20 °C until
analysis.
Preparation of Standard Calibration Curve of Di-
clofenac in Rat Serum: To 100 ␮L of blank serum in
test tubes, 50 ␮L of diclofenac acid in methanol was
added from stock solutions containing 0.05, 0.1, 0.25,
0.5, 1, 2 and 4 ␮g/mL of diclofenac acid. To this 50
␮L of chlorzoxazone (5 ␮g/mL) in methanol and 100
␮L of 2M HCl were added and vortexed for 3 min. To
this 3 mL of chloroform was added and vortexed for 5
min followed by centrifugation at 3000 rpm for 10
min. The organic phase was separated and evaporated
Vol. 66, No. 1, January–February 2012
under reduced pressure in a vacuum oven (Toshniwal,
Mumbai, India). The resulting residue was reconsti-
tuted in 100 ␮L of mobile phase and 20 ␮L was
injected into HPLC column. The mobile phase con-
sisted of methanol: 0.1M ammonium acetate buffer:
acetonitrile (60:30:10). The flow rate was 1 mL/min,
and detection was performed at 280 nm (16). Peak
ratio versus concentration was plotted to get the stan-
dard calibration curve (18).
Estimation of Diclofenac in Serum Samples: To
100 ␮L of test serum, 50 ␮L of methanol, 50 ␮L of
chlorzoxazone (5 ␮g/mL) in methanol, and 100 ␮L of
2M HCl were added and vortexed for 3 min. To this
3 mL of chloroform was added and vortexed for 5 min
followed by centrifugation at 3000 rpm for 10 min.
The organic phase was separated and evaporated under
reduced pressure in a vacuum oven (Toshniwal, Mum-
bai, India). The resulting residue was reconstituted in
100 ␮L of mobile phase, and 20 ␮L was injected into
the HPLC column. The unknown concentrations were
found using the standard calibration curve obtained
with known concentrations of diclofenac acid.
Calculation of Pharmacokinetic Parameters and
Statistical Significance: Pharmacokinetic parameters
such as Cmax, Tmax, area under the curve (AUC), t1/2,
and mean residence time (MRT) were calculated by
using Kinetica software assuming a one-compartment,
open model, IV bolus injection of unchanged drug in
blood. The statistical significance of observed differ-
ences in Cmax, AUC, t1/2, and MRT of different groups
were assessed by the analysis of variance (ANOVA)
test by using Graph Pad Prism software. All data were
expressed as mean ⫾ standard deviation (SD). Two
sample comparisons were performed by using New-
man-Keul’s multiple comparison tests and a P-value
below 0.05 was considered to be statistically signifi-
cant.
3. Results and Discussion
Diclofenac acid was prepared from diclofenac sodium
salt and verified by DSC; the endothermic peak was
observed at 175.8 °C. The LNEs were prepared with
different compositions (Table I) by hot homogeniza-
tion followed by ultrasonication. The amount of oil
phase in the emulsions was fixed at 10% and that of
emulsifying agent at 1.2% to produce stable emulsions
(12); this was used as the basis for the preparation of
LNEs in this study. All the emulsions were character-
ized for the globule sizes and Zps before and after
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TABLE I
Compositions of Prepared LNEs

Stearyl
Amine
Cholesterol Concentration Varied Emulsion
Formulation Ingredients LNE-0 LNE-1 LNE-2 LNE-3 LNE-4
Diclofenac acid (mg) 25 25 25 25 25
Soya bean oil (mg) 1000 1000 1000 1000 1000
Egg lecithin (mg) 120 120 120 120 120
Cholesterol (mg) 0 10 20 30 30
␣-tocopherol acetate (mg) 20 20 20 20 20
Oleic acid (mg) 25 25 25 25 0
Stearyl amine(mg) 0 0 0 0 30
Glycerol (mg) 225 225 225 225 225
Double-distilled water to 10 10 10 10 10
make (mL)

sterilization (Table II). Phospholipids are weak emul-
sifiers (8); the use of cholesterol in the formulation
results in the formation of a more rigid layer, enhanc-
ing the stability of the parenteral emulsion (19) and
might influence the release kinetics of the drug from
the formulation. Cholesterol was included in the
LNE-0 formula in different concentrations, and LNEs
were prepared. There was no significant change in
average Zp and average size of globules with different
concentrations of cholesterol when compared to the
control LNE without cholesterol. Cholesterol concen-
tration in LNEs was optimized at 0.3%, as this resulted
in lower globule sizes and high creaming percent
values of LNEs. The use of cholesterol along with the
1.2% (EPC, Egg Phosphatidyl Choline) produced sta-
ble emulsions.
When these emulsions were subjected to autoclav-
ing, in general there were no significant changes
found in average sizes and the zeta potentials of
globules. However, in the case of LNE-3 (choles-
terol 0.3%), the globule sizes were minimal before
and after the autoclaving (Table II). This indicated
that the presence of cholesterol in these emulsions
imparted stability to the LNEs. Based on the similar
physical characteristics such as size and Zp before
and after sterilization, the LNE-3 was chosen for
further study.
The positive charge inducer stearyl amine was in-
cluded at a concentration of 0.3% in the LNE-3 for-
mulation, substituting oleic acid (negative charge in-
ducer). The inclusion of stearyl amine in LNEs
resulted in formation of globules with size 226 ⫾ 5.5
nm and Zp of 62.1 ⫾ 3.5 mV. There were no big
differences in globule size, PDI and Zp of LNE-4 after
sterilization indicating the prepared LNE-4 was also
stable.

TABLE II
Globule Size, PDI, and Zp of LNEs Before and After Sterilization

Size (nm) PDI ZP (mV)

Sample Before After Before After Before After
Code Autoclaving Autoclaving Autoclaving Autoclaving Autoclaving Autoclaving
LNE-0 217 ⫾ 5.5 212 ⫾ 6.02 0.216 ⫾ 0.1 0.246 ⫾ 0.03 ⫺29.7 ⫾ 1.37 ⫺29.2 ⫾ 1.01
LNE-1 225 ⫾ 5.56 230 ⫾ 4.35 0.183 ⫾ 0.05 0.216 ⫾ 0.015 ⫺28.6 ⫾ 3.09 ⫺27.4 ⫾ 2.62
LNE-2 228 ⫾ 8.0 232 ⫾ 9.6 0.181 ⫾ 0.05 0.223 ⫾ 0.02 ⫺30.3 ⫾ 3.7 ⫺28.4 ⫾ 1.05
LNE-3 215 ⫾ 6.4 219 ⫾ 9 0.112 ⫾ 0.01 0.123 ⫾ 0.02 ⫺29.4 ⫾ 1.7 ⫺29.4 ⫾ 0.97
LNE-4* 226 ⫾ 5.5 196 ⫾ 5.85 0.276 ⫾ 0.04 0.293 ⫾ 0.04 62.1 ⫾ 3.5 58.1 ⫾ 1.9
* Filtration sterilization.

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TABLE III

0.2 ⫾ 0.05
0.25 ⫾ 0.07
0.25 ⫾ 0.06
0.263 ⫾ 0.15
Effect of Centrifugation on Stability of LNEs—

0.17 ⫾ 0.1
LNE-4#
Creaming Volume Percentages of Prepared
Formulations

% Creaming
Formulation Volume
Code (Mean ⴞ SD)

0.13 ⫾ 0.05
0.19 ⫾ 0.02
0.24 ⫾ 0.56
0.2 ⫾ 0.05
0.2 ⫾ 0.06
LNE-3*
PDI
LNE-0 97 ⫾ 0.56
LNE-1 96 ⫾ 2.3
LNE-2 97 ⫾ 1.0
LNE-3 98 ⫾ 1.3
98 ⫾ 0.28

0.15 ⫾ 0.05
0.16 ⫾ 0.02
0.2 ⫾ 0.02
0.22 ⫾ 0.01
0.28 ⫾ 0.05
LNE-4

LNE-0*
Centrifugal stress is a rapid method to determine the
stability of emulsions. Higher creaming volume indi-
cates the greater stability. Stable emulsions possess

50.5 ⫾ 3.5
LNE-4#
very high percent creaming volumes (15). This study

54 ⫾ 3
52.4 ⫾ 2
50.4 ⫾ 3
51.2 ⫾ 4
indicated that all the tested LNEs in general were
stable to the centrifugal stress and that there were no
large differences in creaming percent volumes (Table

Zeta Potential (mV)

III). The percent creaming values after inclusion of

⫺26.7 ⫾ 3.1
⫺30.6 ⫾ 0.7
⫺31.6 ⫾ 0.8
⫺30.7 ⫾ 2.8
⫺30.9 ⫾ 1.7
cholesterol was increased, indicating the stability of

LNE-3*
LNEs due to rigidization of surface of LNEs. LNE-3
and LNE-4 showed relatively increased creaming per-
cent volumes. Hence, they were considered optimized
and studied for the effect of storage along with control
LNE for total drug content, EE, in vitro drug release, ⫺30.9 ⫾ 0.6
⫺31.0 ⫾ 0.9
⫺32.9 ⫾ 1.3
⫺33.8 ⫾ 0.4
⫺31.1 ⫾ 0.5
LNE-0*

and in vivo pharmacokinetics in male Wistar rats.

During storage at RT for a 60 days period, there was
no significant increase in globule size or decrease in
Zps of the optimized LNEs, indicating the physical
stability of emulsions (Table IV).
210 ⫾ 3.5
214.3 ⫾ 4.2
218.3 ⫾ 5.0
223 ⫾ 3.7
Stability of the Optimized LNEs on Storage at RT

LNE-4#

227 ⫾ 4

The dilution of an emulsion may bring about changes

in the rigidity of the surfactant layers at the interface
leading to instability of the system. The stability of an
emulsion can be rapidly checked by measuring the
Zeta Size (nm)

changes in Zp due to dilution. However, stable emul-

213.3 ⫾ 1.5

223.6 ⫾ 2.5
LNE-3*

217 ⫾ 2

226 ⫾ 2
229.6 ⫾ 3

sions do not undergo changes in Zp due to dilution and

withstand higher dilution effect (20). Therefore, the
optimized LNEs (LNE-0, LNE-3, and LNE-4) were
* Sterilized by autoclaving.
# Sterilization by filtration.

subjected to dilution (50 to 5000 times) stress. There

were no significant changes found in Zps of all the
224.3 ⫾ 5.1
228.3 ⫾ 3.5

systems (Table V). Further, due to dilution, there was

LNE-0*
212 ⫾ 4
215 ⫾ 3
220.6 ⫾ 4

no much change in sizes of LNE globules, indicating

that all of these three systems were stable.
TABLE IV

The total drug content of the formulations (n ⫽ 3)

Day

LNE-0, LNE-3 and LNE-4 was found to be 2.48 ⫾

1
15
30
45
60

0.63, 2.36 ⫾ 0.108, and 2.46 ⫾ 0.71 mg/mL, respec-

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TABLE V
Effect of Dilution (Desorption Stress) on Globule Size, and Zp of LNE-0, LNE-3, and LNE-4 Formulations

Formulation Dilution
Code Factor Size (nm) PDI Zp (mV)
LNE-0 1:50 222.3 ⫾ 2.51 0.237 ⫾ 0.004 ⫺28.13 ⫾ 3.15
1:100 230.6 ⫾ 3.05 0.283 ⫾ 0.009 ⫺31.16 ⫾ 2.03
1:200 234 ⫾ 5.56 0.245 ⫾ 0.004 ⫺27.93 ⫾ 3.16
1:500 239 ⫾ 3.6 0.308 ⫾ 0.01 ⫺26.83 ⫾ 2.96
1:1000 242.3 ⫾ 2.51 0.314 ⫾ 0.01 ⫺29.9 ⫾ 4.40
1:5000 245.6 ⫾ 3.05 0.343 ⫾ 0.006 ⫺32.23 ⫾ 2.79
LNE-3 1:50 218.3 ⫾ 6.02 0.22 ⫾ 0.05 ⫺29.13 ⫾ 4.44
1:100 222.3 ⫾ 3.05 0.24 ⫾ 0.03 ⫺28.46 ⫾ 4.29
1:200 227 ⫾ 2.51 0.214 ⫾ 0.02 ⫺29.56 ⫾ 3.92
1:500 230 ⫾ 3.6 0.34 ⫾ 0.01 ⫺30.06 ⫾ 4.21
1:1000 234 ⫾ 2.5 0.35 ⫾ 0.01 ⫺30.12 ⫾ 3.24
1:5000 239 ⫾ 3 0.36 ⫾ 0.05 ⫺29.53 ⫾ 3.78
LNE-4 1:50 215 ⫾ 5.01 0.321 ⫾ 0.04 56.2 ⫾ 1.5
1:100 225 ⫾ 7.4 0.206 ⫾ 0.01 54.3 ⫾ 2
1:200 234 ⫾ 9.5 0.208 ⫾ 0.04 54.0 ⫾ 1.2
1:500 239 ⫾ 6.9 0.32 ⫾ 0.03 52.4 ⫾ 2.5
1:1000 241 ⫾ 4.5 0.19 ⫾ 0.04 51.5 ⫾ 3.4
1:5000 243 ⫾ 2.3 0.212 ⫾ 0.05 49.5 ⫾ 2

tively, and the entrapment efficiencies were 98.8 ⫾
0.2%, 98.87 ⫾ 0.21%, and 97.2 ⫾ 2.51%, respectively
(Table VI). The quantity of drug entrapped in the oil
phase of the LNEs remained almost same with in-
crease in cholesterol concentration. Further, there is
little effect of stearyl amine to influence the drug
loading.
retarded release trend could be due to the rigidity of
monolayer at the interface contributed by the increase
in cholesterol concentration upto 0.3%. On inclusion
of stearyl amine, further retardation of drug release
was observed; this might be due to the charged inter-
actions of diclofenac acid with the stearyl amine moi-
ety at the interface.

The drug release was observed over a period of 12 h

for all the prepared LNEs. The cumulative release
profiles were plotted and are shown in Figure 1. The
release of diclofenac acid from LNE-0, LNE-1,
LNE-2, LNE-3, and LNE-4 after 12 h was found to be
81.28 ⫾ 1.02%, 57.64 ⫾ 0.51%, 54.44 ⫾ 0.78%,
50.68 ⫾ 1.56%, and 41.96 ⫾ 2.58% respectively. This

TABLE VI
Drug Content and EE of the Optimized
Formulations

Formulation Total Drug

Code Content % EE
Figure 1
LNE-0 24.81 ⫾ 6.31 98.8 ⫾ 0.2
LNE-3 23.63 ⫾ 1.08 98.87 ⫾ 0.21
Cumulative percentage drug release from different
LNE-4 24.6 ⫾ 7.18 97.2 ⫾ 2.51 formulations.

34 PDA Journal of Pharmaceutical Science and Technology

 Downloaded from journal.pda.org on February 19, 2015
Figure 2
HPLC chromatogram showing retention times 6.9 min (diclofenac) and 5.0 min chlorzoxazone (internal
standard).
The prepared LNEs were compared for their pharma-
cokinetic properties along with a commercially avail-
able Voveran injection in male Wistar rats. The di-
clofenac extracted from serum samples was estimated
by HPLC (16). The HPLC chromatogram of diclofe-
nac acid and chlorzoxazone (internal standard) ob-
tained is shown in Figure 2. Serum concentration of
diclofenac acid upon IV administration of selected
LNEs is shown in Figure 3. The results indicated that
LNEs showed sustained effect than Voveran威. The
diclofenac serum concentration after administration of
LNEs was higher than that of Voveran威 and was
Figure 3
Serum profiles diclofenac acid upon IV administra-
tion of different formulations in male Wistar rats.
Vol. 66, No. 1, January–February 2012
detectable up to 12 h. The LNE-0 showed higher
diclofenac serum levels (Cmax), AUCtotal, and MRT
than that of Voveran威, LNE-3, and LNE-4. But, when
LNE-3 and LNE-4 were compared to control emulsion
(LNE-0), the drug levels were lower in serum. This
may be attributed to the fact that the cholesterol-
containing emulsions (LNE-3 and LNE-4) might
mimic the endogenous low-density lipoprotein (LDL)
and be endocytosed by LDL receptors present on
different tissues, resulting in preferential accumula-
tion in various organs such as brain, liver, kidney,
spleen, etc., resulting in low diclofenac levels (21).
Further, the lower levels of drug from LNE-4 prepa-
ration may be due to preferential uptake of the posi-
tively charged LNEs by the negatively charged sur-
faces of cell membranes. This is in addition to the
effect of cholesterol, which cumulatively results in the
lowering of drug levels in serum. The therapeutic
availability (TA) of prepared LNEs LNE-0, LNE-3,
and LNE-4 in comparison to Voveran威 were 3.26 ⫾
0.19, 1.73 ⫾ 0.08, and 1.39 ⫾ 0.09, respectively. This
indicated that the therapeutic availability of three se-
lected LNEs was higher than that of Voveran威. How-
ever, the TA of LNE-3 and LNE-4 were 0.54 ⫾ 0.03
and 0.43 ⫾ 0.05, respectively (Table VII). Compari-
son of prepared LNEs by one-way ANOVA analysis
showed that the Cmax, AUCtotal, t1/2, and MRT are
statistically significant. Two sample comparisons by
Newman-Keuls Multiple Comparison (Student’s t test)
35
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TABLE VII
Pharmacokinetic Parameters of Diclofenac Acid in Male Wistar Rats from Different Formulations

Pharmacokinetic P-Value
Parameters Voveran LNE-0 LNE-3 LNE-4 (ANOVA)
Cmax (␮g/mL) 4.22 ⫾ 0.40 6.37 ⫾ 0.39 4.54 ⫾ 0.48 4.53 ⫾ 0.34 0.0001
tmax (h) 0.25 0.25 0.25 0.25 -
AUC total (␮g/mL) h 6.73 ⫾ 0.92 21.6 ⫾ 0.59# 11.7 ⫾ 0.57# 9.36 ⫾ 0.87# 0.0001
t1/2 (h) 1.14 ⫾ 0.5 4.28 ⫾ 0.54# 4.99 ⫾ 0.84# 4.86 ⫾ 0.99# 0.0001
MRT (h) 2.09 ⫾ 0.11 6.145 ⫾ 0.37# 6.25 ⫾ 0.71# 5.89 ⫾ 1.07# 0.0001
TA (therapeutic 1 3.215 ⫾ 0.188* 1.73 ⫾ 0.078* 1.388 ⫾ 0.086* -
availability) - - 0.54 ⫾ 0.033† 0.43 ⫾ 0.048† -
* TA in comparison with Voveran.
† TA in comparison with LNE-0.
Comparison of different groups using ANOVA, showing the P-value less than 0.0001, indicating the significant
difference between different groups for cmax, AUCtotal, t1/2, and MRT.
# Comparison with Voveran injection—Newman-Keul’s Multiple Comparison (Student’s t test) test. P ⬍ 0.05 was
considered to be statistically significant.

showed that Cmax and AUCtotal were significant be-
tween all groups, whereas t1/2 and MRT were signif-
icant only in Voveran威 versus LNE-0, Voveran威 ver-
sus LNE-3, Voveran威 versus LNE-4, but not
significant in LNE-4 versus LNE-3 and LNE-4 versus
LNE-0. Diclofenac in lipid nanoemulsions showed an
improved pharmacokinetic profile with an increase in
AUCtotal, elimination half-life, and MRT in compari-
son to Voveran威 injection, a solution form.
for use of the HPLC facility; Prof. E. Ram Reddy and
Mr. Gopal Kishan Rao and the Central Instrumenta-
tion Centre, Kakatiya University, for their help in
getting DSC curves. We are thankful to All India
Council for Technical Education (AICTE), New Delhi
for Research Promotion Scheme grant, F. No: 8023/
BOR/RID/RPS-155/2008-2009.
Conflict of Interest Declaration

4. Conclusion The author(s) declare that they have no competing

In conclusion, LNEs containing diclofenac were pre-
pared. No changes in the globule sizes and Zps of the
emulsions were found due to autoclaving. The effects
of cholesterol on the size and Zp of the emulsions was
determined. Further, the stability under stress condi-
tions of centrifugation and dilution and changes in the
storage at RT were also measured. The total drug
content and EEs were determined. In general, above
97% of drug was entrapped in oil phase. The opti-
mized formulations were tested for drug release. Fi-
nally, all the optimized emulsions were compared with
marketed diclofenac injection (Voveran威) for the
pharmacokinetic serum profiles in male Wistar rats.
Diclofenac in LNEs showed an improved pharmaco-
kinetic profile with an increase in AUCtotal, elimina-
tion half life, and MRT in comparison to Voveran威.
Acknowledgments
We thank Prof. Y. Madhusudan Rao, University Col-
lege of Pharmaceutical Sciences, Kakatiya University
interests.
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