REVIEW ARTICLE

Nanoemulsion: A Review Microfluidization

Microfluidization is a patented mixing
RK Chouksey 1*, H K Pandey1, A Maithil1; AK Jain 2 technology, which makes use of a device
called microfluidizer. This device uses a high-
Abstracts: In this review, the formation, characterization, properties and applications of nano-
pressure positive displacement pump (500-
emulsions are reviewed and summarized. Nanoemulsions are submicron sized emulsions that
20000 psi), which forces the product through
are under extensive investigation as drug carriers for improving the delivery of therapeutic
the interaction chamber, which consists of
agents. Nanoemulsions are the thermodynamically stable isotropic system in which two
small channels called "microchannels." The
immiscible liquid (water and oil) are mixed to form a single phase by means of an appropriate
product flows through the microchannels on
surfactants. Due to their small droplet size nano-emulsions possess stability against
to an impingement area resulting in very fine
sedimentation or creaming with Ostwald ripening forming the main mechanism of
particles of submicron range. The two
nanoemulsion breakdown. Nanoemulsion droplet sizes fall typically in the range of 20-200nm.
solutions (aqueous phase and oily phase) are
Diameter and surface properties of droplets of nanoemulsion plays an important role in the
combined together and processed in an
biological behavior of the formulation. Nanoemulsion show great promise for the future of
inline homogenizer to yield a coarse
cosmetics, diagnostics, drug therapies, and biotechnologies. The main application of
emulsion. The coarse emulsion is into a
nanoemulsions is the preparation of nanoparticles using a polymerizable monomer as the
disperse phase (the so-called miniemulsion polymerization method) where nanoemulsion microfluidizer where it is further processed
droplets act as nanoreactors. Another interesting application which is experiencing an active to obtain a stable nanoemulsion. The coarse
development is the use of nanoemulsions as formulations, namely for controlled drug delivery emulsion is passed through the interaction
and targeting. chamber of the microfluidizer repeatedly
until desired particle size is obtained. The
Key Words: Nanoemulsion, Microfluidization, microchannels, nanospheres. bulk emulsion is then filtered through a filter
INTRODUCTION equilibrium is single or multiphase.(3) under nitrogen to remove large droplets
Nanoemulsions are a group of dispersed Nanoemulsions possess various advantages resulting in a uniform nanoemulsions. Other
particles used for pharmaceutical and such as (4) method used for nanoemulsions preparation
biomedical aids and vehicles that show great • Nanoemulsions have a much higher is the phase inversion temperature
promise for the future of cosmetics, surface area and free energy than technique. (8)
diagnostics, drug therapies and macroemulsions that make them an
biotechnologies. Nanoemulsions can be effective transport system. Characterization of Nanoemulsions
defined as oil-in-water (o/w) emulsions with • Nanoemulsions do not show the problems Different characterization parameters for
mean droplet diameters ranging from 50 to of inherent creaming, flocculation, nanoemulsion include transmission electron
1000 nm. Usually, the average droplet size is coalescence and sedimentation, which are microscopy, nanoemulsion droplet size
between 100 and 500 nm. The particles can commonly associated with analysis, viscosity determination, refractive
exist as water-in-oil and oil-in water forms, macroemulsions. index, in vitro skin permeation studies, skin
where the core of the particle is either water irritation test, in vivo efficacy study,
• Nanoemulsions can be formulated in
or oil, respectively. The terms sub-micron thermodynamic stability studies, and surface
variety of formulations such as foams,
emulsion (SME) and mini-emulsion are used characteristics. The surface charge of the
creams, liquids and sprays.
as synonyms. Usually, SMEs contain 10 to 20 nanoemulsion droplets has a marked effect
• Nanoemulsions are non-toxic, non-irritant
per cent oil stabilized with 0.5 to 2 per cent on the stability of the emulsion system and the
hence can be easily applied to skin and
egg or soybean lecithin.(1) droplet in vivo disposition and clearance. (9)
mucous membranes.
Phase into an aqueous phase with a high-
• Nanoemulsions do not damage healthy Application of Nanoemulsions in Cosmetics
stress, mechanical extrusion process that is
human and animal cells hence are suitable Nanoemulsions have recently become
Nanoemulsions are made from surfactants
approved for human consumption and for human and veterinary therapeutic increasingly important as potential vehicles
common food substances that are 'Generally purposes. for the controlled delivery of cosmetics and
Recognized as Safe' (GRAS) by the FDA. for the optimized dispersion of active
These emulsions are easily produced in large FORMULATION ASPECTS FOR ingredients in particular skin layers. Due to
quantities by mixing water immiscible oil NANOEMULSIONS their lipophilic interior, nanoemulsions are
available worldwide. (2) Since nanoemulsions have very small particle more suitable for the transport of lipophilic
The Nanoemulsions are also referred as size range, they can be most effectively compounds than liposomes. Nanoemulsions
miniemulsions, ultrafine emulsions and produced using high-pressure equipment.(5) are acceptable in cosmetics because there is
submicron emulsions. Phase behavior no inherent creaming, sedimentation,
studies have shown that the size of the High-Pressure Homogenisation flocculation, or coalescence that are observed
droplets is governed by the surfactant phase This technique makes use of high-pressure with macro emulsions. The incorporation of
structure (bicontinuous microemulsion or homogenizer/piston homogenizer to potentially irritating surfactants can often be
lamellar) at the inversion point induced by produce nanoemulsions of extremely low avoided by using high-energy equipment
either temperature or composition. Studies particle size (up to 1nm). In a high-pressure during manufacturing. (10)
on Nanoemulsions formation by the phase homogenizer, the dispersion of two liquids Nanoemulsions have attracted
inversion temperature method have shown a (oily phase and aqueous phase) is achieved considerable attention in recent years for
relationship between minimum droplet size by forcing their mixture through a small inlet application in personal care products as
and complete solubilization of the oil in a orifice at very high pressure (500 to 5000 potential vehicles for the controlled delivery
microemulsion bicontinuous phase psi), which subjects the product to intense of cosmetics and the optimized dispersion of
independently of whether the initial phase turbulence and hydraulic shear resulting in active ingredients in particular skin layers. (11)
extremely fine particles of emulsion.
1Sri
Homogenizers of varying design are available Antimicrobial Nanoemulsions
Satya Sai College of Pharmacy, Sehore, M. P.,
India. for lab scale and industrial scale production Antimicrobial nanoemulsions are oil-in-
E-mail: chouksey_rajendra@yahoo.com of nanoemulsions. This technique has great water droplets that range from 200 to 600
*Corresponding author efficiency, the only disadvantage being high nm. They are composed of oil and water and
energy consumption and increase in are stabilized by surfactants and alcohol. The
2G. R Medical College, Gwalior, M.P., India. temperature of emulsion during processing.(6,7) nanoemulsionhas a broad-spectrum activity

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 REVIEW ARTICLE
against bacteria (e.g. E. coil, Salmonella s, S.
aureus), enveloped viruses (e.g. HIV, Herpes
simplex), fungi (e.g. Candida,
Dermatophytes), and spores (e.g. anthrax).
The nanoemulsion particles are
thermodynamically driven to fuse with lipid-
containing organisms.
This fusion is enhanced by the
electrostatic attraction between the cationic
charge of the emulsion and the anionic
charge on the pathogen. When enough
nanoparticles fuse with the pathogens, they
release part of the energy trapped within the
emulsion. Both the active ingredient and the
energy released destabilize the pathogen
lipid membrane, resulting in cell lysis and
death. In the case of spores, additional
germination enhancers are incorporated into
the emulsion. Once initiation of germination
takes place, the germinating spores become
susceptible to the antimicrobial action of the
nanoemulsion. As a result, the nanoemulsion
can achieve a level of topical antimicrobial
activity that has only been previously
achieved by systemic antibiotics. (12-13)
As A Mucosal Vaccine
Nanoemulsions are being used to deliver
either recombinant proteins or inactivated
organisms to a mucosal surface to produce
an immune response. The first applications,
an influenza vaccine and an HIV vaccine, can
proceed to clinical trials. The nanoemulsion
causes proteins applied to the mucosal
surface to be adjuvant and it facilitates
uptake by antigen-presenting cells.
Additional research is ongoing to complete
the proof of concept in animal trials for other
vaccines including Hepatitis B and
anthrax.(14) Mice and guinea pigs intranasally
immunized by the application of
recombinant HIV gp120 antigen mixed in
nanoemulsion demonstrated robust serum
anti-gp120 IgG, as well as bronchial, vaginal,
and serum anti-gp120 IgA in mice.
The serum of these animals
demonstrated antibodies that cross-reacted
with heterologous serotypes of gp120 and
had significant neutralizing activity against
two clade-B laboratory strains of HIV
(HIVBaL and HIVSF162) and five primary
HIV-1 isolates. The analysis of gp120-specific
CTL proliferation, INF-g induction, and
prevalence of anti-gp120 IgG2 subclass
antibodies indicated that nasal vaccination in
nanoemulsion also induced systemic, Th1-
polarized cellular immune responses. This
study suggests that nanoemulsion should be
evaluated as a mucosal adjuvant for
multivalent HIV vaccines. (15) Hepatitis B
virus infection remains an important global
health concern despite the availability of safe
and effective prophylactic vaccines.
Limitations to these vaccines include
requirement for refrigeration and three
immunizations thereby restricting use in the
developing world. A new nasal hepatitis B
vaccine composed of recombinant hepatitis B
surface antigen (HBsAg) in a novel
Inventi Rapid: Pharm Tech Vol. 2, Issue 1
[ISSN 0976-3783]
nanoemulsion adjuvant (HBsAg-
nanoemulsion) could be effective with fewer
administrations. Comprehensive pre-clinical
toxicology evaluation demonstrated that
HBsAg- nanoemulsion vaccine is safe and
well tolerated in multiple animal models. Our
results suggest that needle-free nasal
immunization with HBsAg-NE could be a safe
and effective hepatitis B vaccine, or provide
an alternative booster administration for the
parenteral hepatitis B vaccines. This vaccine
induces a Th1 associated cellular immunity
and also may provide therapeutic benefit to
patients with chronic hepatitis B infection
who lack cellular immune responses to
adequately control viral replication. Long-
term stability of this vaccine formulation at
elevated temperatures suggests a direct
advantage in the field, since potential
excursions from cold chain maintenance
could be tolerated without a loss in
therapeutic efficacy. (16)
A novel technique for vaccinating against
a variety of infectious diseases-using an oil-
based emulsion placed in the nose, rather
than needles-has proved able to produce a
strong immune response against smallpox
and HIV in two new studies. Developing
mucosal immunity may be very important for
protection against HIV. In the study, the
nanoemulsion HIV vaccine showed that it
was able to induce mucosal immunity,
cellular immunity, and neutralizing antibody
to various isolates of HIV virus. A protein
used by the team, gp120, is one of the major
binding proteins under study in other HIV
vaccine approaches. (17)
Nanoemulsion As Non-Toxic Disinfectant
Cleaner
A breakthrough nontoxic disinfectant cleaner
for use in commercial markets that include
healthcare, hospitality, travel, food
processing, and military applications has
been developed by EnviroSystems, Inc. that
kills tuberculosis and a wide spectrum of
viruses, bacteria and fungi in 5-10 min
without any of the hazards posed by other
categories of disinfectants. The product
needs no warning labels. It does not irritate
eyes and can be absorbed through the skin,
inhaled, or swallowed without harmful
effects. The disinfectant formulation is made
up of nanospheres of oil droplets #106 mm
that are suspended in water to create a NE
requiring only miniscule amounts of the
active ingredient, PCMX
(parachlorometaxylenol). The nanospheres
carry surface charges that efficiently
penetrate the surface charges on
microorganisms' membranes-much like
breaking through an electric fence. Rather
than "drowning" cells, the formulation allows
PCMX to target and penetrate cell walls. As a
result, PCMX is effective at concentration
levels 1-2 orders of magnitude lower than
those of other disinfectants; hence, there are
no toxic effects on people, animals, or the
environment. Other microbial disinfectants
require large doses of their respective active
ingredients to surround pathogen cell walls,
which cause them to disintegrate,
fundamentally "drowning" them in the
disinfectant solution. The formulation is a
broad-spectrum disinfectant cleaner that can
be applied to any hard surface, including
equipment, counters, walls, fixtures, and
floors. One product can now take the place of
many reducing product inventories and
saving valuable storage space. (18-19)
Nanoemulsions in Cell Culture Technology
Cell cultures are used for in vitro assays or to
produce biological compounds, such as
antibodies or recombinant proteins. To
optimize cell growth, the culture medium can
be supplemented with a number of defined
molecules or with blood serum. The
advantages of using nanoemulsions in cell
culture technology are better uptake of oil-
soluble supplements in cell cultures; improve
growth and vitality of cultured cells, and
allowance of toxicity studies of oil-soluble
drugs in cell cultures. (20)
Nanoemulsion in Cancer Therapy and
Targeted Drug Delivery
The effects of the formulation and particle
composition of gadolinium (Gd)-containing
lipid nanoemulsion (Gd-nanoLE) on the
biodistribution of Gd after its intravenous
(IV) injection in D1-179 melanoma-bearing
hamsters were evaluated for its application
in cancer neutron-capture therapy.
Biodistribution data revealed that Brij 700
and HCO-60 prolonged the retention of Gd in
the blood and enhanced its accumulation in
tumors. Upon dermal application, the drug
was predominantly localized in deeper skin
layers, with minimal systemic escape. This
has amounted to an absolute bioavailability
of 70.62%. Inhibition of P-glycoprotein efflux
by D-tocopheryl polyethyleneglycol 1000
succinate and labrasol would have
contributed to the enhanced peroral
bioavailability of PCL. This investigation
provides direct evidence on the localization
of high-molecular-weight, lipophilic drug,
PCL, in dermis. Further, the nanoemulsion
formulation has enhanced the peroral
bioavailability significantly to more than
70%. The developed nanoemulsion
formulation was safe and effective for both
peroral and dermal delivery of PCL. (22)
Camptothecin is a topoisomerase-I
inhibitor that acts against a broad spectrum
of cancers. However, its clinical application is
limited by its insolubility, instability, and
toxicity. The aim of the present study was to
develop acoustically active nanoemulsions
for camptothecin encapsulation to
circumvent these delivery problems. The
nanoemulsions were prepared using liquid
perfluorocarbons and coconut oil as the
cores of the inner phase. These
nanoemulsions were stabilized by
phospholipids and/or Pluronic F68 (PF68).
The nanoemulsions were prepared at high
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 REVIEW ARTICLE
drug loading of approximately 100% with a
mean droplet diameter of 220-420 nm.
Camptothecin in these systems showed
retarded drug release. Camptothecin in
nanoemulsions with a lower oil
concentration exhibited cytotoxicity against
melanomas and ovarian cancer cells.
Confocal laser scanning microscopy
confirmed nanoemulsion uptake into cells.
Using a 1 MHz ultrasound, an increased
release of camptothecin from the system
with lower oil concentration could be
established, illustrating a drug-targeting
effect. [23]
The scientists have investigated the
nanoemulsion containing risperidone (RSP)
to accomplish the delivery of drug to the
brain via nose. Risperidone nanoemulsion
(RNE) and mucoadhesive nanoemulsion
(RMNE) were characterized for drug content,
pH, percentage transmittance, globule size,
and zeta potential. Biodistribution of RNE,
RMNE, and risperidone solution (RS) in the
brain and blood of Swiss albino rats
following intranasal (i.n.) and intravenous
(i.v.) administration was examined using
optimized technetium-labeled [ (99 m) Tc-
labeled] RSP formulations. Gamma
scintigraphy imaging of rat brain following
i.v. and i.n. administrations were performed
to ascertain the localization of drug in brain.
Higher drug transport efficiency (DTE%) and
direct nose to brain drug transport (direct
transport percentage, DTP%) for
mucoadhesive nanoemulsions indicated
more effective and best brain targeting of
RSP amongst the prepared nanoemulsions.
Studies conclusively demonstrated rapid and
larger extent of transport of RSP by RMNE
(i.n.) when compared to RS (i.n.), RNE (i.n.),
and RNE (i.v.) into the rat brain. (24)
Another study reported the formulation
of filter sterilizable emulsion formulation of
paclitaxel using á-tocopherol as the oil phase
and á-tocopherylpolyethyleneglycol-1000
succinate (TGPS) and poloxamer 407 as
emulsifiers. The formulation exhibited better
efficacy and was more tolerable when
studied in B16 melanoma tumor model in
mice. (25)
Emulsion formulations also show
promise in cancer chemotherapy as vehicles
for prolonging the drug release after
intramuscular and intratumoral injection
(W/O systems) and as a means of enhancing
the transport of anti-cancer drugs via the
lymphatic system.[26] Positively charged
nanoemulsions systems are expected to
interact with negatively charged cell surfaces
more efficiently, and this aspect of the
positively charged nanoemulsions has been
explored for possibility of oligonucleotide
delivery to cancer cells.(27-30) Photodynamic
therapy (PDT) of cancer is based on the
concept that certain photosensitizers can be
localized in the neoplastic tissue, and
subsequently, these photosensitizers can be
activated with the appropriate wavelength
(energy) of light to generate active molecular
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species such as free radicals and singlet
oxygen (1O2) that are toxic to cells and
tissues. (31-33) Various PDT therapies have
reported two different vehicles for
photosensitizers, a cremophor oil emulsion
and DPPC (dipalmitoylphosphatidylcholine)
liposomal vesicles. The reported
pharmacokinetic studies clearly indicate that
the former vehicle yields a significantly
larger selectivity of tumor targeting, mainly
as a consequence of an enhanced
accumulation in the malignant lesion.
Neutron Capture Therapy (NCT) is a binary
radiation therapy modality that brings
together two components that when kept
separate had only minor effects on the cells.
The first component is a stable isotope of
boron or gadolinium (Gd) that can be
concentrated in tumor cells by a suitable
delivery vehicle. The second is a beam of
low-energy neutrons. Boron or Gd in or
adjacent to the tumor cells disintegrates after
capturing a neutron, and the high energy
heavy charged particles produced through
this interaction destroy only the cancer cells
in close proximity to it, leaving adjacent
normal cells largely unaffected. (34) The
success of NCT relies on the targeting of
boron and Gd-based compounds to the
tumor mass and to achieve desirable
intracellular concentrations of these agents.
At the present time, there are two targets
with NCT, namely glioblastoma (malignant
brain tumor) and malignant melanoma.
The perfluorochemical nanoemulsions
(PFCE) have opened interesting
opportunities in cancer therapy. It is
suggested that fluorocarbon emulsions might
find a role in photodynamic therapy, both as
carriers for sensitizing dyes and to maintain
tissue oxygenation in hypoxic regions of solid
tumors. The high solubility of oxygen in
fluorocarbon emulsions maintains solution
oxygen tension, optimizing photo-oxidative
damage. The hydrophobic anti-cancer drugs
can be delivered to the tumor mass by
dissolving them in a hydrophobic core of the
emulsion. Furthermore, PFCE can be used as
an adjuvant to radiation therapy and/or
chemotherapy in the treatment of solid
tumors.(36-37)
The preclinical studies have shown very
positive effects with single dose and
fractionated radiation in several rodent solid
tumor models. Many widely used anticancer
drugs, including anti-tumor alkylating agents
and doxorubicin, have shown improved
response by PFCE coadministration. (38) Also,
local application of toxic doses of PFCEs
resulted in the necrosis of cancer cells. This
is especially promising in the treatment of
cancers of the head and neck regions that are
currently difficult to treat. (39)
Nanoemulsion in the Treatment of
Various Other Disease Conditions
Pharmos' (US-based company) has
developed the nanoemulsion topical
diclofenac cream as a potential treatment for
osteoarthritis (OA) pain. OA is a painful
condition affecting more than 30 million
people in the USA and is the most frequent
cause of physical disability among adults,
mainly elderly. Topical diclofenac is also
being considered as treatment for soft tissue
injuries, sprains, and strains. It is estimated
that 20% of OA patients are not receiving
treatment, mainly due to gastrointestinal
side effects of oral NSAIDs and
cardiovascular risk of COX-2 inhibitors. A
topical NSAID offering adequate pain relief
targeted to the site of injury with an
improved safety profile could become a
treatment alternative for these patients. In
the USA, there are no approved topical
NSAIDs for the treatment of OA. Pharmos' NE
technology consists of an efficient solvent-
free topical vehicle based and drug
entrapment in stable, submicron particles of
oil-in-water emulsions with a mean droplet
size between 100 and 200 nm that are
uniformly dispersed in an aqueous phase.
One of the unique characteristics of the
nanoemulsion technology is the relatively
high percentage of total particle volume
occupied by the internal hydrophobic oil core
of the droplets. This provides high
solubilization capacity for lipophilic
compounds compared to other lipoidal
vehicles such as liposomes. Viscosity-
imparting agents are used for nanoemulsion
thickening to produce creams with the
desired semisolid consistency for application
to the skin. The skin penetrative properties
of the solvent-free nanoemulsion delivery
technology and its low irritancy make this
novel topical nanovehicle a promising
candidate for effective transcutaneous
delivery of lipophilic drugs. Primaquine (PQ)
is one of the most widely used antimalarial
and is the only available drug till date to
combat relapsing form of malaria especially
in case of Plasmodium vivax and Plasmodium
ovale. Primaquine acts specifically on the
pre-erythrocytic schizonts that are
concentrated predominantly in the liver and
causes relapse after multiplication. However,
application of PQ in higher doses is limited
by severe tissue toxicity including
hematological and GI-related side effects that
are needed to be minimized. Lipid
nanoemulsion has been widely explored for
parenteral delivery of drugs. Primaquine
when incorporated into oral lipid
nanoemulsion having a particle size in the
range of 10-200 nm showed effective
antimalarial activity against Plasmodium
bergheii infection in Swiss albino mice at a
25% lower dose level as compared to
conventional oral dose. Lipid nanoemulsion
of primaquine exhibited improved oral
bioavailability and was taken up
preferentially by the liver with drug
concentration higher at least by 45% as
compared to the plain drug. (40)
Nanoemulsion Formulations for Improved
Oral Delivery of Poorly Soluble Drugs
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 REVIEW ARTICLE
Nanoemulsion formulations were developed
to enhance oral bioavailability of
hydrophobic drugs. Paclitaxel was selected
as a model hydrophobic drug. The oil-in-
water (o/w) nanoemulsions were made with
pine nut oil as the internal oil phase, egg
lecithin as the primary emulsifier, and water
as the external phase. Stearylamine and
deoxycholic acid were used to impart
positive and negative charge to the
emulsions, respectively.
The formulated nanoemulsions had a
particle size range of 90-120 nm and zeta
potential ranging from +34 mV to 245 mV.
Following oral administration, a significantly
higher concentration of paclitaxel was
observed in the systemic circulation when
administered in the nanoemulsion relative to
control aqueous solution. The results of this
study suggest that nanoemulsions are
promising novel formulations that can
enhance the oral bioavailability of
hydrophobic drugs. (41)
Coenzyme Q10 (CoQ10), also known as
ubiquinone, is used for energy production
within cells and acts as an anti-oxidant. Since
CoQ10 is highly lipophilic, the topical and
oral bioavailability is very low. Several
attempts have been made to improve
absorption. Latest technical developments
reveal that encapsulation of CoQ10 in
nanoemulsion s results in a significantly
enhanced bioavailability. The application of
CoQ10 has been further improved by the
development of novel CoQ10 double
nanoemulsions containing tocopherol and
CoQ10 in individual nanodroplets. In
addition, the CoQ10 concentration in these
nanoemulsions could be increased by the
development of a supersaturated CoQ10
nanoemulsion . (42)
Nanoemulsions As A Vehicle
From in vitro and in vivo data, it was
concluded that the developed nanoemulsion
s have great potential for transdermal drug
delivery of aceclofenac. (43) The
nanoemulsion of the system containing
ketoprofen evidenced a high degree of
stability. Ketoprofen-loaded nanoemulsions
enhanced the in vitro permeation rate
through mouse skins as compared to the
control. (44)
The study was developed to evaluate the
potential of nanoemulsions for increasing the
solubility and the in vitro transdermal
delivery of carvedilol. The prepared
nanoemulsion s were subjected to physical
stability tests. Transdermal permeation of
carvedilol through rat abdominal skin was
determined with the Keshary-Chien diffusion
cell. Significant increase (P < 0.05) in the
steady state flux (Jss) and permeability
coefficient (Kp) was observed in
nanoemulsion formulations as compared to
control or drug-loaded neat components. The
irritation studies suggested that the
optimized nanoemulsion was a non-irritant
transdermal delivery system. (45)
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Celecoxib, a selective cyclo-oxygenase-2
inhibitor, has been recommended orally for
the treatment of arthritis and osteoarthritis.
Long-term oral administration of celecoxib
produces serious gastrointestinal side
effects. Skin permeation mechanism of
celecoxib from nanoemulsion was evaluated
by FTIR spectral analysis, DSC thermogram,
activation energy measurement, and
histopathological examination. The
optimized nanoemulsion was subjected to
pharmacokinetic (bioavailability) studies on
Wistar male rats. Photomicrograph of a skin
sample showed the disruption of lipid
bilayers as distinct voids and empty spaces
were visible in the epidermal region. The
absorption of celecoxib through
transdermally applied nanoemulsion and
nanoemulsion gel resulted in 3.30- and 2.97-
fold increase in bioavailability as compared
to oral capsule formulation. Results of skin
permeation mechanism and pharmacokinetic
studies indicated that the nanoemulsions can
be successfully used as potential vehicles for
enhancement of skin permeation and
bioavailability of poorly soluble drugs. (46)
Self-Nanoemulsifying Drug Delivery
Systems
Self-nanoemulsifying drug delivery systems
for oral delivery of protein drugs:
Formulation development, in vitro transport
study and in vivo oral absorption study.(47)
The research project was done to develop a
self-nanoemulsifying drug delivery system
(SNEDDS) for non-invasive delivery of
protein drugs. An experimental design was
adopted to develop SNEDDS. Fluorescent-
labeled beta-lactamase (FITC-BLM), a model
protein, was loaded into SNEDDS through the
solid dispersion technique. The experimental
design provided 720 compositions of
different oil, surfactant, and co-surfactant at
various ratios, of which 33 SNEDDS
prototypes were obtained. A SNEDDS was
developed to load FITC- BLM into the oil
phase that can spontaneously form O/W NE
upon the addition of water. Fluorescently
labeled BLM (FITC-BLM), a model protein,
formulated into 16 SNEDDS preparations
through a solid dispersion technique were
studied for transport across monolayer. All
the SNEDDS NEs resulted in higher transport
rate than the free solution. The transport rate
by SNEDDS depends on the SNEDDS
composition. The SNEDDS significantly
increased the transport of FITC-BLM across
MDCK monolayer in vitro. SNEDDS may be a
potential effective delivery system for non-
invasive protein drug delivery. The oral
absorption of BLM in rats when delivered by
such a SNEDDS was investigated and showed
significantly enhance in the oral
bioavailability of BLM. So the SNEDDS has a
great potential for oral protein delivery.(48-50)
CONCLUSION
Nanoemulsion formulations offer several
advantages for the delivery of drugs,
biological, or diagnostic agents. Traditionally,
nanoemulsions have been used in clinics for
more than four decades as total parenteral
nutrition fluids. Although nanoemulsions are
chiefly seen as vehicles for administering
aqueous insoluble drugs, they have more
recently received increasing attention as
colloidal carriers for targeted delivery of
various anticancer drugs, photo sensitizers,
neutron capture therapy agents, or
diagnostic agents. Because of their
submicron size, they can be easily targeted to
the tumor area. It is expected that further
research and development work will be
carried out in the near future for clinical
realization of these targeted delivery
vehicles.
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Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126

INTERNATIONAL JOURNAL OF PHARMACY & LIFE SCIENCES

Development and bioavailability studies of atorvastatin
nanoemulsion
Rajendra Chouksey1*, Anand Kumar Jain2, Harish Pandey3, Ankur Maithil3
1, Gyan Vihar University, Jaipur, (RJ) - India
2, Gajraja Medical College, Gwalior, (M. P.) - India
3, Sri Satya Sai College of Pharmacy, Sehore, (M. P.) - India
Abstract
Poor bioavailability by the oral route can be due to poor solubility, degradation in GI lumen, poor membrane
permeation and presystemic elimination. Any of the approaches, which can alter these characteristics, should help in
improving the bioavailability of the drugs. Atorvastatin is one of the most important hypolipidemic drug available
today and circumventing the major problem of its poor bioavailability remains a bigger challenge of pharmaceutical
scientists.The objective of the present study was to develop and characterize an optimal stable nanoemulsion
formulation of atorvastatin (AT) with an aim to increase its bioavailability. The components selected for the
nanoemulsion were of GRAS category as the safety is the major determining factor for the excipient selection. Thus,
Safsol 218 and Oleic acid mixture was selected as the oil phase, surfactants namely Tween 20 and the co-
surfactants, Carbitol were selected. Pseudo ternary phase diagrams were constructed using aqueous titration method.
From phase diagram different concentrations of oil and surfactant, which formed nanoemulsions were selected based
on the thermodynamic stability and dispersibility test. Optimized formulation was selected for in vivo study on the
basis of higher drug release, optimum globule size, minimum polydispersity value, lower viscosity, and overall
lower surfactant concentration and co-surfactant. The difference in tmax of nanoemulsion formulation was found to
be significant (p<0.05) when compared to API drug suspension whereas the difference was insignificant (p>0.05)
when compared to tablet. The difference in Cmax of nanoemulsion was very significant (p<0.01) when compared
with the tablet suspensions and API drug suspension. The relative bioavailability of nanoemulsion to that of
conventional tablet suspensions was 356.32% whereas to that of API drug suspension was 559.86% respectively.
Thus nanoemulsions could be used effectively to improve the bioavailability of poorly water soluble drugs to
improve their bioavailability.
Key-Words: Nanoemulsion, Hypolipidemic, Polydispersity, Pseudo ternary phase diagrams

Introduction
Nanoemulsions being colloidal nanodispersions of oil
in water (or water in oil), thermodynamically stabilized
by an interfacial film of surfactant(s) and co-
surfactant(s) have revealed tremendous potential in
nanoengineering of various inorganic materials1.
Droplet size in thermodynamically stable
nanoemulsions is usually 10-100 nm2. The
homogeneous systems that can be prepared over a wide
range of surfactant concentrations and oil to water
ratios (1:4 %) are all fluids of low viscosity.
Nanoemulsions provide ultra low interfacial tensions
and large o/w interfacial areas.
* Corresponding Author:
E-mail: chouksey_rajendra@yahoo.com
Mob.: +91-9993815318
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug.: 2011, 982-988
982
Nanoemulsions have a higher solubilization capacity
than simple micellar solutions and their
thermodynamic stability offers advantages over
unstable dispersions, such as emulsions and
suspensions, because they can be manufactured with
little energy input (Low energy emulsification
techniques/heat or mixing) and has a long shelf life.
The nanosized droplets leading to enormous interfacial
areas associated with nanoemulsions would influence
the transport properties of the drug, an important factor
in sustained and targeted drug delivery3-4. The
attraction of o/w nanoemulsion systems lies in their
ability to incorporate hydrophobic drugs into the oil
phase thereby enhancing their solubility3.
Nanoemulsions have been reported to make the plasma
concentration profiles and bioavailability of drugs
more reproducible5-6. The objective of the present study
Research Article
is to prepare nanoemulsion of atorvastatin to improve
its solubility and bioavailability.
Material and Methods
Development of drug containing nanoemulsion
formulation
The drug stock solutions in oil mixture were prepared
in such a way that 10 mg dose was present in each
formulation complying the oil percentage for each
formula as selected from the phase diagram. This was
prepared by dissolving the 1000 mg of drug
individually in the 10, 15, 20 and 25 mL of oily
mixture, which compiles the 10%, 15%, 20% and 25%
oil compositions respectively in the formulae.
Screening of formulations on the basis of
thermodynamic stability studies
Microemulsions are thermodynamically stable systems
and are formed at a particular concentration of oil,
surfactant: co-surfactant mixture and water, with no
phase separation, creaming or cracking. It is the
thermostability which differentiates micro emulsion
from emulsions that have kinetic instability and will
eventually phase separate3. The thermodynamic
stability studies were performed on the basis of
following tests.
Centrifugation study
The selected formulations were centrifuged (REMI,
India) at the 5000 rpm for 30 mins and observed for
phase separation, creaming or cracking. The
formulations which showed maximum stability (no
creaming, cracking, phase separation) were selected
and studied for heating-cooling cycle, freeze-thaw
cycles and Dispersibility tests.
Heating cooling cycles
It is used to see the stressed effect of heating and
cooling on the nanoemulsion`s stability. In this study
the formulations were kept at 450 c and at 0 0C
temperature for not less then 48 h. for each temperature
cycle.
Freeze –thaw cycles (Accelerated ageing)
This test was performed for accelerated stability testing
of nanoemulsion formulations. In this study the
formulations were exposed at two different
temperatures i.e. -210C and 210C for each temperature
cycles not than 24 hrs. For the better estimation of
accelerated stability studies three such cycles should be
run for each batch of formulation. The formulations
which showed the maximum stability were selected for
further study.
Characterization of the nanoemulsion
Nanoemulsion has been characterized using a wide
variety of techniques. The characterization of
nanoemulsion is a difficult task due to their
complexity, variety of structures and components
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
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[Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
involved in these systems, as well as the limitation
associated with each technique, but such knowledge is
essential for their successful commercial exploitation.
The rate of release of sodium salicylate from a lecithin-
based nanoemulsion, is dependent upon their
microstructure7.
A complementarily of methods is generally required in
order to fully characterize these systems. At the
macroscopic level viscosity, conductivity and dielectric
methods provide useful information.
Phase Behavior studies
Phase diagram showing the nanoemulsion region,
information about of different phases as a function of
composition variables can be analyzed from vigorous
study of the phase diagrams.
Viscosity measurements
The viscosity of the prepared nanoemulsion
formulations were determined as such without dilution
by Brookfield DV III ultra V6.0 RV cone and plate
rheometer (Brookfield Engineering Laboratories, Inc,
Middleboro, MA) using spindle # CPE40 at 25 ±0.5°C.
The software used for the viscosity calculations was
Rheocalc V2.6.
Refractive index
Refractive index of selected formulations was
determined using an Abbe type refractrometer. It was
standardized using castor oil.
Dielectric measurements
They are powerful means of probing both structural
and dynamic features of nanoemulsions systems.
Electron microscopic studies
Morphology and structure of the nanoemulsion was
studied using transmission electron microscopy (TEM)
TOPCON 002B operating at 200 KV and of a 0.18 nm
capable of point to point resolution. In order to perform
the TEM, the nanoemulsion formulation was diluted
with distilled water (1/100). A drop of the diluted
nanoemulsion was directly deposited on the Copper
holey film grid and observed after putting fixing agent
and drying it in the filtered air.
Scattering techniques
Dynamic light scattering photon correlation
spectroscopy (PCS) is used to analyze the fluctuations
in the intensity of the scattering by the droplets due to
Brownian motion.
Interfacial tension measurement
The formation and properties of nanoemulsion can be
studied by measuring the interfacial tension. Spinning-
drop apparatus can be used to measure the ultra low
interfacial tension.
Droplet size distribution
Droplet size of the prepared nanoemulsion was
determined by using photon correlation spectroscopy,
 Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
which analyzes the fluctuations in light scattering due
to Brownian movement of the particles8. The Comparative dissolution profile in simulated gastric
formulation (0.1 mL) was dispersed in 50 mL (500 fluid (pH 1.2)
dilution) of distilled water in a volumetric flask and
gently mixed by inverting the flask and measurement 120
done using a Zetasizer (Nano ZS-90, UK). Light 100

% Drug release
scattering was monitored at 25°C at a 90° angle. 80
Refractive index/ Isotropicity 60
Refractive index of nanoemulsions formulations was 40
determined using an Abbe type refractrometer. It 20
basically gives an idea about the isotropicity of the 0
formulations. The isotropic nature of microemulsions 0 20 40 60 80
and their optical clarity makes their study by Time (min)
spectroscopic techniques straightforward, particularly
in comparison to conventional macroemulsions. Self-nanoemulsifying pellets Tablet Avas Tablet Atorlip

In-vitro drug release performance Fig. 1 Comparative dissolution profile

The study was performed by using dialysis bag
method9. The dialysis membrane used in the study was In vitro release test was performed in 250 ml of
Cellulose membrane (Sigma, USA). With a molecular distilled water, which was based on USP XXIV method
weight cut off of 12000 g/mole. (Dissolution apparatus I.P. 2, at 100 rpm and 37 ± 0.5
0
C). 1 ml of nanoemulsion formulation (Single dose
Table 1: In vitro dissolution study of self- containing 10 mg of AT Calcium) was placed in treated
nanoemulsifying pellets and marketed tablet Avas® dialysis bag (MWCO 12,000 g/mole; Sigma, USA) and
(microlab) and Atorlip® (Cipla) in simulated 1 mL samples was withdrawn at regular time intervals
gastric fluid (pH 1.2) (0, 0.5, 1, 1.5, 2,2.5,3,3.5, 4,4.5,5.5, 6,6.5,7, 8,9, 10,
Tablet- 12, 22 and 24 h) and same amount of distilled water
SNE pellet Tablet-Cipla
Time Microlab was replaced10. The withdrawn 1 ml samples were
CR CR CR diluted with 3 ml methanol and analyzed for the drug
min %R %R %R content by using developed RP-HPLC at 247 nm. The
µg/mL µg/mL µg/mL
same method was used for the suspension containing
0 5 00 5 00 5 00 10 mgof AT Calcium in 1 ml distilled water. The
release of drug from different selected nanoemulsion
3 10 35.60 10 30.23 10 22.35 formulations was compared with drug suspension and
finally selected formulation was used for the further
6 15 59.33 15 34.51 15 35.37 study.
10 20 81.14 20 46.42 20 40.84 Bioavailability study of Atorvastatin nanoemulsion
in rats
12 25 99.64 25 51.17 25 58.93 The ultimate test of success of a formulation depends
on its in vivo performance and for the assertion of the
15 30 99.80 30 64.04 30 66.28 same the in vivo studies were conducted in a suitable
animal model. Approval to carry out in vivo study was
20 35 99.45 35 83.54 35 91.57 obtained from Gyan Vihar University, Institutional
Animal Ethics Committee and their guidelines were
25 40 99.38 40 93.82 40 96.37 followed for the studies (173/CPCSEA). The animals
used for in vivo experiments were adult wistar female
30 45 99.32 45 95.70 45 98.38 albino rats (n=6) weighing 180-200 gm obtained from
Central Animal House of Gyan Vihar University,
60 50 99.14 50 97.34 50 99.37 Jaipur, India. The animals were kept under standard
laboratory conditions at a temperature of 25 ± 2°C and
relative humidity (55 ± 5%). The animals were housed
in animal cages, six per cage, with free access to
standard laboratory feed (Lipton feed, Mumbai, India)
and water ad libitum. The nanoemulsion formulation

Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
984
Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
GM1 that showed the highest release profile of drug in Tubes were stored at room temperature, 25 ± 2°C and
in vitro studies was subjected to in vivo studies. 6 relative humidity (55 ± 5%) for 30 minutes. The clotted
mg/kg9 body weight of the Atorvastatin Ca was used in blood was then centrifuged at 5000 rpm for 30 min.
the study.Six tablets with label claim of 10 mg The serum was separated and stored at -21 °C until
(Atorlip®) were taken, crushed in mortar, and mixed drug analysis was carried out using RP-HPLC method.
with 50 ml of double distilled water (1.2 mg/mL). Each The collected serum was extracted with ethyl-acetate (5
rat in this group was given 1.0 ml using oral feeding ml) by using Sodium phosphate buffer (pH 7, 1 ml) as
needle. Same dose of drug suspension and a protein-precipitating agent for analysis and
nanoemulsion was administered. The blood sampling centrifuged at 5000 rpm for 5 min and dried at room
was carried out for around 48 hours and 10 to 12 temperature. After evaporating the ethyl acetate solid
samples of blood were taken from each animal in the residue was reconstituted using 100 µl mobile phase
group. Blood was withdrawn from the juglar vein after and then analyzed by RP-HPLC. The samples were
anesthesia at 0 (pre-dose), 0.25, 0.5, 0.75, 1.0, 1.25, injected and the area was noted down in triplicate. The
1.5, 6, 12, 24, 36 and 48 h in micro centrifuge tubes. mean areas obtained by HPLC are given in the Table 2.
Table 2: Working formula for nanoemulsion (GM1)
Oil :Sefsol 218+ Oleic acid , Surfactant : Tween 20, Cosurfactant: Carbitol
Volume of components for Nanoemulsion Formulation Code
Oil (ml) Surfactant (ml) Co – surfactant (ml) Aqueous phase Atorvastatin (mg)
(ml)
0.10 0.19 0.19 0.52 1.2 GM1
pharmacokinetic (PK) parameters (tmax, Cmax, AUC0→t,
The formulations were given orally using oral feeding AUMC0→t and MRT 0→t) were calculated individually
needle. Dose for the rats was calculated based on the for each subject in the group and the values were
weight of the rats (6 mg/ kg). expressed as mean ±SD. The comparative in-vivo
Pharmacokinetic and statistical analysis bioavailability profiles of nanoemulsion formulation
Pharmacokinetic parameters were calculated by using GM1, API suspension and Tablet suspension are
non-compartmental analysis also called as Model shown in the Table 4.
independent analysis using WinNonLin version 4.0
(Pharsight Corp., Mountain View, CA). All
Table 4: Comparative observation table for in-vivo bioavailability studies in rat animal model (n=6).
Sampling time SNEDDS Tablet suspension API suspension
(Hours) (Mean Conc. ± SD) (Mean Conc. ± SD) (Mean Conc. ± SD)
0 0 0 0
0.25 24719.36 ± 142.54 4747.476 ± 63.76 3636.91 ± 57.56
0.5 35582.73 ± 345.54 5491.985 ± 36.87 4307.00 ± 24.87
0.75 37687.34 ± 733.16 5815.137 ± 62.97 4456.82 ± 54.89
1 41090.76 ± 531.78 6343.85 ± 64.86 4959.85 ± 86.36
1.25 43603.77 ± 632.63 6750.599 ± 145.35 5271.45 ± 75.37
1.5 37508.95 ± 456.56 5801.549 ± 48.25 4345.85 ± 86.74
6 24312.26 ± 731.84 3761.141 ± 65.36 2887.23 ± 75.36
12 12093.38 ± 234.47 1870.322 ± 124.12 1437.17 ± 44.66
24 6370.94 ± 32.47 987.3089 ± 32.85 765.24 ±75.05
36 3851.75 ± 42.74 599.0169 ± 24.86 548.16 ± 36.87
48 2811.38 ± 57.38 436.0885 ± 14.60 338.09 ± 23.21

Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
985
Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126

Table 5: Relative pharmacokinetic parameters of different formulations containing AT Calcium (n = 6).

Formulation tmaxa Cmaxb AUC0 → tc Rel. BAf (%)
(hrs) (ng/ml) (ngh/ml)
GM1 0.95 ± 0.13 43603.77 ± 632.63 383453.2 ± 2352.2 357.23± 43.3

Tablet Suspension 2.24 ± 1.74 6750.599 ± 145.35 35432.4 ± 1143.53 765.86 ±32.7
API drug 2.52 ± 1.93 5271.45 ± 75.37 68232.5 ± 6734.46 893.57 ±84.2
Suspension
a
time of peak concentration; b peak of maximum concentration;e area under the concentration time profile curve until
last observation; fRelative bioavailability of formulations

little energy input (heat or mixing) and has a long shelf

Compa ra tive bioa vaila bility profile life thus scale up technology is easy. The nanosized
in Ra t droplets leading to enormous interfacial areas
60000 associated with nanoemulsions would influence the
transport properties of the drug, an important factor in
Concentration (ng/ml)

40000 sustained and targeted drug delivery. The droplet size

SNEDDS
20000 Formulations
Tablets
0 Suspension
0 API
0.75 1.5 Suspension
24
Time (Hrs)
Fig 2: Comparative In-vivo absorption profile of
atorvastatin using different formulations in rat
model
Results and Conclusion
Poor oral bioavailability has the consequence of more
variable and poorly controlled plasma concentration
and drug effects. Poor bioavailability by the per-oral
route can be due to poor solubility, degradation in GI
lumen, poor membrane permeation and presystemic
elimination. Any of the approaches, which can alter
these characteristics, should help in improving the
bioavailability of the drugs.
The design of effective formulation for drugs has long
been a major challenge, because drug efficacy can be
severely limited by instability or poor solubility in the
vehicle. Nanoemulsions being a versatile technology
have the potential to increase the bioavailability of
drug in many ways. They act as supersolvents for
poorly soluble drugs. Nanoemulsions have a higher
solubilization capacity than simple micellar solutions
and their thermodynamic stability offers advantages
over unstable dispersions, such as emulsions and
suspensions, because they can be manufactured with
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
986
of nanoemulsion is between 14 to 100 nm, and can be
sterilized by filtration. The use of nanoemulsion as
drug delivery systems can improve the efficacy of
drug, allowing the total dose to be reduced and thus
minimizing side effects.
Atorvastatin, a synthetic cholesterol-lowering agent, is
a HMG-CoA (3 hydroxy-3-methylglutaryl-coenzyme
A) reductase inhibitor. The calcium salt of atorvastatin
is used in the treatment of primary
hypercholesterolemia and dyslipidemia. Atorvastatin is
highly lipophillic in nature (logP (octanol/water),
6.36), freely soluble in methanol and soluble in
dimethylsulphoxide(DMSO) and dimethyl formamide.
It is soluble in aqueous medium with a pH of less than
4.0. It is very slightly soluble in distilled water,
phosphate buffer (7.4) and acetonitrile; while it is
slightly soluble in ethanol. 20.4 µg/ml (pH 2.1), 1.23
mg/mL (pH 6.0). Owing to its poor water-solublity the
drug, it gets metabolized in the GI tract, with absolute
bioavailability of mere 14%. Atorvastatin is without
question one of the most important hypolipidemic drug
available today and circumventing the major problem
of its poor bioavailability remains a bigger challenge of
pharmaceutical scientists. With this background in
mind, the objective of the present study was to develop
and characterize an optimal stable nanoemulsion
formulation of atorvastatin with an aim to increase its
bioavailability.
The components selected for the nanoemulsion were of
GRAS category as the safety is the major determining
factor for the excipient selection. They offer
formulative and physiological advantages and their
degradation products resemble the natural end products
of intestinal digestion. Thus, Safsol 218 and Oleic acid
Research Article
mixture were selected as the oil phase for the
development of the formulation because of the highest
solubility of Atorvastatin (130.06± 2.68mg/mL) in it.
The higher solubility of the drug in the oil phase is
important for the nanoemulsion to maintain the drug in
solubilized form. In the present study, surfactants
namely Tween 20 and the co-surfactants, Carbitol were
selected. AT Calcium is very slightly soluble in water
at a pH below 4, therefore different buffers were used
to prepare the calibration curve for preliminary studies.
The relationship between the phase behaviour of a
mixture and its composition can be captured with the
aid of a phase diagram. Pseudo ternary phase diagrams
were constructed using aqueous titration method10.
After taking observation, pseudo ternary phase
diagrams were constructed based on the observations
marked during titration. Thus while studying the phase
diagrams, it could be seen that the free energy of
nanoemulsion formation can be considered to be
dependent on the extent to which the surfactant lowers
the surface tension of the oil-water interface and the
change in dispersion entropy. Therefore,
nanoemulsification is specific to the nature of the oil
and surfactant pair, the surfactant concentration and
oil/surfactant ratio. Thus, a negative free energy of
formation is achieved when large reduction in surface
tension is accompanied by significant favourable
entropic changes. In such case nanoemulsion formation
is spontaneous and the resulting dispersion is
thermodynamically stable. While going through
different pseudo ternary phase diagrams, oil could be
solubilized up to the extent of 25% v/v. Therefore,
from phase diagram different concentrations of oil,
which formed nanoemulsions, were selected at a
difference of 5% (10, 15, 20 and 25%) and for each
percentage of oil selected; only those formulations
were taken from the phase diagram, which used least
concentration of Smix, irrespective of Smix ratio used.
These formulations passed the thermodynamic stability
and dispersibility test showing that they can be diluted
in GI fluids still maintaining the nanosized character
without drug precipitation that will lead to higher
solubility.
Droplet size analysis of the selected formulations was
carried out. The difference in the droplet size between
the formulation was not statistically significant. There
was only a marginal difference in the mean droplet size
of formulations but polydispersity (0.237) was
minimum in the case of formulation, GM1, containing
10% of oil suggesting uniformity in the globule size
(42.8 nm) of the formulation.
It was observed that the viscosity of all the
formulations is less than 45 mP. Formulation, GM3,
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
987
[Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
has the minimum viscosity (10.03 ± 0.91 mP), as
compared to the other formulations. The viscosity of
the selected formulation GM1 was found to be
intermediate showing numeric value of 27.51 ± 1.01
mP.
In vitro drug release was carried out using a dialysis
bag and the selected formulations were compared for
the drug release with API drug suspension. The release
of drug from nanoemulsion formulations was high
when compared to drug suspension The drug release
was found to highest (99.65±0.75%) in case of GM1 as
compared to the API drug suspension (13.77±0.214) in
6.5 hrs.
Therefore, the optimized formulation, GM1, having
higher drug release (99.65±0.75%), optimum globule
size (42.8 nm), minimum polydispersity value (0.237),
lower viscosity (27.51 ± 1.01 mP), and overall, lower
surfactant concentration (19%) and co-surfactant (19%)
was selected for in vivo study.
The in vivo study was performed to quantify AT
Calcium, after oral administration of formulations
containing drug. The plasma concentration time
profiles of AT Calcium in adult female albino wistar
rats following oral administration of the nanoemulsion
(GM1) formulations, marketed tablet (AtorlipTM 10)
suspensions and API drug suspensions formulations
were compared. It was found that the Cmax in serum for
nanoemulsion formulation (45729.33 ±13689.15
ng/mL) represents greater improvement than the
marketed formulations (8158.26 ±1568.84 ng/mL) or
simple API drug suspension (5009.16 ±1339.59
ng/mL). It was also observed that AUC0→t GM1
formulation was 394520.05 ±87932.39 ng.h/mL and
thus the difference were highly significant (p<0.01) as
compared to AUC0→t ( 110271.4± 47224.84) and
(70467.13 ± 26350.34) highly significant (p<0.01)
tablet suspension and API suspension (Table 7.3)
formulations respectively.
Statistically, the difference in tmax of GM1
Nanoemulsion formulation was found to be significant
(p<0.05) when compared to tmax of API drug
suspension where as the difference was insignificant
(p>0.05) when compared to tablet. The difference in
Cmax of GM1 formulation was very significant (p<0.01)
when compared with the Tablet suspensions and API
drug suspension. It was also observed that AUC0→t of
GM1 formulation was ng.h/m L, thus the difference
were very significant (p<0.01) as compared to Tablet
suspensions and API drug suspension formulations.
Both the values of GM1were very significant (p<0.01)
as compared to drug suspension. The relative
bioavailability of GM1Nanoemulsion to that of
conventional Tablet suspensions was 356.32% where
Research Article
as to that of and API drug suspension was 559.86%
respectively.
Therefore, based on higher drug release, optimum
globule size, minimum polydispersity, lower viscosity,
lower surfactant concentration, higher solubility as well
as higher bioavailability without variable absorption
has been optimized. The composition of optimized
formulation (GM1) was Sefsol 218 (5% v/v), Oleic
acid (5% v/v), Tween-20 (19% v/v), Carbitol (19%v/v)
and distilled water (52% v/v) as oil, surfactant,
cosurfactant and aqueous phase respectively,
containing 10 mg of AT Calcium.
The in vivo studies revealed significantly greater extent
of absorption than the conventional tablet suspension
and API drug suspension formulation. The absorption
of AT Calcium from Tween 20 nanoemulsion resulted
in 3.56 fold increase in bioavailability as compared to
conventional Tablets suspension and 5.59 fold to that
of API drug suspension. Furthermore, this result
attributed that the presence of surfactants in
nanoemulsion system might have caused changes in
membrane permeability of the GI tract and the
inhibition of an apically polarized efflux system, which
could lead to enhancement of the oral absorption.
Acknowledgements
Author is thankful to Lupin Pharmaceuticals Pvt. Ltd.
for providing drug sample. We are also thankful to
Medical Superintendent, G. R. Medical College,
Gwalior for kind cooperation, guidance and help
during my tenure of my research work. I am also
thankful to Dean Research, Gyan Vihar University,
Jaipur for all the support and guidance.
References
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strategies for engineering drug nanoparticles.
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delivery systems. Adv. Drug Deliv. Rev., 45(1):
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Masuda K. (2002). Microemulsion formulation
for enhanced absorption of poorly soluble
drugs II. J. Control. Release, 81: 75-82.
6. Constantinides P. P. (1995). Lipid
microemulsions for improving drug dissolution
and oral absorption and biopharmaceutical
aspects. Pharm. Res., 12(11): 1561-1572.
7. Khoshnevis P., Mortazavi S. A., Lawrence M.
J. and Aboofazeli R. (1997). In-vitro release of
sodium salicylate from water-in-oil
phospholipid microemulsions. J. Pharm.
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8. Attwood D., Mallon C. and Taylor C. J.
(1992). Phase studies of oil-in water
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9. Shen H. R. and Zhong M. K. (2006).
Preparation and evaluation of self
microemulsifying drug delivery system
(SMEDDS) containing atorvastatin. Pharmacy
and Pharmacology., 58: 1183-1191.
10. Shafiq S., Faiyaz S., Sushma T., Farhan J. A.,
Khar R. K. and Mushir A. (2007).
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ramipril nanoemulsion formulation. Eur. J.
Pharm. Biopharm., 66(2): 227-243.
 International Journal of Pharmacy and Pharmaceutical Sciences
 
ISSN- 0975-1491 Vol 3, Issue 3, 2011

Research Article 

PREPARATION AND EVALUATION OF THE SELF EMULSIFYING DRUG DELIVERY SYSTEM 
CONTAINING ATORVASTATIN HMG­COA INHIBITER 
 
RAJENDRA CHOUKSEY1*, HARISH PANDEY2, A. K. JAIN3, HIMESH SONI4, G. K. SARAOGI4 
1 Gyan Bihar University, Jaipur (Rajasthan), 2 Sai Satya Sai College of Pharmacy, Sehore (M.P.), 3 GR Medical College Gwalior (M.P.),                    
4 Lakshmi Narain College of Pharmacy, Bhopal (M.P.) India Email: himeshsoni@rediffmail.com 

 Received: 28 March 2011, Revised and Accepted: 26 April 2011 

ABSTRACT 
As a consequence of modern drug discovery techniques, there has been a steady increase in the number of new pharmacologically active lipophilic 
compounds that are poorly water‐soluble. It is a great challenge for pharmaceutical scientists to convert those molecules into orally administered 
formulations with sufficient bioavailability. Among the approaches to improve the oral bioavailability of these molecules, the use of self‐emulsified 
drug  delivery  systems  (SEDDS)  has  been  shown  to  be  reasonably  successful  in  improving  the  oral  bioavailability  of  poorly  water‐soluble  and 
lipophilic  drugs.  In  the  present  study  we  formulate  self‐emulsified  drug  delivery  systems  (SEDDS)  through  the  nanoemulsion  for  the  effective 
delivery of Atorvastatin which is a HMG‐COA Inhibiter.  
Keywords: Atorvastatin, Self emulsifying drug delivery systems, Oral drug delivery, Bioavailability. 
  
INTRODUCTION  catabolism of LDL, its also reduces LDL production and the number 
of LDL particles. The absolute bioavailability of atorvastatin (parent 
The  oral  route  is  one  the  most  commonly  used  method  for  drug)  is  approximately  14%  and  the  systemic  availability  of  HMG‐
administration  of  drugs  and  drug  delivery.  The  only  disadvantages  CoA  reductase  inhibitory  activity  is  approximately  30%  this  low 
of  this  method  are  sluggish  onset  time,  the  possibility  of  erratic  systemic  availability  is  ascribed  to  presystemic  clearance  in 
absorption,  degradation  of  some  specific  drugs  by  digestive  gastrointestinal mucosa and/or hepatic first‐pass metabolism.  
enzymes.  The  poorly  water‐soluble  drugs  such  as  HIV  protease 
inhibitors,  glycoprotein  inhibitors  and  anticancer  drugs  have  The  food  decreases  the  rate  and  extent  of  drug  absorption  by 
problems to produce and retain a good solubility in gastrointestinal  approximately 25% and also responsible for reduction in C max and 
tract. The drug delivery industry scientists are used a wide range of  AUC,  LDL‐C  level.  A  blood/plasma  ratio  of  approximately  0.25 
methods  to  improve  the  dissolution  rate  of  poorly  water‐soluble  indicates  poor  drug  penetration  into  red  blood  cells.  Based  on 
drugs,  including  formulations  containing  nanoparticles,  a  solid  observations in rats, atorvastatin calcium is likely to be secreted in 
solution  formulation  or  self  emulsifying  drug  delivery  system  human  milk.  So  it  is  aimed  to  formulate  a  SEDDS  containing  a 
(SEDDS),  and  stable  amorphous  form  of  the  drug  1‐5.  SEDDS  is  an  lipophilic  drug,  atorvastatin  and  to  explore  the  potential  of  this 
isotropic mixture of oil, surfactant and/or co‐surfactant can be used  carrier  for  improvement  of  the  oral  bioavailability  of 
for  formulations  to  improve  the  absorption  of  drugs  in  atrovastatin.11 
gastrointestinal  tract  and  solve  the  solubility  problems.  SEDDS  can 
produce  fine  oil/water  emulsion  after  dilution  in  gastrointestinal  MATERIALS AND METHODS 
fluids  and  provide  large  interfacial  area  for  drug  partitioning 
Materials 
between oil and water phases and so increase in solubility rate and 
extent of absorption  6‐10. Atorvastatin, a 3‐hydroxy‐3‐methylglutaryl  Atorvastatin was obtained from Lupin Laboratory, Aurangabad as a 
coenzyme  A  (HMG‐CoA)  reductase  inhibitor  (a  statin),  is  a  lipid  gift sample.  
regulating  drug  with  actions  on  plasma  lipids  similar  to  those  of 
simvastatin.  It  is  used  to  reduce  LDL‐cholesterol,  apolipoprotein  B,  Formulation of self emulsifying drug delivery system (SEDDS) 
and  triglycerides,  and  to  increase  HDL‐cholesterol  in  the  treatment 
of  hyperlipidaemias  ,  including  hypercholesterolaemias  and  The drug stock solutions in oil mixture were prepared in such a way 
combined  (mixed)  hyperlipidaemia  (type  IIa  or  IIb  that  10  mg  dose  is  present  in  each  formulation  complying  the  oil 
hyperlipoproteinaemias),  hypertriglyceridaemia  (type  IV),  and  percentage for each formulae selected from the phase diagram. This 
dysbetalipoproteinaemia  (type  III).  Atorvastatin  lowers  plasma  was prepared by dissolving the 1000 mg of drug individually in  the 
cholesterol and lipoprotein levels by inhibiting HMG‐CoA reductase  10, 15, 20 and 25 mL of oily mixture, which complies the 10%, 15%, 
and cholesterol synthesis in the liver and by increasing the number  20%  and  25%  oil  compositions  respectively  in  the  formulae.  The 
of  hepatic LDL receptors on the cell‐surface to enhance  uptake  and  drug stock is shown in Table no 1. 
 
Table 1: Preparation of drug stock for each formula selected in phase diagram 
S.NO  Oil percentage in formulations  Amount of drug  Volume of oil  Final concentration 
(mg)  (mL)  (mg/μL) 
1  10%  1000 10 10 mg/100 μL
2  15%  1000 15 10 mg/150 μL
3  20%  1000  20  10 mg/200 μL 
4  25%  1000  25  10 mg/250 μL 
 
Construction of pseudo­ternary phase diagrams 15‐16  were  chosen  in  increasing  concentration  of  cosurfactant  with 
respect to surfactant and increasing concentration of surfactant with 
Surfactant  and  co‐surfactant  (Smix)  in  each  group    were  mixed  in  respect to cosurfactant for detailed study of the phase diagrams for 
different volume ratios (1:0, 1:1, 1:2, 1:3, 2:1, 3:1, 4:1) and the stock  the nanoemulsions formation.  
of 100 mL of each groups was prepared (Table 2). These Smix ratios 

 
 Chouksey et al. 
Int J Pharm Pharm Sci, Vol 3, Issue 3, 2011, 147­152 

Table 2: Different volumes of surfactant and co­surfactant taken to make a stock Smix ratio 
S.No  Volume of Surfactant  Volume of Cosurfactant  Ratio of Smix 
(mL)  (mL) 
1  100  0  1:0 
2  50  50  1:1 
3  33.3  66.7 0.5:1 or 1:2
4  25  75 1:3 
5  66.7  33.3  2:1 or 1:0.5 
6  75  25  3:1 
7  80  20 4:1 
 
For  each  phase  diagram,  oil  and  specific  Smix  ratio  was  mixed  diagrams,  the  oil  phase  (oleic  acid:  Sefsol,  1:1)  was  mixed  with 
thoroughly  in  different  volume  ratios  from  1:9  to  9:1  in  different  different  ratio  of  surfactant  and  cosurfactant  (Tween  20  and 
small glass test tubes. Sixteen different combinations of oil and each  Carbitol®  respectively) and mixture was titrated with distilled water 
Smix, 1:9, 1:8, 1:7, 1:6, 1:5, 2:8 (1:4), 1:3.5, 1:3, 3:7 (1:2.3), 1:2, 4:6  until  it  turned  turbid.  Examine  each  and  every  point  I  detailed  and 
(1:1.5),  5:5  (1:1),  6:4  (1:0.7),  7:3  (1:0.43),  8:2(1:0.25),  9:1  (1:0.1)  note it down. Pseudo ternary phase diagrams were drawn by using 
were  made  so  that  maximum  ratios  were  covered  for  the  study  to  data obtained in aqueous titration method as shown in Figure.1 (A‐
delineate  the  boundaries  of  phases  precisely  formed  in  the  phase  G).  The  amount  of  water  added  to  give  water  concentration  in  the 
diagrams.  range  of  5‐95%  of  total  volume  at  5%  intervals.  After  every  5% 
addition  of  the  water  to  the  oil  and  Smix  mixture,  visual 
For  the  determination  of  existence  zone  of  microemulsion,  observations  were  made  as  shown  in  Figure  1.  The  ratio  of 
pseudoternary  phase  diagrams  were  constructed  using  water  surfactant and co surfactant (Tween 20 and Carbitol ®) were used for 
titration  method  [17‐19].  To  construct  pseudoternary  phase  the titration  
        
A              B 
Water Water
S:CoS-2:1
Ratio-1:2

Oil Smix Oil Smix

       
C              D 
Water Water

S:CoS-3:1 S:CoS-1:3

Oil Smix Oil Smix

         
E 
Water
S:CoS-4:1

Oil Smix
 

148 
 Chouksey et al. 
Int J Pharm Pharm Sci, Vol 3, Issue 3, 2011, 147­152 

 
 
F                                                                                                                                           G 
Water
Water
Ratio-1:1 Ratio-1:0

Oil Smix
                                      Oil Smix
 
Fig. 1:  A­G Phase diagram of Smix containing the surfactant and co surfactant in ratio 1:0, 1:1, 1:2, 1:3, 2:1 3:1and 4:1 respectively 
  
Self emulsifying capacity  Heating cooling cycles 
For evaluation of self‐emulsification properties of formulations, 1 ml  It  is  used  to  see  the  stressed  effect  of  heating  and  cooling  on  the 
of  each  formulation  was  added  to  0.1N  hydrochloric  acid  (50  mL)  nanoemulsion`s stability. In this study the formulations were kept at 
under  continuous  stirring (60  rpm) at  37°C and then spreadability,  450  c  and  at  0  0C  temperature  for  not  less  then  48  hrs  for  each 
tendency  to  emulsifying  and  progress  of  emulsion  droplets  were  temperature cycle. 
observed. Formulation were categorized as clear, non clear, stable or 
unstable. In the other hand, refractive index of different formulations  Freeeze –thaw cycles (Accelerated ageing) 
were measured and compared with 0.1N hydrochloric acid. 
This  test  was  performed  for  accelerated  stability  testing  of 
Solubility determination in the various oils, surfactants and co­ nanoemulsion  formulations.  In  this  study  the  formulations  were 
surfactants  exposed  at  two  different  temperatures  i.e  ‐21 0C  and  210C  for  each 
temperature  cycles  not  than  24  hrs.  For  the  better  estimation  of 
2 ml of different oils was taken in small vials separately and  excess 
accelerated stability studies three such cycles should be run for each 
amount  of  the  drug  was  added  to  each  vial.    The  vials  were  tightly 
batch of formulation .The formulations which showed the maximum 
stopper  and  were  continuously  stirred  for  72  hrs  in  mechanical 
stability were selected for further study. 
shaker at 250C and after that, oils were centrifuged. The supernatant 
was  separated  and  dissolved  in  methanol  and  solubility  was  Dispersibility tests 
quantified  by  UV‐Spectroscopy  (Shimadzu  1701,  Japan)  method  at 
247 nm after appropriate dilution with methanol.  The  efficiency  of  self‐emulsification  of  oral  nanoemulsion  was 
assessed using  a standard  USP  XXII dissolution apparatus  2. [20]. 
Screening  of  formulations  on  the  basis  of  thermodynamic  One ml of each formulation was added to 500 mL of distilled water 
stability studies 
and  in  0.1N  HCl  respectively  at  37  ±  0.5  oC.  A  standard  stainless 
The thermodynamic stability studies were performed on the basis of  steel  dissolution  paddle  rotating  at  50  rpm  provided  gentle 
following tests.  agitation.  The  in  vitro  performance  of  the  formulations  was 
visually  assessed  using  the  following  grading  system  (Table  5). 
Centrifugation study  Those  formulations  that  passed  the  thermodynamic  stability  and 
The  selected  formulations  were  centrifuged  (REMI,  India)  at  the  also  dispersibility  test  in  Grade  A  were  taken  for  the  further 
5000 rpm for 30 mins and observed for phase separation, creaming  studies. Further from each Smix Group one formulation is selected, 
or cracking. The formulations which showed maximum stability (no  having  the  least  Smix  concentration  irrespective  of  Smix  ratio 
creaming, cracking, phase seperation) were selected and studied for  used, but passing dispersibility test in Grade A in distilled water as 
heating‐cooling cycle, freeze‐thaw cycles and Dispersibility tests.  well as in 0.1N HCl. 
 
Table 3:  Observation table of dispersibility study 
S.No.  Observation  Grade 
1  Rapidly forming (within 1 min) nanoemulsion, having a clear or slight bluish A
2  Rapidly forming, slightly less clear microemulsion, in  bluish colour B
3  Fine milky emulsion that formed within 2 minutes  C 
4  Dull, grayish white emulsion having slightly oily appearance that is slow to emulsify (longer than 2 min).  D 
5  Formulation, exhibiting either poor or minimal emulsification with large oil globules present on the surface.  E
 
Viscosity   Table 4: Observation table of viscosity measurements 
The  viscosity  of  the  prepared  nanoemulsion  formulations  were  Code  Zero time  After 120 sec 
determined as such without dilution by Brookfield DV III ultra V6.0  Mean viscosity ± SD (cps)  Mean viscosity ± SD (cps) 
RV cone and plate rheometer (Brookfield Engineering Laboratories,  GM1 27.51  ± 1.01 27.21  ± 0.93
Inc,  Middleboro,  MA)  using  spindle  #  CPE40  at  25  ±1.0°C.  The  GM2 21.32 ± 1.1 20.85 ± 0.64
software  used for the viscosity  calculations was Rheocalc V2.6. The  GM3  10.3 ± 0.91  10.02 ± 0.63 
results are shown in Table 6.4.  GM4  28.22 ± 0.91  27.76 ± 1.75 

149 
 Chouksey et al. 
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Droplet size analysis (Particle size distribution)  200  KV  and  of  a  0.18  nm  capable  of  point  to  point  resolution. 
Combination of bright field imaging at increasing magnification and 
Droplet size of the prepared nanoemulsion was determined by using  of  diffraction  modes  was  used  to  reveal  the  form  and  size  of  the 
photon correlation spectroscopy, which analyzes the fluctuations in  nanoemulsion.  In  order  to  perform  the  TEM  an  observation,  the 
light  scattering  due  to  Brownian  movement  of  the  particles  .  The  nanoemulsion formulation was diluted with distilled water (1/100). 
formulation  (0.1  mL)  was  dispersed  in  50  mL  (500  dilution)  of  A  drop  of  the  diluted  nanoemulsion  was  directly  deposited  on  the 
distilled  water  in  a  volumetric  flask  and  gently  mixed  by  inverting  Copper  holey  film  grid  and  observed  after  putting  fixing  agent  and 
the flask and measurement done using a Zetasizer (Nano ZS‐90, UK).  drying it in the filtered air.  
Light scattering was monitored at 25°C at a 90° angle.    

Table 5: Droplet size of the various emulsion formulations 
S. No  Formulation Code  Particle Size 
1 GM1  42.8±5.2 nm
2  GM2  195±10.5 nm 
3  GM3  107±9.7 nm 
4 GM4 81.6±6.1 nm
 

In vitro drug release study 
In  vitro  release  test  was  performed  using  Dialysis  bag  method  and 
release  study  was  carried  out  in  250  ml  of  distilled  water,  1  ml  of 
emulsion formulation (Single dose containing 10 mg of AT Calcium) 
was  placed  in  treated  dialysis  bag  (MWCO  12,000  g/mole;  Sigma, 
USA) and 1 mL samples was withdrawn at regular time intervals (0, 
0.5, 1, 1.5, 2,2.5,3,3.5, 4,4.5,5.5, 6,6.5,7, 8,9, 10, 12, 22 and 24 h) and 
same  amount  of  distilled  water  was  replaced.[21‐22].  The 
withdrawn  1  ml  samples  were  diluted  with  3  ml  methanol  and 
  analyzed for  the  drug  content  by  using  developed  UV‐spectroscopy 
at  247  nm.  The  same  method  was  used  for  the  suspension 
Fig. 2: TEM of formulation GM1 
containing 10 mg of AT Calcium in 1 ml distilled water. The release 
Transmission electron microscopy  of  drug  from  different  selected  nano  emulsion  formulations  was 
compared  with  drug  suspension  and  finally  selected  formulation 
Morphology  and  structure  of  the  nanoemulsion  were  studied  using  was used for the further study i.e. for preparation of solid self‐nano 
transmission electron microscopy (TEM) TOPCON 002B operating at  emulsifying drug delivery system (SSNEDDS). 

 
 
Comparative dissolution profile of different formulations

120

100
% Cumulative release

80

60

40

20

0
0 5 10 15 20 25 30
Time (Hrs)

GM1 GM2
GM3 GM4
GM5 API suspension
 
Fig. 3:  In­vitro Drug Release of AT nano emulsion by dialysis bag method in distilled water 
 
RESULTS AND DISCUSSION  although the free energy required  to form an emulsion is very low, 
the formation is thermodynamically spontaneous.   
The SEDDS of the drug Atorvastatin were prepared by emulsification 
method.  Constructing  a  phase  diagram  is  one  of  the  primary  steps  The relationship  between the phase  behaviour  of  a mixture  and  its 
and makes a backbone for the SEEDS, particularly when the aim is to  composition can be selected with the aid of a phase diagram. Sefsol 
accurately  delineate  a  phase  boundary.  Observations  are  made  218 (oil) and oleic acid (oil), Tween 20, Tween 80 (surfactants) and 
carefully  to  separate  metastable  systems  from  phase  boundary,  Carbitol (co surfactants), were used to study the phase diagrams in 

150 
 Chouksey et al. 
Int J Pharm Pharm Sci, Vol 3, Issue 3, 2011, 147­152 

detail. The systems were observed for visual  clarity and  flowability  is  seen  using  TEM.  Some  equally  distributed  droplet  sizes  are 
caracteristics.  Those  which  did  not  show  a  change  in  the  miniscus  measured  using  TEM,  as  it  is  capable  of  point‐to‐point  resolution. 
after  tilting  to  an  angle  of  90°  were  classified  as  nanogels  a  The  droplet  size  is  in  agreement  with  the  results  obtained  from 
metastable  system  were  not  selected.  After  taking  observation,  droplet size analysis.  
pseudoternary  phase  diagrams  were  constructed  based  on  the 
observations  marked  during  titration.  Phase  diagrams  were  Dissolution study was performed by dialysis bag method in distilled 
constructed separately for each ratio of Smix prepared  so that  o/w  water for the final selection of formulation for further development 
nanoemulsion  regions  could  be  identified.    In  the  phase  diagrams  of  solid  self‐nanoemulsifying  dosage  forms.  The  dissolution  study 
(Figure.  6.1,  A‐G)  only  o/w  nanoemulsion  region  is  shown.  After  was  performed  for  24  hrs.  Drug  dissolution  from  formulation  GM1 
building  the  backbone  of  the  nanoemulsion  delivery  system,  was  very  fast  as  99.65±1.29%  of  drug  released  in  6.5  hrs;  while 
different  formulations  were  selected  at  different  point  from  the  formulations GM2, GM3, GM4 and GM5 showed comparatively slow 
phase diagram justifying the drug dose.   release  i.e.  82.36±2.12,  93.97±0.56,  72.69±0.81  and  61.06±1.31  in 
6.5  hrs.  In  contrast  to  this  drug  released  from  API  suspension  was 
Vigorous  studies  were  done  for  the  phase  diagram  construction,  found  to  be  very  low  i.e.  13.77±0.98%  in  6.4  hrs.  This  result  was 
phase  boundry  were  plotted  on  the  ternary  phase  diagram.  After  attributed to the fact that formulation GM1 is having comparatively 
building  the  backbone  of  the  nanoemulsion  delivery  system,  smaller  size  (42.8  nm)  of  oil  droplets  and  hence  the  larger  surface 
different  formulations  were  selected  at  different  point  from  the  area  for  dissolution  as  justified  by  droplet  size  distribution  and  by 
pseudophase diagram which justified the drug dose considering the  TEM  photograph.  The  another  possible  reason  was  the  oil 
drug solubility in the oils phase. As per saturation solubility studies  concentration  in  formulation  GM1  (10%)  as  compared  to  the  GM2 
of  AT  Calcium  in  oily  mixture,  Sefsol  218:  Oleic  acid  (1:1),  around  (15%), although having the same concentrations of the Smix, which 
130  mg  of  drug  can  be  solubilized  per  mL.The  concentration  10%  was not sufficient to emulsify the increased amount of oil in GM2.  
(100 μL) of oil in 1 mL formulation is just able to solubilize 10 mg of 
AT  Calcium.  Therefore  10%  was  selected  as  the  least  oil  CONCLUSION  
concentration  to  be  taken  for  one  mL  formulation  from  the  phase 
diagram.  The  selected  formulations  shown  were  screened  on  the  The  formulation  was  found  to  be  the  optimized  formulation  on  the 
basis of the thermodynamic stability studies.   base  of  results  of  pseudo  ternary  phase  diagram,  in  vitro  drug 
release,  droplet  size  and  other  parameters.  The  present  study  was 
Nanoemulsions  are  thermodynamically  stable  systems  and  are  clearly indicated that the usefulness of SEDDS in the improvement of 
formed  at  a  particular  concentration  of  oil,  surfactant  and  water,  the dissolution rate and there by oral bioavailability of poorly water 
with  no  phase  separation,  creaming  or  cracking.    It  is  the  soluble  drugs  like  ATV  without  incompatibility  between  the 
thermostability  which  differentiates  nano  or  micro  emulsion  from  ingredients. 
emulsions  that  have  kinetic  stability  and  will  eventually  phase 
separate. Thus, the selected formulations were subjected to different  ACKNOWLEDGEMENT 
thermodynamic  stability  by  using  heating  cooling  cycle,  The authors are thankful to Suresh Gyan vihar  University, Jaipur for 
centrifugation  and  freeze  thaw  cycle  stress  tests.  Those  providing  necessary  support  to  this  project  and  also  thankful  to 
formulations,  which  survived  thermodynamic  stability  tests,  were  Lupin Lab for the gift sample of atorvastatin. 
taken  for  dispersibility  test.  Those  formulations,  which  survived 
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Bulletin of Pharmaceutical Research 2011;1(2):xx-xx
An Official Publication of Association of Pharmacy Professionals
ISSN: 2249-6041 (Print)

RESEARCH ARTICLE

IN VIVO ASSESSMENT OF ATORVASTATIN NANOEMULSION

FORMULATION
Rajendra Chouksey1*, Anand Kumar Jain2, Harish Pandey3 and Ankur Maithil3
1
Department of Pharmaceutics, School of Pharmacy, Gyan Vihar University, Jaipur-302 017, Rajasthan, India
2
Department of Pharmacy, Gajaraja Medical College, Gwalior-474 006, Madhya Pradesh, India
3
Department of Pharmaceutics, Sri Satya Sai College of Pharmacy, Sehore-466 001, Madhya Pradesh, India

*E-mails: chouksey_rajendra@yahoo.com
Tel.: +91-9993815318

Received: June 30, 2011 / Revised: July 22, 2011 / Accepted: July 25, 2011

Atorvastatin is one of the most important hypolipidemic drug available today and circumventing the
major problem of its poor bioavailability remains a bigger challenge to pharmaceutical scientists.
The objective of the present study was to carry out pharmacokinetic studies of nanoemulsion
formulation of atorvastatin (AT) in order to assess the in vivo performance of developed formulation.
Optimized formulation was selected for in vivo study on the basis of higher drug release, optimum
globule size, minimum polydispersity value, lower viscosity, and overall lower surfactant and co-
surfactant concentration and exhibited better bioavailability as compared to pure drug.
Key words: Nanoemulsion, Atorvastatin, In vivo studies, Bioavailability.

INTRODUCTION
Nanoemulsions being colloidal nanodispersions
of oil in water (or water in oil),
thermodynamically stabilized by an interfacial
film of surfactant(s) and co-surfactant(s) have
revealed tremendous potential in
nanoengineering of various inorganic materials
(Date and Patravale, 2004). Droplet size in
thermodynamically stable nanoemulsions is
usually 10-100 nm (Sintov and Shapiro, 2004).
The homogeneous systems that can be prepared
over a wide range of surfactant concentrations
and oil to water ratios (1:4) are all fluids of low
viscosity. Nanoemulsion provides ultra low
interfacial tension and large o/w interfacial
areas. Nanoemulsions have a higher
solubilization capacity than simple micellar
solutions and their thermodynamic stability
offers advantages over unstable dispersions,
such as emulsions and suspensions, because
they can be manufactured with little energy
input (low energy emulsification
techniques/heat or mixing) and has a long shelf
life. The nanosized droplets leading to enormous
interfacial areas associated with nanoemulsions
would influence the transport properties of the
drug, an important factor in sustained and
targeted drug delivery (Swarbrick and Boylan,
1994). The attraction of o/w nanoemulsion
systems lies in their ability to incorporate
hydrophobic drugs into the oil phase thereby
enhancing their solubility. Nanoemulsions have
been reported to make the plasma concentration
profiles and bioavailability of drugs more
reproducible (Kawakami et al 2002;
Constantinides, 1995; Lawrence and Rees,
2000).
The design of effective formulation for drugs has
long been a major challenge, because drug
efficacy can be severely limited by instability or
poor solubility in the vehicle. Although solid
dispersion is one of the most widely used
technique for solubility enhancement (Pabreja
and Dua, 2011; Dahiya and Kaushik, 2010),
nanoemulsions being a versatile technology have
the greater potential to increase the
bioavailability of drug in many ways. They act as
supersolvents for poorly soluble drugs.
Nanoemulsions have a higher solubilization
capacity than simple micellar solutions and their
thermodynamic stability offers advantages over

1
Chouksey et al
unstable dispersions, such as emulsions and
suspensions, because they can be manufactured
with little energy input (heat or mixing) and has
a long shelf life thus scale up technology is easy.
The nanosized droplets leading to enormous
interfacial areas associated with nanoemulsions
would influence the transport properties of the
drug, an important factor in sustained and
targeted drug delivery. The droplet size of
nanoemulsion is between 14 to 100 nm, and can
be sterilized by filtration. The use of
nanoemulsion as drug delivery systems can
improve the efficacy of drug, allowing the total
dose to be reduced and thus minimizing side
effects. Atorvastatin, a synthetic cholesterol-
lowering agent, is a HMG-CoA (3-hydroxy-3-
methylglutaryl-coenzyme A) reductase inhibitor.
The calcium salt of atorvastatin is used in the
treatment of primary hypercholesterolemia and
dyslipidemia. Atorvastatin is highly lipophillic in
nature (log P (octanol/water), 6.36), freely
soluble in methanol and soluble in
dimethylsulphoxide (DMSO) and dimethyl
formamide. It is soluble in aqueous medium with
a pH of less than 4.0. It is very slightly soluble in
water, phosphate buffer (7.4) and acetonitrile;
while it is slightly soluble in ethanol with
absolute bioavailability of mere 14%.
Atorvastatin is drug of choice among
hypolipidemic drug available today and
circumventing the major problem of its poor
bioavailability remains a bigger challenge of
pharmaceutical scientists. With this background
in mind, the objective of the present study was to
develop and characterize an optimal stable
nanoemulsion formulation of atorvastatin with
an aim to increase its bioavailability. The in vitro
assessment of nanoemulsion formulation of
atorvastatin has been published.
MATERIALS AND METHODS
Development of nanoemulsion formulation
The drug stock solutions in oil mixture were
prepared in such a way that 10 mg dose was
present in each formulation complying the oil
percentage for each formula as selected from the
phase diagram. This was prepared by dissolving
the 1000 mg of drug individually in the 10, 15,
20 and 25 ml of oily mixture, which compiles the
10%, 15%, 20% and 25% oil compositions
respectively in the formulae.
Evaluation of nanoemulsion
The selected formulations were centrifuged
(REMI, India) at the 5000 rpm for 30 min and
2
Bull. Pharm. Res. 2011;1(2)
observed for phase separation, creaming or
cracking. The formulations which showed
maximum stability (no creaming, cracking, phase
separation) were selected and studied for
heating-cooling cycle, freeze-thaw cycles and
dispersibility tests.
Accelerated ageing studies
This test was performed using freeze-thaw
cycles for accelerated stability testing of
nanoemulsion formulations after ageing. In this
study the formulations were exposed at two
different temperatures i.e. -21°C and 21°C, for
each temperature cycles was not for more than
24 h. For the better estimation of accelerated
stability studies, three such cycles run for each
batch of formulation. The formulations which
showed the maximum stability were selected for
further study.
Viscosity measurements
The viscosity of the prepared nanoemulsion
formulations were determined as such without
dilution by Brookfield DV III ultra V6.0 RV cone
and plate rheometer (Brookfield Engineering
Laboratories, Inc, Middleboro, MA) using spindle
# CPE40 at 25 ±0.5°C. The software used for the
viscosity calculations was Rheocalc V2.6.
Droplet size distribution
Droplet size of the prepared nanoemulsion was
determined by using photon correlation
spectroscopy, which analyzes the fluctuations in
light scattering due to Brownian movement of
the particles (Attwood et al 1992). The
formulation (0.1 ml) was dispersed in 50 ml
(500 dilution) of distilled water in a volumetric
flask and gently mixed by inverting the flask and
measurement done using a Zetasizer (Nano ZS-
90, UK). Light scattering was monitored at 25°C
at a 90° angle.
In-vitro drug release performance
The study was performed by using dialysis bag
method (Shafiq et al 2007). The dialysis
membrane used in the study was Cellulose
membrane (Sigma, USA) with a molecular weight
cut off of 12000 g/mol. In vitro release test was
performed in 250 ml of distilled water, which
was based on USP XXIV method at 100 rpm and
37±0.5°C. One ml of nanoemulsion formulation
(single dose containing 10 mg of AT Calcium)
was placed in treated dialysis bag and 1 ml
samples were withdrawn at regular time
intervals (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6,
Chouksey et al
6.5, 7, 8, 9, 10, 12, 22 and 24 h) and same
amount of distilled water was replaced. The
withdrawn 1 ml samples were diluted with 3 ml
methanol and analyzed for the drug content by
using developed RP-HPLC method at 247 nm.
The same method was used for the suspension
containing 10 mg of AT Calcium in 1 ml distilled
water.
Bioavailability study of Atorvastatin
nanoemulsion in rats
Approval to carry out in vivo study was obtained
from Gyan Vihar University, Institutional Animal
Ethics Committee and their guidelines were
followed for the studies (173/CPCSEA). The
animals used for in vivo experiments were adult
wistar male albino rats (n=6) weighing 180-200
g obtained from Central Animal House of Gyan
Vihar University, Jaipur, India. The animals were
kept under standard laboratory conditions at a
temperature of 25±2°C and relative humidity
(55±5%). The animals were housed in animal
cages, six per cage, with free access to standard
laboratory feed (Lipton feed, Mumbai, India) and
water ad libitum. The selected nanoemulsion
formulation was subjected to in vivo studies. The
dose of 6 mg/kg (Shen and Zhong, 2006) body
weight of the Atorvastatin Ca was used in the
study. Six tablets with label claim of 10 mg
(Atorlip®) were taken, crushed in mortar, and
mixed with 50 ml of double distilled water (1.2
mg/ml). Each rat in this group was given 1.0 ml
using oral feeding needle. Same dose of drug
suspension and nanoemulsion was
administered. The blood sampling was carried
out for around 48 hours and 10 to 12 samples of
blood were taken from each animal in the group.
Blood was withdrawn from the jugular vein after
anesthesia at 0 (pre-dose), 0.25, 0.5, 0.75, 1.0,
1.25, 1.5, 6, 12, 24, 36 and 48 h in micro
centrifuge tubes. Tubes were stored at room
temperature, 25±2°C and relative humidity
(55±5%) for 30 min. The clotted blood was then
centrifuged at 5000 rpm for 30 min. The serum
was separated and stored at -21°C until drug
analysis was carried out using RP-HPLC method.
The collected serum was extracted with ethyl-
acetate (5 ml) by using sodium phosphate buffer
(pH 7, 1 ml) as a protein-precipitating agent for
analysis, centrifuged at 5000 rpm for 5 min and
dried at room temperature. After evaporation,
the ethyl acetate solid residue was reconstituted
using 100 µl mobile phase and then analyzed by
RP-HPLC. The samples were injected and the
area was noted down in triplicate.
3
Bull. Pharm. Res. 2011;1(2)
Pharmacokinetic and statistical analysis
Pharmacokinetic parameters were calculated by
using non-compartmental analysis using
WinNonLin version 4.0 (Pharsight Corp.,
Mountain View, CA). All pharmacokinetic (PK)
parameters (tmax, Cmax, AUC0→t,) were calculated
individually for each subject in the group and the
values were expressed as mean±SD.
RESULTS AND DISCUSSION
Droplet size analysis of the selected formulations
was carried out and it was indicated that the
difference in the droplet size between the
formulation was not statistically significant.
There was only a marginal difference in the
mean droplet size of formulations but
polydispersity (0.237) was minimum in the case
of formulation, GM1, containing 10% of oil
suggesting uniformity in the globule size (42.8
nm) of the formulation. The viscosity of the
selected formulation GM1 was found to be
intermediate showing numeric value of
27.51±1.01 mP. In vitro drug release was carried
out using a dialysis bag and the selected
formulation was compared for the drug release
with drug suspension. The release of drug from
nanoemulsion formulations was high when
compared to drug suspension The drug release
was found to highest (99.65±0.75%) in case of
GM1 as compared to the API drug suspension
(13.77±0.214) in 6.5 h. Therefore, the optimized
formulation, GM1, having higher drug release
(99.65±0.75%), optimum globule size (42.8 nm),
minimum polydispersity value (0.237), lower
viscosity (27.51±1.01 mP), and overall, lower
surfactant concentration (19%) and cosurfactant
(19%) was selected for in vivo study.
The results of in vivo studies in rats are
summarized in Table 1. It was found that the
Cmax in serum for nanoemulsion formulation
(43603.77±632.66 ng/ml) represented greater
improvement than the marketed formulations
tablet (6750.599±145.35 ng/ml) or simple API
drug suspension (5271.45±75.37 ng/ml). It was
also observed that AUC0→t GM1 formulation was
394520.05±87932.39 ng.h/ml and thus the
differences were highly significant (p<0.01) as
compared to AUC0→t (110271.4±47224.84) and
(70467.13±26350.34) for tablet suspension and
API suspension formulations respectively (Table
2, Figure 1).
Therefore, formulation GM1 was found to
possess higher drug release, optimum globule
size, minimum polydispersity, lower viscosity,
lower surfactant concentration, as well as higher
Chouksey et al Bull. Pharm. Res. 2011;1(2)

rate and extent of absorption as compared to v/v), Carbitol (19% v/v) and distilled water
marketed formulations. The composition of (52% v/v) as oil, surfactant, co-surfactant and
optimized formulation (GM1) was safsol 218 aqueous phase respectively, containing 10 mg of
(5% v/v), Oleic acid (5% v/v), Tween-20 (19% AT Calcium.

50000

40000
Concentration (ng/ml)

30000
20000
10000
0
0 0.5 1 1.5 12
36

SNEDDS Formulations Time (Hrs)

Tablets Suspension API Suspension

Fig. 1. Comparative in vivo absorption profile of AT calcium using

different formulations in rat model

Table 1. Comparative observation table for in vivo bioavailability studies in rat animal model*
Sampling time GM1 Tablet suspension API suspension
(h) (Mean±SD) (Mean±SD) (Mean±SD)
0 0 0 0
0.25 24719.36±142.54 4747.476±63.76 3636.91±57.56
0.5 35582.73±345.54 5491.985±36.87 4307.00±24.87
0.75 37687.34±733.16 5815.137±62.97 4456.82±54.89
1 41090.76±531.78 6343.85±64.86 4959.85±86.36
1.25 43603.77±632.63 6750.599±145.35 5271.45±75.37
1.5 37508.95±456.56 5801.549±48.25 4345.85±86.74
6 24312.26±731.84 3761.141±65.36 2887.23±75.36
12 12093.38±234.47 1870.322±124.12 1437.17±44.66
24 6370.94±32.47 987.3089±32.85 765.24±75.05
36 3851.75±42.74 599.0169±24.86 548.16±36.87
48 2811.38±57.38 436.0885±14.60 338.09±23.21
*(n=6)

Table 2. Relative pharmacokinetic parameters of different formulations

tmaxa Cmaxb AUC0 → tc
Formulation
(h) (ng/ml) (ng.h/ml)
GM1 0.95 ± 0.13 43603.77 ± 632.63 383453.2 ± 2352.2
Tablet suspension 2.24 ± 1.74 6750.599 ± 145.35 35432.4 ± 1143.53
API drug suspension 2.52 ± 1.93 5271.45 ± 75.37 68232.5 ± 6734.46
a time of peak concentration, b peak of maximum concentration, c area under the concentration time profile curve until last observation.

CONCLUSION absorption than the marketed conventional

The nanoemulsion formulation of AT calcium tablet and API drug suspension formulation.
exhibited significantly greater extent of Furthermore, these results might be attributed
4
Chouksey et al
to the presence of surfactants in nanoemulsion
system that have caused changes in the
membrane permeability of the GI tract and the
inhibition of an apically polarized efflux
system which could lead to the enhancement
of oral absorption.
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