Formulation of Rutin trihydrate Liposomes for Topical Delivery

International Journal of Pharmaceutical Research | July – Sept 2015 | Vol 7 | Issue 3|29
Research Article
Formulation and Characterization of Rutin trihydrate
Liposomes for Topical Delivery
APARAJITA VARSHNEYA
AND PADMINI RAVIKUMAR
Department of Pharmaceutics, SVKM’s Dr. Bhanuben Nanavati College of Pharmacy,
Gate No: 1, Mithibai College Campus, V.M. Road, Vile Parle (W), Mumbai-400 056, India
ABSTRACT
In the present study, liposomes were formulated as drug carriers for enhancing the delivery of an antioxidant Rutin
trihydrate for topical administration. Formulation of liposomes was done by thin film hydration method. Optimization
was achieved by varying the lipid to cholesterol ratio and gradually increasing the amount of drug until maximum
entrapment efficiency was achieved. The formulation and process parameters were optimized to attain multilamellar
liposomes with homogeneous size and good entrapment. The optimized batch gave entrapment efficiency of 88%.
Gels were prepared by using Carbopol 940 as the gelling agent. The in vitro and ex vivo release profile of liposomal gels
was compared with conventional gel formulations. The liposomal gel showed prolonged drug release upto 12 h and
also exhibited anti-elastase activity. The safety of liposomal gels was ensured by conducting skin irritation studies on
albino Wistar rats.
Keywords: Liposomes, Rutin trihydrate, topical delivery, gels.

Address for correspondence
Aparajita Varshneya, SVKM’s Dr. Bhanuben Nanavati College of Pharmacy, Gate No.1, Mithibai, College Campus, V.M.
Road, Vile Parle (West), Mumbai ‐ 400 056, India.
Phone no: +91‐9870687261 e‐mail: aparajita.varshney@yahoo.com
Received:18/02/15, Revised:02/04/15, Accepted:18/04/15
INTRODUCTION
Topical drug delivery has long been sought after, as
it is coupled with well-established advantages like
localised drug delivery, avoidance of first pass
metabolism, improved patient compliance owing to
its non-invasive nature and reduced systemic side
effects [1]. Despite these merits, the brick and mortar
structure of stratum corneum i.e. the horny layer
serves as the principal barrier to the percutaneous
absorption of drugs [2,3]. Owing to these barrier
properties of the skin, the delivery of active
substances from conventional formulations is
generally compromised. Thus, there arises the need
for a suitable carrier to enhance drug delivery [4].
Liposomes
Liposomes were first described by British
haematologist Dr. Alec D Bangham in early 1960’s
[5]. Liposomes are highly organized structures,
composed of concentric bi-layered vesicles in which
an aqueous volume is enclosed by a membranous
lipid bilayer [6]. The main advantages that have
made these vesicular systems favourable for drug
delivery are their tissue compatibility,
biodegradability, safety and their ability to entrap
almost any drug in the bilayer core or in the outer
domain. Additionally, liposomal drug formulations
can be used to overcome a drug’s non-ideal
properties and unfavourable pharmacokinetic
profiles [7]. Regarding topical application, liposomes
offer a wide range of advantages including
moisturization and prolonged dermal release [8].
Topical use of antioxidants has been adopted as an
important strategy in cosmeceutical industry to avoid
wrinkling of the skin and protect it from degenerative
effects such as photoaging, sunburn,
photocarcinogenesis etc. [9]. Antioxidants help in
preventing and repairing damage caused due to
oxidative stress. They scavenge free radicals which
are postulated to be the mediators in skin aging [10].
They are found to be useful in delaying the signs of
aging such as fine lines, wrinkles and age spots.
Antioxidants are promising in photoprotection with
negligible side effects at physiological concentrations
[11]. Of the various antioxidants, bioflavonoids are
one of the most powerful antioxidants. They are a
class of plant secondary metabolites possessing a
broad range of pharmacological activities. Rutin
trihydrate was chosen as the active molecule as it is a
powerful antioxidant which inhibits free-radical
mediated cytotoxicity and lipid peroxidation. It is
sparingly soluble in water (0.125 g/L). This limits its
use in topical delivery. Thus liposomes were chosen
as drug carriers as they are amphiphilic in nature
and lipophilic molecules get embedded in their
concentric bilayers. Liposomes being physiologically
similar to cell membrane, are nontoxic in nature and
are easily re-absorbed from the epidermis into the
deepest layers [12]. Thus, the objective of the study
was to enhance the accumulation of drug at the site
of administration and achieve prolonged release by
means of liposomal formulation.

Aparajita Varshneya et al /Formulation and Characterization of Rutin Liposomes
30 | International Journal of Pharmaceutical Research | July – Sept 2015 | Vol 7 | Issue 3
MATERIALS AND METHODS
Phosphatidylcholine (Leciva S-70) was a generous
gift sample from VAV Life Sciences, Mumbai, India.
Rutin trihydrate was purchased from Loba Chemie,
Mumbai and Cholesterol was procured from
Qualigens, Mumbai. Carbopol 940, Glass beads
(2.5 mm-3.5 mm) and all other AR grade reagents
were purchased from S D Fine-Chem Ltd., Mumbai.
Dialysis membrane 150 was purchased from
HiMedia Laboratories Pvt. Ltd., Mumbai and porcine
skin was procured from local market. Healthy male
albino Wistar rats (Rattus norvegicus sp.) were
purchased from Bharat Serum, Mumbai, India.
Liposomes were prepared using Rotary Evaporator,
Roteva, Equitron and UV-visible spectrophotometer,
Shimadzu 1800 was used for evaluation.
I. Preparation of Rutin Trihydrate Liposomes:
Multilamellar vesicles were prepared by thin film
hydration technique as described by Bangham et al.
[13]. Phosphatidylcholine, Cholesterol and Rutin
trihydrate constituted the lipid mixture. This lipid
mixture was dissolved in chloroform and methanol
(2:1) in a round bottom flask (RBF) and 25 g glass
beads were added for homogeneous film formation.
The RBF was attached to the rotary flash evaporator
(Roteva, Equitron) and allowed to rotate at 80 r/min
in a thermostated water bath at 40°. The organic
solvents were then removed by slow application of
vacuum leading to lipid film formation on the walls
of the flask. The film was allowed to dry for 60 min
to ensure complete removal of solvent. The dried
lipid film was hydrated with phosphate buffer (pH:
5.4) at 150 r/min and 46° that is above the transition
temperature of lipid. Rotation was continued for 30
min. The Rutin trihydrate liposomal suspension was
kept at room temperature for 2 h for complete
hydration and for annealing structural defects.
The liposomes produced by this method are large
and heterogeneous in size, thus they were downsized
by bath sonication for 20 min. The suspension was
also vortexed for few minutes. The liposomal
suspension was characterized for physical
appearance, settling time, re-dispersion
time, entrapment efficiency and vesicle size by optical
microscopy. It was then filled in amber colored glass
bottles and stored at 4° until further use.
II. Characterization of Liposomes:
Rutin trihydrate liposomes were characterized for
following parameters:
Optical Microscopy
A drop of the liposomal suspension was placed on a
clean glass slide and observed under high power
45x and 100x of the optical microscope (Motic
microscope). Multilamellar vesicles (MLV) were
clearly identified (Figure 1). Microscopic observations
were used to study presence of aggregates,
precipitation or leakage of drug. Size and shape of
the vesicles was noted for all liposomal batches.
Figure 1: (a) Large multilamellar vesicles as
visible under 100x magnification
Determination of Vesicle Entrapment Efficiency
Entrapment efficiency for the batches prepared
during the optimization process was estimated by the
following procedure:
The liposomal pellet i.e. the drug loaded liposomes
were separated from the aqueous phase by
centrifugation at 15,000 r/min using Microspin
centrifuge for 30-45 min until a clear supernatant
was obtained. The amount of drug loaded was
estimated by both direct and indirect method.
Direct method: After centrifugation, the supernatant
was decanted. The formed pellet was washed with
phosphate buffer and vortexed, in order to remove
the free drug adsorbed on the surface of liposomes.
This step was repeated thrice to completely separate
the unentrapped drug. The resulting pellet was then
dissolved in methanol and sonicated for 20 min.
(This causes rupturing of pellet and release of
entrapped drug in the solution). The resulting
methanolic solution was then analyzed by UV
spectrophotometry using the developed analytical
method.
% Entrapment Efficiency (E.E.) =

(Amount of drug quantified in the pellet) x 100
Total amount of drug added
Indirect method: The supernatant of the liposomal
dispersion decanted after centrifugation and the
solutions collected after washing of pellet were
combined and analyzed by developed UV
spectrophotometric method for the amount of drug
present. % Entrapment efficiency was estimated as
follows:
% Entrapment Efficiency (E.E) =
(Total amount of drug added - Amount of drug
detected in the supernatant) x 100_
Total amount of drug added
Determination of vesicle size and size distribution
Vesicle size was determined by dynamic light
scattering using a computerized inspection system
(Malvern particle size analyzer). Freshly prepared
batches of vesicles were diluted ten-fold with filtered

distilled water and then analyzed at 25° using ten
runs at each scan. Optimization time was fixed at
120 s. Vesicle size and polydispersity index were
measured for the liposomal formulation. The
measurements were done in triplicate.
Zeta potential measurement
Liposomal suspension was diluted with filtered
distilled water and zeta potential was measured in
triplicate with the help of Malvern zetasizer.
Transmission Electron Microscopy (TEM)
TEM analysis was carried out for optimized drug
loaded liposomal suspension. A drop of the diluted
formulation was placed with the aid of a micropipette
on a coated copper grid 3 mm in diameter. Negative
staining was carried out by uranyl acetate. The grid
was allowed to dry under an IR lamp for about 20
min. The grid was then loaded on a probe which was
inserted in the slot designed for it in the electron
microscope. The grid containing the sample was then
bombarded with electrons accelerated at 200 kV and
the sample visualized over a fluorescent stage.
Vesicle size of the sample was measured and marked
and its morphology observed on the computer
(Figure 2).
Figure 2: Negative staining TEM imaging of Rutin
trihydrate liposomesMagnification 100,000x (size
approx. 600 nm)
III. Optimization of Process and Formulation
Parameters:
Many factors influence the formation of liposomes
and its characteristics. Therefore, the following
process parameters were optimized to obtain
liposomes with maximum entrapment efficiency,
homogeneous size and stability.
a. Process Parameters
Speed of the rotary evaporator
It is desirable to obtain a thin and uniform film as it
governs the final output of liposomal preparation.
The speed of rotation was varied from 60 r/min to
150 r/min during film formation as well as during
hydration.
The ratio and volume of solvent system
The solvent system was optimized by taking various
combinations of organic solvents i.e. chloroform and
methanol. The ratios 1:1, 2:1 and 3:1 were tried and
the film was evaluated in terms of its uniformity.
Quantity of glass beads
Thin film hydration technique is readily employed for
its feasibility on a laboratory scale, but problems
related to limitations of surface area exist. Glass
beads are commonly used for this purpose which
helps to increase the surface area to provide a thin
film. Liposomal dispersions were prepared using
different ratios of glass beads (diameter: 2.5 mm-3.5
mm) and solvent volume such as 0:1, 2:1, 3:1 and
4:1. The formulations were evaluated in terms of
uniformity of film and separation of vesicle
aggregates (microscopic observation).
Vesicle sizing technique
Size reduction of the vesicles is done to obtain a
homogeneous population of vesicles with low
polydispersity index. The various techniques studied
for size reduction were bath sonication using
ultrasonic bath, high pressure homogenization and
by using ultra turrax. Bath sonication was carried out
at room temperature for 20-30 min. Size reduction
using Ultra turrax was done at 6000 r/min for 15
min. High pressure homogenization was also tried by
carrying out approximately 10 cycles at a pressure of
5000 psi for 10 min. The effect of these techniques
was studied by estimating entrapment efficiency,
vesicle size and polydispersity index.
pH of the hydrating media
The effect of pH of the phosphate buffer was studied
on the formulation. The effect of pH on the
entrapment of the drug is related to its pKa. Hence
the pH of the hydrating buffer was adjusted at values
closer to the pKa of the drug and entrapment
efficiency was estimated. Distilled water, phosphate
buffer pH 5.4, 6.8 and 7.4 were used as hydrating
media and formulations were studied in terms of
colour, sedimentation, redispersibility and
entrapment efficiency.
Table 1: Formula optimization based on lipid concentration
Lipid Conc. (%) Consistency Settling rate Redispersibility
Vesicle size
(μ)
0.5 Thin suspension +++ + 1-4
1
Moderate, smooth
suspension
++ +++ 0.25-3
2 Thick suspension + ++ 2-6

Settling rate: + Slow ++ Moderate +++Fast Redispersibility + Poor ++ Good
+++Excellent
Table‐2: Formula optimization based on Drug: Lipid and PC: CH ratio
Batch No.
PC:CH
(molar
ratio)
Total lipid: 100 mg
Globule Size
(μ)
EE
(%)Amount of PC
(mg)
Amount of CH
(mg)
B1 1:0 100 0 0.5-5 56
B2 1:0.2 90.75 9.25 0.5-1.5 88
B3 1:0.5 79.68 20.32 0.5-3 79
B4 1:1 66.21 33.79 0.25-3.5 74
B5 1:0.1 95.15 4.85 1-4 49
B6 1:0.2 90.75 9.25 1-3 53
B7 1:0.5 79.68 20.32 2-4.5 58
B8 1:1 66.21 33.79 1-5 51
B9 1:0.1 95.14 4.85 1-5 33
B10 1:0.2 90.75 9.25 2-5 41
B11 1:0.5 79.68 20.32 2-4 35
B12 1:1 66.21 33.79 1-6 39
PC: Phosphatidylcholine, CH: Cholesterol, EE: Entrapment Efficiency
Formulation parameters
The basic components of formulation such as
lipid concentration, drug to lipid ratio and
phosphatidylcholine to cholesterol ratio were
optimized to obtain reproducibility in the quality
of liposomes. The key parameters considered for
optimization of formula were entrapment
efficiency and vesicle size. Percent drug
entrapment i.e. the amount of drug inside the
vesicles is the most important parameter for
liposomal formulations.
Lipid Concentration
Liposomal formulations were prepared with
increasing concentration of phosphatidylcholine
0.5%, 1% and 2% i.e. the quantity of lipid taken
was 50 mg, 100 mg and 200 mg for 10 ml
liposomal batch. Rutin trihydrate at low, fixed
concentration was incorporated in the
formulations. The formulations were studied on
the basis of physical observations such as
consistency, settling rate, redispersibility and
vesicle size.
Drug to Lipid ratio
This is an important factor that determines the
amount of drug entrapped within the liposomes.
It varies as per the drug, its molecular size,
charge and physical properties. For lipid soluble
drugs, larger quantities of drug can be
encapsulated and hence drug to lipid ratio can be
large. Water soluble drugs have limitations of
encapsulation efficiency, as comparatively smaller
volume of aqueous portion is available for
entrapment.
Phosphatidylcholine to cholesterol ratio
Liposomes were formulated in different molar
ratios of phosphatidylcholine and cholesterol, with
increasing concentration of drug and parameters
such as % entrapment efficiency and globule size
were estimated.
IV. Preparation of Liposomal Gels
Rutin trihydrate lipogel was prepared by using
Carbopol 940 gel base. Carbopol is a pH
sensitive polymer and gel formation occurs after
inducing neutralization. Definite amount of
Carbopol 940 was kept for hydration in double
distilled water containing preservatives overnight.
Liposomal pellet (obtained after centrifugation
and separation of unentrapped drug) was
dispersed in distilled water by sonication and
added to hydrated Carbopol solution with stirring.
Gelling was induced by neutralization using
triethanolamine. The gels were evaluated for their
visual appearance, colour, texture, feel upon
application (like greasiness, smoothness), pH,
viscosity, spreadability, drug content and in
vitro/ex vivo drug release.

Figure 3: Comparative in vitro drug release
through dialysis membrane
Figure 4: Comparative ex vivo drug release
through porcine ear skin
in vitro / ex vivo drug release from lipogel and
conventional gel
in vitro / ex vivo drug release studies were
performed using Franz diffusion cells having
surface area of 3.91 cm2
and receptor
compartment having a capacity of 22 ml.
Phosphate buffer pH 5.4 was used as receptor
fluid. The pH of the buffer approaches the natural
pH value of human skin.
in vitro: Dialysis membrane filters (molecular
weight cut off 12 000 to 14 000) which were pre-
hydrated by soaking in buffer overnight were
mounted on the cells. Receptor compartment was
filled with buffer as diffusion medium (37±0.5°).
Reservoir solution was stirred at 300 r/min using
magnetic bead. 0.5 g gel was placed in the
donor compartment on the membrane. Aliquots
were collected at 1, 2, 3, 4, 5, 6, 7, 8 &
24 h intervals and analyzed by UV-
spectrophotometry (Figure 3).
ex vivo: Porcine ear skin was shaved and carefully
separated. Subcutaneous fat was removed using
a scalpel. Skin sections were soaked in buffer
overnight. Skin sections thus obtained were
mounted on Franz Diffusion cells. Epidermal side
of the skin was exposed to ambient condition
while dermal side was kept facing the receptor
solution. Receptor compartment was filled with
buffer as diffusion medium (37±0.5°). Reservoir
solution was stirred at 300 r/min using magnetic
bead. 0.5 g gel was placed in the donor
compartment on the skin. All bubbles were
carefully removed between the underside of the
skin and solution in the receiver compartment.
Aliquots were collected at 1, 2, 3, 4, 5, 6, 7, 8 &
24 h intervals and analyzed by UV-
spectrophotometry (Figure 4).
Kinetic modeling
The release data was analysed using zero order,
first order, Higuchi, Hixson-Crowell and
Korsmeyer-Peppas models, to evaluate the
mechanism of drug release from the liposomal
gel. Mathematical models aid in predicting the
drug release rate and diffusion behaviour, thus
reducing the number of experiments needed. It
also helps in understanding the physics of a
particular drug transport phenomenon, thus
enabling the development of new formulations
[14,15] .
Figure 5: Pharmacokinetic release profile
of lipogel (ex vivo)
Anti-elastase assay
Elastase is the only enzyme that is capable of
breaking down elastin, an insoluble elastic fibrous
protein that, together with collagen, determines
the mechanical properties of connective tissue.
Several studies have demonstrated that both skin-
aging and anti-wrinkle effects are significantly
correlated with decreased elastase activity [16].
Therefore anti-elastase assay was carried out of
Rutin trihydrate liposomal gel to confirm its utility
as an anti-wrinkle product. Porcine pancreatic
elastase enzyme was incubated with the sample in
the cuvette. The amount of enzyme left
uninhibited was detected by reacting it with
substrate N-Succ-Ala-Nitroanilide that gives p-
nitroaniline as the final product. This was read
spectrophotometrically at 405 nm. Higher the
amount of product formed, lower is the inhibitory
effect of the sample on the enzyme.

% Elastase inhibition = (Absorbance of
control –Absorbance of test) x 100
Absorbance of control
Figure 6: Anti‐elastase inhibition by Rutin
trihydrate
V. Skin Irritation Studies
Healthy male albino Wistar rats (Rattus norvegicus
sp.), 180-220 g, 3 months old were selected as
test models. Six animals were used for the study,
each animal served as its own control. The
research project protocol was approved by Local
Institutional Animal Ethics Committee (Approval
No. CPCSEA/IAEC/BNCP/P-10/2014).
All the animals were allowed to acclimatize to
laboratory conditions prior to study. Hair was
removed from the back side of rats and an area
of 1 cm2
was marked on both the sides. One side
served as control while the other as test. Control
and test formulations (200 mg/rat) were applied
on each side and the site was covered with cotton
bandage. Observations for sensitivity and
reaction were made and graded as follows
Grade 1: No reaction, Grade 2: Slight, patchy
erythema, Grade 3: Slight but confluent or
moderate but patchy erythema, Grade 4:
Moderate erythema, Grade 5: Severe erythema.
RESULTS AND DISCUSSION
Method of preparation of liposomes
Thin film hydration method was selected for
preparation of multilamellar liposomes. The
reason behind the selection of thin film hydration
method was that Rutin trihydrate is lipophilic in
nature and has low aqueous solubility, so
multilamellar vesicles are more capable of
loading a higher mass of a hydrophobic drug
than are unilamellar vesicles. Thin film hydration
method was found to give excellent drug loading
with efficiency over 80%. Hence, this method was
used for preparation of liposomes.
Optimization of process parameters
a. Speed of rotation
Uneven film was observed at higher speed of
rotation (150 r/min) during film formation. This
may be due to improper distribution of heat at
higher speed. During film formation, lower speed
increases the time of contact of the film with the
hot water in the water bath. At higher speed,
since sufficient time is not available for the lipid to
form film, liquid to gel transitions of the lipid does
not occur. Based upon these observations 80
r/min was considered optimum for formation of
uniform film.
But, during hydration of lipid film sufficient energy
and vigorous shaking is required for the dried
film to get hydrated and form liposomes, thus
100-150 r/min was found adequate to form
uniform liposomal dispersion.
b. The ratio and volume of solvent blend
The choice of solvent is usually chloroform owing
to its low boiling point (61.15°) and easy removal
under vacuum leading to thin film deposition on
the walls of the flask. Rutin trihydrate is insoluble
in chloroform but soluble in methanol (Boiling
point 64.7°) and thus methanol was used in
combination with chloroform. Chloroform:
methanol in a 2:1 ratio was selected. This ratio
gave an even, uniform film. When the volume of
solvents is low (5 ml or less), solvents evaporate
rapidly under vacuum leading to improper film
formation. On the other hand when the volume
of solvent is high (10 ml or more), inadequate
film drying was observed. The presence of
organic solvent often leads to emulsion
formation. Volume of the solvent system used was
7.5 ml (5 ml Chloroform and 2.5 ml methanol).
c. Quantity of glass beads
Optimum quantity of glass beads is required to
obtain a thin uniform film on the surface of RBF. It
was observed that in the absence of glass beads,
the organic solvents evaporated rapidly even
under slow application of vacuum which lead to
poor film deposition on the walls of RBF, whereas
excessive bead quantity resulted in attrition and
inability of the solvents to evaporate. Glass beads
to solvent ratio 3:1 was found to be optimum as it
gave thin uniform film with no visible aggregates.
22 to 25 g glass beads were found to be
optimum for homogeneous film formation.
d. Vesicle sizing technique
Thin film hydration method gives large,
heterogeneous multilamellar vesicles. Vesicle size
analysis revealed Z-average (d-nm) size of 1360
nm with high PDI of 0.98, but good entrapment
efficiency was achieved. Techniques such as Ultra
Turrax and high pressure homogenization (HPH)

were tried to obtain size reduction and narrow
size distribution i.e. lower PDI. The results
indicated satisfactory size reduction and low PDI
but affected the entrapment efficiency adversely
which could be due to vesicle rupturing at high
pressure and speed. Hence, bath sonication was
used for further batches which gave vesicles in the
size range of 100-500 nm with PDI of 0.6 without
loss in drug entrapment.
e. pH of the hydrating media
According to Handerson Hasselbalch equation,
pKa is defined as the extent to which a drug is
available in the ionized form at a given pH.
pH = pKa + log (ionized/unionised)
Hence, the extent of ionization is influenced by
the pKa of any drug. Thus, entrapment of any
drug depends on its pKa to a considerable extent.
The pKa of Rutin trihydrate is cited as 6.4 in the
literature. Maximum ionization (~90%) would
hence occur at pH values ± one unit of the pKa
value. Phosphate buffer pH 5.4 was selected as it
yielded uniform suspension with minimal
sedimentation and maximum entrapment
efficiency. Sedimentation was observed when pH
of the buffer was raised beyond 7 whereas phase
separation was observed towards acidic pH.
Hence, further batches were prepared at pH value
of 5.4.
Formula optimization
a. Lipid concentration was gradually increased
from 0.5 to 2%. Increase in concentration of lipid
phase leads to increase in viscosity and thus the
settling rate was decreased. Liposomal
suspension with 0.5% lipid was a thin suspension
which exhibited faster settling rate. After 24 h, the
sediment formed took time to redisperse.
Formulation containing 2% lipid was much thicker
and showed less settling even after 24 h. But the
microscopic evaluation showed presence of larger
vesicles and aggregates. In comparison 1% lipid
suspension was found to give satisfactory results
in terms of settling, redispersion and microscopic
observation (Table 1).
b. Drug to lipid ratio was changed from 1:10,
2:10 and 3:10. The molecular weight of Rutin
trihydrate is high (664.56). This factor is of
importance as the amount of drug that enters the
lipid bilayer is also governed by its molecular
weight. The bilayer can only incorporate certain
amount of drug after which the drug starts
sedimenting immediately upon formation.
Microscopic observation also confirms this fact as
the unentrapped drug is clearly visible outside
vesicles. The threshold for Rutin trihydrate was
found to be 1 part of drug per 10 parts of lipid
employed. Hence, 1:10 drug to lipid ratio (10 mg
drug in 100 mg lipid) was found to be optimum
as maximum entrapment efficiency was obtained
by this ratio and optical microscopy showed
minimal unentrapped drug.
c. Phospahatidylcholine: Cholesterol ratio: In
principle, liposomes can be prepared using only
phosphatidylcholine. However, cholesterol is
added to improve stability and other structural
properties. Cholesterol has the effect of making
the membrane less permeable by filling up holes
or disruptions. Liposomes formed only with
phosphatidylcholine gives porous membranes
resulting in drug leakage whereas cholesterol acts
a cementing material thus improving drug
loading capacity of vesicles. Highest entrapment
was obtained with the batches having
Phosphatidylcholine: Cholesterol ratio as 1:0.2.
This could be because, the addition of cholesterol
in this ratio provides optimum rigidity to the
bilayer. Increase in the quantity of cholesterol
causes decrease in entrapment. This can be
explained by the fact that cholesterol might be
replacing Rutin trihydrate in the bilayer (Table 2).
Vesicle Size and Zeta Potential Measurement
MLV’s of size 0.5 to 1 μm were visualized easily
for all the batches using optical microscope (Motic
microscope) at magnification of 100x. TEM
imaging of optimized formulation showed drug
loaded liposomes of somewhat spherical shape.
Vesicles showed inner dark spherical core,
surrounded by comparatively faint background.
Vesicle size of optimized batch was approximately
600 nm (Figure 2).
The zeta potential was used as an indicator of the
stability of vesicles formed by thin film hydration
method. The zeta potential of the optimized batch
was -30.7 which indicates sufficient stability.
Liposomal gels
Liposomal suspensions were converted to gel
formulations since simple mixing of liposomal
dispersions with polymers gives the corresponding
gels. This is advantageous because the fusion of
vesicles can be effectively minimized or avoided
as the polymer molecules serve as a spacer
between the liposomes. Moreover, the size of the
vesicles is not affected because it is a controlled
liposome/polymer interaction process. Liposomal
creams were not chosen as stability problems are
reported in emulsion-based products due to
presence of excess oil and surfactant which may
interact with the vesicles. Various properties like
viscosity can be easily controlled by varying the
amount and type of polymer. Thus liposomal gels
were formulated as they circumvent the stability
issue, provide controlled release, have aesthetic
appeal as skin care cosmetics and are easy to
prepare [15].
Lipogels were pale yellow (colour of Rutin
trihydrate) and opaque. 1% w/w Carbopol gel

was found to be of good consistency and
acceptable feel with smooth appearance devoid
of any aggregation. Drug content for the gels
was found to be in the range of 97% to 101%.
Viscosity of gels was in the range of 8500 cps to
9500 cps. Spreadability of gels was around 5-7
g.cm/s.
in vitro / ex vivo drug release
The stratum corneum is built like a wall with
protein bricks and lipid mortar. The intercellular
lipids are important in controlling the
percutaneous absorption. In the case of
liposomes, the phospholipids may mix with the
intercellular lipids and thereby cause the swelling
of lipids without altering the multiple bilayer
structure of the stratum corneum. These swollen
lipids cause accumulation of the drug and thereby
form an intracutaneous depot. Although the
mechanism of enhancement using topically
applied liposomes is not fully understood, drug
disposition is primarily dependent on lipid
composition, liposome lamellarity and surface
charge. Studies have indicated topical drug
delivery is also influenced by the size of liposomes
[17,18].
in vitro and ex vivo application of the liposomal
gel formulation showed sustained release effect.
The drug release was sustained upto 12 hrs in
comparison to 5-6 hrs of conventional gel (Figure
3, Figure 4). This could be due to depot effect of
large multilamellar vesicles. ex vivo drug release
profile of lipogel showed that it follows zero order
kinetics as its R2
value was closest to unity (Figure
5).
Anti-elastase assay
The 0.1 % Rutin trihydrate lipogel showed a mean
anti-elastase activity of 32.01 ± 1.40 % at a
concentration of 3000 μg/ml. 32% inhibition at
0.1% concentration of Rutin trihydrate indicates
good activity of the formulation (Figure 6).
Skin Irritation Study
Skin irritation study conducted on albino Wistar
rats when observed for sensitivity and reaction at
the end of 24, 48 and 72 h showed absence of
erythema or edema for both the formulations
(control as well as test). This indicates that the gels
do not cause any skin irritation as no sensitivity
reaction (redness/ erythema) was observed.
CONCLUSION
Liposomes have been reported to be efficient
colloidal carriers for the delivery of therapeutics
into skin. This work too confirms the promising
role of liposomal topical formulation.
Multilamellar vesicles with good entrapment
efficiency were successfully prepared by thin film
hydration method and the drug containing
liposomes were formulated into a gel. The
liposomal gel showed prolonged drug release
upto 12 h and also exhibited anti-elastase activity.
Due to the striking similarity between liposome
components and skin lipids, they are safe and
effective. In addition to delivering the drug in
higher concentration into skin layers, they
enhance skin hydration making them an ideal
vehicle for anti-aging remedies. Thus liposomes
can be considered as effective carriers for better
topical delivery.
ACKNOWLEDGEMENTS
The authors are thankful to VAV Life Sciences Pvt.
Ltd., Mumbai for gift sample of Leciva-S70
(soya lecithin). They also sincerely thank NIRRH,
Mumbai for TEM imaging, Dr. Kshitij Vasant,
Kelkar College, Mumbai for anti-elastase assay
and NMIMS’s SPTM College, Mumbai for helping
in carrying out skin irritation studies.
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