JPRHC

Research Article

PREPARATION AND EVALUATION OF NIOSOMES OF BRIMONIDINE TARTRATE AS OCULAR DRUG

DELIVERY SYSTEM

P. PRABHU*, MARINA KOLAND, K. VIJAYNARAYAN, NM. HARISH, D. GANESH, RN CHARYULU,

D. SATYNARAYANA.
For author affiliations, see end of text
This paper is available online at www.jprhc.in

ABSTRACT kinetics. The intra ocular pressure lowering activity of

prepared formulations were determined and compared
Nioosomes of brimonidine tartrate were prepared by with pure drug solution. It was found that intra ocular
film hydration method. The prepared vesicles were pressure lowering action was sustained for longer
evaluated for photomicroscopic characteristics, period of time. Stability study data revealed that the
entrapment efficiency, in vitro, ex- in vitro drug formulations were found to be stable when stored at
release, in vivo intra ocular pressure lowering activity. refrigerator temperature (2 °C to 8 °C) and at 25 °C
Methods employed for the preparation of vesicles with no change in shape and drug content. Results of
were found to be simple and reproducible, produced the study indicated that it is possible to develop a safe
vesicles of acceptable shape and size with unimodal and physiological effective topical niosomal
frequency distribution pattern. The in vitro, ex-in vitro formulation which is patient compliance.
drug release studies showed that there was a slow and KEYWORDS: Niosome, film hydration,
prolonged release of drug which followed zero order bromonidine tartrate, intra ocular pressure.

INTRODUCTION medication is required at the site of action which is

The main objective of drug delivery system to the eye patient incompliance1. Vesicular drug delivery
is to improve existing ocular dosage forms and exploit systems allows the entrapment of drug molecule into
newer drug delivery system for improving the lipid bilayer or surfactant vesicles and thus increase
therapeutic efficiency. Topical application of eye drug concentration at the site of application with
drops is the most common method of administering sustained drug delivery of medicament, which results
drugs to the eye in the treatment of ocular diseases. in improved bioavailability. Such vesicles (liposome
Topical and localized applications are still an and niosome) acts as carrier for controlled ocular drug
acceptable and preferred route, such dosage forms are delivery by preventing metabolism of drug from
no longer sufficient to overcome the various ocular enzymes present at the corneal epithelial surface.
diseases like glaucoma due to poor bioavailability, due Vesicle entrapped drug can be easily administered in
to the efficient mechanism protecting the eye from liquid dosage forms such as eye drops with patient
harmful materials and agents. This includes reflex, compliance, modulated drug release profile and high
blinking, lachrymation, tear turnover, and drainage of drug pay load. Niosomes can encapsulate both
tear results in the rapid removal of the drug from eye lipophilic and hydrophilic drugs and protect against
surface. Similarly frequent instillation of concentrated acidic and enzymatic effects in vivo. They offer

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several advantages over liposomes such as higher the storage, which includes vesicles aggregation,
chemical stability, intrinsic skin penetration enhancing fusion, leaking or hydrolysis of encapsulated drugs.
properties and lower costs. However, there may be This may affect the shelf life of the niosomes 2
problems of physical instability of niosomes during
,3
.
temperature about 60 °C, till the lipid film was
Brimonidine tartrate is α2-adrenergic agonist indicated formed. Dried lipid film obtained was hydrated with
in open angle glaucoma, which is common form of aqueous phase of phosphate buffer pH 7.4 (10 ml)
glaucoma. Glaucoma is group of diseases of optic containing drug. The flask was shaken for 1 h to get
nerve involving loss of retinal ganglion cells. niosomal formulation. Niosomal formulations
Increased intra ocular pressure (IOP) is significant risk prepared were coded as NF1, NF2 and NF3. Once a
factor for the development of glaucoma. At present the stable suspension was produced, subjected to ultra
eye drops (0.2%) of the said drug is available in the probe sonication by transferring the colloidal
market all over the world. However, the drug has to be suspension on to a glass vial. The probe tip of the ultra
4, 5
instilled into the eye 3-4 times a day . To avoid such sonicator was just dipped into the suspension (care
frequent administration of the drug, in the present should be taken such that the probe tip does not touch
study an attempt was made to develop a niosomal drug the bottom of the glass vial during sonication).
delivery system of brimonidine tartrate for ocular Sonication was done in 2 cycles. First the niosomal
administration and investigated its intraocular pressure suspension was sonicated at 80% amplitude with a
lowering activity. pulse of 0.5 cycles per second for a period of 3 min,
followed by 3 min rest (excess heat may be generated
MATERIALS AND METHODS during probe sonication, which may damage the
Materials lipids). After 3 min, second cycle was processed for 3
min at 80% amplitude with 0.5 sec pulse for another 3
Brimonidine tartrate was a gift sample from FDC Ltd, min.
Aurangabad, India. Cholesterol, Span 60 were Photomicroscopic study of niosomes:
obtained from CDH laboratories Ltd, New Delhi. The niosomal suspensions was subjected to
Diethyl ether, chloroform, methanol, potassium size analysis under a microscope (10×400
dihydrogen phosphate, disodium hydrogen phosphate magnification) fitted with a calibrated ocular
were obtained from E-Merck India Ltd, Mumbai. micrometer. The shape of prepared liposomes was also
Methods studied.
Preparation of niosomes:
In the present study three niosomal
formulations of brimonidine tartrate were prepared by
film hydration method6 as described by Bangham et.al.
(1965). All the lipid components including surfactant,
span 60, as per the formula were taken in round
bottom flask and dissolved into sufficient quantity (10
ml) of organic solvent (chloroform). Organic solvent
was evaporated under reduced pressure, at a

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Drug entrapment efficiency determination: suspension was obtained and 1 ml of this suspension was
Entrapment efficiency of brimonidine tartrate in taken in a micro-pipette and transferred to a test tube. To
the niosomes was determined as follows: After sonication, this 5 ml methanol was added which resulted in a clear
1 ml of niosomal suspension (SUVs) was taken in a 1 ml solution, this was further vertexed in a vertex mixer for 2
micro-centrifuge tube. Centrifuged at 20,000 rpm for 1 h, at min such that to ensure that the niosomes are lysed
4 ºC in a cold centrifuge to get a white pellet. This was completely to release the drug7. This solution (1 ml) was
settled at the bottom of the centrifuge tube. Supernatant further diluted with methanol and the absorbance was
was separated as it contains unentrapped drug which is determined using a UV spectrophotometer (Jasco V-530).
highly soluble in PBS 7.4, using a micro-pipette. To the The entrapment efficiency (EE) was calculated using the
remaining pellet in the centrifuge tube 500 µl of 0.1 N following formula:
NaOH (as drug is highly soluble in 0.1N NaOH) was added Entrapped drug (mg)
Percentage entrapment (%EE) = ---------------------------- × 100
and vertexed thoroughly for 3 min. After vertexing a white
Total drug added (mg)

In vitro drug release study: Ex- in vitro drug release study of prepared niosomes was
In vitro drug release study of niosomal studied by membrane diffusion technique. In this study in
formulations was studied by membrane diffusion vitro diffusion cell was made using porcine cornea as
technique7. In vitro diffusion cell was made using semipermeable membrane. All the procedures followed
cellophane membrane as a semipermeable membrane. The were similar to that explained under in vitro drug release
diffusion cell consists of a beaker, magnetic stirrer with study, except the cellophane membrane was replaced by
temperature control and test tube with both ends open. One fresh porcine cornea7.
end of test tube was closed using treated cellophane In vivo intra ocular pressure lowering activity:
membrane as semi permeable membrane and other end was In vivo intra ocular pressure lowering activity of
open to introduce the niosomal formulation. The diffusion selected niosomal preparation (NF3) of brimonidine
medium was freshly prepared phosphate buffer pH 7.48 tartrate was studied in normotensive male albino rabbits
solution (100 ml) equilibrated at 37± 0.5°C temperature. weighing 1.2 to 2.5 Kg. This study was conducted in
The niosomal formulation (5 ml) was placed inside the accordance with CPCSEA (guidelines and the experimental
diffusion cell through open end of test tube on the protocol was approved by Institutional Animal Ethics
cellophane membrane. The diffusion medium of freshly Committee (K.S. Hegde Medical Academy). The animals
prepared phosphate buffer pH 7.4 solution (100 ml) was were housed under well controlled conditions of
placed inside the beaker such way that the lower surface of temperature (22± 2 °C), humidity (55±5%) and 12/12 – h,
cellophane membrane makes contact with the buffer. The light-dark cycle, were given access to food and water. The
temperature of buffer solution was maintained at 37± 0.5 rabbits were divided into three groups, each containing of
°C and stirred with magnetic stirrer throughout the study single male albino rabbit. The protocol of the experiment
period. Aliquots (5 ml) of the medium was withdrawn was approved by the Institutional Animal Ethics
every hour and replaced with fresh diffusion medium of Committee. To induce acute glaucoma, 5% dextrose
phosphate buffer pH 7.4, to maintain constant volume (sink solution (15 ml/kg) was intravenously infused through
condition). The samples were analyzed marginal ear vein. The basal intraocular pressure was
spectrophotometrically for concentration of brimonidine measured by tonometere. The drug formulations (20 µl,
tartrate at 320 nm. drug equivalent to pure drug solution 0.2%) were
Ex- in vitro drug release study administered to rabbits in different sequence. In sequence

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1: Drug formulations were administered 30 min before the vs. square root of time), and zero order (cumulative amount
administration of dextrose solution. In sequence 2: Drug of drug released vs. time) pattern11
formulations and dextrose solution were administered
together. In sequence 3: Drug formulations were RESULTS AND DISCUSSION
administered 30 min after the administration of dextrose Niosomes were prepared by thin film hydration
solution. The intraocular pressure (IOP) changes were method as per the method described by Bangham et al.,
recorded every 30 min till the pressure difference between 1965. The molar ratios of surfactant and cholesterol were
the control eye and treated eye is zero. Formulation was dissolved in 2 ml of a mixture of choloroform: methanol
instilled on to corneal surface of one eye and contra lateral (2:1) in a 250 ml round bottom flask. The powder particles
eye was remaining as control. Intraocular pressure (IOP) of lipid mixture don’t seem to dissolve readily in the
was measured by tonometry method with the help of chloroform: methanol solution. So the flask was rotated for
Schniotz tonometere and mean was taken at three times 15 min over the water bath (at a temperature above the
fixed interval. All IOP measurements were carried out by transition temperature of the lipids) before starting the
the same operator, using same tonometere. Each rabbit was vacuum pump. A very low nitrogen flux (through a
given washout period of three days after every treatment. nitrogen cylinder connected to the evaporator by an inlet
The ocular hypotensive activity was expressed as the rubber pipe) was set up during the preparation of niosomes
average difference in IOP between the treated and control to prevent too much oxygen to get dissolved. Gradually the
eye of the same rabbit, according to the equation ∆ IOP = nitrogen pressure was raised at the cylinder head until there
8, 9
IOP of Treated Eye – IOP of control Eye . was no pressure difference between the inside and outside
Stability study: of flask. The pressure release valve between the cylinder
For stability testing, the sonicated niosomal suspension of and the evaporator prevents the build up of pressure inside
was stored away from light in sealed 2 ml micro centrifuge the apparatus. If this flux is too high the solvent may
eppendroff tubes in refrigerator (4-8 ºC) and at room evaporate. Some of the solvent evaporates inevitably during
temperature (25 ºC) for 3 months. Sampling was done by this period, but the solution thermalizes and the lipids get
withdrawing 100 µl of the supernatant using a micro- dissolved. These formulations were characterized in Table
nd th th
pipette at different time intervals of 2 day, 4 day, 10 1. The size of niosomal formulations ranged from 8.00 µ to
th th th th th th
day, 20 day, 40 day, 45 day, 60 day, 80 day and 90 9.37 µ and showed unimodal normal symmetrical
day respectively. Suitable dilutions were made with PBS frequency distribution patterns. All the vesicles were found
7.4 whenever sample was withdrawn and UV absorbance to be spherical in shape (Fig 1). Further, the sonication,
was determined. The entrapment efficiency was calculated. resulted in much smaller vesicles, which is very essential in
from the regression equation In the present work, stability avoiding the irritation to the eye. The size of particles in
study was carried out for selected formulation NF3, at room ophthalmic dosage forms apart from influencing
temperature and refrigerator (2 °C to 8 °C), for 3 months bioavailability, plays important role in the irritation
10
and evaluated for the drug content . potential of formulation, hence it is recommended that
Determination of drug release kinetics: particles of ophthalmic solution should be less than 10 µ to
To know the mechanism of drug release from minimize irritation to the eye 12. Further, the size of
these formulations, the data were treated according to first- sonicated niosomes was found to be 245 nm (Fig. 2). The
order (log cumulative percentage of drug remaining vs. results shows that the amount of drug entrapped in
time), Higuchi’s (cumulative percentage of drug released niosomes ranged between 32.33 % to 43.4 %.

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Table 1: Characterization of niosomal formulations

Percentage drug
Formulation Formulation Ratio (drug: Average particle
entrappement
code cholesterol: span 60) size µ (micron)
efficiency

NF 1 1:1:1 8.03 32.33

NF 2 1:1:2 8.27 39.20

NF 3 1:1:3 9.37 43.40

Table 2: Comparative In vitro dissolution profile of different formulations

Time Formulation NF
Pure Drug Solution
(h)
NF1 NF2 NF3

1 68.00 ± 1.44 10.69 ± 0.99 14.13 ± 0.99 4.46 ± 0.99

2 77.40 ± 1.28 19.29 ± 0.98 22.35 ± 1.09 12.86 ± 0.98

3 78.80 ± 1.32 20.28 ± 0.97 24.91 ± 1.16 14.40 ± 0.99

4 78.30 ± 1.28 21.42 ± 0.94 26.52 ± 0.98 14.58 ± 0.98

5 80.00 ± 1.32 22.69 ± 0.99 27.99 ± 0.98 16.67 ± 0.97

6 79.64 ± 1.46 23.19 ± 0.98 29.30 ± 0.99 16.83 ± 0.98

7 81.54 ± 1.67 24.19 ± 0.99 31.11 ± 0.97 16.98 ± 1.08

8 83.42 ± 1.13 27.19 ± 1.05 33.85 ± 0.98 18.92 ± 1.10

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Table 3: Comparative Ex-In vitro dissolution profile of different formulations

Formulation
Time
(h) Formulation Formulation Formulation
Pure Drug Solution
NF1 NF2 NF3
1 68.00±1.43 12.89±0.99 07.89±0.99 06.37±0.99

2 77.41±1.29 15.68±0.98 13.04±0.98 13.12±0.98

3 78.80±1.31 18.92±0.97 13.36±0.99 14.31±1.09

4 78.30±1.28 24.76±0.94 14.00±0.98 15.87±1.17

5 80.00±1.33 27.56±0.99 15.64±0.97 16.59±0.99

6 79.64±1.45 27.89±0.98 17.56±0.99 18.57±0.98

7 81.54±1.69 28.56±0.99 17.76±1.05 20.55±0.99

8 83.42±1.17 29.81±1.07 18.89±1.10 22.56±0.98

Fig 1: Photomicrograph of niosomes Fig. 2. Change in IOP. Sequence-1. Drug

formulations was administered 30 min
before the administration of dextrose solution.

0
0 100 200 300
IOP Difference

-5 Pure drug
solution
-10
Niosomal
-15 formulation

-20
Time (min)

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Fig. 3. Change in IOP. Fig.4. Change in IOP.

5
5

0
0
0 100 200 300
0 100 200 300 -5 Pure drug
-5 solution
Pure drug solution
-10
Niosomal
Niosomal formulation
-10 -15 formulation

-20
-15
-25
-20

The comparative in vitro drug release profile summarized profile. The in vitro and ex- in vitro drug release studies
in Table-3, for pure drug solution and for each formulation. showed that, there was slow and prolonged release of drug
It was observed that pure drug solution released from all the formulations and followed zero order kinetics.
approximately 78% of drug within 2 h, while niomal This indicated that the drug release was independent of
formulations NF1, NF2 and NF3 showed 18.92 %, 22.50 % concentration of drug entrapped.
and 29% drug release respectively in 8 h. The result of in To study the in vivo performance of prepared formulations,
vitro drug release profile of formulations showed that intraocular pressure lowering activity was determined. It
niosomal formulations provides the prolonged release of was found that in sequence 1, where drug formulations
drug when compared to pure drug solution. Similarly, the were administered 30 min before the administration of
comparative ex - in vitro drug release profile was dextrose solution (Fig 2), intraocular pressure lowering
summarized in Table 3, for pure drug solution and for each activity with liposomal formulation was sustained for
formulation. It was observed that pure drug solution longer period (3-4 h). However marketed product though
released major amount of drug within 1 h, while the showed activity within 30 min, but could not sustain for
niosomal formulations NF3 and NF2 showed 18.89 % and more than 60 min. It was found that the IOP difference
22.56 % drug release respectively in 8 h. Hence, from produced between pure drug solution and niosomes is very
comparative in vitro and ex-in vitro drug release data of significant. Niosomal formulations sustained the action for
brimonidine tartrate from liposomes and pure drug solution, prolonged period (Fig 2) of time (240 min). Hence, the
it has been observed that the amount of drug release difference in IOP lowering activity with pure drug solution
remained similar. Further the delayed drug release rate may did not last long and sustainability of action was also not
be attributed largely to the drug transport by diffusion observed. In sequence 2, formulation and dextrose solution
controlled mechanism resulting in prolonged drug release were administered together (Fig 3), and sustained action

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was observed compared to pure drug solution. Further, rate and extent of absorption resulting in reduction of IOP
extent of IOP lowering activity was found to be better in for prolonged period of time.
comparison to marketed product. Whereas for marketed Result of stability study was found to be satisfactory and
product, the effect was observed immediately. The duration acceptable. The niosomes stored at refrigerator (2 °C to 8
of intraocular pressure lowering activity remained more or °C), and room temperature, found to be sufficiently stable
less similar to that of sequence 1. In sequence 3, dextrose with no change in shape and no significant difference in
solution was administered before the administration of drug content.
niosomal formulation and marketed product (Fig 4) and CONCLUSION:
intraocular pressure lowering activity with niosomal Niosomes of brimonidine tartrate allowed a
formulation was not found to be significant. However, significant vesicular carrier system for therapeutic
better duration of action was visible to some extent with effectiveness in terms of duration of action and decrease in
niosomes. It was also observed that at the end of 240 min, dose frequency. The in vitro and ex- in vitro drug release
the effect of all the formulations was found to be nil. This studies showed that, there was slow and prolonged release
may be due to the fact that the induced IOP by injecting 5% of drug from all the formulation and followed zero order
dextrose solution did not last long. Nevertheless, the kinetics. The in vivo intraocular pressure lowering activity
experimental data justified the sustained action of of niosome formulation was found to be significant and
liposomal formulation in comparison to marketed eye sustained for long period of time which encourages its
drops. This may be the reason why in sequence 3, the effect physiological effectiveness. Thus niosomes offer a
of niosomal formulation was not observed to the greater promising avenue to fulfill the need for an ophthalmic drug
extent as in the case of sequence 1 and sequence 2. The delivery system that not only has the convenience of a
drug formulations were administered 30 minute after the drop, but that can localize and maintain drug activity at its
induction of glaucoma. site of action for a longer period of time thus allowing for a
However, the better reduction in IOP with niosomes may sustained action; minimizing frequency of drug
probably due to the better partitioning of drug between administration with patient compliance.
vesicle and eye corneal surface. Further, it is believed that ACKNOWLEDGEMENTS:
the release of drug from niosome will increase the local The authors wish to acknowledge Nitte University,
concentration at corneal surface, after the release from Mangalore (Karnataka) India, for providing the necessary
vesicle depending on passive diffusion of drug molecule facilities and financial support to carry out this project
across the corneal barrier. The longer contacts time of
vesicles at corneal surface, leads to higher bioavailability of
drug. Thus the niosome acts as drug carrier, which changes

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