April 2014541

Regular Article Biol. Pharm. Bull. 37(4) 541551 (2014)

Potential Use of Niosomal Hydrogel as an Ocular Delivery System for

Atenolol
Irhan Ibrahim Abu Hashim,a Marwa Salah El-dahan,a Rehab Mohammed Yusif,a
Abd-ElGawad Helmy Abd-ElGawad,a and Hidetoshi Arima*,b,c
a
Department of Pharmaceutics, Faculty of Pharmacy, Mansoura University; Mansoura 35516, Egypt: bDepartment
of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University; and cProgram
for Leading Graduate Schools HIGO (Health Life Science: Interdisciplinary and Glocal Oriented) Program,
Kumamoto University; 51 Oe-honmachi, Chuo-ku, Kumamoto 8620973, Japan.
Received September 16, 2013; accepted December 24, 2013

Niosomes have been reported as possible approach to improve the low corneal penetration and bioavail-
ability characteristics for many drugs. The purpose of this study was to prepare and characterize an effec-
tive ocular niosomal hydrogel containing 0.5% (w/v) atenolol which is 1 adrenoceptor blocker for treatment
of glaucoma. Thin lm hydration method was used for the preparation of niosomes using Span 60 and cho-
lesterol at different molar ratios. Niosomes were characterized using laser diffraction particle size analyzer,
transmission electron microscopy, and differential scanning calorimetry. The results showed that higher
entrapment efciency (80.7%1.2) was obtained from niosomes prepared using Span 60/cholesterol at a 2 : 1
molar ratio. Stability study revealed that a fairly high retention of atenolol inside vesicles (83.1%2.35) up
to a period of 3 months at 4C. It was found that niosomal hydrogel formulation using carbopol 934P signi-
cantly exhibited sustained in vitro release of the drug compared with free drug solution and other polymeric
hydrogels. The intraocular pressure (IOP) lowering activity of selected atenolol formulations was determined
and compared with that of atenolol solution. It is worth noting that niosomal hydrogel formulation was found
to show the most signicant prolonged decrease in IOP, suggesting that niosomal hydrogel could be a promis-
ing delivery system for atenolol.
Key words atenolol; niosomal hydrogel; carbopol 934P; sustained release; intraocular pressure; glaucoma

Eye is the most important and sensitive organ; in fact, it is the need for an ophthalmic drug delivery system that has the
the window of our soul. Drug delivery in ocular therapeutics convenience of a drop, and at the same time can localize and
is a challenging problem due to physiological constrains im- maintain drug activity at its site of action for longer period of
posed by the unique anatomic structure and efcient protec- time thus allowing for a sustained action.10)
tive mechanism of the eyes.1) The majority of the ophthalmic From a technical point of view, nonionic surfactant vesicles
drugs are administrated topically in the form of conventional (niosomes) are considered promising drug carriers as they
eye drops.2) However, the rapid turnover of lacrimal fluid and possess greater stability and lack of many disadvantages as-
extensive nasolacrimal drainage3) along with eyes blinking sociated with phospholipid vesicles (liposomes), such as high
reflex rapidly eliminate the administrated eye drops.4,5) This cost, stringent storage condition and the oxidative degradation
causes short pre-corneal residence time, which limits effective of phospholipids.16)
transcorneal drug absorption. Thus, frequent instillation of eye Niosomes are formed from the self-assembly of non-ionic
drops is required to achieve therapeutic effect.6) In addition, amphiphiles in aqueous media resulting in closed bilayer
the topically applied drugs could enter the systemic circulation structures17) which can entrap both hydrophilic and lipophilic
via conjunctiva and nasal mucosa,7) which may result in some drugs either in the aqueous layer or in the vesicular mem-
undesirable side effects.8) Ophthalmic ointment formulations brane.18) Moreover, sparingly soluble drugs can be entrapped
demonstrated substantially improvement in drug bioavail- in the vesicles.19)
ability when compared with their eye drops counterparts. Glaucoma is a disease characterized by higher level of
However, they suffered from some disadvantages that include intraocular pressure (IOP) which might progressively hurt vis-
sticky sensation, blurred vision and induced reflex blinking ibility. The chronic glaucoma with open angle poses a major
which also caused patient non-compliance.9) problem of public health and it is the second leading cause of
To overcome these problems, different approaches such as blindness in the world.20) It is estimated that the number of
in situ forming gel,10) micro and nanocarrier systems,11,12) in- people with glaucoma will be nearly 79.6 million worldwide
serts,13) and vesicular systems14) have been adopted. In recent by 2020.21) This alarmingly large number of anticipated pa-
years, vesicular drug delivery systems used in ophthalmics tients requires urgent improvement in the current therapeutic
such as liposomes and niosomes help in providing prolonged approaches adopted for the treatment of this disease. Even
and controlled action at the corneal surface and preventing though, the currently available eye drops for glaucoma treat-
the metabolism of the drug by the enzymes present at the ment reduce its disability. Their long-term effectiveness and
tear/corneal epithelial surface.15) Drug enclosed in the vesicles efcacy are being questioned due to poor patient compliance.
allows improved partitioning and transport through the cor- Atenolol is a 1 adrenoceptor blocker. It is reported to
nea. Moreover, vesicles offer a promising avenue to fulll lack intrinsic sympathomimetic activity and membrane-
stabilizing properties.22) This suggests that its action in
The authors declare no conflict of interest. reducing IOP must in some way be mediated by the inhibi-
 To whom correspondence should be addressed. e-mail: [email protected]
* 2014 The Pharmaceutical Society of Japan
542 Vol. 37, No. 4

tion of the 1-receptors in the eye. It remains to be seen Inc., Rancho Dominguez, Canada).
whether this inhibition causes an increase in outflow facility Methods. Preparation of Atenolol Niosomes Niosomes
or impairs the production of aqueous humour.23) Oral admin- were prepared by thin lm hydration method.40) Briefly, ac-
istration of atenolol has been shown to reduce the IOP.2426) curately weighed quantities of Span 60 and CH at differ-
P-glycoprotein (P-gp) is a 170 kDa membrane protein encoded ent molar ratios, viz. 1 : 1, 2 : 1, 3 : 1 and 1 : 2 (Table 1), were
by the multidrug resistance gene (MDR1) and functions as placed in a 100 mL round bottomed flask and dissolved in
an energy-dependent efflux pump.27) P-gp is typically local- 10 mL chloroform. The organic solvent was evaporated under
ized at the apical surface of the epithelial cells and has been reduced pressure at 60C using a rotary evaporator (Buchi
shown to reduce the transepithelial permeation of diverse Rotavapor, Switzerland) till the formation of a thin lipid lm.
drugs.28) In the eye, P-gp is expressed in the retinal capillary The excess organic solvent was removed by leaving the flask
endothelial cells,29) retinal pigmented epithelial cells,30) ciliary in a desiccator under vacuum overnight. The lipid lm was
nonpigmented epithelium,31) conjunctival epithelial cells,32) then hydrated with 10 mL of distilled water containing ateno-
corneal epithelium33,34) and iris and ciliary muscle cells.29) lol in a concentration equivalent to 10 mg/mL at 55C, which
Role of P-gp as a barrier to corneal delivery of drugs has been is above the gel-liquid transition temperature (Tc) of sorbitan
reported earlier.33,35) monoesters.41,42) The resulting niosomal suspension was
Yang et al.28) investigated the role of P-gp in transepithelial mechanically shaken for 1 h using a horizontal mechanical
transport and uptake of propranolol in conjunctival epithelial shaking water bath at 55C. Then, the vesicle suspension was
cells cultured on Transwell lters in the presence and absence sonicated for 20 min in a bath type sonicator (Ultrasons, Se-
of P-gp competing substrates. It was found that not only hy- lecta, Barcelona, Spain). The niosomal suspension was left to
drophilic atenolol but also moderately lipophilic metoprolol mature overnight at 4C and stored at refrigerator temperature
and highly lipophilic alprenolol failed to affect propranolol (4C) for further studies.
uptake. This nding indicated that such -blockers are non Characterization of Niosomes. Entrapment Efciency
P-gp substrates. In addition, another study examined the effect (EE%) Niosomes containing atenolol were separated from
of elacridar as a P-gp inhibitor on the permeability of atenolol unentrapped drug by cooling centrifugation at 18000 rpm
across the blood brain barrier in mice and rats. The authors for 60 min at 4C. Niosomal pellets were resuspended in
reported that atenolol, a non P-gp substrate, exhibited poor distilled water and then centrifuged again. This washing pro-
brain penetration in the presence or absence of elacridar in cedure was repeated two times under the same conditions to
both species.36) ensure that the unentrapped drug was no longer present in
In late 1970 s, earlier studies have shown that topically the void volume between the niosomes.43) The supernatant
applied atenolol eye drops lowered the IOP in patients with was separated each time from niosomal pellets and assayed
ocular hypertension after short duration therapy.23,37,38) Other spectrophotometrically for free atenolol at 274 nm using UV/
researchers have attempted to overcome the drawbacks as- VIS spectrophotometer (JASCO, V-530, Japan). Amount of
sociated with the conventional eye drops through inclusion of entrapped drug was obtained by subtracting amount of unen-
atenolol in gel formulations.39) trapped drug from the total drug incorporated.44)
Still, it is important to develop a suitable long-term ocular entrapment efficiency (%)
delivery system to control the IOP within an appropriate dura-
tion, minimizing dosing frequency, reducing systemic absorp- amount of entrapped drug (mg)
= 100
tion and optimizing therapeutic effect. Thus, niosomes could total amount of drug (mg)
be a useful ocular drug delivery system for the treatment of Determination of Vesicle Size The average size of the
glaucoma. prepared niosomes was determined using laser diffraction
The objective of the present study was to prepare and particle size analyzer (Malvern Instruments Ltd., Worcester-
characterize niosomal hydrogels containing atenolol. The fac- shire, U.K.). Before measurement, samples were dispersed in
tors influencing the encapsulation of atenolol into niosomes distilled water.19)
were investigated. Characterization of the prepared niosomes Transmission Electron Microscopy (TEM) The mor-
regarding physical morphology, particle size, in vitro drug phology of the prepared niosome formulations was determined
release, and stability study was performed. Moreover, the IOP by TEM (JEOL 100 CX Transmission electron microscope at
lowering activity of the selected atenolol niosomal formula- 80 KV): a drop of the dispersion was diluted 10-fold using
tions was evaluated. deionized water, then a drop of the diluted dispersion was
applied to a carbon-coated 300 mesh copper grid and left for
MATERIALS AND METHODS 1 min to allow some of the niosomes to adhere to the carbon
substrate. The remaining dispersion was removed by absorb-
Chemicals Atenolol was kindly supplied by European
Egyptian Pharm. Ind., Alexandria, Egypt. Sorbitan monostea- Table 1. Composition and Characterization of Niosomal Formulations
rate (Span 60) and cholesterol (CH) were purchased from Sig-
Formulation Span 60 : CH Particle size Entrapment
ma-Aldrich (St. Louis, MO, U.S.A.). Sodium alginate (BDH
code (molar ratio) (nm) efciency (%)
Chemicals, Ltd., Poole, U.K.), hydroxypropylmethyl cellulose
(Dow Chem. Co., Midland, MI, U.S.A.), carbopol 934P (BF F1 1:1 56.56.8 60.92.1
Goodrich, U.S.A.), chitosan (Loba Chemie, Mumbai, India). F2 2:1 94.28.1 80.71.2
All other chemicals and solvents were of analytical reagent F3 3:1 155.311.6 73.61.9
grade. Spectra/Por dialysis membrane (1200014000 molecu- F4 1:2 133.410.1 32.91.5
lar weight cutoff) was purchased from (Spectrum Laboratories Each value represents the meanS.D. (n=3).
April 2014543

ing the drop with the corner of a piece of lter paper. After Table 2. The Composition of Different Gel Formulations
twice rinsing the grid (deionized water for 35 s) a drop of
Polymer
2% aqueous solution of uranyl acetate was applied for 1 s. Gel formulation Atenolol
The remaining solution was removed by absorbing the liquid code Type Conc. (%w/w) 0.5% (w/w)
with the tip of a piece of lter paper and the sample was air G1 HPMC 2% Free drug
dried.45) G2 HPMC 2% Niosomes
Differential Scanning Calorimetry (DSC) The thermal G3 Sodium alginate 2% Free drug
properties were analyzed using differential scanning calo- G4 Sodium alginate 2% Niosomes
rimetry (DSC; Shimadzu, DSC-60 with TA-60 WS thermal G5 Chitosan 2% Free drug
analyzer, Tokyo, Japan) calibrated with indium. Thermograms G6 Chitosan 2% Niosomes
were analyzed using Shimadzu TA-60 software. The lyophi- G7 Carbopol 934P 1% Free drug
lized pellets of the niosomal formulations were used for the G8 Carbopol 934P 1% Niosomes
investigation. Span 60, CH, and atenolol were also investigat-
ed. A sample (4 mg) of powder was placed in a hermetically
sealed aluminum pan and scanned at a rate of 10C/min over zero-order kinetics (cumulative % drug released vs. time),
a temperature range of 20350C under nitrogen atmosphere. rst-order kinetics (log % drug retained vs. time), Higuchi
Alumina powder was used as the reference material in the model (cumulative % drug released vs. square root of time),
DSC runs. and KorsmeyerPeppas equation (log amount of drug released
In Vitro Drug Release Studies In vitro release experi- vs. log time). The correlation coefcient (r) values were calcu-
ments were assessed for the selected niosomal formulation lated for the linear curve obtained by regression of the above
showing highest drug entrapment level (F2). The in vitro plots.
release of atenolol from solution or niosomal dispersion was Evaluation of Niosomal Stability The optimized nio-
carried out using dialysis method. Spectra/Por dialysis mem- somal formulation (F2) was tested for stability by storing it at
brane (1200014000 molecular weight cutoff) was washed 41C and at ambient room temperature. Vesicles size and %
several times with distilled water and soaked in simulated tear atenolol retained were assessed before and after storage for 3
fluid pH 7.4 for 24 h before the experiment. The membrane months. Size of the vesicular system was determined by laser
was stretched over the open end of 3 cm diameter glass tube diffraction particle size analyzer. In addition, % drug retained
and was made water tight by a rubber band. A 3 mL sample, was evaluated by taking samples after 3 months and estimated
either of the freshly puried niosomal dispersion or of free spectrophotometrically at 274 nm.
atenolol solution (0.5% w/v equivalent 15 mg atenolol), was In Vivo Studies Adult male albino normotensive rabbits,
placed in the tube. The tube was then immersed upside- each weighing 1.52.0 kg were used in the experiments. All
down in a beaker containing 50 mL simulated tear fluid pH animals were healthy and free of clinically observable ab-
7.4 which is preheated and maintained at 370.5C using normalities. Animals were housed singly in a standard cages,
thermostatically controlled water bath (Hilab, GLF 3202, Ger- in a light controlled room (12-h light and 12-h dark cycles)
many). The tube height was adjusted, so that the membrane at 2024C, with no restriction to food or water.46) The ex-
was just below the surface of the release medium. The rotary perimental procedures conform to the ethical principles of the
shaker was adjusted to a rate of 25 strokes/min. At predeter- scientic committee of the Faculty of Pharmacy, Mansoura
mined time intervals of 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h, University, Egypt for the use of experimental animals. The
aliquots of 1 mL were withdrawn from the release medium rabbits were divided into four groups, each consisting of six
and replaced by equivalent volume of the buffer solution. The rabbits: group I received atenolol solution, group II received
released amounts of the drug were analyzed spectrophoto- atenolol niosomal dispersion composed of Span 60 : CH in a
metrically at 274 nm. 2 : 1 molar ratio, group III received free atenolol/carbopol gel,
Preparation and in Vitro Release of Atenolol from Nio- and group IV received atenolol niosome/carbopol gel. A single
somal Hydrogels Different polymers used as bases for pre- dose of 100 mg of ophthalmic drug solution or other investi-
paring niosomal hydrogels are listed in Table 2. The weighed gated formulations (0.5% atenolol) were applied directly into
amount of the polymer was dispersed in distilled water in the lower conjunctival sac of the right eye of rabbits while
which 0.01% benzalkonium chloride as a preservative was the left eyes served as a control. At certain time intervals,
previously dissolved. The aqueous dispersion was allowed the IOP was measured before and after application of the
to hydrate for 45 h. The selected niosome dispersion (F2) formulations for both control and tested eyes using Schiotz to-
with drug concentration of 0.5% (w/w) was added and gently nometer (Winters, Eichtabelle, Germany).47) All measurements
stirred (Polamed magnetic stirrer, model MM5, Poland) till were done three times at each interval, and the mean values
obtaining a homogenous mixture. The niosomal gel was left were used to calculate the percentage decrease in IOP.48) The
overnight at 4C for air removal. Free atenolol 0.5% (w/w) gel pharmacodynamic parameters taken into consideration were
formulations were also prepared following the same proce- maximum percentage decrease in IOP (Emax), time for maxi-
dure. The viscosity of the prepared gels was determined using mum response (Tmax), area under percentage decrease in IOP
Rotary viscometer (Hakke Inc., Germany). The release of versus time curve (AUC08 h).
atenolol from free and niosomal gel preparations was studied Statistical Analysis The data are represented as mean
using the same method as niosomal dispersion. S.D. Statistical analysis of the data was carried out using
Kinetic Analysis of Drug Release Data To investigate one way ANOVA followed by TukeyKramer multiple com-
the mechanism of drug release, the in vitro release data were parisons test at a level of signicance of p<0.05 with Instat
analyzed mathematically according to the following models: Graphpad prism software (version 4.00; Graphpad software,
544 Vol. 37, No. 4

Fig. 1. Transmission Electron Microphotographs of Atenolol Niosomal Formulations Composed of Span 60 : Cholesterol at Different Molar Ratios
a) 1 : 1; b) 2 : 1; c) 3 : 1; d) 1 : 2.

San Diego, CA, U.S.A.). cles. They were well identied and present in a nearly perfect
sphere-like shape having a large internal aqueous space and a
RESULTS AND DISCUSSION smooth vesicle surface. In addition, they existed in disperse
and aggregate collections. Particle size analysis of the freshly
Vesicles Preparation and Characterization To obtain prepared niosomes is presented in Table 1. The size of nio-
the desired vesicle size and the highest encapsulation ef- somal formulations ranged from 56.5 to 155.3 nm. Sonication
ciency, the process variables such as, organic solvent, speed of may be responsible for the breakdown of the multilamellar
rotation of flask, hydration medium, hydration time, agitation vesicles to form much smaller unilamellar vesicles which
method, and time of sonication were investigated and opti- is very essential in avoiding the irritation to the eye.53,54)
mized. Nanoparticles less than 200 nm are considered accepted for
Entrapment Studies The effect of different molar ratios passive drug targeting and for in vivo study.55,56)
of Span 60 : CH on the entrapment efciency % (EE%) of at- Vesicle size is an important parameter that influences the
enolol in niosomes is illustrated in Table 1. Signicant differ- biopharmaceutical feature of the carrier.57) The size of par-
ence in EE% between all niosomal formulations (p<0.05) was ticles in ophthalmic dosage forms apart from influencing bio-
observed. The results revealed that F2 showed the maximum availability, plays an important role in the irritation potential
entrapment efciency (80.7%1.2) at 2 : 1 Span 60 : CH molar of the formulation, hence it is recommended that particles of
ratio. Span 60 showed the maximum entrapment efciency ophthalmic solution should be less than 10 m to minimize
at this molar ratio, as it has a long saturated alkyl chain that irritation to the eye.58)
decreased the amount of CH needed to form niosomes.49) CH Differential Scanning Calorimetry (DSC) DSC thermo-
is one of the common additives included in the formulation grams of atenolol loaded niosomes composed of Span 60 : CH
in order to prepare stable niosomes. It stabilizes bilayers, pre- (2 : 1) molar ratio, plain niosomes, and their individual com-
vents leakage and retards permeation of solutes enclosed in the ponents are illustrated in Fig. 2. Span 60, CH, and atenolol
aqueous core of these vesicles.50) Moreover, drug partitioning showed sharp endothermic peaks at 58.16C, 148.70C, and
will occur more easily in highly ordered systems of surfactant 155.61C, respectively. Characterization by DSC showed that
and CH.51) It was observed that further increase in CH con- a change in the phase transition temperature of the main
tent (formula F4) had a signicant decrease (p<0.05) in the constituents is generally observed in the DSC thermograms
entrapment efciency of sorbitan ester niosomes (32.9%1.5). of plain niosomes. An obvious change in the phase transition
This could be due to the fact that CH beyond a certain con- temperature of Span 60 from 58.16 to 49.57C was observed
centration can disrupt the regular bilayered structure of ve- with clear changes in the enthalpy in addition to a consider-
sicular membranes leading to loss of drug entrapment levels.52) able peak broadening. A second peak was generally noticed
Morphology and Size Analysis of Niosomes Figure 1 which could represent other components such as CH.59) DSC
shows the TEM micrographs of atenolol niosomes. As ob- thermogram of atenolol loaded niosome interestingly showed
served, niosomes appeared as homogenous unilamellar vesi- disappearance of the melting endothermic peak of atenolol and
April 2014545

Fig. 3. In Vitro Release Proles of Atenolol from Free Solution and

Niosomal Dispersion in Simulated Tear Fluid pH 7.4
Each point represents the meanS.D. (n=3).

8 h, no signicant difference was observed on the release of

atenolol at various concentrations of the polymers. The same
behavior was noticed on the release of the drug from niosomal
hydrogels. We found that the release of atenolol from niosomal
hydrogels was signicantly (p<0.05) sustained compared with
Fig. 2. DSC Thermograms of a) Span 60 Alone; b) Cholesterol Alone; its release from free hydrogels (Fig. 4). In addition, increasing
c) Atenolol Alone; d) Plain Niosome; e) Atenolol Niosome the polymer concentration was associated with an increase in
the viscosity of the gel (data not shown). This may have some
the major endothermic peaks of the lipid bilayer components disadvantages including difculty in topical application to the
were also shifted with the same manner as observed in plain eye, sticky sensation, blurred vision and reflex blinking. In
niosome. This may be due to entrapment of atenolol in the view of the above mentioned data, we selected the appropri-
vesicular system. ate concentrations of the polymers (2% sodium alginate, 2%
In Vitro Release Study Figure 3 illustrates the drug re- hydroxypropyl methylcellulose (HPMC), 2% chitosan and 1%
lease proles from its free solution and niosomal dispersion. carbopol).
Free drug solution appeared to exhibit a signicant (p<0.05) Figure 5 shows the in vitro release prole of atenolol from
higher and faster release in the rst 0.5 h than that from the different polymeric gels compared to its release from the
niosomal dispersion. The drug solution showed 40.2% release niosomal hydrogels. It is obvious that incorporation of nio-
after 0.5 h, whereas the niosomal drug dispersion showed somes into a structured gel vehicle resulted in a signicant
only 11.2% drug release after 0.5 h. The drug release from the (p<0.05) slower release of the drug compared with free drug
free solution began to plateau after 2 h, whereas the release gel formulations possibly because of the diffusion restriction
from the niosomal dispersion was continued for 8 h without imposed by the polymeric network of the gel.6163) It is also
reaching plateau. These results pointed to sustained release clear that atenolol release was signicantly (p<0.05) sustained
characteristics with a Higuchi pattern of drug release, where and more extended in case of niosomal carbopol gel (47.4%)
niosomes act as a reservoir system for continuous delivery of compared with other niosomal gels (67.0%, 60.8%, 67.0% for
the drug.53) CH reduces the leakage or permeability of encap- HPMC, chitosan, and sodium alginate, respectively) after 8 h.
sulating drug by decreasing the niosomal membrane fluidity.50) This may be due to the higher viscosity of niosomal carbopol
Yoshioka et al.60) found that the release rate of carboxyfluo- gel, which provides an extra barrier for atenolol release.64)
rescein, a water soluble compound, from niosomes prepared The viscosity values were found to be 1017, 1035, 1097, and
with Span 60 was slower than the release rate from other Span 1220 mPas for 2% sodium alginate, 2% HPMC, 2% chitosan,
formulations (Span 20, 80, and 85). This result could be due to and 1% carbopol, respectively.
the fact that at 25C, the molecules of Span 60 are in the or- Kinetic Studies of the Release Data Table 3 summarizes
dered gel state, but those of other Spans are in the disordered the release kinetic parameters and correlation coefcients (r2)
liquid crystalline state. calculated for the investigated formulations. The in vitro re-
In Vitro Drug Release from Niosomal Hydrogels From lease results showed that the release of atenolol from niosomal
our experimental data, F2 formulation was selected because of gels as well as niosomal dispersion is most tted to diffusion-
its reasonable size (94.28.1 nm), high entrapment efciency controlled mechanism (Higuchi model).61,65,66) These results
(80.7%1.2), as well as good release properties. This niosomal pointed to sustained release characteristics with a Higuchi
formulation was then incorporated in different polymeric gels. pattern of drug release, where niosomes act as a reservoir
As a preliminary study, we investigated the in vitro release system for continuous delivery of the drug.53) In this study, the
proles of atenolol from different concentrations of polymeric KorsmeyerPeppas equation was utilized to interpret the at-
gels compared to its release from niosomal hydrogels. After enolol release kinetics. It can give more insights on other drug
546 Vol. 37, No. 4

Fig. 4. In Vitro Release Proles of Atenolol from Free and Niosomal Hydrogels in Simulated Tear Fluid pH 7.4 at Various Concentrations of Poly-
mers
Each point represents the meanS.D. (n=3).

Fig. 5. In Vitro Release Proles of Atenolol from Free and Niosomal Hydrogels in Simulated Tear Fluid pH 7.4
Each point represents the meanS.D. (n=3).
April 2014547

Table 3. Kinetic Analysis of the Release Data of Atenolol from Niosomal Dispersion and Different Gel Formulations

Formula Zero order First order Higuchi model Release KorsmeyerPeppas

code 2
Correlation coefcient (r ) mechanism r 2
Release exponent (n)

F2 0.9690 0.9960 0.9970 Diffusion 0.9855 0.6634

G1 0.8606 0.8448 0.9531 Diffusion 0.9706 0.5074
G2 0.9495 0.9702 0.9812 Diffusion 0.9919 0.6026
G3 0.9072 0.9486 0.9783 Diffusion 0.9885 0.5878
G4 0.9347 0.9712 0.9856 Diffusion 0.9841 0.6234
G5 0.8880 0.9650 0.9840 Diffusion 0.9872 0.5018
G6 0.8679 0.9258 0.9796 Diffusion 0.9884 0.5089
G7 0.8996 0.9445 0.9790 Diffusion 0.9798 0.5330
G8 0.9301 0.9535 0.9779 Diffusion 0.9750 0.5414

Fig. 6. Percentage Decrease in IOP after Administration of Atenolol Solution and Other Selected Formulations
Each point represents the meanS.D. (n=6).

release mechanisms such as Fickian (diffusion), non-Fickian represent a metastable state in that the vesicles possess excess
(anomalous), and erosion-mediated (zero-order) release. Addi- of energy bilayer phospholipids, which can undergo chemi-
tionally, this equation was successfully used to explain release cal degradation such as oxidation and hydrolysis. Due to this
mechanisms from thin lm, cylindrical, disc, and spherical change, vesicular systems maintained in aqueous dispersion
controlled release devices.67) The n values were in the range may aggregate/fuse, and encapsulated bioactive material may
between 0.5 and 1 (0.5<n<1) suggesting the non-Fickian tend to leak out from the bilayer structure during storage.69)
(anomalous) release mechanism for the drug i.e. both ero- At 41C, a minimum loss of the drug was observed, which
sion and diffusion.68) may be attributed to the regidization of the vesicles at low
Evaluation of Niosomal Stability In the present study, temperature that reduced the permeability of the drug through
the stability of the vesicles was determined by measuring the the membrane.69) Thus, it is worth noting that the prepared
vesicle size and % drug retained before and after 3 months at vesicular systems are more stable at 41C, as compared to
41C and at ambient room temperature. Mean vesicle size storage at room temperature in terms of mean vesicle size and
was found to increase on storage after 3 months. The increase % drug retained.
in vesicle size was more in the formulation stored at room In Vivo Studies Atenolol niosomal dispersion (ATN)
temperature than at 41C. The vesicle size of 128.903.85 and atenolol niosome/carbopol gel (ATNG) were selected for
and 155.339.1 nm was recorded at storage temperature of 4 in vivo studies as they showed slower and sustained in vitro
1C and ambient room temperature, respectively, compared release of the drug. The chosen formulations were then com-
to initial size of 94.28.1 nm. At 41C, % drug retained pared with atenolol solution (ATS) and free atenolol/carbopol
was 83.22.35%, but marked decrease in % drug retained gel (ATG) in their IOP lowering efcacy. The mean % de-
was found when formulation was stored at room temperature crease in IOP proles after the instillation of atenolol solution
(39.14.45%). or application of its selected formulations (ATG, ATN, and
Lipid vesicles are self assembles of amphiphiles into closed ATNG) into the rabbits eye until 8 h following administration,
bilayer structures. Hydrated bilayer vesicles, however, are not are shown in Fig. 6. Also, the relevant pharmacodynamic data
considered to be thermodynamically stable and are thought to are listed in Table 4.
548 Vol. 37, No. 4

Table 4. In Vivo Pharmacodynamic Parameters after Administration of sustained sufciently and diminished after 6 h. ATN showed
Atenolol Solution and Other Selected Formulations a signicant effect which was sustained for up to 7 h. On the
other hand, ATNG showed promising results as it signicantly
Formula Pharmacodynamic parameters
decreased the IOP to maximum value (49.80%) after 4.33 h
code Tmax (h) a)
Emax (%)b) AUC (% h)c) of drug administration, and the effect was sustained and pro-
ATS 1.000.00 29.836.05 57.1412.79 longed for the time of the experiment up till 8 h. The AUC
ATG 2.000.00* 40.174.02* 126.9312.84* after application of atenolol formulations until 8 h were 2.22,
ATN 2.660.57* 41.805.27* 163.0018.68* 2.85, 4.58-fold higher than that of atenolol solution for ATG,
ATNG 4.330.58*, 49.801.21*, 261.9325.43*, ATN, and ATNG, respectively.
Each value represents the meanS.D. (n=6). * p<0.05 versus atenolol solution The better reduction in IOP with niosomes may probably
(ATS). p<0.05 ATNG versus ATN. a) Time for maximum response. b) Maximum be due to the better partitioning of drug between vesicle and
percentage decrease in IOP. c) Area under percentage decrease in IOP versus time
curve up to 8 h post-administration.
eye corneal surface. Furthermore, the release of drug from
niosomes will increase its local concentration at the corneal
surface; however, after release from the vesicles, drug mol-
Gross examination of the rabbit eyes during this study ecules rely on passive diffusion to cross the corneal barrier.
showed no signs of abnormal lachrymation or increased blink- The corneal penetration enhancing effect of niosomes could
ing upon instillation of any of atenolol formulations. No irrita- be attributed to many factors. Disrupting the tight junctions
tion with the niosomal formulation was observed. The same of the corneal epithelium is partly responsible for increasing
nding was reported by other authors.70) Additionally, Guin- corneal uptake. Other possible reasons are their better spread-
edi et al.71) reported that, no major changes were observed in ing ability on the lipophilic corneal surface and favorable
histological photomicrographs of control corneal tissues and rheological properties.80) The longer the contact time at the
corneal tissues following instillation of multilamellar niosomal corneal surface, the higher the bioavailability of the drug.81)
formulations composed of Span 60 and CH. This may be due This will also reduce the amount of drug and the dose fre-
to the fact that the irritation power of surfactants decreases in quency necessary for therapeutic effect.82) Thus, niosomes
the following order: cationic>anionic>ampholytic>non-ionic, act as drug carriers which change the rate and extent of drug
so the non-ionic surfactants are preferred for ocular delivery.72) absorption resulting in the reduction of IOP for prolonged pe-
Previous studies demonstrated that in vitro cytotoxicity is riod of time.46)
indicative of irritation potential.73) Cheong et al.74) compared Since, vesicular systems offer a great deal of advantages
the in vitro cytotoxicity, using the 3-(4,5-dimethylthiazol- over the conventional systems, various pharmaceutical ap-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, of eight proaches can be tried to render their nal formulation more
clinically available -blockers (propranolol, alprenolol, ateno- effective. The best way to achieve this would be to enhance
lol, labetalol, metoprolol, pindolol, timolol, and bisoprolol) on the precorneal retention. One such approach is the use of com-
human corneal epithelial and retinal pigment epithelial cell binatorial drug delivery systems which are a promising trend
lines. Primary and immortalized corneal and retinal cell lines in ophthalmic research, with the great potential of combining
were compared for their susceptibility to the cytotoxic effect the advantages of various systems and overcoming their limi-
of the drugs. The cytotoxicity of -blockers was also evalu- tations.15) Mucoadhesive polymers when used in combination
ated on human skin keratinocytes and broblasts in order to with vesicular systems provide vesicles with the necessary
investigate susceptibility differences as a function of the tis- site adherence and site retention to achieve carrier and drug
sue origin. Comparison of the IC50 of the eight -blockers for targeting in topical ocular therapy and endow them with the
different cell lines after 16 h demonstrated that atenolol was ability to be mucoadhesive.58,44) Carbopols is an important
the least cytotoxic drug.74) Moreover, the cytotoxic effect of class of ocular bioadhesives.79) Due to the low viscosity of
niosomal Ciclopirox olamine composed of Span 60 and CH colloidal suspensions of vesicles that does not allow sufcient
was evaluated on KB (oral cancer), PC3 (prostate cancer), retention time of the dosage form in the eye upon instillation,
Siha (cervical cancer) and Vero (kidney epithelial) cell lines different hydrogel matrices such as carbopol have been used
using MTT assay. The results indicated that blank niosomes to increase the viscosity of topical preparation and to increase
(not containing Ciclopirox olamine) did not show any evidence the retention time of the formulation at the site of administra-
of cytotoxicity on the cell lines chosen.75) In addition, Zarei et tion due to good bioadhesive properties.83)
al.76) studied the cytotoxicity of paclitaxel niosome composed Our results have shown that 0.5% (w/v) atenolol niosomal
of Span 60 and CH on MCF-7 cell line after 48 h by MTT hydrogel extended the duration of action for 8 h with 4.33 fold
assay. The authors found that the higher concentration of increase in the AUC than that of 0.5% (w/v) atenolol solution.
niosome devoid of drug did not affect the cell line, thereby, The IOP lowering activity of 1% (w/v) atenolol gel formula-
it was considered to be safe.76) In this study, no change in tions was previously studied.39) It was found that, increasing
IOP was observed in the untreated eye during the course of the viscosity of the polymers was associated with the IOP
measurement in any of the formulations, thus indicating that lowering effect. The AUC after application for the higher
these formulations exerted a local action within the eye and concentrations of the polymers were 3.2 and 5.4 fold higher
that the observed IOP lowering activity is not because of any than that of 1% (w/v) atenolol solution with duration of action
systemic absorption.7779) of 6 and 8 h for 15% sodium alginate and 3% carboxymethyl
After instillation of ATS, the Tmax was reached after 1.00 h cellulose, respectively.39) Based on these data, it is clear that,
of instillation, followed by a rapid decline of the percentage our formulation is signicantly better considering that similar
decrease in IOP indicating its short duration of action. In case effect is obtained at half the concentration of the drug; 0.5%
of ATG, Tmax was observed at 2.00 h, but the effect was not w/v versus 1% w/v reported earlier.39) This will lead to reduc-
April 2014549

ing the amount of the drug and the dose necessary for the 10) Abraham S, Furtado S, Bharath S, Basavaraj BV, Deveswaran R,
therapeutic effect with subsequent limited systemic absorption Madhavan V. Sustained ophthalmic delivery of ofloxacin from
an ion-activated in situ gelling system. Pak. J. Pharm. Sci., 22,
and side effects. This study indicated that niosomal hydrogel
175179 (2009).
combined two important features, long retention and sustained
11) Losa C, Alonso MJ, Vila JL, Orallo F, Martinez J, Saavedra JA,
drug release which is essential and fruitful approach to pro- Pastor JC. Reduction of cardiovascular side effects associated with
vide a steady and prolonged release of atenolol into the eye. ocular administration of metipranolol by inclusion in polymeric
nanocapsules. J. Ocul. Pharmacol., 8, 191198 (1992).
CONCLUSION 12) Maurice DM. Prolonged-action drops. Int. Ophthalmol. Clin., 33,
8191 (1993).
In the last couple of years, continuous research has been 13) Nadkarni SR, Yalkowsky SH. Controlled delivery of pilocarpine.
going on for better delivery of anti-glaucoma drugs with the 1. In vitro characterization of Gelfoam matrices. Pharm. Res., 10,
aim of more localized drug delivery and minimization of dos- 109112 (1993).
ing frequency. An ophthalmic delivery system should prefer- 14) Davies NM, Farr SJ, Hadgraft J, Kellaway IW. Evaluation of mu-
coadhesive polymers in ocular drug delivery. II. Polymer-coated
ably release the drug at a controlled rate to prolong the effect
vesicles. Pharm. Res., 9, 11371144 (1992).
in reducing IOP and should be non toxic and comfortable
15) Kaur IP, Garg A, Singla AK, Aggarwal D. Vesicular systems in
for patient use. Our ndings have shown that higher EE% of ocular drug delivery: an overview. Int. J. Pharm., 269, 114 (2004).
(80.7%1.2) was obtained from niosomes prepared using Span 16) Vora B, Khopade AJ, Jain NK. Proniosome based transdermal
60/CH at 2 : 1 molar ratio with particle size diameter of 94 delivery of levonorgestrel for effective contraception. J. Control.
8.1 nm. Niosomal hydrogel formulation using carbopol 934P Release, 54, 149165 (1998).
signicantly exhibited sustained in vitro release of atenolol 17) Uchegbu IF, Vyas SP. Non-ionic surfactant based vesicles (nio-
compared with free drug solution and other polymeric hydro- somes) in drug delivery. Int. J. Pharm., 172, 3370 (1998).
gels. In vivo study proved that niosomal hydrogel was found 18) Carafa M, Santucci E, Alhaique F, Coviello T, Murtas E, Riccieri
to show the most signicant prolonged decrease in IOP com- FM, Lucania G, Torrisi MR. Preparation and properties of new
unilamellar non-ionic/ionic surfactant vesicles. Int. J. Pharm., 160,
pared with other drug formulations of the same concentration.
5159 (1998).
In conclusion, niosomal hydrogel could be a promising deliv-
19) Arunothayanun P, Bernard MS, Craig DQ, Uchegbu IF, Florence
ery system for atenolol with improved ocular bioavailability AT. The effect of processing variables on the physical character-
and prolonged drug release proles. istics of non-ionic surfactant vesicles (niosomes) formed from a
hexadecyl diglycerol ether. Int. J. Pharm., 201, 714 (2000).
Acknowledgments The authors would like to express 20) Resnikoff S, Pascolini D, Etyaale D, Kocur I, Pararajasegaram R,
their heartfelt thanks to Prof. Dr. Osama Abd El-Azeem Soli- Pokharel GP, Mariotti SP. Global data on visual impairment in the
man, the head of Pharmaceutics Department, Faculty of Phar- year 2002. Bull. World Health Organ., 82, 844851 (2004).
macy, Mansoura University, Egypt for his support in measur- 21) Quigley HA, Broman AT. The number of people with glaucoma
ing the rabbits IOP. worldwide in 2010 and 2020. Br. J. Ophthalmol., 90, 262267
(2006).
22) Barrett AM, Carter J, Fitzgerald JD, Hull R, Le Count D. A
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