Journal of Pharmaceutical Investigation
DOI 10.1007/s40005-014-0121-8
RESEARCH ARTICLE
Effects of surfactant type and cholesterol level on niosomes
physical properties and in vivo ocular performance using timolol
maleate as a model drug
Hamdy Abdelkader Usama Farghaly
Hossam Moharram
Received: 5 January 2014 / Accepted: 18 February 2014
The Korean Society of Pharmaceutical Sciences and Technology 2014
Abstract This study aimed at investigating the roles of
both Span surfactant type (having a gel/liquid transition
temperature range from\10 to 55 C), and cholesterol level
on in vitro characteristics and in vivo performance of
timolol maleate niosomes. Span 20, Span 40 and Span 60
niosomes were prepared using the thin film hydration
method. Span:cholesterol levels employed were 7:3 mol/mol
(a minimum concentration for stable niosomes) and
1:1 mol/mol (a maximum concentration accommodated by
niosomes). Niosomes were characterized for size, percent-
age entrapment efficiency, morphology, in vitro release and
intra-ocular pressure (IOP) lowering activity in rabbits. The
order of the area under IOP-time curve (AUC) was ranked
as follows: Span 40 [ Span 20 [ Span 60. The AUC and
IOPmax values for Span 40:cholesterol (7:3) niosomes were
superior due to having a good compromise between thermo-
responsiveness and efficient capacity for loading a signifi-
cant drug cargo, compared with Span 20 and Span 60.
Keywords Niosomes  Intraocular pressure 
Timolol maleate  Ocular delivery  Span surfactants
Introduction
Vesicles consisting of one or more surfactant bilayers
enclosing aqueous spaces are called non-ionic surfactant
H. Abdelkader (&)  U. Farghaly
Department of Pharmaceutics, Faculty of Pharmacy,
Minia University, Minia, Egypt
e-mail: hamdy2002m@yahoo.com; h.abdelkader@mu.edu.eg
H. Moharram
Department of Ophthalmology, Faculty of Medicine,
Minia University, Minia, Egypt
vesicles or simply niosomes. Niosomes are considered of
particular interest as they offer several advantages over lip-
osomes. Niosomes are more stable chemically (Uchegbu and
Florence 1995; Kaur et al. 2004). They incur lower produc-
tion cost due to the availability of starting materials (Sahin
2006). They are biodegradable and non-immunogenic (Kaur
et al. 2004). Niosomes do not require expensive handling
(storing in a freezer and preparation under nitrogen gas).
Over the last three decades, niosomes of different
compositions used for topical ophthalmic drug delivery
have increased/prolonged the therapeutic effect while
minimizing toxic effects (Abdelbary and El-gendy 2008;
Abdelkader et al. 2013). Niosomal sizes of [10 lm have
been reported to be optimum for ocular delivery, in order to
carry a significant amount of payload, resist lacrimal
flushing effect and stay longer on the surface of the eye
(Uchegbu and Vyas 1998; Abdelkader and Alany 2012).
Cholesterol, a bilayer membrane stabilizer, is reported to
increase membrane stability, decrease the fluidity of the
membrane and alter membrane permeability (Uchegbu and
Florence 1995; Uchegbu and Vyas 1998). However, the
impact of these effects on the in vivo behavior upon
instillation onto the ocular surface is yet to be resolved.
Alkyl esters, such as sorbitan fatty acid esters (Span)
have been widely used in ocular delivery (Aggarwal et al.
2004; Abdelbary and El-gendy 2008; Kaur et al. 2008) and
we have recently reported on their good conjunctival and
corneal tolerability (Abdelkader et al. 2012).
Timolol maleate (TM), a water soluble model drug, was
used in this study. TM has been the first topical non-
selective cardio-selective beta-blocker and has remained
the milestone treatment for glaucoma therapy (Schenker
et al. 1999; Sweetman 2009).
Niosomes encapsulating TM were prepared using the
reverse phase evaporation method and coated with 0.5 % w/v
123

chitosan solution. The TM niosomes achieved a signifi-
cantly (1.7 times) higher TM concentration in the aqueous
humor than that of the drug solution. This was attributed to
better improvement of corneal penetration and prolonged
precorneal residence time compared with the TM aqueous
solution (Kaur et al. 2010).
However, no plausible explanation has yet to be pro-
vided on the effects of surfactant types or the cholesterol
levels on drug uptakes by ocular tissues. Therefore, this
report aims at studying the effects of surfactant types and
cholesterol levels on in vitro and in vivo performance of
ocular niosomes for the anti-glaucomic drug TM. Ocular
bioavailability could be simply monitored through moni-
toring intra-ocular pressure (IOP) of rabbits, this technique
has been employed elsewhere to study the ocular bio-
availability of different glucocorticoids from nano-sus-
pensions (Kassem et al. 2007). Specific objectives will look
at the effects of both surfactant types and cholesterol levels
on size, Entrapment efficiency percentage (EE %), mor-
phology and IOP lowering activities for the tested
niosomes.
Materials and methods
Materials
Timolol maleate (TM) was a generous gift from EIPICO
Pharmaceuticals (Cairo, Egypt). Span 60, Span 40, Span
20, cholesterol, and cellulose membrane, molecular weight
cut-off 12,00014,000, were purchased from Sigma-
Aldrich, St. Louis, USA. All other solvents and buffer salts
were of analytical grade and used as received.
Methods
Thin film hydration method (TFH)
Niosomes were prepared by adopting the TFH method
(Azmin et al. 1985). Briefly, a specified amount
(900 lmol) of Span 60, Span 40 or Span 20: cholesterol at
a molar ratio of 1:1 and 7:3 was dissolved in chloroform
and rotary evaporated (Heidolph, Laborota 4000, GmbH,
Germany) to form a thin lipid film. The dried surfactant/
lipid film was kept overnight in order to remove any
residual traces of the organic solvent. The lipid film was
then hydrated with either 8 ml of phosphate buffer saline
(PBS) pH 7.4 or TM solution (5 mg/ml) in PBS at 200 rpm
for 2 h. The selected hydration time at 2 h was based on
preliminary studies. Longer hydration time did not show
further improvement in terms of TM loading. The resultant
niosomal dispersion was set aside for at least 2 h at room
temperature, to allow the vesicles membrane to anneal.
123
H. Abdelkader et al.
The formed niosomes were stored in a fridge for sub-
sequent analyses.
Entrapment efficiency percentage (EE %)
The EE % determination method used relied on exhaustive
dialysis of the prepared niosomes and subsequent quanti-
fication of the released drug molecules. Accordingly, 2 ml
of niosome dispersion was transferred into a dialysis bag
and allowed to exhaustive dialysis in 100 ml of PBS twice
for 4 h at 4 C. The dialysate solutions were collected and
analyzed for TM content spectrophotometrically at 295 nm
using a UVVis spectrophotometer (Spectronic, Genesys
2PC, USA).
The drug EE % was expressed as a percentage and
calculated using Eq. 1 (Nasr et al. 2008):
Ao  A
EE %  100
Ao
where A is the amount of TM in the dialysate solution and
Ao is the initial amount of TM used.
Niosome size measurements
The size of the prepared niosomes was determined by the
laser diffraction technique using a particle size analyzer
(Quantachrome CLIAS00, France). The samples were
properly diluted with PBS and measured at 25 C. Nio-
somes size was expressed in terms of volume diameter and
the measurements were done in triplicate and the average
values were used.
Transmission electron microscopy (TEM)
Selected niosomal systems prepared, were examined under
TEM. A drop of the niosome sample was transferred into
the copper mesh grids. After the sample was adsorbed
(about 1520 min), the staining dye (potassium phospho-
tungstate) was dripped onto the film. The staining time was
about 12 min. After drying the copper mesh grids, the
morphology of the investigated niosomes was visualized
(Liu and Guo 2007).
In vitro release studies
A sample of 2 ml of the prepared niosomal dispersions was
placed on a semi-permeable standard cellophane mem-
brane (previously immersed in PBS for 24 h). The loaded
membrane was stretched over the lower end of an open
glass tube of 3 cm diameter and fixed with an elastic band.
The glass tube was then immersed in a 50-ml beaker
containing 20 ml PBS (NaCl 137 mM, Na2HPO4 10 mM,
KH2PO4 1.47 mM, KCl 2.68 mM) at pH 7.4 in such a
Effects of surfactant type and cholesterol level on niosomes
Table 1 Summary of the rabbit groups recruited in the study
Group Right eye Left eye
Group 1 TM solution (0.25 % w/v) Span 20 7:3
Group 2 Span 40 7:3
Group 3 Span 40 1:1
Group 4 Span 60 7:3
Group 5 Span 60 1:1
manner that the membrane was located just below the
surface of the buffer solution. This in-house designed dif-
fusion unit was placed in a thermostatically controlled
shaking water-bath adjusted at 35 0.1 C with a constant
shaking at 100 rpm to avoid any development of a con-
centration gradient. At appropriate sampling intervals an
aliquot of 2 ml was collected and replaced by an equal
volume of the fresh release medium at the same tempera-
ture. The samples were analyzed for TM spectrophoto-
metrically at 295 nm. The kinetics of drug release was
studied by fitting the release data through three common
mathematical models (zero-order, first-order and Higuchi
diffusion models).
Ocular bioavailability study
Pigmented rabbits, weighing between 1.0 and 1.5 kg, were
employed in the experiments. The rabbits were fed bal-
anced diet and maintained on 12 h/12 h light/dark cycle in
an air-conditioned room, at 28 C before the experiment.
The experimental procedures conformed to the ethical
guidelines of Minia University Animal Ethics Committee
(Minia, Egypt).
The rabbits were divided into five groups, 5 each, and
the basal IOP was measured for both right and left eyes of
all rabbits to give a basal IOP value at time zero using a
pre-calibrated Schoetz tonometer (Riester, Germany).
An instilled dose of 2025 ll of TM solution or noisome
dispersions, equivalent to TM concentration of 0.25 % w/v,
was instilled into the right eye surface of the rabbit,
whereas the same dose of the tested niosomes were
instilled into the left eyes, so that the five groups are shown
in Table 1.
Pharmacokinetic parameters such as Tmax and %
maximum reduction of IOP (IOPmax) were determined for
the tested niosomes and the control TM solution.
Tmax is the time to maximum reduction in IOP (IOPmax).
% maximum reduction of IOP %IOPmax
IOP0  IOPmax
IOP0
where IOP0 is the intra-ocular pressure at 0 time, IOPmax is
the intra-ocular pressure at Tmax.
Statistical analysis
To compare mean values of Tmax, IOP and IOPmax for the
prepared niosomes and the control, unpaired t test and
analysis of variance (ANOVA) were carried out at a 5 %
significance level using Graph Pad Software, USA) (1995).
Results and discussion
Span 20, Span 40 and Span 60 surfactants, having melting
points of 10, 45, and 54 C respectively, were investigated
for their ability to form niosomes in combination with two
different levels of cholesterol at molar ratios of 7:3 and
1:1 mol/mol, forming a cholesterol concentrations in
bilayers of 3050 % mol/mol respectively. In a previous
report, a cholesterol level of 30 % mol/mol in Span: cho-
lesterol niosomes of 7:3 was sufficient to stabilize niosomal
bilayer membranes and imparting a residual gel/liquid
transition enthalpy a property known as thermo-respon-
siveness (Abdelkader et al. 2010), whereas a cholesterol
concentration of 50 % mol/mol (1:1 niosomes) was capa-
ble of abolishing gel/liquid transition of the bilayer mem-
branes (Uchegbu and Florence 1995; Abdelkader et al.
2010). It is worthy to mention that TM has no appreciable
effects on the gel/liquid transition temperature of niosomes,
as TM is a water soluble drug and preferentially encapsu-
lated in the aqueous compartments of niosomes without
affecting the gel/liquid transition of niosomes.
Entrapment efficiency (EE %) of TM
Effect of surfactant type
The EE % values for the prepared niosomes ranged from
30 % 1.5 to 65 % 3 (Table 2). Span 40 and Span 60
established the highest EE %, whereas the lowest EE %
values were recorded for Span 20. The main factors
affecting the entrapment of small water-soluble drug mol-
ecules in niosomes are the permeability of the bimolecular
membranes and the structure continuity of the hydrocarbon
chain of the bilayer-forming surfactant (Grant et al. 2001).
Span 40 and Span 60 have long saturated acyl chains
[palmityl (C-16) and stearyl (C-18) chains respectively]
which are in a gel state, compared with a liquid surfactant
of short acyl chains [lauryl (C12)] at ambient conditions
(Grant et al. 2001). Such features render Span 40- and Span
60-based niosomes less likely to leak the encapsulated
water-soluble drug molecules (TM). In contrast, Span
20-based niosomes are fluid at room temperature and, even
in the presence of high levels of cholesterol, are disorga-
nized due to the trans-gauche conformations of their acyl
chains (Israelachvili et al. 1980). The EE % values for the
123

prepared niosomes can be summarized as follows: Span
60 [ Span 40 [ Span 20. These results support the
hypothesis that the higher the transition temperature of the
surfactant, the higher the EE % recorded for niosomes
(Yoshioka and Florence 1994; Abdelkader et al. 2010).
Effect of cholesterol concentrations
Table 2 shows the effects of different cholesterol levels
used on EE %. Irrespective of surfactant type, the higher
the concentration of cholesterol incorporated into nio-
somes, the lower the EE % attained. This effect was
significantly recorded with Span 20-based niosmes
(shortest hydrophobic chain length); EE % for Span 20
niosomes of cholesterol levels 30 and 50 % mol/mol was
45 and 30 % w/w respectively. Increases in cholesterol
concentration could increase the rigidity of the bilayer
membranes, by virtue of cholesterols rigid structure and
characteristic inverted cone shape (Israelachvili et al.
1980). This results in disruption of the vesicles bilayer
structure which compromises their ability to entrap TM
(Manosroi et al. 2003). This could lead to a decrease in
the total number of bilayer vesicles available for encap-
sulating the drug. Consequently, there is a decrease in the
ability of the niosomes to entrap such water-soluble drug
molecules.
Niosomal sizes
Table 2 shows the mean volume diameter of the prepared
niosomes composed of various surfactants and contained
two cholesterol levels (30 and 50 % mol/mol). The mean
diameter values of the prepared niosomes ranged from
5.0 0.01 to 9.67 0.01 lm. The size measurements
were repeatable and reproducible from batch to batch
(acceptable deviations from the average values). These
sizes are well accepted for ocular administration onto
surface of the eye, niosomal sizes of [10 lm were rec-
ommended for ocular delivery especially for water soluble
drugs for better precorneal residence time and significant
Table 2 Physical properties estimated for the prepared niosomes
Surfactant:cholesterol Ratio (mol/mol) Cholesterol level (%mol/mol)
Control (PBS)
Span 20:cholesterol 7:3 30
1:1 50
Span 40:cholesterol 7:3 30
1:1 50
Span 60:cholesterol 7:3 30
1:1 50
123
H. Abdelkader et al.
drug payload, compared with submicro-sized niosomes
(Uchegbu and Vyas 1998; Abdelkader and Alany 2012).
Effect of surfactant type
The diameter values of Span 60-based niosomes were
significantly smaller than both Span 40-based and Span
20-based ones. Lipid aggregation and self-assembly in an
aqueous medium is mainly controlled by two opposing
electrostatic forces. These are hydrophobic attraction for-
ces between the hydrocarbon tail chains and electrostatic
repulsion of the polar head groups (Tanford 1973). The
surface free energy increases with any decrease in hydro-
phobicity (Yoshioka and Florence 1994). Hence, surfac-
tants of higher hydrophilicity (higher HLB) should yield
larger vesicles.
Effect of cholesterol levels
The increases in the mean diameter values were insignifi-
cant (P [ 0.05), when cholesterol level ranged from 30 %
(7:3) to 50 % (1:1) mol/mol. Similar results were reported
elsewhere (Essa 2010). Cholesterol is a rigid molecule with
an inverted cone shape. It can be intercalated between the
fluid hydrocarbon chains of the amphiphile when hydrated
at a temperature above the gel/liquid transition tempera-
ture. It can also produce larger vesicles (Uchegbu and
Florence 1995; Essa 2010). Cholesterol is a membrane
stabilizer and can be accommodated and work coopera-
tively with the surfactant bilayer membranes.
Transmission electron microscopy (TEM)
The microstructures of the prepared niosomes were studied
using TEM (Fig. 1). The micrographs captured revealed
spherical-shaped niosomes of smooth surfaces. However,
the diameters were not correlated with those obtained from
laser diffraction measurements.
The steps involved in the sample preparation (e.g. dur-
ing replica formation on the copper grid and staining) for
TEM are likely to promote the formation of smaller sizes
EE % w/w Volume diameter (lm) T3h (% w/w)
100 1.3
45 2.3 5.0 0.01 68 2.0
30 1.5 11 .2 61 4.0
58 2.2 4.9 0.03 50 1.7
52 2.0 8.0 0.01 36 4.7
65 3.0 4.8 0.02 51 4.0
56 1.4 9.67 0.01 36 6.0
Effects of surfactant type and cholesterol level on niosomes
and artifacts during sample preparation and replica creation
(Egelhaaf et al. 2003; Perrie et al. 2007).
In vitro release studies
The in vitro release characteristics of TM from the pre-
pared niosomes were studied on in-house designed diffu-
sion cells as mentioned in the method section. An aqueous
solution of PBS was used as a release medium, stirred and
equilibrated at 35 C 0.5. The tested niosomes in the
donor compartment were separated, by a cellulose mem-
brane, from the receptor compartment that was filled with
20 ml of the release medium.
Figure 2 shows various release profiles of TM from the
aqueous TM solution and the prepared niosomes. Per-
centage TM released at 3 h was denoted asT3h; the esti-
mated T3h for TM solution was 100 %, whereas that for all
other niosomal formulations was significantly lower
(P \ 0.05). This could be ascribed to encapsulating the
water soluble molecules within the aqueous compartments
of niosomes. Accordingly, more time was required for TM
molecules to partition out of the bilayer membranes. These
results indicated that niosomes can successfully control
TM release.
With regard to effect of surfactant type, the values of
T3h calculated for Span 20-, Span 40- and Span 60-based
niosomes (7:3) were 68, 50 and 51 % respectively. These
results could be attributed to the gel/liquid transition tem-
perature (Tm) of niosomes forming surfactant. Span 20
molecules are always fluid and their hydrocarbon chains
Fig. 1 Micrographs of niosomes composed of Span 60: cholesterol 1:1 molar ratio
undergoing trans-gauch conformation all the time, whereas
Span 40 and Span 60 are gel and their hydrocarbon chains
are under restricted rotation. Accordingly, the encapsulated
small drug molecules are easily partitioning out from fluid
bilayer membranes than gelled ones.
Cholesterol can be considered as mortar for niosomal
bilayer membranes. Increasing the concentration of cho-
lesterol is always associated with decreasing the perme-
ability of the encapsulated water soluble drug molecules
and release rate.
The TM release data obtained from the prepared nio-
somes were fitted into three mathematical models (zero-
order, first-order and Higuchi diffusion models) in order to
elucidate the best fitting model which can predict the
mechanism of TM release. Table 3 summarizes the corre-
lation coefficients (r) and release rate constants (k) for the
prepared niosomes. With the exception of Span 60-based
niosomes, it was obvious that the highest r values were
recorded for the Higuchis model indicating that the best
fitting model was Higuchis and the general release
mechanism for TM from the prepared niosomes was the
diffusion throughout the bilayer membranes of the
uncharged conventional niosomes. On the other hand, the
drug release from Span 60-based niosomes was dependent
on time rather than square root of time. These results are in
a good agreement with those reported elsewhere (Guinedi
et al. 2005; Bayindir and Yuksel 2010). Further, the release
rate constants calculated for the TM solution and the tested
niosomes were in a good agreement with the above men-
tioned in vitro release parameter (T3h) suggesting that
123
 H. Abdelkader et al.

Fig. 2 In vitro release profiles

of timolol maleate (TM) from
TM solution and different
niosomal formulations

Table 3 Release kinetics parameters calculated for TM solution and niosomes

Formulation Zero order First order Higuchi
-1 -1
r K (%.h ) r K (h ) r K (%.h-0.5)

Control 0.975 36.40 0.915 1.40 0.990 7.64

Span 20:cholesterol 7:3 0.940 19.15 0.927 0.57 0.992 5.65
Span 20:cholesterol 1:1 0.942 17.22 0.974 0.37 0.990 5.07
Span 40:cholesterol 7:3 0.968 15.10 0.978 0.27 0.983 4.42
Span 40:cholesterol 1:1 0.954 10.58 0.984 0.15 0.988 3.11
Span 60:cholesterol 7:3 0.957 13.94 0.998 0.22 0.988 4.10
Span 60:cholesterol 1:1 0.964 10.26 0.993 0.14 0.987 3.00
r correlation coefficient, k release rate constant
Underlined data indicate the best fitting release mechanisms

niosomes successfully controlled the TM release and this
effect is significantly dependent on the type of the surfac-
tant used and the cholesterol level in the bilayer
membranes.
Ocular bioavailability study
Figure 3 shows the IOP-time curves for TM solution and
niosomes composed of Span 20, Span 40 and Span 60.
The time to trough IOP is called Tmax which is the time
for maximum reduction of IOP (IOPmax). Tmax for TM
solution and niosomes were 2 and 3 h respectively. In
general, niosomes exhibited significantly (P \ 0.05) more
sustained ([7 h) reduction of the IOP compared with that
for TM solution (less than 5 h). Also, the extent of IOP
reduction (as seen from the area under the curve, AUC) is
significantly greater than that for TM solution. This is
evident from maximum percentage reduction of IOP
(IOPmax) (Fig. 3).
Effect of surfactant type
Figure 4 displays the effect of surfactant type on the IOP
lowering profiles for the prepared niosomes. The values of
IOPmax were in the following order Span 40 [ Span
20 [ Span 60. This behavior is not correlated with the
in vitro release profiles (Span 60 [ Span 40 [ Span 20).
Further, it could not be explained due to noisome size
differences, as there were no significant differences in the
mean diameters for Span 20 7:3, Span 40 7:3 and Span 60
7:3 (Table 2). Other plausible explanation should be sought
such as gel/liquid transition and the drug cargo capacity of
niosomes. Therefore, the superior efficacy of Span
40-based niosomes could be accordingly explained. On one
hand, the transition temperature (Tm) of Span 40 (45 C) is
less than that for Span 60 (55 C), this could impart rela-
tively more flexibility of the hydrophobic bilayer mem-
branes, compared with that for Span 60-based niosomes.
On the other hand, Span 40-based niosomes have

123
Effects of surfactant type and cholesterol level on niosomes

Fig. 3 Effect of surfactant type

on the ocular bioavailability
(IOP-time AUC) of Span-based
niosomes

Fig. 4 Summary of the effects

timolol maleate (TM) niosomes
of different surfactant types and
cholesterol concentrations on
the maximum percentage
reduction of IOP (IOPmax).
*P \ 0.05 indicates significant
differences. **P [ 0.05
indicates no significant
differences

Fig. 5 Representative graphs

for the effect of cholesterol
levels on ocular bioavailability
(IOP-time AUC) form Span
40-based niosomes

significantly greater capacity of the drug cargo than Span compromise between sufficient thermo-responsiveness and
20-based niosomes. Therefore, the highest IOPmax for Span capacity to load an efficient drug cargo, compared with
40-based niosomes could be attributed to having a good Span 20- and Span 60-based niosomes.

123

Effect of cholesterol levels
Figure 5 shows representative curves of the effect of cho-
lesterol concentrations on the IOP lowering activity for
niosomes composed of Span 40: cholesterol at 7:3 and
1:1 mol/mol. The AUC and IOPmax for Span 40 7: 3 nio-
somes were significantly higher than those for Span 40 1:1.
Cholesterol at a molar ratio of 30 % mol/mol imparts a
residual gel liquid transition which is likely to allow nio-
somes to transit from gel to fluid at ocular surface tem-
perature (a property know as thermo-responsiveness),
whereas cholesterol concentration of 50 % mol/mol is
reported to completely abolish gel/liquid transition of
niosomes. These finding support the hypothesis that a
residual gel/liquid transition is favorably required for better
ocular drug uptake.
Conclusion
Span-based niosomes (Span 20, Span 40 and Span 60) having
different gel/liquid transition temperatures of \10, 45 and
55 C respectively, and two levels (30 and 50 % mol/mol) of
cholesterol were prepared, characterized and in vivo evalu-
ated. It was found that both gel/liquid transition temperatures
of the surfactants and the drug-cargo properties (EE %) of
niosomes contribute significantly to the ocular bioavailability.
This study can be considered a platform to study the influ-
ences of other parameters such size, the polar head group of
the surfactants and membranes additives on the ocular
bioavailability.
Acknowledgments This article dose not contain any studies with
human and animal subjects performed by any of the authors. All
authors (H. Abdelkkader, U. Farghaly, H. Moharram) declare that
they have no conflict of interest. The authors alone are responsible for
the content and writing of this article.
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