Journal of Microencapsulation

Micro and Nano Carriers

ISSN: 0265-2048 (Print) 1464-5246 (Online) Journal homepage: http://www.tandfonline.com/loi/imnc20

Novel in situ gelling ocular inserts for

voriconazole-loaded niosomes: design, invitro
characterisation and invivo evaluation of the
ocular irritation and drug pharmacokinetics

Marwa Hassan Shukr

To cite this article: Marwa Hassan Shukr (2016): Novel in situ gelling ocular inserts for
voriconazole-loaded niosomes: design, invitro characterisation and invivo evaluation
of the ocular irritation and drug pharmacokinetics, Journal of Microencapsulation, DOI:
10.3109/02652048.2015.1128489

To link to this article: http://dx.doi.org/10.3109/02652048.2015.1128489

Published online: 06 Jan 2016.

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 JOURNAL OF MICROENCAPSULATION, 2016
http://dx.doi.org/10.3109/02652048.2015.1128489

RESEARCH ARTICLE

Novel in situ gelling ocular inserts for voriconazole-loaded niosomes: design,

in vitro characterisation and in vivo evaluation of the ocular irritation and
drug pharmacokinetics
Marwa Hassan Shukr
National Organization for Drug Control and Research, Cairo, Egypt

ABSTRACT ARTICLE HISTORY

This work aimed to develop voriconazole in situ gelling ocular inserts loaded with niosomal suspension. Received 1 May 2015
Niosomes and mixed niosomes were prepared using span 40 and span 60 with pluronic L64 and pluronic Revised 28 September 2015
F127. The entrapment efficiency percentages (EE%), mean vesicle size, polydispersity index (PI), zeta Accepted 9 November 2015
potential and in vitro drug release of these niosomes were evaluated. F3-mixed niosomes prepared with Published online
span 60 and pluronic L64 was selected, due to its highest EE; optimum vesicle size with smallest PdI and 31 December 2015
suitable release pattern of the drug (63% after 8 h). In situ ocular inserts were prepared using sodium
Downloaded by [Gazi University] at 05:48 24 January 2016

KEYWORDS
carboxymethylcellulose (CMC Na) and sodium alginate (ALG) and characterised for surface morphology, In situ gel; niosomes; ocular
surface pH, water uptake, mucoadhesion and in vitro release. ALG in situ ocular insert (S2) was selected for insert; voriconazole
further in vivo evaluation of the ocular irritation and drug pharmacokinetics in the aqueous humour of
rabbits eyes. S2 in situ gelling ocular insert was non-irritant and showed significantly (p50.01) higher Cmax,
delayed Tmax and increased bioavailability.

Introduction
Fungal keratitis is a serious corneal disease that can impair vision or
lead to total blindness, if improperly treated (Liang et al., 2009).
Voriconazole (VCZ), a second-generation anti-fungal agent, pos-
sesses phenomenal characteristics like broad-spectrum activity,
activity against resistant fungal species and acceptable tolerability
(Wiebe and Karriker, 2005; Lat and Thompson, 2011). Studies
suggested an excellent efficacy of VCZ against several ocular
mycoses following topical administration (Gupta et al., 2011; de Sa
et al., 2015).
Topical ocular drug delivery is the preferred route for the
treatment of disorders of the ocular surface and anterior segment,
such as keratitis, conjunctivitis, glaucoma and diabetic keratopathy
(Abdelkader et al., 2011a, 2011b; Khutoryanskaya et al., 2014).
Recent studies focussed on delivering the antifungal drugs in
sufficient therapeutic concentrations to the cornea via several drug
delivery systems like surfactant-based elastic vesicles (Kaur et al.,
2012), mucoadhesive nanoparticles (Bhatta et al., 2012), liposomes
(Dai et al., 2013), in situ gels (Gratieri et al., 2011), nanosuspensions
(Luschmann et al., 2013), solid lipid nanoparticles (Basaran et al.,
2010), nanostructured lipid carriers (Shen et al., 2010) and
microemulsions (Kapoor and Chauhan, 2008).
Over the last three decades, niosomes of different compositions
used for topical ophthalmic drug delivery have increased/prolonged
the therapeutic effect while minimising toxic effects (Abdelkader
et al., 2013). Niosomes have been reported as possible approach to
improve the low corneal penetration and bioavailability character-
istics for many drugs. The corneal penetration enhancing effect of
niosomes could be attributed to many factors. Disrupting the tight
junctions of the corneal epithelium is partly responsible for
increasing corneal uptake. Other possible reasons are their better
spreading ability on the lipophilic corneal surface and favourable
rheological properties. Locally applied niosomal drug formulations
controlled ocular delivery and protected against drug metabolism
by the enzymes present in the lachrymal fluid (Abdelbary and El-
gendy, 2008).
The clinical efficacy of antifungal drugs depends on the
achievement of an optimal drug concentration in the ocular tissues
(Kaur and Kakkar, 2010), and as a result, frequent dosing is usually
required. To overcome the side effects of pulsed dosing, attempts
have been made to combine the advantages of a solid, single unit
dosage for ocular drug delivery by using in situ gelling inserts. The
application of solid structures in the eye has gained some interest
over the years (Ahmed and Aboul-Einien, 2007). The inserts
prepared by lyophilisation or freeze-drying method that hydrate
and gel quickly when exposed to lachrymal fluid will serve as an
alternative to conventional niosomal eye drops and allow for
simpler administration in the hope of increasing patient
acceptability.
Ideally, the formulation should be able to control the drug
release, remain in contact with the front of the eye for extended
period of time and avoid foreign body sensation (Bertram et al.,
2010; Farid et al., 2013).
The proper choice of polymers can allow mucoadhesion and
controlled drug release and due to dissolution of the gel, there
would be no need to remove the insert after it is depleted of drug
(Werner, 2003). Therefore, ocular inserts have high potential as
ocular drug delivery system.
The aim of the study was to develop a novel niosomal solid
topical ocular drug delivery system. A number of non-ionic

CONTACT Marwa Hassan Shukr marwa_25_2001@hotmail.com National Organization for Drug Control and Research, Cairo, Egypt

2016 Taylor & Francis

 2 M. H. SHUKR

amphiphiles have been used in niosomal formulations to improve
their properties such as stability and drug-loading capacity.
Mixed niosomes were developed using pluronics F127 (EO100
PO65EO100) and L64 (EO13PO30EO13) with span 40 and span 60.
Pluronics are widely used triblock copolymers consisting of hydro-
philic poly (ethylene oxide) (PEO) and hydrophobic poly (propylene
oxide) (PPO) groups. These polymers were chosen due to their
ability to interact with the body membranes and hydrophobic
surfaces, thus enhancing the drug transport across cellular barriers
(Tavano et al., 2013). The addition of cholesterol into the niosomal
formulation results in increasing hydrophobicity (Bernsdorff et al.,
1997) and stability (Gregoriadis and Davis, 1979) of the bilayer but
decreasing membrane permeability (Kirby et al., 1980) which
ultimately leads to improvement in encapsulating the lipophilic
drug into bilayers as vesicles assembled. Higher entrapment
efficiency was achieved at 1:1 cholesterol/surfactant molar ratio
compared with the higher cholesterol molar ratio or niosomal
formulations without any added cholesterol (Balakrishnan et al.,
2009). In this study, the ratio of surfactant and cholesterol was
adjusted to 1:1.
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dried thin lipid film was then hydrated with 10 ml of phosphate
buffered saline (PBS, pH 7.4), by rotating the flask in a water bath
using a rotavapour under normal pressure in order to ensure
complete hydration of the film. The niosomal suspension was left to
mature overnight at 4  C (Bayindir and Yuksel, 2010).
Determination of drug encapsulation efficiency (% EE)
The amount of drug entrapped into niosomal vesicles was
determined by ultracentrifugation, 1 mL of the niosomal suspension
was placed into centrifuge tubes and ultracentrifuged for 1 h at
15 000rpm at 4  C (Ultra centrifuge 5417R, Eppendorf, Hamburg,
Germany), in order to separate unentraped drug. The niosomes
were separated from the supernatant and 0.1 ml of the supernatant
was added to 10 ml methanol in a 10 ml volumetric flask and the
concentration of VCZ was determined spectrophotometrically at
256 nm. The % EE was calculated according to the following
equation (Aggarwal and Kaur, 2005):
% EE Ct  Cf =Ct   100

Materials and methods where Ct is the concentration of total VCZ and Cf is the concen-
Materials tration of free VCZ.

VCZ (99%) was kindly provided by Pfizer. Diazepam (as an internal Vesicle size, size distribution and zeta potential
standard) was kindly provided by Amoun Pharmaceutical Industries
Co., Cairo, Egypt. Span 40, span 60 and cholesterol (CH) were The niosomes size and polydispersity index (PI) were determined
purchased from Sigma Chemical Co. (St. Louis, MO). Pluronic L64, using Master sizer (Malvern Instruments, Malvern, Worcestershire,
pluronic F127 and sodium alginate (ALG) were purchased from UK). The diluted niosomal suspension (1 ml) was vortex mixed for
Sigma Aldrich (St. Louis, MO). Sodium carboxy methylcellulose (CMC 5 min then placed in the cell of the master sizer and the size and
Na) medium viscosity: Biochemica, Baden-Dattwil, Switzerland. polydispersity index (PI) were determined. The zeta potential was
Spectra Por semi-permeable membrane tubing (molecular measured with photon correlation spectroscopy (PCS) using
weight cut off of 12 00014 000) was purchased from Spectrum Malvern Zetasizer (Malvern, UK). Each sample was measured in
Medical Industries (Los Angeles, CA). Chloroform, sodium bicarbon- triplicate.
ate, calcium chloride, potassium chloride and sodium chloride were
from El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt. Acetonitrile Transmission electron microscopy (TEM)
and methanol (HPLC grade) were purchased from Sigma Aldrich
Chemical Co. (St. Louis, MO). All other chemicals were of analytical The morphological features of the prepared niosomes were examined
grade and used as received. via TEM. One drop of each drug-loaded niosomal formulation was
loaded onto a copper grid and the excess was removed with a filter
paper. The formulation was negatively stained with one drop of
Preparation of niosomes
phosphotungstic acid aqueous solution (2%, w/v) for 3 min.
Drug-loaded niosomes were prepared using film hydration tech- Examination of the grid was achieved via a transmission electron
nique. In a flask of a rotary evaporator (Buchi R-110 Rotavapor, microscope (TEM; Tecnai G20, FEI, Hilsboro, OR, Super twin, double tilt).
Flawil, Switzerland), the mixture of surfactants (pluronic F127,
pluronic L64, span 40 and span 60), drug and cholesterol were In vitro release
dissolved in chloroform at the molarities shown in Table 1. Then the
previous solution was completely dried in a rotary evaporator at a The in vitro drug release studies of the prepared niosomes were
rotating speed of 70 rpm and water bath maintained at 50  C. The performed, in triplicate, using a USP dissolution tester (Apparatus I,
Hanson SR6, Chatsworth, CA), at 34 0.5  C to simulate the ocular
Table 1. Composition of the prepared niosomes. surface temperature (Miyazaki et al., 2001). One millilitre of each
formula was placed in a glass cylindrical tube (2 cm internal
Formula Span Span Pluronic Pluronic
Code 60 40 L64 F127 Cholesterol VCZ diameter and 5 cm length) tightly covered with a pre-soaked
Spectra Por semi-permeable membrane tubing from one end. The
F1 mM 10 10 10
mg 44.17 39.57 35.7 loaded tubes were attached to the shafts of the USP Dissolution
F2 mM 5 5 10 10 tester apparatus from the other end (Abd-Elbary et al., 2008). The
mg 22.08 643.32 39.57 35.7 shafts rotated at a speed of 25 rpm in simulated tear fluid (pH 7.4,
F3 mM 5 5 10 10 100 ml). The simulated tear fluid (based on the electrolyte compos-
mg 22.08 147.83 39.57 35.7
ition of tear fluid) was prepared using 6.8 g of NaCl, 2.2 g of NaHCO3,
F4 mM 10 10 10
mg 41.14 39.57 35.7 0.084 g of CaCl22H2O and 1.4 g of KCl in 1 L of ultrapure water.
F5 mM 5 5 10 10 These amounts are equal to 142 mM of Na+, 19 mM of K+ and 0.6 mM
mg 20.57 643.32 39.57 35.7 of Ca2+ with an osmolality of 288 5 mmol/kg (Paulsson et al., 1999).
F6 mM 5 5 10 10 Periodically, 1 ml samples were withdrawn and replaced with fresh
mg 20.57 147.83 39.57 35.7
medium at predetermined time intervals of 0.25, 0.5, 1, 1.5, 2, 3, 4, 5,

6, 7 and finally at 8 h. The drug-released percentages were
determined spectrophotometrically at 256 nm.
Ocular insert preparation
Polymer solutions were prepared by dissolving specified amounts of
ALG and CMC Na in distilled water to produce final concentration of
2% w/v. The selected drug-loaded niosomeal formulation (drug
equivalent to 0.25%w/v) was added. The formed polymeric solutions
were sonicated to remove air bubbles. Aliquots of 0.4 ml were
poured into each of the pockets of a PVC blister with a diameter of
10 mm and frozen at 25  C for 24 h. The frozen inserts were placed
then in a lyophiliser for 24 h using a Novalyphe-NL 500 Freeze Dryer
(Savant Instruments, Holbrook, NY) with a condenser temperature
of  45  C and a pressure of 7  10  2 mbar. The lyophilised inserts
were stored in desiccators over calcium chloride (0% relative
humidity) at room temperature until further use (Bertram and
Bodmeier, 2012).
Characterisation of the inserts loaded with VCZ niosomes
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Water uptake and mass loss of the inserts
A sponge with dimensions of 5 cm  6.5 cm  3 cm was fully soaked
in the hydration medium STF and placed in a petri-dish filled with
the same medium to a height of 1 cm to keep the sponge soaked
during the experiment. Round filter paper (d 55 mm, Schleicher
Schuell GmbH, Dassel, Germany) was also soaked in medium and
positioned on the top of the sponge. The experimental set up was
equilibrated for 30 min. Accurately weighed inserts were placed on
the filter paper and the water uptake was determined as weight
increase of the insert (weight of hydrated insert and wet filter paper
minus weight of wet filter paper) over time normalised to the initial
dry insert weight (Bertram and Bodmeier, 2006).
Surface pH
The inserts were left to swell for 2 h on the surface of agar plate.
Agar solution was prepared by dissolving 2%w/v agar in STF by
heating under stirring, then poured into a petri dish for gel
formation at room temperature. Surface pH was measured by means
of a pH paper placed on the surface of the swollen inserts (Nafee
et al., 2003). The measurements were performed in triplicate.
Insert mucoadhesion properties
Mucoadhesion was performed by adapting the displacement
method of Bertram and Bodmeier (2006). Agar/mucin solution
(1%w/w, and 2% w/w, respectively) in STF was casted on a petri
plate and left to gel at 48  C in a refrigerator for 3 h. The inserts
were placed on the top of the agar/mucin gel, casted on a glass
plate in an incubator at 37  C. The inserts were moved downward
due to the gravity after the glass plate was turned into a vertical
position. The displacement in centimetre was measured after 2 h
(n 3). The adhesive potential was inversely related to the
displacement of the insert.
Scanning electron microscopy (SEM)
The shape and the surface morphology of selected ocular insert
were performed by SEM (JXA-840 A, Tokyo, Japan). Inserts were cut
with a razor blade to expose the inner structure, fixed on supports
JOURNAL OF MICROENCAPSULATION 3
and coated with goldpalladium under an argon atmosphere using
a gold sputter module in a high-vacuum evaporator and then the
surface was examined. The morphological features of the hydrated
inserts were examined via TEM.
In vitro drug release
The in vitro release of VCZ from different ocular inserts was carried
out in triplicate, using a USP dissolution tester (Apparatus I, Hanson
SR6, Chatsworth, CA), at 34 0.5  C to simulate the ocular surface
temperature (Miyazaki et al., 2001). One insert was placed in a glass
cylindrical tube (2 cm internal diameter and 5 cm length) tightly
covered with a pre-soaked Spectra Por semi-permeable mem-
brane tubing from one end. The procedure was continued as
previously mentioned.
Statistical analysis
A one-way analysis of variance (ANOVA) followed by Tukeys
pairwise test at 5% significance level was used to test for statistical
significance between the prepared formulations using GraphPad
Software Version 3.05 (GraphPad Software Inc., San Diego, CA).
In vivo evaluation of VCZ-loaded in situ gelling ocular inserts
The potential ocular irritancy and/or damaging effects of the
selected insert were evaluated by observing them for any redness,
inflammation or increased tear production, upon application to the
eyes of albino rabbits. The selected inserts were pre-sterilised prior
to application by irradiation with a short-wavelength UV beam
(254 nm) on both sides for 10 min using a hand-held UV lamp
(UVGL-58 UVP, Upland, Cambridge, UK). Insert was inserted into
the conjunctival sac in the right eye of three rabbits and the left
eye was left without any treatment as a control. Both eyes of the
rabbits under test were examined for any irritation signs, such as
conjunctival corneal oedema and/or hyperaemia on the basis of
direct visual observation using a slit lamp, before treatment, and 1,
24 and 48 h after insertion (Tamilvanana and Benitab, 2004; Colo
et al., 2001).
Estimation of VCZ in the rabbit aqueous humour
The in vivo study was carried out to compare the pharmacokinetics
of VCZ in the rabbit aqueous humour following ocular instillation of
a drug suspension and selected insert. The experimental design was
in agreement with the ethical principles of EU Directive 2010/63/EU
for the use of animals in scientific researches. Ten New Zealand
albino rabbits weighing 22.5 kg were used in this study and were
randomly divided into two groups. The lower conjunctival sac of the
right eye of each rabbit in the first group was treated with the drug
suspension (1%, w/v, 50 ml) in two instillations at 5 min intervals
using a micropipette without touching the eyes or irritating the
corneal surface. While those of the other group were treated with
the selected formulation containing the same amount of VCZ in the
in situ gelling insert. The rabbits were systemically anaesthetised
with intramuscular injections of ketamine hydrochloride (50 mg/kg)
in combination with a relaxing agent xylazine (10 mg/kg) (El-Laithy
et al., 2011). They were locally anaesthetised with benoxinate HCl
(0.4%, w/v, 2 drops) at 0.5, 1, 2, 4, 8 and 10 h post-instillation,
aqueous humour samples (0.15 ml) were withdrawn using an insulin
syringe (1.0 ml) fitted with a 29 G needle. The samples were
collected and stored at  20  C until analysed.
 4 M. H. SHUKR
Sample preparation
The thawed aqueous humour samples were spiked with Diazepam,
mixed (0.10 ml) with acetonitrile (0.20 ml) and vortexed for 30 s.
After centrifugation for 15 min at 2000  g, the organic phase in the
upper layer was collected. A portion of 20 ml was used for HPLC.
HPLC analysis of VCZ in rabbit aqueous humour
A previously reported HPLC method for the determination of VCZ in
human plasma was adopted for the determination of the drug
pharmacokinetics in the aqueous humour of the rabbits eyes
(Khoschsorur et al., 2005). A Hypersil C18 column (4.6 mm  250 mm,
5 mm, Thermo Scientific, Uppsala, Sweden) was used as the analytical
column maintained at 40  C. The mobile phase consisted of 50 mM
phosphate buffer, pH 6.0 (adjusted with 1 M KOH), acetonitrile, and
methanol (35:45:20) delivered at 1.7 mL/min. UV-detection was
performed at 255 nm and Diazepam was employed as the internal
standard (1 mg/ml), and the injected volume was 30 ml.
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Pharmacokinetic analysis
The non-compartment model is used to evaluate pharmacokinetic
parameters of VCZ using WinNonlin1 5.3 software (Pharsight
Corporation, Mountain View, CA). The maximum VCZ concentration
in the aqueous humour of the rabbits eyes (Cmax, mg/ml) and the
time to reach Cmax (Tmax, h) were calculated from the individual
aqueous humour concentrationtime curves. The trapezoidal rule
method was adopted to calculate the area under curve from zero to
10 h (AUC010, mg h/ml). The increase in the ocular bioavailability
(folds) was calculated from the AUC010 of the S2 insert and that of
the drug suspension.
Results
Optimisation of niosomal formulation
Vesicle size, polydispersibility index (PI) and zeta potential
The average vesicle size and zeta potential of niosomes are shown in
Table 2. Formulations F1 and F4 (prepared with span 60 and span 40
alone) showed the largest vesicle size (478 nm and 415 nm,
respectively) with polydispersity indexes of 0.36 and 0.67, with a
comparable zeta potential of 36.5 mV and 35.5 mV, respectively.
When a mixture of span 60 and pluronic L64 was used (F3), it was
observed that both the size of the vesicles and PI were decreased to
214 nm and 0.28, respectively. However, the zeta potential was
increased to 38.5 mV. F2 formulation (prepared with span 60 and
pluronic F 127) showed the smallest zeta potential (22.4 mV) and
largest PI (0.68) with an average size of 127.8 nm.
On the other hand, for the mixture of span 40 with pluronic F127
(F5) and pluronic L64 (F6), it was observed that the size of the
Table 2. Entrapment efficiency, mean vesicle size, polydispersibility index (PI) and
zeta potential of different niosomes (results are expressed as mean values SD,
n 3).
Formula Vesicle size
Code (nm) Zeta Potential EE%
F1 478.0 2.3  36.5 0.2 66.6 1.5 0.36 0.01
F2 127.8 1.8  22.4 0.13 58 1.7 0.68 0.012
F3 214 1.9 38.5 0.25 75.6 1.3 0.28 0.012
F4 415 3.2 35.5 0.23 61.5 1.7 0.67 0.023
F5 122.70 2.1 29.9 0.19 53 1.6 0.28 0.011
F6 157.40 1.83 35.7 0.23 49.7 1.8 0.28 0.01
vesicles decreased to 122.7 and 157.4 nm, respectively, with a
polydespersity index of 0.28. However, the zeta potential was
29.8 mV and 38.5 mV.
The results (Table 2) revealed that the mean vesicles size of
niosomes significantly decreased (p50.05) in the order of span
604span 404span 60 with pluronic L644span 60 with pluronic F
1274span 40 with pluronic L 644span 40 with pluronic F 127.
Entrapment efficiency (EE%)
Table 2 shows the results of the percent entrapment efficiency. The
entrapment efficiency followed the trend Sp 60 with pluronic
L644Sp 604Sp 404Sp 60 with pluronic F1274Sp 40 with pluronic
L644Sp 40 with pluronic F127. From the results tabulated, it is clear
that niosomes prepared with span 60 alone exhibited higher EE
(64.6%) than niosomes prepared with span 40 alone (56.5%). The
highest EE (75.6%) was exhibited with mixed niosomes prepared
with span 60 and pluronic L64.
TEM
Figure 1(a) and (b) shows that the niosomes were homogenous and
had a spherical shape with a smooth surface.
In vitro release
In vitro release profiles of VCZ from different formulations are
illustrated in Figure 2. F4 formulation prepared with span 40 alone
exhibited the highest percentage released (85%) after 480 min,
followed by F1 prepared with span 60 alone (80%), then mixed
niosomes prepared with span 40 and span 60 with pluronic F127
(77% and 75%, respectively), and finally mixed niosomes prepared
with span 40 and span 60 with pluronic L64 (74% and 63%,
respectively). From the previous results, F3 mixed niosomes
prepared with span 60 and pluronic L64 was selected for the
preparation of ocular inserts.
Characterisation of the inserts loaded with VCZ niosomes
Figure 3 shows the sponge-like structure of the ocular inserts.
Surface pH measurements were in the range of 7 0.2 (Table 3). TEM
was used to investigate the morphology of the niosomes within the
inserts after hydration. Figure 4(a) shows that no change was
observed in the shape of the niosomes. However, the vesicle size of
the niosomes (Figure 4b) was significantly increased (p50.05) from
niosomal suspension to niosomes within insert after hydration.
Figure 5 shows the results of water uptake studies, over the study
period (5 h), the two ocular inserts hydrated without dissolution and
CMC Na insert exhibited higher moisture uptake than AlG insert.
CMC Na and ALG inserts showed high water uptake.
The vertical displacement of inserts on an agar/mucin gel was
used as a measure of bioadhesion potential. ALG shows significantly
(p50.05) stronger adhesive forces (0.4 cm) compared with CMC
(0.7 cm). The release profiles of VCZ in STF pH 7.4 from the two in
PI situ gelling ocular insert are shown in Figure 6. The two formulation
exhibited prolonged drug release for at least 8 h.
In vivo evaluation of VCZ-loaded in situ gelling ocular inserts
It was observed that over the study period (48 h), the selected
formulation (S2) produced non-irritant effect on the rabbits eyes, it
 JOURNAL OF MICROENCAPSULATION 5

Figure 1. Transmission electron micrographs of F3 niosomal formulation.

Downloaded by [Gazi University] at 05:48 24 January 2016

Figure 2. In vitro drug release from niosomal formulation (a) prepared with span 60 and (b) with span 40 in simulated tear fluid (pH 7.4) at 34 0.5  C (mean SD, n 3).

Figure 3. Scanning electron micrographs of the ocular insert prepared from Na CMC (S1) and ALG (S2).

can be accomplished that the tested formulation was well tolerated Table 3. Concentration of the corresponding polymer solutions, pH and
mucoadhesion of the prepared ocular inserts.
showing no evidence of redness, inflammation, or increased tear
production effects to the ocular surface. Insert Polymer concentration pH mean SD Displacing (cm)
The drug pharmacokinetics in the aqueous humour follow- code (% w/w) (n 3) mean SD (n 3)
ing ocular insertion of S2 in situ gelling ocular insert and S1 2% Na CMC 6.8 0.31 0.7 0.1
the drug suspension was evaluated in rabbits (Figure 7). S2 2% Na alginate 6.9 0.25 0.4 0.1
 6 M. H. SHUKR
Figure 4. Transmission electron micrographs of S2 in situ gelling ocular insert after hydration.
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Figure 5. Water uptake profiles of the in situ gelling VCZ ocular inserts.
Figure 6. In vitro drug release from in situ gelling ocular inserts in simulated tear
fluid (pH 7.4) at 34 0.5  C (mean SD, n 3).
The differences between the two treatments were mathematically
evaluated by the calculation of Cmax, Tmax and AUC(010). The Cmax
and Tmax values representing the drug suspension and the in situ
gelling ocular insert were 1170.26 ng/ml after 2 h and 2203 ng/ml
after 1 h, respectively. The differences between the two treatments
were statistically significant (p50.01). These differences were
proved to be statistically significant, p50.01. The increase in the
ocular bioavailability of VCZ, judged from the AUC(010), was 5.91-
fold.
Figure 7. Aqueous humour concentrationtime profiles of VCZ following insertion
of S2 in situ gelling ocular insert and instillation of the drug suspension in rabbits
(mean SD, n 5, p50.01).
Discussion
Preparation of niosomes requires a deep understanding of the
characteristics of the principal ingredients and their effects on the
physicochemical properties and stability of the resultant niosomes.
A variety of non-ionic surfactants and their combinations have been
reported to have great potential to accommodate many drugs in
niosomes (Giddi et al., 2007).
The results are in good agreement with the literature (Kaur et al.,
2008), regarding stability of small niosomes with optimum zeta
potential560 mV. It also indicates the advantage of using pluronic
F127 and pluronic L 64/span 60 and span 40 mixture (1:1) in
producing smaller and physically stable niosomes. All the formula-
tions showed negative zeta potential values, which could reflect
adsorption of counter ions or preferential adsorption of hydroxyl
ions at the niosomal surface (Shahiwala and Misra, 2002). The
highest zeta potential was observed for the niosomes prepared with
span 60 and pluronic L 64, indicating that the mixed niosomes (F3)
were more stable against aggregation and fusion.
The hydrophilelipophile balance (HLB) values of non-ionic
surfactants can be a representative of the vesicle forming ability.
Surfactants of HLB values between 4 and 8 (e.g. span surfactants)
were reported to be compatible with vesicle formation (Uchegbu and
Vyas, 1998). Addition of applied cholesterol completes the lipophilic
moiety of non-ionic surfactants with high HLB values to form vesicles
(Masotti et al., 2010). For surfactants with higher HLB values, the
minimum amount of cholesterol necessary to form vesicles increases
in order to compensate the larger hydrophilic head group which
increases the lipophilic behaviour of the lipid bilayer (Kumarn and

Rajeshwarrao, 2011). This may explain the observed smaller particle
size for niosomes prepared with span 60 or span 40 and pluronic
F127 or pluronic L64 than those prepared with span 60 and span 40
alone. The HLB values were 6.7, 4.7, 22 and 8 for span 40, span 60,
pluronic F127 and pluronic L64, respectively.
The decrease in the vesicle size that was observed with mixed
niosomes prepared with span 40 and span 60 with pluronics (L64
and F 127) could be explained as the concentration of added
cholesterol was more efficient with niosomes prepared with span 60
and span 40 alone than with mixed niosomes and smaller vesicle
sizes were obtained. As the concentration of cholesterol increases,
the vesicle size also increases; this may be attributed to the fact that
cholesterol increases the width of vesicular bilayers and conse-
quently increases the vesicle size (Hosny, 2010).
EE is one of the important parameters in the design of vesicular
formulations. The higher EE exhibited with span 60 may be
explained due to the effect of HLB of the surfactant, the lower the
HLB the higher will be the drug EE and stability. Because higher HLB
values reflect a low hydrocarbon chain volume in comparison with a
hydrophilic surface area, they result in niosomes that are difficult to
form (Parthasarathi et al., 1994). Cholesterol is commonly used as an
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additive in niosomal systems, to influence the stability and
permeability of the membrane, which is mainly due to interaction
between surfactant and cholesterol, through hydrogen bonding
between its hydroxyl group and surfactants hydrocarbon chain
which increase the mechanical stiffness of the membranes and
membrane cohesion (Aripin et al., 2012).
The highest EE observed with F3 mixed niosomes (span 60 and
pluronic L64) may be due to hydrogen-bonding association
between the straight-chain structures of pluronic (L64) and span
60 (C16) during the formation of noisome vesicles. This interaction
seemed to occur at lower extent between span 60 with pluronic
F127 (it has a larger molecular weight of 11 500) and span 40 with
and pluronics.
TEM was used to investigate the morphology of the niosomes.
The images also confirmed that the niosomes were of nanometre
size.
The results of drug release were in agreement with the results of
EE%, F3-mixed niosomes prepared with span 60 and pluronic L64
exhibited the highest EE % and showed the lowest percent of drug
release. This can be explained as previously mentioned due to
hydrogen-bonding association between the straight-chain struc-
tures of pluronic L64 and span 60 (C16) during the formation of
noisome vesicles.
It was apparent that the incorporation of VCZ in mixed niosomes
led to significant slower release profiles (p50.05) compared with
niosomes prepared with span 60 and span 40 alone (F1 and F4,
respectively). The acceleration of the early portion of the release curve
is the result of VCZ desorption from the surface of niosomes while the
drug release in the slower phase was regulated by diffusion through
the swollen niosomal bilayers (Pardakhty et al., 2007).
F3 mixed niosomes were selected due to their highest encap-
sulation efficiency; optimum particle size with smallest variation in
size, which indicated by smallest PI and suitable prolonged release
pattern of the drug.
Ocular inserts are prepared by freeze-drying of aqueous solutions
containing polymeric carriers and drug. This production procedure
ensures the formation of highly porous polymeric sponges into
which VCZ niosomes are embedded.
The sponge-like structure of the ocular inserts is an important
parameter to ensure rapid hydration and gelation of the insert at the
ocular mucosa resulting in a larger surface/contact area and
increased water uptake by capillary forces and to a reduced foreign
body sensation when compared with other solid dosage forms.
JOURNAL OF MICROENCAPSULATION 7
The values of surface pH indicating that the prepared inserts is
compatible with the physiological pH of the surface of the eye, this
minimises any possible ocular surface irritation, tearing and reflex
blinking.
Increasing the vesicle size of niosomes might be attributed to a
fusion of vesicles. The swelling behaviour of in situ gelling insert
governs its bioadhesion and drug release pattern. The faster the
swelling of the polymer, the faster the initiation of diffusion and
formation of adhesive bonds resulting in faster initiation of
bioadhesion (Singh et al., 2010). The uptake of water by in situ
gelling inserts is a crucial step for the transformation into the gel
and for adhesion to the mucosa.
The high water uptake observed for CMC Na and ALG inserts
could be greatly attributed to the ionic nature of the polymers,
allowing high tendency towards water uptake.
CMC Na has an abundance of hydroxyl and ether groups along
their length, which are responsible for the higher water uptake
properties (Mansour et al., 2008). When CMC Na is exposed to an
aqueous medium, it undergoes rapid hydration and chain relaxation
to form a viscous gelatinous layer (gel layer).
For a polymer to show good adhesive characteristics to the
mucoploysaccharide components of the mucin produced by the
conjunctival goblet cells, the polymer must demonstrate one or
more of the following characteristics: hydrogen-binding capacity,
anionic charge, high molecular weight, sufficient chain flexibility and
spreading ability on mucus coat (Gilhotra et al., 2009).
Wetting has been reported to be essential for establishment of
intimate contact between the mucoadhesive and mucin/tissue to
develop strong adhesive bond (Wang and Bazos, 1983) and
prolonged ocular drug delivery. Both ALG and CMC have free
hydroxyl groups available for hydrogen binding with the conjunc-
tival mucin coat.
Furthermore, the stronger adhesive forces for ALG could be
ascribed to the conformational arrangements of D-mannuronic acid
(M blocks) and L-guluronic acid (G blocks) of ALG chains that are
favourable for interaction with the mucin coat, as well as the higher
density of carboxylate groups and higher molecular weight
(100 kDa) for ALG, compared with CMC (90 kDa) (Skjak-Brak, 1992).
The polymeric materials become adhesive with hydration while
excessive moisture uptake leads to reduced mucoadhesiveness
because water molecules bind to polymer groups required for
adhesion (Deasy and ONeill, 1989).
ALG (S2) absorbs a significant amount of STF to hydrate, swells
and forms a stable hydrogel upon exposure to the divalent cations
Ca + 2 present in STF. The drug which is embedded in the ALG insert
is now immobilised in the polymer, therefore, the in situ gel-forming
insert acts as a reservoir for the drug (Mishra and Gilhotra, 2008).
Drug release from CMC Na ocular insert (S1) was relatively slower
than from ALG ocular insert (S2) (Figure 6).
This may be explained by referring to the water uptake data,
where CMC Na exhibited higher water uptake (Figure 5). Although
the marked increase in surface area during swelling can promote
drug release, the increase in diffusional path length of the drug may
paradoxically delay such release. Further, the thick gel layer formed
on the swollen insert surface is capable of preventing matrix
disintegration and controlling additional water penetration
(Rodriguez et al., 2000).
According to the above results in situ ocular insert S2 was chosen
depending on their superior mucoadhesive properties, appropriate
water uptake and suitable prolonged release profile and hence was
subjected to further in vivo evaluation.
The aqueous humour concentrationtime profiles of VCZ clearly
indicate the ability of the in situ gel to control the drug release rate
upon insertion. The delay in Tmax (from 1 to 2 h) for the drug
 8 M. H. SHUKR
suspension and the in situ gelling inserts, respectively, could
indicate the controlled release characteristics of the latter. The
higher ocular bioavailability of the in situ gelling ocular insert could
be related to its ability to prolong the residence time on the corneal
epithelial layer. This would enable better transcorneal drug pene-
tration to the ocular internal tissues.
Conclusion
A novel in situ gelling ocular inserts loaded with VCZ niosomes was
developed and characterised in this study. Niosomes made up of
mixed surfactants (prepared with span 60 and pluronic L64: choles-
terol at a molar ratio of 1:1) proved to be effective carriers and
exhibited high EE and relatively small vesicle size and prolonged drug
release. The niosomal suspension was further developed to form in
situ gelling ocular insert by the freeze-drying method. The morph-
ology, surface pH, mucoadhesion and release study were evaluated
for the VCZ-loaded niosomal ocular insert. ALG in situ gelling insert
(S2) showed prolonged drug release with superior mucoadhesion and
appropriate water uptake, and it was selected for further in vivo
Downloaded by [Gazi University] at 05:48 24 January 2016
evaluation. The sterilised S2 in situ gelling ocular insert was non-
irritant to rabbits eyes, succeeded in controlling the rate of VCZ
release in the rabbit aqueous humour and improved the ocular drug
bioavailability, relative to the drug suspension.
The VCZ in situ gelling niosomal inserts could prove to be a good
alternative to conventional niosomes formulations, combine the
advantages of solid, single unit dosage forms, such as high stability
and dosing accuracy, with those of gel preparations, which avoid
foreign body sensation and allow prolonged drug delivery.
Declaration of interest
The author reports no conflicts of interest. The author alone is
responsible for the content and writing of the article.
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