Drug Delivery

ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: https://www.tandfonline.com/loi/idrd20

Ocular ketoconazole-loaded proniosomal gels:

formulation, ex vivo corneal permeation and in vivo
studies

Ghada A. Abdelbary, Maha M. Amin & Mohamed Y. Zakaria

To cite this article: Ghada A. Abdelbary, Maha M. Amin & Mohamed Y. Zakaria (2017) Ocular
ketoconazole-loaded proniosomal gels: formulation, ex�vivo corneal permeation and in�vivo studies,
Drug Delivery, 24:1, 309-319, DOI: 10.1080/10717544.2016.1247928

To link to this article: https://doi.org/10.1080/10717544.2016.1247928

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ISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, 2017; 24(1): 309–319

! 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
DOI: 10.1080/10717544.2016.1247928

ORIGINAL ARTICLE

Ocular ketoconazole-loaded proniosomal gels: formulation, ex vivo

corneal permeation and in vivo studies
Ghada A. Abdelbary1, Maha M. Amin1, and Mohamed Y. Zakaria2
1
Department Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt and 2Department Pharmaceutics and
Industrial Pharmacy, Faculty of Pharmacy, Sinai University, Sinai, Egypt

Abstract Keywords
Context: Vesicular drug carriers for ocular delivery have gained a real potential. Proniosomal Proniosomal gel, ketoconazole, ocular
gels as ocular drug carriers have been proven to be an effective way to improve bioavailability delivery, ex vivo corneal permeation,
and patient compliance. ocular keratitis
Objective: Formulation and in vitro/ex vivo/in vivo characterization of ketoconazole (KET)-loaded
proniosomal gels for the treatment of ocular keratitis. History
Materials and methods: The effect of formulation variables; HLB value, type and concentration of
non-ionic surfactants (Tweens, Spans, Brijs and Pluronics) with or without lecithin on the Received 26 August 2016
entrapment efficiency (EE%), vesicle size and in vitro KET release was evaluated. An ex vivo Revised 10 October 2016
corneal permeation study to determine the level of KET in the external eye tissue of albino Accepted 10 October 2016
rabbits and an in vivo assessment of the level of KET in the aqueous humors were performed.
Results and discussion: In vivo evaluation showed an increase in bioavailability up to 20-folds
from the optimum KET proniosomal gel formula in the aqueous humor compared to drug
suspension (KET-SP). The selected formulae were composed of spans being hydrophobic
suggesting the potential use of a more hydrophobic surfactant as Span during the formulation
of formulae. Factors that stabilize the vesicle membrane and increase the entrapment efficiency
of KET (namely low HLB, long alkyl chain, high phase transition temperature) slowed down the
release profile.
Conclusions: Proniosomal gels as drug delivery carriers were proven to be a promising approach
to increase corneal contact and permeation as well as retention time in the eye resulting in a
sustained action and enhanced bioavailability.

Introduction surface both are resulted from the frequent use of concentrated
solutions (Topalkara et al., 2000; Kaur et al., 2004).
The use of vesicular systems is considered as an alternative
Absorption of drugs into the eye requires good corneal
way to overcome many problems associated with ocular drug
penetration and a prolonged contact time with the corneal
delivery and to enhance the topical controlled delivery of
tissue. Various attempts were investigated for prolonging the
ophthalmic drugs with respect to traditional eye drops. The
contact time between the drug and corneal–conjunctival
limited extent of ocular absorption caused by physiological
epithelium in order to increase its bioavailability. In spite of
constraints, such as induced lacrimation, normal tear turnover
increasing the corneal–conjunctival contact time by adopting
and rapid precorneal clearance, leading to a significant drug
various delivery systems, certain disadvantages were noted,
loss is still remaining the main challenge facing ocular drug
including poor patient compliance, side effects such as
delivery system (Lee & Robinson, 1986). That is also the
blurring of vision, sticky sensation and induced reflex
reason of the limited bioavailability of ophthalmic solutions,
blinking due to irritating properties (Kaur et al., 2004).
where the therapeutic effect is achieved by daily frequent
In vesicular drug delivery systems, the drug is encapsu-
instillation of the solution. Nevertheless, the systemic absorp-
lated in lipid vesicles, which can cross cell membrane.
tion of the drug drained through the nasolacrimal duct systems
Vesicles, therefore, as drug carriers can enhance both the
can cause side effects and the drastic damage in the ocular
bioavailability and the disposition of the drug. Vesicular
systems provide prolonged duration of action at the corneal
Address for correspondence: Ghada A. Abdelbary, 8 Avenue Bertie surface by preventing ocular metabolism by enzymes in the
Albrecht, 75008 Paris, France. Tel: (+33) 6 76 91 71 44. Fax: (+33) 1 40 lacrymal fluid (Allam et al., 2011). Vesicular drug delivery
70 08 31. Email: gabdelbary@gmail.com systems used in ophthalmic preparations broadly include
This is an Open Access article distributed under the terms of the Creative liposomes and niosomes. Niosomes have gained popularity in
Commons Attribution License (http://creativecommons.org/Licenses/by/
4.0/), which permits unrestricted use, distribution, and reproduction in ocular drug delivery research and area potential delivery
any medium, provided the original work is properly cited. system for the effective treatment of glaucoma (Paul et al.,
310 G. A. Abdelbary et al. Drug Deliv, 2017; 24(1): 309–319

2010) and various other conditions. Allam et al. (2011) emulsion solvent evaporation technique aiming ocular appli-
reported that acyclovir-loaded niosomes were effective for the cation (Demirel & Genc, 2015). Solid lipid nanoparticles
treatment of herpes simplex keratitis, a condition that can lead (SLN) ocular dispersion of KET comprising Compritol 888
to blindness. Similarly, gentamicin-loaded niosomes provided ATO and PEG 600 matrix were prepared using hot high-
controlled, opthalmic delivery (Abdelbary & El-Gendy, 2008) pressure homogenization (Kakkar et al., 2015).
and brimonidine-loaded niosomes were therapeutically effect- In vivo behavior of proniosomes-derived niosomes showing
ive with a long duration of action due to slow and prolonged advantages as drug carriers, comprising lower cost and toxicity,
zero-order release of drug (Prabhu et al., 2010). easy storage and handling as well as increased stability.
Proniosomal gels are liquid crystalline (gel) vesicular Encapsulation of drug in niosomal formulations reduces the
structures produced from nonionic surfactants having the toxicity in various therapies and applications and also prolongs
ability to entrap both hydrophilic and lipophilic drugs, they the encapsulated drug circulation time and changes drug
can promote adherence to the corneal/conjunctival surface distribution in the body. Niosomes as drug delivery vesicles
when used as ophthalmic preparations. They can be trans- increases absorption of some drugs through cell membranes
formed easily into niosomes immediately upon hydration and cellular uptake via endocytosis and so confines the drug in
(Gupta et al., 2007). Recently, various studies proved the tissues and targeted organs (Yasam et al., 2014).
reliability of proniosomal gels in promoting the ocular Based on the aforementioned, the aim of this work is to
bioavailability of different drugs. Ocular proniosomal gels improve the ocular bioavailability of KET through the prepar-
of lomefloxacin HCl were prepared using different types of ation of ocular KET mucoadhesive proniosomal gels using
nonionic surfactants solely and as mixtures with Span 60 in different types of nonionic surfactants (Spans, Tweens, Brijs
order to improve its ocular bioavailability for the management and Pluronics) with or without lecithin. Furthermore, the
of bacterial conjunctivitis (Khalil et al., 2016). Tacrolimus- prepared proniosomal gel formulae were characterized regard-
loaded proniosomes containing poloxamer 188 and lecithin as ing encapsulation efficiency percent (E.E%), vesicle size
surfactants, cholesterol as a stabilizer and minimal amount of analysis and in vitro drug release. An ex vivo corneal
ethanol were prepared and characterized regarding the permeation study of the selected gel formulae was performed
occurrence of corneal allograft rejection and the median to determine the level of KET in the external eye tissue of albino
survival time of corneal allografts (Li et al., 2014). rabbits after topical application. Finally, an in vivo character-
Many advantages of proniosomes are presented over ization of the optimum proniosomal gel formula was performed
niosomes, namely physical stability (avoiding aggregation, through evaluating the level of KET in the aqueous humors of
fusion, leakage), shielding for the entrapped drug from thirty-six albino rabbits using a validated HPLC method.
hydrolysis (Hu & Rhodes, 2000). The ease of preparation of
proniosomes by simply dissolving the surfactant in the least Materials and methods
amount of organic solvent (Vora et al., 1998) can solve the Materials
problems arising from other tedious preparation methods of
niosomes such as reversed-phase evaporation and ether or Ketoconazole (KET) powder was kindly supplied by El Nile
ethanol injection methods (Weiner, 1994). pharmaceutical company (Cairo, Egypt). Span 20, Span 60,
Ketoconazole (KET) is abroad spectrum antifungal, highly Span 65, Span80, Tween 80, Brij 35, Brij 72, Brij 92, Pluronic
lipophilic molecule (log p ¼ 4), belonging to imidazoles, its F68, Pluronic L121, L-a-phosphatidylcholine (PC) from egg
absorption is highly dependent on gastric pH and its oral yolk, Cholesterol (CH) powder were purchased from Sigma
administration causes many side effects (nausea, vomiting, Chemicals Co. (St. Louis, MO). Methanol, sodium chloride,
gastrointestinal disturbance, hepatitis, gynecomastia and potassium chloride, sodium bicarbonate, calcium chloride,
adrenal cortex suppression) (O’Brien, 1999). The mode of sodium hydroxide, magnesium chloride, potassium dihydro-
action of KET is to inhibit synthesis of ergosterol and to gen orthophosphate and absolute ethanol were purchased
increase fungal cellular permeability. Ketoconazole (KET) from El-Nasr Chemical Co. (Cairo, Egypt). Spectra/PoreÕ
has been used for various types of ocular fungal species such dialysis membrane (12 000–14 000 molecular weight cutoff)
as Aspergillus species, Candida species and some Fusarium was purchased from Spectrum Laboratories Inc (Los Angeles,
species (Zhang et al., 2008). CA).
The topical use of KET is characterized by a short
Preparation of KET proniosomal gels
residence and ocular half-life (Zhang et al., 2008). In spite of
KET lipophilicity, its high molecular weight (531.44 Da) The composition of the different prepared KET proniosomal
offers an obstacle against its transport across the biological gels is shown in Table 1. Proniosomal gels were prepared by
membrane, so passage through corneal stroma is hampered. the coacervation-phase method previously reported by
Therefore, a suitable carrier system for KET is required (Mishra et al., 2012) with some modifications. In this
(Barar et al., 2008). Furthermore, it is desired to attain a high method, the accurately weighted amount of drug, surfactant
concentration of KET crossing cornea reaching the posterior cholesterol/or lecithin (as permeation enhancer) were trans-
segment of the eye for an effective treatment of ocular fungal ferred to tightly closed glass vials to which absolute ethanol
infections; fungal keratitis, candidal chorioretinitis that are (0.4 mL) was added. The vials were transferred to water bath
caused by Candida albicans (Ahuja et al., 2008). at (55–60  C) for 5 min with continuous shaking till complete
Some trials were performed aiming to incorporate the dissolution of cholesterol. To each of the formed transparent
fungistatic molecule KET into polymeric (EudragitÕ RS 100) solutions, about 0.15 mL of hot distilled water (55–60  C) was
and solid lipid (GelucireÕ 44/14) nanoparticles by quasi- added while keeping in water bath for 3–5 min till a clear or
DOI: 10.1080/10717544.2016.1247928 Ex vivo and in vivo studies of ocular ketoconazole-loaded proniosomal gels 311
Table 1. Composition of the different prepared ketoconazole-loaded proniosomal gels.

Formula Span20 Span60 Span65 Span80 Tween80 Brij35 Brij72 Brij92 Pluronic (F68) Pluronic (L121) Lecithin
F1 500 mg
F2 250 mg 250 mg
F3 500 mg
F4 250 mg 250 mg
F5 500 mg
F6 250 mg 250 mg
F7 500 mg
F8 250 mg 250 mg
F9 500 mg
F10 250 mg 250 mg
F11 500 mg
F12 250 mg 250 mg
F13 500 mg
F14 250 mg 250 mg
F15 500 mg
F16 250 mg 250 mg
F17 500 mg
F18 250 mg 250 mg
F19 500 mg
F20 250mg 250 mg

translucent solution was produced. The mixtures were Vesicle size measurement
allowed to cool down to room temperature.
The vesicle size of the prepared KET niosomes after
hydration was determined by light scattering based on
Hydration step and formation of niosomes LASER diffraction using the Malvern Mastersizer X
Niosomes were prepared by hydration of the previously LASER scattering instrument (detection limit 0.1–2000 mm)
prepared proniosomal gels as described by Mokhtar et al., (Malvern Instruments Ltd., Worcestershire, UK) (Agarwal
(2008). About 7 mL of Sorensen’s phosphate buffer (pH 7.4) et al., 2001).
was added to a certain weight (100 mg) of the gel from each
vial followed by heating at a temperature of 40–50  C for In vitro release of KET from the prepared proniosomal gels
approximately one minute with the aid of Vortex (Maxi mix,
M 36710 mixer, Barnstead International, Dubuque, IA). The The in vitro release of KET from the prepared proniosomal
final volume was adjusted to 10 mL using Sorensen’s gels was determined using the membrane diffusion tech-
phosphate buffer (pH 7.4). nique; (Junyaprasert & Manwiwattanakul, 2008). A certain
weight (200 mg) of the prepared proniosomal gel equivalent
In vitro characterization of the prepared KET to 2 mg KET was transferred to a glass cylinder having the
proniosomal gels length of 10 cm and diameter of 2.5 cm fitted at its lower
end with presoaked cellulose membrane on which the gel
Entrapment efficiency (EE%) was spread over (Spectra/Pore dialysis membrane 12 000–
The entrapment efficiency of KET from the prepared 14 000 Mwt cutoff). The glass cylinder was attached to the
niosomes was calculated following ultra-centrifugating of shaft of the dissolution apparatus and then suspended in the
1 mL of the niosomal suspension in Sorensen’s phosphate dissolution flask of a USP dissolution apparatus (VK 7000
buffer (pH 7.4) at 15 000 rpm for one hour using cooling Dissolution Testing Station, Vankel Industries, Inc., NJ)
centrifuge at 4  C (Beckman, Fullerton, Canada). The containing 100 mL simulated lacrimal fluid (SLF, pH 7.4)
niosomes were separated from the supernatant, washed kept at a temperature of 37 ± 0.5  C (Abdelbary et al.,
twice, with 1 mL Sorensen’s phosphate buffer each time and 2008). The glass cylinder was allowed to rotate at a
recentrifuged again for 30 min. The amount of entrapped KET constant speed (25 rpm). For comparison, the in vitro
was determined after lysis of the separated vesicles by release of an equivalent amount of KET suspension (KET-
sonication (Model 275 T (Crest Ultrasonics Corp., Trenton, SP) was carried out adopting the same procedure as
NJ) with methanol; (Maestrelli et al., 2005). The concentra- previously described.
tion of the entrapped drug was determined spectrophotomet- At predetermined time intervals (1, 2, 3, 4, 6, 8, 12 and
rically (Shimadzu, model UV-1601 PC, Kyoto, Japan) at max 24 h), aliquots were withdrawn and the drug content was
295 nm against methanol as blank. determined spectrophotometrically at 295 nm, the mean
The percent entrapment efficiency was calculated as values of three runs (± SD) were calculated.
follows: Based on the above tests, selection of KET proniosomal
gel formulae was based on the highest E.E%, with the lowest
amount of KET entrapped in vitro release at Q8hrs. The selected formulae were further
KET %EE ¼  100 ð1Þ
total amount of KET investigated through ex-vivo corneal permeation.
312 G. A. Abdelbary et al. Drug Deliv, 2017; 24(1): 309–319

Ex vivo corneal permeation of the selected KET standard cages, in a light-controlled room at 20 ± 1  C and
proniosomal gels 50 ± 5% relative humidity, with no restriction of food or
water. During the experiments, the rabbits were placed in
Ex vivo corneal permeability study of the selected KET
restraining boxes, where they could move their eyes and heads
proniosomal gels had been approved by Cairo University
freely. All experiments were carried out under veterinary
Research Ethics Committee. Permeability studies were per-
supervision, used in full compliance with local, national,
formed using Franz diffusion cell consisting of two-limbed
ethical and regulatory principles for animal care. Each group
reservoir (Aggarwal et al., 2004) having a donor compartment
was divided into six subgroups corresponding to withdrawal
of about 15 mm orifice diameter with effective diffusion area
time intervals (three rabbits in each subgroup). The rabbits
of 0.78 cm2 and a receptor volume (7 mL). The isolated
were kept under anesthesia throughout the experiment using
cornea together with each of the selected KET proniosoma gel
sodium pentobarbital (30 mg/kg) injected into the marginal
formulae was mounted on one limb, and the other limb was
ear vein.
used as the sampling port. The permeation study was
KET-proniosomal gel was applied to one group of animals
maintained at a constant temperature (37 ± 0.2  C) using a
whereas KET-SP was instilled into the eyes of the second
magnetic stirrer (Wisestir magnetic stirrer, China).
group. A dose of (50 mL) of KET-proniosomal gel or KET-SP
Rabbit corneas used in this study were separated from
was instilled into the lower cul-de-sac of the eye of each
male New Zealand albino rabbits. Rabbits were killed by
rabbit. Aqueous humor was withdrawn with a 26-G needle
injection of an overdose of sodium phenobarbital and the
attached to a tuberculin syringe at 0.5, 1, 2, 4, 6 and 8 h.
corneas were excised from the globes. The cornea used in the
Samples were stored until they were extracted. The extraction
experiment was immersed in simulated lacrimal fluid (pH
was performed in accordance to (Zhang et al., 2008), First,
7.4) for 30 min before the conduction of the experiment in
100 mL of aqueous humor was transferred into a glass test tube
order to simulate the same physiological conditions of the
and 100 mL of phosphate buffer saline (PBS) was added. The
eye. Extreme care was taken not to produce any wrinkles or
mixture was vortexed and 2 mL of dichloromethane was
folding of the membrane before mounting the cornea onto the
added. The mixture was vortexed for 2 min and then
ring of the diffusion apparatus. A dose of 50 mL of KET-
centrifuged at 3000 rpm for 5 min.
proniosomal gel from the selected proniosomal gel formulae
The upper layers were aspirated and discarded, and the
(F2, F3, F4, F5 and F6) was placed on the isolated cornea in
organic layer was transferred to the other cone glass test tube.
5 mL freshly prepared simulated lacrimal fluid (pH 7.4).
The organic layer was evaporated to dryness. The residue was
Aliquots of the medium were withdrawn from the sampling
reconstituted in a 100 mL of mixture of methanol and
port after specified time intervals; 1, 2, 4, 6, 8 and 10 h and
deionized water (50:50 v/v). The mixture was vortexed for
were replaced with equal volume of fresh medium to maintain
1 min and centrifuged at 4000 rpm for 15 min. Then, 20 mL
a constant volume.
aliquot of each supernatant was directly injected into HPLC to
Samples were analyzed using a validated HPLC method
be analyzed. No internal standard was required as the peaks
(Zhang et al., 2008). The mobile phase consisted of a mixture
were separated from that of aqueous humor and no noise or
of potassium dihydrogen phosphate (0.02 mol/L) aqueous
overlapping occurred.
solution and methanol in the ratio of (25:75 v/v) (pH was
adjusted to 3.0 using phosphoric acid) with a flow rate of
Occular irritancy test
1.5 mL/min. The determination was performed at 235 nm
using HPLC instrument (Hitachi LaChrome Elite, Tokyo, An ocular irritancy testing was also performed in order to
Japan). verify the safety of the optimum proniosomal gel. The
HPLC instrument was equipped with a model series potential ocular irritancy and/or damaging effects of the tested
L-2000 organizer box, L-2300 column oven, L-2130 pump proniosomal gel formula (F2) was evaluated by observing any
with built in degasser, Rheodyne 7725i injector with a 20 mL redness, inflammation or increased tear production, upon
loop and a L-2455 photodiode array detector (DAD), application to the eyes of albino rabbits. The formulation was
separation and quantitation were made on a 250  4.6 mm tested on three albino rabbits. The experiment was performed
(i.d.), 5 mm ODS column (Inertsil, Tokyo, Japan). The HPLC by a single instillation (50 mL) of the proniosomal preparation
was operated by EZ chrom Elite version 3.3.2 SP1 by Agilent. under test into the conjunctival sac of one eye, while the
contralateral eye served as control. Both eyes of the rabbits
In vivo study of the optimum proniosomal gel formula under test were examined for any sign of irritation, such as
conjunctival corneal edema and/or hyperhemia upon direct
Determination of KET level in aqueous humors of rabbits
visual observation using a slit lamp, before treatment and 1, 8
The in vivo characterization of the optimum proniosomal gel and 24 h following drug instillation (Colo et al., 2001).
formula (F2) was performed through evaluating the level of
KET in the aqueous humors of 36 albino rabbits. This was Results and discussion
accomplished by comparing the level of KET from the
optimum proniosomal gel formula with 1% KET ophthalmic Preparation of proniosomal gels
suspension (KET-SP), respectively, after topical application. Proniosomal gel formulae of KET were prepared efficiently
Thirty-six healthy New Zealand albino male rabbits, adopting coacervation-phase method using different types of
weighing about 2.0–3.0 kg, were divided randomly into two nonionic surfactants as; Spans (sorbitan fatty acid esters),
groups (18 rabbits in each group). The animals were housed in Tweens (polyoxyethylene sorbitan esters), Brijs
DOI: 10.1080/10717544.2016.1247928 Ex vivo and in vivo studies of ocular ketoconazole-loaded proniosomal gels 313

(polyoxyethylene alkyl ethers) and Pluronics (polyoxyethy- Table 2. Physical evaluation of the prepared ketoconazole-loaded
proniosomal gels.
lene-polyoxypropylene block copolymers) with or without
lecithin in a ratio of S.A.A:lecithin (1:1 w/w) together with a %Drug Z-average
constant amount (50 mg) of cholesterol per each formulation Formula entrapped ± S.D (d.nm)±S.D PDI ± S.D
(Table 1).
F1 57.90 ± 2.90 1322 ± 82 0.09 ± 0.00
Nonionic surfactants are the most common type of F2 87.10 ± 1.19 590.80 ± 34 0.14 ± 0.06
surface-active agents used in preparing vesicles due to F3 85.20 ± 1.67 855 ± 57 0.49 ± 0.13
their superior benefits with respect to stability, compatibility F4 86.60 ± 1.80 535.20 ± 28 0.15 ± 0.08
F5 83.80 ± 2.30 339 ± 19 0.61 ± 0.18
and toxicity. They are generally less toxic, less hemolytic
F6 93.00 ± 1.10 559.10 ± 22 0.60 ± 0.20
and less irritating to cellular surfaces and tend to maintain F7 47.00 ± 1.25 2695 ± 98 0.61 ± 0.10
near physiological pH in solution (Kumar & Rajeshwarrao, F8 79.50 ± 3.10 413.60 ± 24 0.11 ± 0.03
2011). Cholesterol must be added to the surfactant in order F9 37.50 ± 1.15 97.56 ± 12 0.37 ± 0.11
F10 49.50 ± 2.34 140.60 ± 10 0.21 ± 0.09
to form a bilayered vesicle, also cholesterol enhances the F11 40.60 ± 1.55 4432 ± 105 1 ± 0.42
stability of the prepared vesicles. The addition of cholesterol F12 49.90 ± 1.25 452.70 ± 63 0.49 ± 0.11
enables more hydrophobic surfactants to form vesicles, F13 60.60 ± 1.21 2271 ± 79 1 ± 0.37
suppresses the tendency of the surfactant to form aggregates F14 74.90 ± 0.65 550 ± 72 1 ± 0.50
F15 62.90 ± 2.25 1096 ± 89 1 ± 0.39
and provides greater stability to the lipid bilayer by F16 72.70 ± 2.96 1140 ± 99 0.34 ± 0.13
promoting the gel liquid transition temperature of the F17 41.60 ± 1.31 1961 ± 88 0.87 ± 0.29
vesicle (Lawrence et al., 1996). F18 51.70 ± 2.96 805 ± 63 0.36 ± 0.09
Lecithin is generally named depending on its source of F19 51.40 ± 3.85 663.40 ± 92 0.77 ± 0.34
F20 70.70 ± 2.70 510.30 ± 49 0.62 ± 0.23
origin such as soya lecithin from soya beans and egg lecithin
from egg yolk. Phosphatidyl choline is such a major
component of lecithin. In the vesicular system, it plays a Increasing the alkyl chain number leads to an increase in
number of important roles: (a) it acts as permeation enhancer; entrapment efficiency. It is known that Sp 65 has three alkyl
(b) enhances the percent drug entrapment due to high Tc chains (stearate alkyl chain) with an HLB value of 2.1.
(phase transition temperature); (c) leads to vesicles of smaller Consequently, it may play a role in decreasing the perme-
size due to increase in hydrophobicity which results in the ability of the membrane and increases the encapsulation
reduction of vesicle size; (d) prevents the leakage of drug efficiency. Similar results were obtained by (Hao et al., 2002)
(Rawat et al., 2011). who reported that the lower the HLB of the used surfactant,
Finally, the addition of water leads to swelling of bilayer the higher the entrapment efficiency of colchicine within the
which is due to the interaction between water and the polar prepared niosomes.
groups of the surfactants leading to the formation of The HLB values of Sp 60, Sp 65 and Sp 80 are equal to
multivesicular, multilamellar and spherical shaped structures 4.7, 2.1 and 4.3, respectively, compared to 8.6 in case of Sp
(Rawat et al., 2011). 20. The fact that lower HLB spans exhibit the highest E.E%
was attributed to many reasons such as; being solid at room
Entrapment efficiency (EE%) temperature with higher phase transition temperature (Tc), the
higher (Tc) of surfactants, they are more involved in a more
Effect of surfactant HLB
rigid bilayers highly ordered gel formation, leading to a higher
Table 2 shows the entrapment efficiency (EE%± SD) of the entrapment efficiency.
different prepared KET proniosomal gel formulae. It is clear The gel transition temperature of spans increases as the
that results ranged from 37.50 ± 1.15 to 93.00 ± 1.10%. length of the alkyl chain increases. Thus, sorbitan mono-
Regarding formulae prepared using different grades of laurate (Sp20) (C12) is liquid at room temperature
Spans (F1-F8), The significantly highest (p50.05) EE% (Tc ¼ 16  C); sorbitan monostearate (Sp60) (C18) has a gel
(93.00 ± 1.10%) was obtained from formula F6 prepared using transition temperature of 54  C and about 53  C for sorbitan
Sp 65 (HLB ¼ 2.1) and lecithin in a ratio of (Sp 65: lecithin tristearate (Sp65) (C54) chain (Bouwstra et al., 1997). In
1:1 w/w). It is clear that the EE% from formulae prepared addition, the lowest transition temperature of Sp 80 (C18)
using different grades of Spans followed the order of: Sp (Tc ¼ 12  C) among all tested Spans was the main reason of its
654Sp604Sp204Sp 80. lowest E.E% among other spans (Kibbe, 2000). As possessing
It is known that the head groups are similar in all Spans, the highest phase transition temperature (Tc) in Spans
while the alkyl hydrocarbon chains are different. In spite of provides the highest entrapment for the drug and vice versa
the same head groups and same carbon atoms (C18) in the as lower (Tc) surfactants are more prone to form less packed
alkyl chain of Sp 60 (HLB ¼ 4.7) and Sp 80 (HLB ¼ 4.3) ordered liquid form (Hao et al., 2002).
having almost the same HLB value, they differ in the structure In the current study, the E.E% of formulae prepared using
of the alkyl chain. The presence of double bonds in the alkyl T80 surfactant (F9–F10) showing 37.50 ± 1.15 and
chains of Sp 80 leads to a markable increment in the 49.50 ± 2.34 of KET entrapped for F9 and F10 which were
permeability of bilayer of niosomes, thus possibly justifying significantly less (p50.05) than their corresponding ones
the lower entrapment efficiency of Sp 80 formulations; (F7–F8) having 47.00 ± 1.25 and 79.50 ± 3.10, respectively,
47.00 ± 1.25 and 79.50 ± 3.10 for F7 and F8, respectively using Span surfactant having the same alkyl chain length (Sp
(Table 2). Surfactants of longer saturated alkyl chains showed 80). T80 being hydrophilic surfactant with high HLB value of
higher entrapment efficiency (Guinedi et al., 2005). 15 compared to 4.3 in case of the hydrophobic Sp 80, this
314 G. A. Abdelbary et al. Drug Deliv, 2017; 24(1): 309–319

probably elucidates the lower entrapment efficiency of T80 concentration because they have relatively large hydrophobic
compared to Sp 80 formulations as previously discussed (Hao moieties with low water solubility (Manosroi et al., 2003). On
et al., 2002). the other hand, other Span grades, Brij 35, Tween 80 and
Brij surfactants are polyoxyethylene alkyl ethers that differ Pluronic F68 were not able to form niosomes in the presence
in the number of hydrophilic oxyethylene groups and length of small amounts of cholesterol, this might be attributed to
of hydrophobic alkyl chain. Regarding the E.E% of their high HLB values, solubilizing property and therefore
proniosomal gel formulae prepared using different grades of micelle formation ability that dissolves the small amounts of
Brijs (F11-F16); Brij 35 (polyoxyethylene (23) lauryl ether), cholesterol (Pardakhaty et al., 2007), this might also explain
Brij 72 (polyoxyethylene (2) stearyl ether) and Brij 92 the low entrapment ability of the proniosomal formulae
(polyoxyethlene (2) oleyl ether) having the corresponding prepared by these surfactants.
HLB values of 16.9, 4.9 and 5, respectively. According to the
results shown in Table 2, both long-chain surfactants (C18),
Effect of cholesterol
namely Brij 72 and Brij 92, with HLB value of approximately
five had significantly (p50.05) higher E.E% compared to that In order to enhance drug-loading capacity, cholesterol content
of Brij 35 (C12). This could be attributed to the increased should be increased during the preparation of niosomal
bilayer hydrophobicity due to their longer alkyl chains and systems. Also there is a great influence on vesicle stability
lower HLB values leading to effective encapsulation of the and permeability upon addition of cholesterol (Gregoriadis,
drug within the hydrophobic core of the bilayer (Abdelbary & 1993). The influence of changing cholesterol ratio within the
Aburahma, 2012). lipid composition on KET entrapment efficiency was
Concerning proniosomal gel formulae (F17–F20) prepared determined. It was found that changing surfactant:cholesterol
using different grades of Pluronics (F68 and L121) which ratio from 10:1 to 5:1 led to a significant increase in E.E% as
belong to a group of surfactants formed of triblock copoly- reported by (El-Laithy et al., 2011). Furthermore,
mers composed of a central hydrophobic polyoxypropylene (Mohammed & Perrie., 2005) studied the effect of cholesterol
(POP) fragment and similar hydrophilic chains of polyox- incorporation into liposomes on the entrapment efficiency of
yethylene (POE) on either sides. Variation of the length of the poorly soluble drug ibuprofen. It was suggested that
each of the blocks enables the modulation of the copolymer increasing cholesterol leads to the enhancement in drug
properties (Abdelbary & Aburahma, 2012). loading capacity but upon exceeding a certain limit, a great
Pluronic F68 is composed of 75 POE units and 30 POP reduction in drug incorporation occurred, this might be due to
units, while Pluronic L121 consists of two POE units and two conflicting factors:
4500 POP units (Moghimi & Hunter, 2000) with an HLB of (1) With increasing cholesterol, the lipophilicity and perme-
25 and 0.5, respectively. The entrapment efficiencies of KET ability of the bilayer decreased and rigidity increased
within the niosomal vesicles prepared using Pluronics were leading to the lipophilic drug to be trapped efficiently
significantly different (p50.05) as the lower HLB L121 gave into bilayers as vesicles formed.
rise to a significantly higher drug entrapment of: 51.40 ± 3.85 (2) In contrast, higher amounts of cholesterol may compete
and 70.70 ± 2.70 for F19 and F20 prepared with Pluronic with the drug for filling in the space within the bilayer. It
L121 compared to 41.60 ± 1.31 and 51.70 ± 2.96 for F17 and was suggested that decreasing the entrapment efficiency
F18 using Pluronic F68 respectively. Pluronics are suggested with increasing cholesterol ratio above certain limit may
to stabilize the lipid membranes of the vesicles in the presence be due to the distruption in the regular linear structure of
of cholesterol by adsorption on the membrane and through vesicular membranes occurred on increasing cholesterol
selective incorporation into low lipid density regions of the beyond a certain concentration. In addition, the ratio of
membrane, holding lipid molecules to pack firmly on the cholesterol may influence the ability of proniosomal gel
vesicles phospholipid membrane retarding the drug leakage formation. These results were in accordance with
(Wu et al., 2004). (Ibrahim et al., 2008) who found that there is no ability
Based on the above results, vesicles formation ability of to form proniosomal gels in the presence of Sp 20 and Sp
nonionic surfactant depends on its structure and hydrophilic– 80 at cholesterol concentration less than 20% being
lipophilic balance which are considered as good indicators of liquids at room temperature. Other surfactants such as;
the entrapping efficiency of any surfactant and its vesicle- Brij 35, Brij 92, Tween 80 and Pluronic F68 require also
forming ability. a higher concentration of cholesterol which might be
The critical packing parameter CPP ¼ (v/lc ao) of a given attributed to their high HLB values, solubilizing property,
surfactant depends on the balance between the critical leading to micelle formation that dissolves the small
hydrophobic group length (lc), hydrophobic group volume amount of cholesterol (Yoshioka et al., 1994).
(v) and the area of the hydrophilic head group (ao) (Uchegbu On the other hand, proniosomal gels can be produced in
& Vyas., 1998). A value of CPP lying between 0.5 and 1 case of Brij 72 (HLB 4.9, Tc ¼ 44  C), Sp 60 (HLB 4.7,
indicates that the surfactant is more prone to form vesicles. A Tc ¼ 54  C), Sp 65 (HLB 2, Tc ¼53  C) even at low cholesterol
value of CPP below 0.5 indicates the spherical micelle content as they are solids at room temperature (Uchegbu &
formation and a CPP of surfactant above one would lead to Vyas, 1998). Moreover, below transition temperature, chol-
inverted micelles formation (Uchegbu & Florence., 1995). esterol made the membrane less ordered and increasing
It was reported that Both Sp 65, Brij 72 and Pluronic L121 cholesterol has been found to increase membrane fluidity to
were able to form vesicular structure with high entrapment the extent where the phase transition is abolished
efficiency even in the presence of low cholesterol (Arunothayanun et al., 2000).
DOI: 10.1080/10717544.2016.1247928 Ex vivo and in vivo studies of ocular ketoconazole-loaded proniosomal gels 315

Effect of lecithin inverse relationship between particle size and E.E% in

proniosomes prepared with different Span derivatives.
Results shown in Table 2 revealed that the addition of lecithin
Regarding proniosomal gel formulae prepared using the
generally led to a significant increase in E.E% in all the
hydrophilic T80 (F9–F10) (HLB ¼ 15), on contrary to Sp80
prepared KET proniosomal gel formulae. This might be due
(HLB ¼ 4.3), the smallest the particle size of 97.56 ± 12 and
to its high Tc (phase transition temperature), decrease in
140.60 ± 10 nm for F9 and F10 compared to 2695 ± 98 and
membrane permeability therefore preventing drug leakage,
413.60 ± 24 for F7 and F8 containing the hydrophobic Sp80.
hence the increase in KET content within the prepared
A direct relationship correlates the particle size and EE% in
vesicles.
case of T80, as the vesicles size might depend on the
Vesicle size analysis properties of the molecules entrapped in the hydrophobic area
of the vesicle bilayer. The vesicle size depends on the distance
The mean particle size and size distribution of the freshly between the bilayers, which increased due to the inclusion of
prepared hydrated niosomes are demonstrated in Table 2. It drug molecules within (Hathout et al., 2007).
can be noted that the vesicle size ranged from 97.56 ± 12 nm The average vesicle size of proniosomal gel formulae
to 4432 ± 105 nm indicating that the average particle size of prepared using different grades of Brijs was41000 nm except
the measured hydrated niosomal suspension varies from the for the two formulae F12 and F14, having 452.70 ± 63 and
nanometer to submicron range. The particle size distribution 550 ± 72 nm respectively. According to the results shown in
of all the tested formulae demonstrated unimodal normal Table 2, both long-chain surfactants (C18), namely Brij 72
symmetrical frequency distribution patterns (PDI  1). and Brij 92, with HLB value of aproximate 5 had significantly
The prepared proniosomal gels will help in ocular delivery (p50.05) higher E.E% compared to that of Brij 35 (C12) as
by improving corneal permeation, prolonging ocular mean previously discussed. The presence of high lecithin ratio in
residence time, thus increasing corneal contact time with the both F12 and F14 contributes in further reduction of
formula, it has been reported that ophthalmic preparations hydrophobicity resulting in smaller vesicle size.
should have particle size less than 10 000 nm in order not to From Table 2, it is clear that the mean average vesicle size
cause ocular irritation (Hecht, 2001). Regarding ocular drug of proniosomal gel formulae prepared using different Pluronic
delivery systems, smaller particles are characterized by grades; Pluronic F68 and L121 with an HLB of 25 and 0.5
greater surface area available for conjugation between the respectively varied according to the HLB of the given
cornea and proniosomal gel formula (Yoncheva et al., 2005). surfactant, as increasing the hydrophobicity attributes to a
Furthermore, the prepared nano to submicron vesicles will decrease in free energy resulting in smaller vesicles.
help in crossing the ocular physiological and anatomical
constraints. Kassem et al. (2007) reported that as the particle
In vitro KET release
size in the drug suspension decreased, this leads to an increase
in the mean residence time of drugs on the ocular surface. Results of in vitro release of KET from the different
Therefore, a smaller size could be an added advantage for prepared proniosomal gels are shown in Figure 1(A–D). The
treatment of superficial fungal infections. Upon variation of percentage of KET released from the different prepared
amounts of proniosomal ingredients, the following effects on proniosomal gels after 2 h (Q 2 h) and 8 h (Q 8 h) are shown
vesicle size were observed. Increasing surfactant/lipid ratio in Figure (2). It is clear that KET release after 2 h (Q 2 h)
led to an increase in vesicle size and was attributed to the ranged from 20.7%± 0.15 to 68.5% ± 4.7, while the release
increase in the overall degree of hydrophilicity. Also, the after 8 h (Q 8 h) ranged from 81.16% ± 1.178 to
increase in mean vesicle size by increasing lecithin content 101.97% ± 1.2, respectively.
can be also accepted if we considered the long hydrocarbon The release profiles of KET from the different prepared
chain of lecithin molecules (18  C). The opposite held true proniosomal gel formulae were found to be biphasic release as
with increasing the cholesterol amount that was associated reported by Mokhtar et al. (2008). A rapid drug leakage was
with a decrease in the hydrophilicity of bilayers, thus limiting observed in the initial phase, where about 25–55% of the
the water intake to the vesicles core and resulted in a entrapped drug was released within the first few hours of
subsequent decrease in mean vesicle size. Finally, the increase proniosomal incubation in 100 mL of simulated lacrimal fluid
in mean vesicle size with increasing drug load was attributed (pH 7.4). While in the second phase, a slow release of KET
to the drug entrapped in the hydrophobic domain of the was observed from the different proniosomal formulations.
vesicle, causing the bilayer molecules to become apart from The initial rapid phase might be due to the unentrapped drug,
each other leading to an increase in vesicle size (Hathout which is mainly present between the large hydrocarbon chains
et al., 2007). in the lipid bilayers of proniosomal vesicles which leads to a
Concerning formulae (F1–F8) prepared using different rapid leakage from the vesicles in large simulated lacrimal
grades of Spans (Sp20, Sp60, Sp65 and Sp80), it is clear that fluid (pH 7.4) until reaching equilibrium. Moreover, it has
the largest vesicle size was obtained from F1 (Sp20) and F7 been reported that, this drug explosion occurs as a result that
(Sp80) having an average diameter of 1322 ± 82 and the highly ordered lipid particles cannot accommodate large
2695 ± 98 nm together with lowest E.E% of 57.90 ± 2.90 amounts of drug (Wissing et al., 2004).
and 47.00 ± 1.25% respectively. Both formulae (F1 and F7) Accordingly, factors that stabilize the vesicle membrane
were composed of S.A.A: lecithin ratio of 10:1 w/w, it is clear and increase the entrapment efficiency of a hydrophobic drug
that decreasing lecithin content will decrease the hydropho- such as KET (namely low HLB, long alkyl chain, high phase
bicity and hence contributed to larger vesicle size. There is an transition temperature) will slow down the release profile.
316 G. A. Abdelbary et al. Drug Deliv, 2017; 24(1): 309–319

Figure 1. In vitro release profile of the different prepared KET-loaded proniosomal gel formulae prepared with: (A) Spans (B) Tween 80 (C) Brijs (D)
Pluronics.

Figure 2. In vitro release of the different prepared KET-loaded proniosomal gel formulae at Q 2 h and Q 8 h.
DOI: 10.1080/10717544.2016.1247928 Ex vivo and in vivo studies of ocular ketoconazole-loaded proniosomal gels 317

The release profiles of the proniosomes revealed a significant the least amount of KET permeated, this might be attributed
increase (p50.05) in the percentage drug released with the to the transition temperature of the used surfactant, where the
increase in HLB since hydrophilic surfactants have higher high transition temperature of Sp65 (53  C) made the
solubilizing power on hydrophobic solutes in aqueous proniosomes in a more packed ordered gel state at the
medium compared to hydrophobic (Pardakhaty et al., 2007). specified permeation temperature (37  C) (Vora et al., 1998).
Increasing lecithin content resulted in an increase in phase On the other hand, the lower transition temperature of Sp20
transition temperature (Tc) together with a more intact lipid (16  C) in formula (F2) allows the proniosomes to be in a
bilayer with low permeability which hindered drug leakage completely fluid state at the specified permeation tempera-
leading to a significant slow release profile of entrapped drug ture. F2 was selected as the optimum formula showing
from the vesicles (p50.05) compared to lecithin-free significantly higher (p50.05) permeability coefficient and
proniosomes (Guinedi et al., 2005; AbdElbary et al., 2008). steady state flux of 0.000244 cm2/h and 2.44 mcg/cm2h,
The addition of cholesterol (50 mg) in the preparation of respectively.
KET proniosomal gel formulations resulted in further
decrease in KET release due to the decrease in leakage and In vivo study of the optimum proniosomal gel formula
permeability of niosomal vesicular membrane in the presence Determination of KET level in aqueous humor
of cholesterol. (Cocera et al., 2003) suggested that the
presence of cholesterol resulted in an optimum lipophilicity Based on the aforementioned results, formula F2 composed
which decreased the formation of the transient hydrophilic of 250 mg span 20, 250 mg lecithin together with 50 mg
holes by the incorporation of cholesterol, this was done by cholesterol) was selected for in vivo evaluation; where KET
decreasing membrane fluidity, responsible for drug release levels in the aqueous humor were determined following
through liposomal layers. topical application of KET-proniosomal gel (F2) and KET-
Based on the previous results, Formulae (F2, F3, F4, F5 SP at different intervals (Figure 4). Table 4 shows the
and F6) were selected for further investigations, having E.E% different pharmacokinetic parameters (Cmax, Tmax, AUC, k,
of not less than 80%, mean vesicle size less than 1000 nm with MRT and T1/2) calculated for both formula and drug
P.D.I value less than 1 and less than 40% KET released within suspension. Following topical application of KET-Gel
the first 2 h. It is clear that all the selected formulae were (Cmax, 18.8 mg/mL) was attained after 4 h which was 22
composed of spans being hydrophobic compared to other times greater than that of KET-SP (Cmax, 0.896 mg/mL)
used surfactants (Tween, Brijs and Pluronics) suggesting the reached after 2 h, respectively. The KET concentrations in
potential use of a more hydrophobic surfactant as Span during aqueous humor post-6 and 8 h instillation of KET-Gel were
the formulation of proniosomal gel formulae. 66 and 73 times higher than that of KET-SP, respectively,
with about 20 fold increase in KET-proniosomal gel (F2)
Ex vivo corneal permeation bioavailability in the aqueous humor over KET-SP (Table 4).
Topical administration of KET-Gel to rabbits with an
Figure (3) shows the cumulative amounts of KET permeated
intact epithelium resulted in a significant increase (p50.05)
from the selected proniosomal gel formulations through
in KET level in cornea exceeding the minimum inhibitory
isolated corneal rabbit as described before. It is clear that a
concentration (MIC) of ocular isolates of fungi; Filamentous
significant higher amount (p50.05) of KET permeated from
fungi and Yeast, whose MIC (50.8 mg/mL) (Therese et al.,
formulae F2 and F3 compared to other formulae (F4, F5 and
2006). The elevated KET levels in the cornea and aqueous
F6). Furthermore, F2 and F3 showed the highest steady-state
humor following the administration of KET-Gel might be due
flux and permeability coefficient as shown in Table 3. It is
to the increase in the amount of KET dissolved in the
clear that proniosomes prepared using Sp65 (F5, F6) showed
precorneal area leading to high concentration gradient,
favoring good permeation, together with higher contact time
with the corneal area.

Ocular irritancy test

The ocular irritancy testing revealed that the tested formula
KET-Gel (F2) did not show any sign of redness, inflammation
or increased tear production over the study period (24 h),
Therefore, it could be concluded that the proniosomal gel

Table 3. Permeability parameters of the selected ketoconazole-loaded

proniosomal gels.

Permeability Steady-state Correlation

Formula coefficient (cm2/h) flux (mcg/cm2hr) coefficient (R)
F2 0.000244 2.44 0.993
F3 0.0002009 2.009 0.99
F4 2.36  105 0.236 0.91
F5 3.18  106 0.0318 0.88
Figure 3. In vitro corneal permeation of the selected KET-loaded
F6 6.39  106 0.0639 0.924
proniosomal gel formulae.
318 G. A. Abdelbary et al. Drug Deliv, 2017; 24(1): 309–319

Figure 4. Concentration-time curve of KET-proniosomal gel formula (F2) and KET-SP in aqueous humor of albino rabbits.

Acknowledgements
Table 4. Pharmacokinetic parameters of ketoconazole after topical
administration of KET-Gel optimum formula and KET-SP rabbit’s eye. The authors express special thanks to Dr. Mahmoud el
khoudary, Analytical chemistry department, Faculty of
Pharmacokinetic parameters KET-Gel (F2) KET-SP
Pharmacy, Sinai University, for assisting with the HPLC
Cmax (mcg/ml) 18.8 ± 1.23 0.896 ± 0.052 analysis.
Tmax median tmax (h)a 4 2
AUC 0–8 (mcg.h/mL) 60.1 ± 3.04 3.15 ± 0.27
AUC 0–1 (mcg.h/mL) 62.48 ± 3.56 3.18 ± 0.25 Declaration of interest
K (h1) 0.625 ± 0.045 0.682 ± 0.251
T1/2 (h) 1.1 ± 0.07 1.01 ± 0.264 Authors report no conflicts of interest. The authors alone are
MRT (h) 3.88 ± 0.1 2.5 ± 0.0305 responsible for the content and writing of this article.

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