Journal of Liposome Research

ISSN: 0898-2104 (Print) 1532-2394 (Online) Journal homepage: http://www.tandfonline.com/loi/ilpr20

Enhancement of lomefloxacin Hcl Ocular

efficacy via niosomal encapsulation: in- vitro
characterization and in- vivo evaluation.

Rawia M. Khalil, Ghada A. Abdelbary, Mona Basha, Ghada E. A. Awad &

Hadeer A. Elhashemy

To cite this article: Rawia M. Khalil, Ghada A. Abdelbary, Mona Basha, Ghada E. A. Awad &
Hadeer A. Elhashemy (2016): Enhancement of lomefloxacin Hcl Ocular efficacy via niosomal
encapsulation: in- vitro characterization and in- vivo evaluation., Journal of Liposome Research,
DOI: 10.1080/08982104.2016.1191022

To link to this article: http://dx.doi.org/10.1080/08982104.2016.1191022

Accepted author version posted online: 31

May 2016.
Published online: 31 May 2016.

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 Enhancement of lomefloxacin Hcl Ocular efficacy via niosomal encapsulation: in- vitro

characterization and in- vivo evaluation.

Rawia M. Khalil1, Ghada A. Abdelbary 2, Mona Basha1, Ghada E. A. Awad 3

and Hadeer A.

Elhashemy1
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1 Pharmaceutical Technology Department, National Research Centre, Cairo, 12622 Egypt.

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2 Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

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3 Chemistry of Natural and Microbial Product Department, National Research Centre, Cairo,
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12622 Egypt.
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Corresponding author: Rawia M. Khalil
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Tel.:+20106935895; fax:+203370931.

E-mail address: rawia khalil@yahoo.com

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 Abstract

The aim of this study is to develop and evaluate niosomal dispersions loaded with the

hydrophilic drug; Lomefloxacin Hcl (LXN) for the management of ocular bacterial

conjunctivitis. LXN loaded niosomes were prepared by thin film hydration method following a

full factorial formulation design. Two independent variables were evaluated; the type of

surfactant (X1) and the surfactant: cholesterol ratio (X2). The dependent variables comprised
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entrapment efficiency (EE%: Y1), particle size (PS: Y2) and zeta potential (ZP: Y3). The

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optimum formulation; N-LXN14 (Tw60: CH, 1:1) was spherical in shape exhibited EE% of

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68.410.07, PS of 176.00.98 and ZP of -40.702.20 with a sustained release profile over 8

hours following Higuchi model. N-LXN14 proved good physicochemical stability under
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refrigeration up to 3 months. Ocular irritancy test showed no signs of ocular toxicity confirming

the safety and suitability for ocular application. Microbiological evaluation of the antibacterial
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effect of N-LXN14 was conducted using susceptibility test and through the induction of topical
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conjunctivitis by Staphylococcus aureus (S. aureus) followed by topical therapy. Susceptibility

test manifested significantly higher percent inhibition of S. aureus and higher AUC 0-12h of N-

LXN14 (604.59 0.05) compared to the commercial product (126.25 0.049). Both clinical
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observation and colony count of the infected eyes after eight days of treatment demonstrated
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significant improvement in therapeutic response. The infected eyes were perfectly healed with

complete eradication of S. aureus. In conclusion, the results showed that LXN niosomal

dispersions may serve as a promising superior ocular delivery system in treatment of bacterial

conjunctivitis.

Keywords: Lomefloxacin Hcl, niosomes, Ocular Delivery, Microbiological Evaluation,

Conjunctivitis.
 1. Introduction

The eye is a very complex sensory organ (Diebold and Calonge, 2010). The complicated

structure and the protective mechanisms of the eye represent an obstacle in front of

pharmaceutical scientists in designing effective ocular drug delivery systems (Fangueiro et al.,

2014, Kakkar and Kaur, 2011, Patel et al., 2013). The defense mechanisms of the eye represent a

compact barrier hindering optimum drug absorption into the intraocular area resulting in low
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bioavailability of administered ocular medications. Such mechanisms include; extensive drug

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loss by tear dilution and high tear turnover, drainage through the nasolacrimal duct, non-

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productive absorption in addition to the impermeability of the corneal epithelium which restricts

the entry of foreign substances (Kompella et al., 2013, Dong et al., 2015). As a result, very low
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percentage of administered drug penetrates the cornea reaching the intraocular tissues thus

achieving low ocular bioavailability usually ranging from 0.15% (Ruponen and Urtti, 2015).
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Thus, there is an urgent need for a suitable ocular delivery system capable of increasing the
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contact time of the drug at the eye surface and facilitating the transport of drug molecules into

the eye tissue.

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In order to overcome these obstacles, some interesting approaches have been adopted by the

pharmaceutical researches. Colloidal drug delivery systems, such as non-ionic surfactant

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vesicles; niosomes have been studied extensively for potential ocular application. Niosomes

offer several advantages compared to other conventional carriers; (i) they are biodegradable,

biocompatible with low toxicity due to their non-ionic nature; (ii) they can entrap both lipophilic

and hydrophilic drugs either into the vesicular bilayers or in the aqueous compartments; (iii)

being mainly water-based dispersions, niosomes can be easily instilled as eye drops onto the

surface of the eye acquiring high patient compliance; (iv) they are targeted carriers protecting
 drug molecules from biological environment to be localized in targeted tissues resulting in

sustained drug delivery with improved therapeutic performance (Abdelkader et al., 2012, Maiti

et al., 2011); from the industrial point of view, their lower cost in addition to greater stability

make niosomes more attractive for numerous applications in the pharmaceutical field

(Abdelbary and AbouGhaly, 2015). Various drug molecules, such as naltrexone hydrochloride

(Abdelkader et al., 2011, Abdelkader et al., 2012), timolol maleate (Kaur et al., 2010),

acetazolamide (Guinedi et al., 2005) have been exploited in niosomal formulations manifesting
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the successful use of niosomes as a potential ocular drug delivery system.

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Lomefloxacin Hcl (LXN) is a third generation fluoroquinolone antibacterial agent (Ying et al.,

2013, Attia, 2008, Sattar et al., 2014), it is well known for its bactericidal effect (Beberok et al.,
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2013). Having a broad spectrum of action against a wide range of gram-positive and gram-

negative organisms, it also has the advantage of being effective against some anaerobic bacteria
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(Ying et al., 2013, Krustev et al., 2013, Attia, 2008, Sattar et al., 2014). The bactericidal action
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of LXN is based upon the interference with the activity of the bacterial enzymes DNA gyrase

and topoisomerase IV, which are needed for the transcription and replication of bacterial DNA

(Drlica and Malik, 2003, Attia, 2008), resulting in rapid bacterial death (Beberok et al., 2013,
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Aldred et al., 2014). LXN is widely used in clinical practice to treat infections of the respiratory
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or urinary tracts (Symonds and Nix, 1992), as well as in ophthalmology and dermatology

(Thompson, 2007). Concerning ocular diseases, LXN is considered as a promising ophthalmic

pharmaceutical agent for the treatment of bacterial conjunctivitis (Thakur et al., 2009).

Conjunctivitis, a corneal inflammation, is one of the most painful and acute conditions (Yasin et

al., 2012) that is caused by a variety of aerobic and anaerobic bacteria (Comstock et al., 2010).

For the treatment of bacterial conjunctivitis, the recommended dosage regimen of LXN is to be
 instilled in the affected eyes every two hours up to eight times during the first two days, then

every four hours up to four times for next five days. A previous study showed that LXN is

characterized by a rapid and very short elimination half-life in aqueous humor (Sato et al., 1996)

As a hydrophilic agent with low log P value of 0.146, the corneal permeation of LXN is likely

the rate limiting step for its ocular absorption leading to potential decrease in LXN

bioavailability. Therefore, an optimum delivery system for LXN is required to enhance drug

penetration localizing its effect in the targeted tissues and also to maintain its stability and
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reduce its undesirable side effects.

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The aim of this study is to design and formulate LXN niosomal colloidal dispersions of various

compositions. The effect of formulation factors such as type of surfactant and ratio of cholesterol
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content on the characteristics of the niosomal vesicles was evaluated by employing a full

factorial design. Moreover, the microbiological activity of the selected niosomal formulation
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compared with a commercially available LXN eye drops has been investigated using an animal
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model.

2. Materials and methods

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2.1. Materials
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Lomefloxacin Hcl (LXN) was kindly donated as a gift sample by Sigma Co. Menofya, Egypt.

Span 20 (sorbitan monolaurate), Span 40 (sorbitan monopalmitate), Span 60 (sorbitan

monostearate), Tween 80 (polyoxyethylene sorbitan monooleate) were purchased from Sigma

Aldrich, St. Louis, USA.Tween 40 (polyoxyethylene sorbitan monopalmitate) and Tween 60

(polyoxyethylene sorbitan monostearate), were obtained from Scharlau Chemie S.A., Barcelona,

Spain. Cholesterol PB (95%) was supplied by Panreac, Barcelona, Spain. Methanol, HPLC
 Isocratic, Fisher Scientific, UK.Chloroform, HPLC, Alpha Chemical, India.Cellulose membrane,

molecular weight cut-off 12,00014,000 purchased from SigmaAldrich, ST. Louis, and

experimental conditions USA.Potassium dihydrogen phosphate, disodium hydrogen phosphate

and sodium chloride were of analytical grade. Commercial 0.3 % Lomefloxacin Hcl eye drops

(Orchacin), Orchidia Pharmaceutical Ind., Egypt. Staphylococcus aureus ATCC 29213

purchased from ATCC.

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2.2. Methods

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2.2.1. Experimental factorial design

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LXN loaded niosomal dispersions were prepared using a 61 31 full factorial experimental
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design in order to investigate the joint influence of formulation variables using Design-Expert

8 software. This design is composed of 2 variables; the type of surfactants studied at 6 levels
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(X1) and the ratio between surfactant: cholesterol (X2) studied at 3 levels (Table1) which were
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evaluated to reach maximum experimental efficiency using the least number of experiments. The

design required a total of 18 possible experimental combinations. The established dependent

variables were the entrapment efficiency (EE %) (Y1), particle size (Thompson) (Y2), and zeta
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potential (ZP) (Y3). The experiments were carried out in a random order in triplicates and the

data were analyzed using one way ANOVA test to evaluate the level of significance of the tested
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factors on the selected responses as well as the interactions between these factors.

2.2.2. Preparation of LXN-loaded niosomes

LXN loaded niosomes were prepared using the conventional thin film hydration technique

(TFH) (Abu Hashim et al., 2014). Briefly, accurately weighed amounts of surfactant (SAA) and
 cholesterol (CH) were dissolved in 10 ml chloroform in a long neck quick fit rounded bottom

flask. The lipid mixture was slowly evaporated under reduced pressure at an adjusted

temperature (60 C) using a rotary evaporator (Heidolph Rotary Evaporator, WB 2000 and VV

2000, Germany) rotating at a fixed speed of 150 rpm. The flask was partially immersed in a

water bath until a dried thin film appeared on the inner wall of the rotating flask. The dried thin

film was subsequently hydrated with 10 ml of LXN buffered solution (pH 7.4) using a rotary

evaporator under normal pressure for about 1 hour. In order to ensure the complete hydration of
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the film forming milky dispersion, the formulation was left overnight at 4C for maturation

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(Abdelbary and El-Gendy, 2008). The composition of the tested niosomal formulae are reported

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in Table 2. EP
2.2.3. Characterization of LXN loaded niosomes

2.2.3.1. Determination of LXN entrapment efficiency (EE %)

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1 ml of the investigated formulation was centrifuged at 9000 rpm and a temperature of - 4C for

1 h using a cooling centrifuge (Union 32R, Hanil Science Industrial, Korea). The vesicles were

separated from the supernatant and were washed twice with PBS (pH 7.4), and re-centrifuged
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again for 30 min. The amount of entrapped LXN was determined by lysis of the separated

vesicles using methanol. The amount in mg of LXN was detected spectrophotometrically

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(Shimadzu UV Spectrophotometer (2401/PC), Japan) at a wavelength of 289 nm against a blank.

The entrapment efficiency percent (EE %) was calculated from the following equation:

EE % =

2.2.3.2. Particle size analysis

 The niosomal vesicle size was determined by photon correlation spectroscopy (PCS) (Malvern

Zetasizer, UK). The samples were diluted to 10 ml with distilled water and measured at room

temperature in triplicates.

2.2.3.3. Zeta potential analysis ()

Zeta potential was measured by PCS in triplicates. About 1 ml of niosomal dispersion was

diluted to 10 ml with distilled water. After shaking, the suspension was transferred into a
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standard cuvette for zeta potential measurement. The sample temperature was maintained

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constant at 25C.

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2.2.4. Optimization of formulation factors EP
The designed formulation factors were selected depending on their effect on the defined

response parameters. Selected formulations were subjected for more investigations.

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2.2.5. In vitro release study

The release of LXN from niosomes was determined according to the method reported by Li et al
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(Li et al., 2012) using the membrane diffusion technique but with slight modifications (Singh et

al., 2014). On the separated niosomal vesicles (equivalent to 2 mg drug) 0.3 ml phosphate
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buffered saline (pH 7.4) was added and transferred to a bottomless glass cylinder having the

length of 10 cm and diameter of 2.5 cm fitted at its lower end with presoaked cellulose

membrane ( 12,000- 14,000 mwt). Then, the glass tube was attached to the shaft of the USP

dissolution tester (apparatus one, Hanson Research, Chatsworth, USA) containing 100 ml PBS

(pH 7.4) instead of the basket (Abdelbary and Aburahma, 2014) .The glass cylinder was allowed

to rotate at a constant speed (50 rpm) and the PBS medium was maintained at a temperature of
 37 0.5 C throughout the whole study. About 5 mL aliquots were withdrawn at predetermined

time intervals for a total period of 8 h and replaced with the same amount of fresh release

medium at the same temperature to maintain a constant volume throughout the study. The

samples were analyzed for the drug content spectrophotometrically at 280 nm against blank

(Ambati et al., 2000). The cumulative percent of drug released was plotted against time, the data

was analyzed using linear regression equations and the order of drug release from the different

formulations was determined.

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2.2.6. Transmission electron microscopy (TEM)

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The morphology of the hydrated selected niosomal dispersion was examined by TEM, (Model

JEM-1230, Jeol, Tokyo, Japan). A drop of the dispersion was placed onto a carbon coated
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copper grid and left to adhere on the carbon substrate for about 1 min. The dispersion in excess

was removed by a piece of lter paper. A drop of 1% phosphotungestic acid solution was added
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to act as a negative staining agent and again, the solution in excess was removed by a tip of lter
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paper. After being stained, samples were allowed to dry at room temperature for 10 minutes for

investigation.
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2.2.7. pH measurement
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The pH value of the selected formula was measured using digital pH meter (JENWAY 350, UK)

at 25C and standardized using buffer solution at pH 7.0 before use. All measurements were

performed in triplicate.

2.2.8. Differential scanning calorimetry (DSC)

 Differential scanning calorimetry (DSC) analysis was carried out for the drug powder, the freeze

dried formula and its individual components (Shimadzu DSC-50, Kyoto, Japan). The sample (5

mg) was placed into a standard aluminum pan, crimped and scanned from 40 C to 400 C at a

heating rate of 5C /min with continuous purging of nitrogen (20 ml/min). An empty sealed pan

was used as reference. All measurements were performed in triplicate.

2.2.9. Stability study

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The selected formula was sealed in glass vials and stored under refrigeration temperature (4 2

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C) for a period of 3 months. Physical appearance was assessed and the formulation was

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analyzed with respect to drug entrapment efficiency and particle size after 1, 2 and 3 months of

storage and compared with the freshly prepared formula.

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2.2.10. In vivo Microbiological evaluation
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2.2.10.1. Animals
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Total of twenty seven adult male albino rabbits weighing 2-3 kg were kept in individual cages

under well-defined and standardized conditions (humidity and temperature controlled room; 12-
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h light and 12-h dark cycle), where three rabbits were assigned for ocular irritancy test, nine

rabbits were assigned for susceptibility test and nine were assigned for topical therapy after pre-
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induced bacterial conjunctivitis. They were fed with standard dry food, water and libitum. All

eyes were initially examined with a hand-held slit lamp. Only animals showing no sign of ocular

inflammation were included in this study.

 The animal experiments were conducted in full compliance with local, national, ethical and

regulatory principles for animal care and were approved by the Cairo University Animal Care

Committee (Tamilvanan and Benita, 2004).

2.2.10.2. Ocular irritancy test

The potential ocular irritancy and/or damaging effects of the selected niosomal formulation were

evaluated by observing any signs of redness, inflammation, or increased tear production, upon
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application to the eyes of six albino rabbits by visual observation using a slit lamp, before

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treatment, and 1, 12, 24 and 48 h after instillation (Basha et al., 2013). The formulation was

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tested on three albino rabbits; the experiment was performed by a single instillation (50 l) of

the investigated preparation into the conjunctival sac of one eye, whilst the contralateral eye
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served as control.
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2.2.10.3. Susceptibility test

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Susceptibility test was carried out on albino rabbits for the selected LXN niosomal formulation

and the commercially available LXN eye drops Orchacin. Sterile 6mm diameter filter paper
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discs (Whatman no. 1) were placed under the eyelid of each rabbit for 30 seconds at specific

time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h) following a single installation (50 l)

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of the investigated formulae each in the conjunctival sac of the right eye of three rabbits (Basha

et al., 2013). The discs were then placed in the nutrient broth (du Plessis et al.) tubes inoculated

with 100l of bacterial suspension. The tubes were then incubated at 370.5 C for 24 h. The

growth inhibition of the inoculated bacteria was evaluated by measuring the cultures optical

density at 600 nm using UV spectrophotometer. Percentage of inhibition, which is related to the

level of drug in the eye tears following the topical application of tested drug formulae, was
 calculated using the NB medium inoculated with S. aureus ATCC 29213 as control (Basha et al.,

2013).

The experimental groups each consisting of three rabbits was as follow:

Group 1: assigned for control.

Group 2: assigned for LXN niosomal formulation.

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Group 3: assigned for commercial LXN eye drops (Orchacin).

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The area under the curve from 0 to 12 h (AUC 0-12 h) was estimated by the linear trapezoidal

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method which is used to predict and compare the antibacterial effect of the tested formulation as
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well as commercial Orchacin eye drops.

2.2.10.4. Induction of ocular conjunctivitis and topical therapy:

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For conjunctivitis induction, bacterial suspension of 24 h old S. aureus ATCC 29213 was
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adjusted to contain 106 CFU/ml with sterile physiological saline. In order to induce

conjunctivitis, each rabbit was infected with 100 l bacterial suspension of S. aureus. The
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experimental groups each consisting of three rabbits was as follow:

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Group 1: control, infected and untreated.

Group 2: Infected and treated using selected LXN niosomal formulation.

Group 3: Infected and treated using commercial LXN eye drops (Orchacin).

After 48 h of infection, bacterial conjunctivitis was confirmed when the following signs were

observed: redness, inflammation and excessive tearing. Topical treatment was started with the
 selected niosomal formulation and Orchacin while the control group did not receive any

treatment. Treatment consisted of a single instillation (50 l containing 0.3 % LXN) every 12 h

for 8 days (total 16 doses). The eyes of rabbits were examined for signs of bacterial

inflammation (conjunctivitis, blepharitis, iritis, corneal oedema, and corneal infiltrates). Samples

were taken by placing a sterile 6mm diameter filter paper discs (Whatman no. 1) under the

eyelid of the rabbit for 30 seconds at specific time intervals. The discs were then placed in 5 ml

of fresh and sterile NB for examination of bacterial presence. The inoculated medium was
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incubated at 37C for 24h. The bacterial growth was evaluated by measuring the cultures optical

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density at 600 nm using a UV spectrophotometer.

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2.2.11. Statistical analysis
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Experiments were performed in triplicate and results were reported as a mean SD. Statistical

analysis of the results was computed with the SPSS software using one-way analysis of
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variance (ANOVA) followed by the least-significant difference test (LSD). Differences were
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considered to be statistically significant when the p values were less than 0.05.

3. Results and discussion

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3.1. Analysis of factorial design

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The production and selection of niosomal dispersions requires extensive literature research

followed by formulation studies. The choice of the surfactant, its concentration as well as the

amount of the added cholesterol can affect the characteristics of the produced niosomes, hence

these are considered to be very crucial factors in order to obtain a stable and functional niosomal

system (Moghassemi and Hadjizadeh, 2014). The physicochemical properties of LXN loaded
 niosomes obtained from the experimental design were analyzed. The experimental runs, with the

selected variables and their effect on the measured responses are depicted in Table 2.

3.1.1. The effect of formulation variables on the EE% of LXN-loaded niosomes

3.1.1.1. The Effect of surfactant type:

As shown in Table 2, the EE % of LXN-loaded niosomes formulated with Spans varied from

40.69 3.97 % with Sp60 (N-LXN 7) to 59.291.83 % with Sp20 (N-LXN 3). The results
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indicated that the EE % of LXN loaded niosomes was higher with Sp20 compared to other

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Spans. This might be due to the high HLB value of Sp20 (8.6) (Abdallah et al., 2013) compared

to Sp40 and Sp60 with HLB values of 6.7 and 4.6 respectively. Based on the previous data,
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LXN EE % decreased in the following order Sp20 > Sp40> Sp60 (Abdallah et al., 2013).

Despite their high HLB value; Tweens series can form niosomes in the presence of cholesterol
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(Santucci et al., 1996, Ramos-Cabrer and Campos, 2013). The EE % in case of the formulations
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based on Tweens were within the range; 41.64 1.74 % in case of Tw40 (N-LXN 12) and

68.41 0.07 % with Tw60 (N-LXN 14). Moreover, the EE % of LXN-loaded niosomes formed

with Tw60 (C18) was significantly higher (P < 0.05) compared to other Tweens which may be
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due to its longest chain length (Kayser et al., 2005). These results were in agreement with those

found by Dharashivkar et al. who reported that the length of alkyl chain is a crucial factor of
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entrapment ability, thus, longer chain surfactants have higher entrapment efficiency

(Dharashivkar et al., 2014). It is reasonable to speculate that the reason behind the higher EE %

of Tw60 over Tw80 (the only Tween having an unsaturated alkyl chain) despite having the same

chain length is the introduction of double bond which in turn increases the chain fluidity and

hence increases the particle permeability to aqueous phase (Abdelbary and El-Gendy, 2008).
 Thus, the membrane formed was more permeable, which possibly explains the lower EE % of

the Tw80 formulations compared to Tw60 (Kayser et al., 2005). Abdelbary and El-Gendy

(Abdelbary and El-Gendy, 2008), found analogous results, where the EE % of Gentamycin

sulphate, also a hydrophilic drug, was higher with Tw60 compared to Tw80 when entrapped in

niosomal formulation for ocular application. From the above results, it can be concluded that

among Spans and Tweens series, Sp20 and Tw60 niosomal formulations showed the highest

LXN EE %.
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3.1.1.2. The Effect of surfactant:CH ratio

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The SAA: CH ratio used, plays a decisive role in determining the properties and the behavior of

the bilayer of the niosomes (Sankhyan and Pawar, 2013). Hence, there is a need for examining
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the consequences of varying the CH ratio in formulating LXN-loaded niosomes. An inverse

relation was found between LXN-EE% and CH ratio in niosomes formulated using Spans, where
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EE % decreased upon increasing the CH amount. This behavior could be due to the fact that CH
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competes with the drug for the packing space within the bilayer (Moribe et al., 1999) as well as

to the fact that its presence in the environment does not favor drug entrapment (Abdelbary,
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2011). These results were in harmony with those reported by Muzzalupo et al. where the best

EE% was obtained when niosomes were made up of surfactant and CH at the molar ratio of 2:1
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(Muzzalupo et al., 2005). Same results were reported by Abdelbary and Al-Mahallawi et al.

where a decrease in EE % of ciprofloxacin hydrochloride was revealed upon increasing CH.

However, the opposite observation was detected with LXN-loaded niosomes formulated with

Tweens, where increasing the amount of CH up to a ratio 1:2 SAA: CH, resulted in a higher

LXN EE %. This could be related to the CH ability for increasing the viscosity of niosomal
 dispersion and imparting rigidity to the flexible bilayer (Abdelbary and El-Gendy, 2008), which

resulted in the formation of highly ordered structures of SAA with CH embedded in the bilayer

(Guinedi et al., 2005, Ruckmani and Sankar, 2010). This further facilitates partitioning of the

drug in the bilayer and increases the entrapment of the drug in niosomal vesicles (Ruckmani and

Sankar, 2010). These findings were in agreement with results mentioned by Guinedi et al. where

incorporation of cholesterol into niosomes was found to increase the EE% of acetazolamide

(Guinedi et al., 2005). Ruckmani and Sankar, reported same results about increasing the EE % of
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the drug in the vesicles with adding CH in niosomal vesicle (Ruckmani and Sankar, 2010).

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3.1.2. The effect of formulation variables on the PS of LXN-loaded niosomes

3.1.2.1. The Effect of surfactant type:

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The prepared niosomal vesicles were found to be within the nano-size ranged from 96.0 0.86
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nm (N-LXN 8) to 369.0 9.23 nm (N-LXN 1) (Table 3). The size of LXN-loaded niosomes
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prepared with Spans was found to be dependent on the HLB value of the studied surfactants

(Tavano et al., 2014). As observed, increasing hydrophobicity resulted in smaller vesicles, while

decreasing the hydrophobicity of the surfactant resulted in an increase of the vesicles size
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(Tavano et al., 2014, Nagarwal et al., 2009). The size significantly decreased (P<0.05) according

to the following order: Sp20 (HLB=8.6) > Sp40 (HLB=6.7) > Sp60 (HLB=4.6), where the
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smallest size measured (96.0 0.86 nm) was obtained when using Sp60 (N-LXN 8) while the

largest particle size (369.0 9.23 nm) was reported when using Sp20 (N-LXN 1). These results

are consistent with that reported by Abdallah et al. Kamboj et al. and Jadon et al. (Kamboj et al.,

2014, Jadon et al., 2009, Abdallah et al., 2013). The effect of HLB values of the surfactants on

the vesicles size could be explained as follows; the surface energy increased with increasing the
 hydrophilicity (Nagarwal et al., 2009); also the water uptake of the surfactants increased with the

HLB values moving towards hydrophilic region and both factors contribute to larger size of the

vesicles (Abdelbary and Aburahma, 2014, Manosroia. A et al., 2003, Nagarwal et al., 2009).

On the other hand, the particle size of LXN-loaded niosomal vesicles prepared using Tweens

could be influenced by the chain length of the surfactants used (Agarwal et al., 2001, Abdelbary

and Aburahma, 2014), as it was reported, surfactants with longer alkyl chains, generally resulted
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in larger vesicles (Abdelbary and El-Gendy, 2008, Manosroia. A et al., 2003). Generally,

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niosomal vesicles formulated with Tw80 were larger than those with Tw60 which in turn were

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larger than Tw40 niosomes (Agarwal et al., 2001). Similar to the previous reported data, the

particle size of LXN-niosomes decreased significantly according to the following order (P<0.05)
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Tw80 > Tw60 >Tw40 as described in table 2. These findings were similar to results reported by

Abdelbary and Aburahma upon using different Tweens as non- ionic surfactants (Abdelbary and
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Aburahma, 2014) . Additionally, Bayindir and Yuksel mentioned identical results when they
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prepared paclitaxel niosomes using Tweens for oral drug delivery (Bayindir and Yuksel, 2010).

3.1.2.2. The Effect of SAA: CH ratio

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Data described in Table 2 shows that, generally, LXN-loaded niosomes particle size decreased

linearly with decreasing CH concentration. Such results were in agreement with results found by
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Taha et al. (Taha et al., 2014) where the particle size of ciprofloxacin liposomes decreased with

decreasing CH concentration.

3.1.3. The effect of formulation variables on the ZP of LXN-loaded niosomes

 Zeta potential is the overall charge a particle acquires in a special medium. The significance of

ZP is based on its value that can be related to the stability of vesicular formulations. It is well

known that particle aggregation is less likely to occur for charged particles having high zeta

potential (|30| mV) because of electrostatic repulsion (Heurtault et al., 2003, Kamel et al., 2013,

Moghassemi et al., 2015). Table 2 shows the measured zeta potential values of the prepared

LXN-niosomes. All formulations were negatively charged, with ZP values ranging from -28.0

0.97 mV (N-LXN 10) to -61.00 0.99 mV (N-LXN 3) indicating good physical stability of
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niosomal dispersions. Such findings come in accordance with other reports observing negative

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zeta-potential values in non-ionic surfactant vesicles (Junyaprasert et al., 2008, Manosroi et al.,

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2010). LXN-loaded niosomes composed of Spans were found to have higher zeta potential

values, ranging from -49.05 2.19 with Sp60 (N-LXN 8) to -61.00 0.99mV with Sp20 (N-LXN
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3) compared to the vesicles manufactured with Tweens, showing lower zeta potential values

ranging from -28.0 0.97 for Tw40 (N-LXN 10) up to-41.80 1.88 for Tw40 (N-LXN 15).
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These results confirm the significant effect of the type of SAA and SAA: CH ratio used on the
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measured ZP values; however there was no significant difference between ZP values of Sp20,

Sp40 and Sp60 (P > 0.05) (Table 2) at the different studied SAA: CH ratios. Factorial design
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charts describing the effect of changing type of SAA, changing SAA: CH ratio and their

interaction on EE %, particle size & zeta potential values of LXN loaded niosomes. The charts
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shown in Fig.1confirm the effect of independent parameters on the dependent factors of LXN

loaded niosomes.

Analysis of variance (ANOVA), applied at a 5% signicance level, indicated that the postulated

regression model was statistically signicant and valid. This proved that type of SAA, SAA: CH
 ratio and their interaction have a significant effect on the EE% of LXN niosomes and PS as well

as ZP.

3.2. Selection of LXN-loaded niosomes

In the present study, factorial design was used to choose a formulation with appropriate

physicochemical properties for the incorporation of LXN; a hydrophilic drug for optimum ocular

delivery. To achieve the desired target, the choosen formulation should possess high EE% as
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well as high ZP to ensure efficient encapsulation of the drug within the vesicles with maximum

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stability. On the other hand, PS values have to be small enough such that the niosomal

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dispersions is well tolerated in the ocular mucosa avoiding eye irritation and at the same time

facilitating the transport and uptake from the cornea (Araujo et al., 2009). LXN loaded niosomes
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were selected for the responses Y1 (EE %), Y2 (Thompson) and Y3 (ZP). The desirable range of

these responses was set to: Y1 50 %, Y2 180 nm and Y3 |-30| mV, respectively. The
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optimum values of the variables were obtained by graphical and numerical analyses using the
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Design-Expert 8 software and based on the criterion of desirability (Basalious et al., 2010).

Based on the previous results, five niosomal formulations (N-LXN3, N-LXN5, N-LXN14, N-
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LXN15 and N-LXN17) were selected and were included in further investigations.

3.3. In vitro release study

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In vitro release profile of the selected LXN niosomal formulations is shown in Fig.2. As

illustrated in the figure, LXN niosomal formulations were able to release LXN in a sustainable

manner where the percentage of drug released after 8 hours ranged from 71.16 1.63 % (N-LXN

5) to 87.44 0.80 % (N-LXN 15), while 50% of LXN diffused into the release medium from an

aqueous solution of the drug (as a control) within the first hour and about 95% released within
 the second hour. Higher release of LXN from the solution may be due to the presence of LXN as

a non-encapsulated drug, while the incorporation of the drug within niosomal vesicles hinders

the drug release showing slower release pattern. The niosomal dispersions exhibited biphasic

release behavior characterized by an initial rapid release phase where approximately 25-30 % of

LXN is released during the first hour, followed by a slower release phase over the next eight

hours. The initial fast rate of release is commonly ascribed to drug detachment from niosomal

surface while the later slow release results from sustained drug release from the inner lamellae of
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the prepared vesicles (Nounou et al., 2006).

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Considering all selected formulations, percent drug released from Tween based niosomes (N-

LXN14, N-LXN15 and N-LXN17) was higher than that from Span based niosomes (N-LXN3,
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N-LXN5). This result can be attributed to the structural differences in niosomes assembly. In

case of Tweens based niosomes, the bilayer structure upon hydration of the lm would be looser
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and more exible due to high aqueous solubility of Tweens, leading to an increased permeability
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to solutes (Girigoswami et al., 2006). It has been stated that more hydrophobic Spans form more

compact niosomes when hydrated in presence of cholesterol (Girigoswami et al., 2006) which

could allow lower drug diffusion to the release medium (Kamel et al., 2013). Bayindir and
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Yuksel, reported the same result where Paclitaxel release from niosomes composed of Tweens
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was faster than from those composed of Spans (Bayindir and Yuksel, 2010).

The in vitro release data obtained for the selected niosomal formulations were fitted into various

kinetic models like zero-order, first-order and Higuchis model in order to calculate the

correlation coefficients (R2) and determine the release mechanism (Nounou et al., 2006). The

release of LXN from niosomal dispersions fitted best to Higuchi square root release kinetics

showing the highest correlation coefficients (R2) indicating a diffusion controlled release model
 (Nounou et al., 2006), hence, confirming the ability of the formulated niosomes to act as a

reservoir for a sustained delivery of LXN (Abdelbary and Aburahma, 2014).

Based upon the obtained results, N-LXN 14 (Tw60: CH, 1:1) was choosen for further

investigations having higher drug EE% value as well as sustainable drug release capability.

3.4. Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) was conducted to investigate the morphology of LXN
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loaded niosomes. Fig.3. shows the micrographs of N-LXN 14 revealing well identied vesicles

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presented in a nearly perfect sphere-like shape having a smooth surface. Diameters of the

vesicles observed in the micrographs were in accordance with the data obtained by particle size
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analysis.

3.5. pH evaluation
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The eye has limited buffering capacity as the ocular comfort pH ranges from 6.6 to 7.8 for

topical ocular formulations (Kumar and Sinha, 2014). In some cases, the instillation of a solution

with a pH different from tears causes irritation and painful sensation (Ammar et al., 2009). The
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pH value of N-LXN 14 was 7.3 0.01, being therefore suitable for ocular application.
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3.6. Differential scanning calorimetry (DSC)

Differential scanning calorimetry is a basic method used to provide information about the

thermal behavior, structure changes, crystallinity and interaction between a drug and other

materials (Klajnert et al., 2006, Jin et al., 2013). The DSC thermograms of N-LXN 14 and its

individual constituents: Tw60, CH, and LXN are presented in Fig.4. The thermogram of LXN
 exhibited single endothermic event with peak at 297C, corresponding to its previously reported

melting point, confirming its crystalline nature (Nanjwade et al., 2012). The thermograms of

Tw60 and CH exhibited endothermic peaks at 25.13C and 149.8C respectively, corresponding

to their melting temperatures. The data clearly showed that the melting endotherm of LXN in N-

LXN 14 was not recorded indicating the conversion of crystalline LXN to amorphous form in

the niosomal formulation (Jin et al., 2013).

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3.7. Stability study

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After 3 months of storage at 4 2 C, N- LXN 14 showed a good physical stability without

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sedimentation, or layer separation. The physical characteristics of the selected formula such as

color, shape and smell were not changed throughout the 3 months period. In order to evaluate
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the stability of the niosomal vesicles, EE% and PS were used as characterization tools because

the change in these parameters might influence the niosomal behavior. Table 3 showed the effect
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of the storage on the selected parameters from the first day of preparation and throughout 1, 2
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and 3 months of storage. Statistical analysis of the data revealed that the differences in these

parameters from the same formula when freshly prepared were not significant (p > 0.05).
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The storage of LXN niosomes at refrigerator temperature insured physical stability for at least

three months. Good stability may be attributed to the presence of cholesterol in the bilayers
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which increased the membrane stabilizing ability and dramatically reduced the leakage of

encapsulated drug (Rogerson et al., 1987). Also, cholesterol produced an optimum

hydrophobicity which decreased the formation of the transient hydrophilic holes, by decreasing

fluidity, responsible for drug leakage through the bilayers (Manosroi et al., 2002, Padamwar and

Pokharkar, 2006). At the same time, high zeta potential of these systems might increase
 niosomal stability by reducing the rate of aggregation and fusion due to electrostatic repulsion

(du Plessis et al., 1996).

3.8. Ocular irritancy test

For an ophthalmic drug carrier to be used safely, it is important to be tested for its ocular

tolerability. Therefore, the ocular irritancy test was performed on the selected formulation; N-

LXN 14 where the left eye of each of the three albino rabbits used, received LXN vesicular
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formula and the right eye served as control. Visual observation showed none of the following

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signs: redness, inflammation or increased tear production compared to the contralateral eye

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(control). Thus, confirming the safety of the used non-ionic surfactants to be applied topically in

the eye. Hence, the prepared vesicular formulae were non-irritant to the eye and suitable for
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ophthalmic use.
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3.9. Susceptibility test

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It is known that if a formulation can overcome the rapid elimination mechanisms of the eye, it

may deliver more amount of drug. Therefore, microbiological susceptibility test evaluating the
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level of LXN in the external eye tissue of albino rabbits was performed following the topical

application of N-LXN 14 and Orchacin (Fig. 5). The results showed that N-LXN 14 niosomal
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vesicles resulted in a significantly higher percentage inhibition of bacterial growth compared to

Orchacin. LXN antibacterial activity associated with the application of Orchacin eye drops

reached its peak after nearly 1 hour and then it started to fade afterwards. In contrast, LXN-

vesicular formulation exhibited a noticeable different effect starting from the first hour and

increasing gradually by time. As demonstrated in the Figure, N-LXN 14 reached its highest

effect after nearly 3 hours then the formulation showed a fairly constant antibacterial effect all
 over the study period (12 h). This may be attributed to the potential advantage of vesicular

carriers in providing an intimate contact with the corneal and conjunctival surface, thereby,

increasing the probability of ocular drug absorption (Kaur et al., 2004). The AUC 0-12h for the

tested vesicular formulae as well as Orchacin eye drops was calculated. The AUC 0-12h of N-

LXN 14 was significantly higher (P< 0.05) compared to Orchacin (604.59 0.05 and 126.25

0.049, respectively). It is clear from the obtained results that the prepared LXN vesicular

formula achieved significantly higher residence time compared to that obtained from the
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commercially available eye drops (P< 0.05)

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3.10. Induction of bacterial conjunctivitis and topical therapy

The bacterial conjunctivitis in all experimental rabbit groups was conrmed after 48 h of
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infection and then topical therapy was started. The ocular therapeutic activity of the selected

LXN-vesicular formula was compared to the commercially available LXN eye drops in treatment
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of pre-induced bacterial conjunctivitis in rabbits eyes.

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The corneal colony counts for each group of rabbits are presented in Fig.6 . As illustrated in the

Figure, the therapy with LXN vesicular formula was able to successfully treat the bacterial
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conjunctivitis. N-LXN 14 signicantly reduced the number of viable S. aureus recovered

reducing the signs of infection compared to Orchacin the commercial LXN eye drops and the
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untreated control group (P<0.05). Since the seventh day of treatment, there was not any viable

bacterial count for N-LXN 14 treated groups and after 8 days treatment, S. aureus was

completely eradicated from the eyes of all rabbits.

Figure 7 photographs demonstrate the observation of the infected eyes after treatment with N-

LXN 14, Orchacin and the control group (left untreated), photos were taken at the beginning
 of the study (day 0), after 4 days and at the end of the study period (day 8) (Fig 7). The

photographs obtained after 4 and 8 days of therapy were in accordance with the results of colony

counts. At the end of 8th day, it was observed that the eyes infected with corneal conjunctivitis

completely healed when treated with N-LXN14 compared to the Orchacin treated group,

where the rabbits eyes still showed signs of redness and inflammation. Thus, in vivo studies

investigating the efcacy of LXN vesicular formulations in the treatment of the bacterial

conjunctivitis confirmed the enhanced antibacterial therapeutic efcacy of the vesicular carriers
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compared to the available market product.

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3.11. Conclusion

In the present study, the production of topical LXN loaded niosomes for management of corneal
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conjunctivitis was investigated. A full factorial design was employed to statistically optimize the

studied formulation variables. N-LXN14, the selected niosomal formulation exhibited high drug
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EE%, small PS and high ZP having spherical shaped vesicles. Ocular irritancy test confirmed the
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non-irritant nature of topically applied N-LXN14. The performed in vivo study involving the

treatment of pre-induced bacterial conjunctivitis in rabbits eyes suggested that niosomes

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promoted LXN penetration localizing its effect in the cornea, compared to available market

product. Moreover, complete and fast recovery of the infected eyes was accomplished at the end
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of study period. In conclusion, the obtained results confirmed that LXN loaded niosomes have

significantly efficient therapeutic value in treatment of bacterial conjunctivitis proving the

potential of the investigated niosomes as carrier systems for prolonged and effective ocular

delivery of LXN.

Acknowledgements
 The authors would like to thank Egyptian National Research Centre, Cairo, Egypt, for financial

support.

Declaration of interest

The authors report no declarations of interest.

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 Table 1. Independent and dependent variables of the factorial design

Factors (independent variables) Levels

X1: Type of SAA Sp20 Sp40 Sp60 Tw40 Tw60 Tw80

X2: Ratio of SAA:CH 1:2 1:1 2:1

Responses (dependent variables)

Y1: Entrapment efficiency (EE %)

Y2: Particle size (PS)

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Y3: Zeta potential (ZP)

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 Table 2. Composition of LXN loaded niosomes (n=3)

X1:Type of X2:Ratio
Runs Y2: PS (nm ) * Y3: ZP (mV) *
SAA (SAA:CH) Y1: EE% *
N-LXN 1 1:2 45.14 2.52 369.0 9.23 -60.05 0.79
N-LXN 2 Sp20 1:1 58.54 2.02 264.0 6.02 -55.75 1.87
N-LXN 3 2:1 59.29 1.83 152.0 7.10 -61.00 0.99
N-LXN 4 1:2 42.53 4.64 330.0 5.52 -55.15 0.45
N-LXN 5 Sp40 1:1 50.29 3.92 133.0 7.05 -51.05 0.70
N-LXN 6 2:1 52.49 0.74 205.0 6.36 -57.35 2.47
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N-LXN 7 1:2 40.69 3.97 211.0 2.83 -55.60 2.40

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N-LXN 8 Sp60 1:1 44.86 2.51 96.0 0.86 -49.05 2.19
N-LXN 9 2:1 46.81 6.52 97.0 1.29 -51.05 0.98

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N-LXN 10 1:2 52.35 0.15 200.0 1.41 -28.00 0.97
N-LXN 11 Tw40 1:1 50.48 0.45 199.0 1.07 -39.25 0.99
N-LXN 12 2:1
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41.64 1.74 154.0 2.43 -40.10 1.84
N-LXN 13 1:2 67.56 0.97 300.0 7.42 -37.15 1.56
N-LXN 14 Tw60 1:1 68.41 0.07 176.0 0.98 -40.70 2.20
N-LXN 15 2:1 50.39 0.20 133.0 9.90 -41.80 1.88
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N-LXN 16 1:2 59.01 0.19 338.0 4.53 -29.20 0.31

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N-LXN 17 Tw80 1:1 55.90 1.30 177.0 8.05 -38.35 3.18

N-LXN 18 2:1 46.48 1.76 205.0 7.07 -37.05 1.76
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*Values represent (mean S.D).

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 Table 3: Changes of EE% and vesicle size of the selected N-LXN 14 loaded niosome

Storage period (month) EE % * Vesicle size (nm) *

Freshly prepared 68.41 2.07 176.0 6.98
1 67.01 1.91 179.2 4.31
2 66.23 2.45 181.8 5.32
3 66.43 3.12 182.01 7.21

*Values represent (mean S.D).

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