Journal of Drug Delivery Science and Technology 31 (2016) 22e34

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Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

Research paper

Levofloxacin hemihydrate ocular semi-sponges for topical treatment

of bacterial conjunctivitis: Formulation and in-vitro/in-vivo
characterization
Osama Saher*, Dalia M. Ghorab, Nadia M. Mursi
Department Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to formulate and evaluate lyophilized, long acting, levofloxacin hemihydrate ocular
Received 3 October 2015 semi-sponges that would fit cul-de-sac shape for bacterial conjunctivitis treatment. Formulae were
Received in revised form prepared using casting/freeze drying technique employing a (41
31) full factorial design to examine the
29 October 2015
effects of polymer type (Gelrite, chitosan (low and high molecular weight), and sodium carboxymeth-
Accepted 13 November 2015
Available online xxx
ylcellulose), and concentration (1%, 1.5%, 2%) on viscosity of the formed solutions, quantity of drug
released after 12 h (Q12h) and time for 50% of the drug to be released (T50%). Formulae were evaluated for
weight and content uniformity, surface pH, water uptake, and in vitro drug release with its kinetic
Keywords:
Levofloxacin
analysis. The optimal formula was chosen using Design-Expert® software and subjected to scanning
Casting/freeze drying technique electron microscope imaging, g-sterilization and in-vivo evaluation. Results showed that formula G 2 (2%
Bacterial conjunctivitis w/w Gelrite) had the highest desirability (0.894), a zero order drug release profile, and stability after g-
Sustained release sponge sterilization. Formula G 2 showed longer residence time (12 h) in rabbits' eye fluids compared to the
In vivo study commercial Levoxin® eye drops (4 h) with good correlations between in vitro and in vivo results.
Conclusively, Gelrite ocular levofloxacin hemihydrate semi-sponges are promising drug delivery systems
that would improve both patient compliance and treatment efficacy.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction concentrations, higher clinical efficacy and lower risk of fluo-

Over one third of eye-related conditions reported by the health
service organizations worldwide are either infectious conjuncti-
vitis, or conditions associated with infectious conjunctivitis [1]. It
may be caused by bacteria, viruses or even fungi, but bacterial
conjunctivitis is more prevalent in children [2]. The main threat of
infectious conjunctivitis lies in eye complications that may cause
blindness, if left untreated.
Fortunately, most ocular, bacterial infections can be treated us-
ing topical fluoroquinolones. They inhibit bacterial DNA synthesis
by inactivation of topoisomerases enzymes causing rapid bacterial
cell death [3,4]. Levofloxacin is a third generation fluoroquinolone
antibacterial agent and it has higher solubility in water at neutral
pH than its parent ofloxacin. This allows the use of higher con-
centration of levofloxacin and achievement of higher ocular tissue
* Corresponding author. Department of Pharmaceutics and Industrial Pharmacy,
Faculty of Pharmacy, Cairo University, Kasr El Aini Street, 11562, Cairo, Egypt.
E-mail address: osama.saher@pharma.cu.edu.eg (O. Saher).
roquinolones resistance [5e8]. Levofloxacin has a broad spectrum
against Gram-positive and Gram-negative bacteria with a higher
activity against Streptococci compared to previous generations [9].
The main dosage form used to deliver most of eye medications
(including levofloxacin) is eye drops. They may offer many advan-
tages as simplicity of formulation, favorable cost advantage and
ease of administration [10]. However, eye drops major drawback is
the need for frequent instillations because topically applied drugs
are rapidly washed off from the eye. Typically, not more than 5% of
the topically applied drugs reach their target in the eye [11]. Lev-
ofloxacin eye drops for example, need to be administered up to 8
times in the first two days of bacterial eye infection treatment [12].
This dosage regimen usually leads to patient incompliance, un-
successful treatment and emergence of bacterial resistance.
Sponges are an example of ocular soluble inserts that showed
promising results as controlled drug delivery matrices [13]. Simply,
sponges are a dispersion of air in a solid matrix prepared by
lyophilization. The end product consists of a sponge like hydro-
philic polymer matrix, in which the drug is embedded [14]. Sponges
as ocular solid dosage forms are a newly emerging candidate that

http://dx.doi.org/10.1016/j.jddst.2015.11.004
1773-2247/© 2015 Elsevier B.V. All rights reserved.
 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34
offers more flexibility, easiness of preparation, and less foreign
body sensation when compared with ocular minitables. However,
few studies were concerned to use sponges to deliver eye medi-
cations. The term semi-sponge is derived from the semi-circular
shape of the formulae produced. This semi-circular shape was
intended to ensure the resemblance and fitting of the formulae in
the lower cul-de-sac, and to minimize the foreign body sensation.
In view of the previously mentioned information, our study
aimed to prepare sustained release ocular semi-sponges of levo-
floxacin hemihydrate. The polymers used are known to be biode-
gradable polymers that are approved by FDA for their safety and
they are commonly used in many ocular drug delivery systems. The
prepared formulae would eliminate the need of repetitive admin-
istration aiming to better patient compliance.
2. Materials and methods
2.1. Materials
Levofloxacin hemihydrate was kindly supplied by El-Gomhouria
Company, Cairo, Egypt. Chitosan [low molecular weight
(30e200 cps) and high molecular weight (800e2000 cps)], Gelrite
(phytagel) and sodium carboxy methyl cellulose (1500e4500 cps)
were obtained from Sigma-aldrich Co., St. Louis, USA. Sodium
chloride powder, sodium bicarbonate powder, and calcium chloride
dihydrate were purchased from El Nasr pharmaceutical company
(Cairo, Egypt). Glacial acetic acid was purchased from El Nasr
chemical company (Cairo, Egypt). Spectra/Pore® dialysis membrane
(12,000e14,000 molecular weight cut off) was purchased from
Spectrum Laboratories Inc. (CA, USA).
2.2. DSC study
Differential scanning calorimetry (DSC) studies were executed
for the used excipients namely; low MW Chitosan (LCh), high MW
Chitosan (HCh), sodium carboxy methyl cellulose (NaCMC) and
Gelrite (G). Physical mixtures of equal amounts of levofloxacin
hemihydrate with the excipients were prepared. Samples (3e4 mg)
were placed in the aluminum pan of Shimadzu differential scan-
ning calorimeter (DSC-60, Shimadzu, Kyoto, Japan). They were
heated in the range 10e400  C at a rate of 10  C/min, with indium in
the reference pan, in a nitrogen atmosphere. DSC and studies were
done for the drug, excipients and drug-excipients mixtures.
2.3. FTIR study
Fourier transform infrared spectroscopy (FTIR) spectra deter-
mination was done for 1:1 physical mixtures of the drug and the
excipients according to potassium bromide disc technique. FTIR
spectrophotometer (Model 22, Bruker, UK) was used and the
wavenumber range was between 4000 and 500 cm1. FTIR spectra
were established for the drug, excipients and drug-excipients
mixtures.
2.4. Preparation of levofloxacin hemihydrate semi-sponges using
casting/freeze drying technique
The semi-sponges were prepared using casting/freeze drying
technique. Levofloxacin hemihydrate was dissolved in 1% v/v acetic
acid (in case of using chitosan) or in water for injection (in case of
using other polymers) to give a dose of 0.5 mg levofloxacin hemi-
hydrate per 100 mg solution. Accurately weighed quantities of the
polymers were gradually added to the required amount of the
proper solvent (containing the dissolved drug) with constant stir-
ring (Magnetic stirrer, WiseStir, Wisd Lab. Instruments, USA) to
23
prepare the required concentrations (1, 1.5, 2% w/w). 200 mg of
each polymer solution was poured in each pocket of a specially
designed cylindrical plastic mold (Poly vinyl chloride (PVC blister))
with an internal diameter of 9 mm and a thickness of 3 mm. The
pockets of the mold were divided into two identical halves by the
use of plastic barriers to produce the desired semi-sponges. Plastic
molds were then stored in a freezer at 22  C for 24 h. The frozen
solutions were then placed in a lyophilizer (Novalyphe-NL 500;
Savant Instruments Corp., Holbrook, NY, USA) for 24 h and the
condenser temperature was - 45  C and under vacuum of 7
102
mBAR. To eliminate the possibility of moisture absorbance and to
obtain soft, flexible sponges [15], the produced semi-sponges were
stored in a desiccator containing anhydrous calcium chloride as a
desiccant and stored at room temperature to be used within one
week from their preparation and storage.
g-sterilization was performed for the optimum formula in the
presence of dry ice to avoid the problems that might occur due to
temperature elevation associated with g-irradiation [16]. Irradia-
tion was performed using a 60Co irradiator (National Center for
Radiation Research & Technology, subordinate to Atomic Energy
Authority, Nasr City, Egypt). Irradiation was done at a dose of
25 kGy at a dose rate of 1.88 kGy/hr [17].
2.5. In-vitro evaluation of the prepared levofloxacin hemihydrate
ocular semi-sponges
2.5.1. Physical characterization
Weight variation testing was done using ten randomly selected
semi-sponges from each formula, which were separately weighed
(Electric balance, AND Co, Ltd, Japan) and mean weight was
estimated.
Content uniformity was evaluated for the tested semi-sponge.
To assure complete dissolution, formulae were stirred overnight
in 100 mL simulated tear fluid (STF-pH 7.4). STF was freshly pre-
pared using sodium chloride 0.67 g, sodium bicarbonate 0.2 g,
calcium chloride.2H2O 0.008 g, and purified water added to 100 g
[18]. The solution was then filtered through a 0.45 millipore filter
and analyzed spectrophotometrically at 288 nm after adequate
dilution with STF. The test was done in triplicate and the average
drug content was calculated.
The surface pH of the semi-sponges was also determined. Each
semi-sponge was allowed to swell by keeping it in contact with
2 mL of STF for 2 h at room temperature [19]. The pH was estimated
by bringing a pH strip (Merck Millipore®, Merck KGaA, Darmstadt,
Germany) in contact with the surface of the semi-sponge and
allowing it to equilibrate for 1 min.
2.5.2. Water uptake
Water uptake of the prepared formulae was assessed gravi-
metrically at room temperature [20]. A small filter paper
(d ¼ 15 mm, Whatman® Inc., Piscataway, NJ, USA) was cut into
small circular pieces sufficient to hold tested semi-sponges over
them. Filter paper pieces were then placed over an agar gel plate
(1% w/v). This set-up was equilibrated for one hr and filter paper
pieces were weighed (mfilter). The accurately weighed semi-
sponges (md) were then placed on the upper side of the filter pa-
per pieces in the covered plates. At certain time intervals up to 3 h,
weights of the swollen semi-sponges (mw) were recorded where
(mw ¼ mtotalmfilter). The study was done in triplicate and the mean
was calculated. Percentage water uptake of semi-sponges (W) was
calculated using the following equation:
24 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

(case II) transport (where the release rate is independent of time

mw  md
W¼
100 (1) and involves polymer relaxation), and n > 0.89 suggests a super
md case II transport [31].

2.5.6. Analysis of the factorial design

2.5.3. Rheological characterization
Viscosities of different polymer solutions were determined
before casting using cone and plate viscometer (Brookfield Co.,
Model HBDV-I þ CP, Middleboro, MA, USA). The study tempera-
ture was (35  C þ 0.5) to simulate eye temperature and the
angular velocity was increased gradually from 0.5 to 100 rpm using
CPE-41 spindle. Accurate weight of each tested solution was applied
to the plate and viscosities were determined at minimum and
maximum rate of shear over the aforementioned range of speeds
with 10 s between each two successive speeds. The torque was
confirmed to be within the acceptable range (10e100%) before
taking any results [21]. Rheological data obtained were shear stress
and viscosity at different rates of shear. These data were fitted to
power law model to study the type of flow:
In order to evaluate the effects of polymer type and concen-
tration on water uptake results, viscosity results, and in vitro
release results, a 41
31 full factorial experimental design was
employed using Social Package for Statistical Study software (SPSS
19.0®). In this design (Table 1), polymer type (Lch, Hch, NaCMC,
Gelrite) and concentration (1, 1.5 and 2%) were selected as inde-
pendent variables, whereas percentage water uptake after 3 h,
viscosity at minimum rate of shear, (Q12h), and (T50%) were chosen
as dependent variable. The analysis was done using one way
ANOVA test followed by Post Hoc's least significant difference (LSD)
test was used in order to compare between individual variable
levels and to confirm where the differences occurred. Difference at
P < 0.05 was considered to be significant.

t ¼ Kgn (2) 2.5.7. Selection of optimal formula through the determination of the
desirability factor
Where, t is the shear stress, g is the rate of shear, K is the con-
Desirability factor for the prepared formulae was determined by
sistency index (sec.) and n is the flow index (dimensionless). For
Design Expert® software. The criteria for the optimization were set
shear thinning fluids, n lies between zero and one while it ap-
at the highest polymer solution viscosity, the lowest Q12 and the
proximates one in case of Newtonian systems and exceeds one in
highest T50%.
dilatant systems [22].

2.5.4. In-vitro release studies
In vitro release of levofloxacin hemihydrate from the prepared
semi-sponges and lyophilized drug solution was determined using
membrane diffusion technique [23e25]. A glass cylinder having the
length of 10 cm and diameter of 2.5 cm fitted at its lower end with
cellulose dialysis membrane presoaked in STF. Semi-sponges were
transferred inside the glass cylinder to rest over the inner surface of
the dialysis membrane. The cylinder was attached from the other
end to the shaft of the dissolution apparatus, instead of the baskets,
and then lowered to the vessels of a USP dissolution apparatus
(Hanson Research Corporation, California, USA) containing 50 mL
STF. The shafts were rotated at a constant speed (20 rpm) and the
release medium was kept at a temperature of 35 ± 0.5  C [26e28].
Samples (1 mL) were withdrawn at specific time intervals and
replaced by equivalent volume of fresh STF kept at the same tem-
perature. Spectrophotometric assay for drug content was done at
the predetermined lmax (288 nm) after sufficient dilution with STF.
The test was performed in triplicate and the mean drug released
was estimated.
2.5.5. Kinetic analysis of the release data
The release data was analyzed to determine the mechanism and
the order of drug release from different formulae. Models used for
analysis of the release kinetics were zero order, first order, and
diffusion controlled mechanism according to simplified Higuchi
model [29]. KorsemeyerePeppas model was also chosen to analyze
the drug release kinetics using the following equation (Korsmeyer
et al., 1983):
2.5.8. Scanning electron microscope imaging (SEM)
Scanning electron microscope (SEM) was used to examine sur-
face morphology and cross-sections of the optimum formula. A thin
piece of the selected semi-sponge was fixed on the SEM sample
holder with double-sided adhesive tape and coated with gold using
Edwards Sputter coater under an argon atmosphere to achieve a

film of 150 A thickness. The sample was then examined using SEM
(Jeol, JXA-840A, Tokyo, Japan) [15,32].
2.5.9. Effect of g-sterilization
Physical characteristics and content uniformity were evaluated
for the optimum formula before and after sterilization. Similarity
factor (f2) was calculated to detect any change in the release profile
that might occur by sterilization using this equation:
" 1 #
W 2
f2 ¼ 50
log10 1þ
100 (4)
n
Where W is the sum of squares of differences in the cumulative
percent dissolved between reference (release profile before steril-
ization) and test (release profile after sterilization) and n is the
number of sampling times with a percent 85% [33].
Table 1
41
31 full factorial design independent variables and optimization criteria of the
prepared formulae.
Factors (independent variables) Levels

Mt X1: Polymer type LCh HCh NaCMC G

¼ Kt n (3) X2: Polymer concentration 1% 1.5% 2%
M∞
Responses selected Desirability constraints (optimization
The diffusional exponent (n) depends on mechanism of release criteria)
and drug delivery device shape [30]. For our case, we used pa- Y1: Polymer solution viscosity maximize
rameters of cylindrical tablets. So, n  0.45 corresponds to a Fickian Y2: Q12 minimize
(case I) diffusion, 0.45 < n < 0.89 means an anomalous (non-Fick- Y3: T50% maximize
ian) transport (where a combination of diffusion and polymer Low MW Chitosan (LCh), high MW Chitosan (HCh), sodium carboxy methyl cellulose
relaxation controls the release), n ¼ 0.89 indicates a zero order (NaCMC) and Gelrite (G).
 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34
2.6. In vivo characterization of prepared levofloxacin hemihydrate
semi-sponges
2.6.1. Eye irritation test
The in vivo characterization of safety of the selected formula was
done by performing eye irritation test. Rabbits were selected as
animal models for the test as their eyes showed to be more
vulnerable to the exposure to irritating substances than the eyes of
humans [34]. A group of 3 animals was used for the selected semi-
sponge. The formula was g-sterilized (dose: 25 kGy) prior to
application, and then was inserted into the lower conjunctival sac
of one eye, whilst the other eye served as control. Both eyes of the
rabbits under test were checked for any irritation signs (redness,
inflammation, or tear production increase) based on direct visual
observation using a slit lamp, before treatment, and 1, 4, 8, 12 and
24 h after instillation [28,35,36].
2.6.2. Microbiological susceptibility testing
Assessment of levofloxacin hemihydrate level in the external
eye tissue of rabbits is expected to indicate residence time of the
selected semi-sponge. This assessment was achieved by comparing
the level of levofloxacin hemihydrate from the selected semi-
sponge and commercially available Levoxin® eye drops (0.5% lev-
ofloxacin hemihydrate, Amoun pharmaceutical Company, Egypt),
via employing a microbiological susceptibility testing after topical
application. The study was performed using a parallel design, in
which six male rabbits (weighing 1.5e2 Kg and with no eye dis-
eases) were chosen for the study. All the experimental procedures
with animals were carried out after being approved by the insti-
tutional review board of the Research Ethics Committee of Faculty
of Pharmacy, Cairo University, Egypt and were in agreement with
the ethical principles of EU Directive 2010/63/EU for the use of
animals in scientific ocular researches.
The rabbits were randomly divided into two groups each con-
sisted of 3 rabbits. The optimized formula was applied to the first
group, while the second group took the same semi sponge dose as
100 mL of the commercially available Levoxin® eye drops (0.5%
levofloxacin hemihydrate, Amoun pharmaceutical Company,
Egypt). Sterile 6 mm diameter filter paper discs were placed under
the lower eyelid of the rabbit's eye for only 1 min at specific time
intervals (0.5,1,2,4,6,8,10 and 12 h) after application. Care was taken
during sampling to avoid irritation of either the eyelid or the eye.
Each disc was stored at 20  C in a plastic Eppendorf for 24 h. Discs
were then placed using sterile forceps under aseptic laminar flow
on the surface of Muller Hinton Agar (MHA, Difco) that was pre-
viously inoculated using non-sporulated Staphylococcus aureus
bacteria, being one of the common causative pathogens of bacterial
conjunctivitis. Incubation of the plates was done at 37 ± 0.5  C for
24 h. Average diameters of the inhibition zones around the discs
were recorded and used to compare the antibiotic levels in the
external eye tissue according to the following proposed score
guide: zone diameter 25 mm ¼ “þþþþþ”, 20 mm ¼ “þþþþ”,
15 mm ¼ “þþþ”, 10 mm ¼ “þþ”, 6e10 ¼ “þ”, no inhibition
zone ¼ “” [28].
2.6.3. In vitro e in vivo correlation (IVIVC)
In vitro e in vivo correlation (IVIVC) is setting up a rational
relationship between a dosage form produced biological property,
or even a parameter derived from a biological property, and the
same dosage form characteristic or physicochemical property [37].
Trials done to establish relations between the average diameters of
inhibition zones at different time intervals with in vitro release
results at the same intervals. In case of the optimum formula, first
relation constructed was between log percentage drug released and
square of average diameter of inhibition zone. Second relation was
25
adopted between fraction released and fraction diameter of inhi-
bition zone. In both relations, the linear regression coefficient (R2)
was calculated.
3. Results
3.1. DSC
In the present study, DSC thermograms of levofloxacin hemi-
hydrate showed peaks that were in agreement with the docu-
mented DSC chart under the same heating rate 10  C/min [38]. Pure
levofloxacin hemihydrate exhibited four endothermic peaks the
first at ~91  C due to the dehydration of levofloxacin hemihydrate,
the second at ~227  C due to the melting of the g form, the third at
~231  C due to the melting of the b form and the last one at ~234  C
due to the melting of the a form. The observed exothermic peaks
might be due to the crystallization of an amorphous form that
resulted partially from levofloxacin dehydration [38]. Endothermic
values of levofloxacin hemihydrate showed no considerable
changes when mixed with other excipients compared to that of
pure levofloxacin hemihydrate.
3.2. FTIR
The FTIR spectra confirmed the absence of any chemical in-
teractions with the used excipients as the DSC studies. Pure levo-
floxacin hemihydrate showed characteristic peaks for the eOH
group of the eCOOH moiety at 3265 cm1 and eC e O peak at
1724 cm1. The aromatic CeH peaks were also observed at
2935 cm1 [39]. There were no significant changes in these peaks in
the prepared mixtures.
3.3. In-vitro evaluation of the prepared levofloxacin hemihydrate
ocular semi-sponges
3.3.1. Physical characteristics, weight variation and content
uniformity
Freeze-dried semi-sponges were white in colour with sponge
like structure. Semi-sponges were hard enough to withstand
handling and at the same time not too hard to avoid hurting the
eye. The average thickness 2.7 ± 0.21 mm and average radius was
4.4 ± 0.13.
Average weights of semi-sponges ranged from 1.63 ± 0.17 to
3.2 ± 0.3 mg. The weight of the prepared semi-sponges increased
with increment in the used polymer concentration.
The average percent drug content of all semi-sponges lied
within 92.6 ± 0.03 to 111.8 ± 0.04% of the labeled claim. This
complied with the pharmacopoeial limits (85%e115%) and proved
there was homogenous drug distribution in the formulae.
The pH of all formulae ranged from 7 to 8. The safe pH range for
eye preparations is from 6.5 to 8.5. Within this range, there is no
chance for eye irritation or damage. These results revealed that all
formulae had an acceptable pH and would not produce any local
irritation or corneal damage upon application.
3.3.2. Water uptake
Percentage water uptake values of the prepared formulae after
3 h of the study varied from 1553 ± 92% to 3612 ± 45.29%. Results of
the 41
31 full factorial design showed that the type of the polymer
had significant effect (p < 0.05) on water uptake (Fig. 1a). LSD test
revealed that the water uptake results of the sponges prepared
from NaCMC was significantly higher from that of the Lch, Hch, or
Gelrite sponges. Oppositely, water uptake values of the sponges
prepared using Lch, Hch, or Gelrite were not significantly different
from each other.
26 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

Fig. 1. Effect of (a) polymer type and (b) polymer concentration on water uptake values. “Lch: Low chitosan, Hch: High chitosan, NaCMC: Sodium carboxymethyl cellulose, G:
Gelrite”.

Regarding the polymer concentration, results reveal that it had a
significant effect (p < 0.05) over values of water uptake (Fig. 1b).
LSD test showed that the water uptake result of the sponges con-
taining 2% of the polymer was significantly lower than the values of
Lch, Hch, or Gelrite sponges. However, water uptake values of the
sponges prepared using 1% or 1.5% showed no significant difference
from each other.
3.3.3. Rheological characterization
All tested solutions exhibited pseudoplastic behavior associated
with thixotropy, which is vital to decrease interference with
blinking. Viscosities of polymer solutions at minimum and
maximum rate of shear are recorded in Table 2.
Results of the 41
31 full factorial design showed that the type
of the polymer had significant effect (p < 0.05) on viscosity results
(Fig. 2a). LSD test revealed that all polymer types were significantly
different from each other. The order of viscosity of polymer solu-
tions according to polymer type was NaCMC > Gelrite > Hch > Lch.
Regarding polymer concentration, It worth noting that
increasing the concentration caused a significant increase
(p < 0.05) in viscosity values (Fig. 2b). LSD test revealed that all
concentrations were significantly different from each other.
3.3.4. In-vitro release studies
The results of the in-vitro release of levofloxacin hemihydrate
from the different ocular semi-sponges were graphically repre-
sented in Fig. 3. In order to understand the barrier presented by the
dialysis membrane, the in vitro release study for plain lyophilized
drug solution of the same concentration was carried out in the
same manner. It was clear from the figure that the release of lev-
ofloxacin hemihydrate from the lyophilized drug solution was
markedly faster than that from the prepared formulae.
Results of the 41
31 full factorial design were analyzed (Fig. 4),
and revealed that both polymer type and concentration had sig-
nificant effect (p < 0.05) on (Q12h) and (T50%). Subsequent LSD test
showed that both Hch and NaCMC semi-sponges were not signifi-
cantly different from each other.
(Fig. 5) shows the combined effects of the type and concentra-
tion of the polymer on both (Q12h) and (T50%). From the figure,
there was significant interaction (p < 0.05) between the two factors.
Formula containing 2% Gelrite was different from the other
formulae, having the highest (T50%) and the lowest (Q12h) among
all formulae. In addition, the figure reveals the minor significance of
polymer type on the values of (Q12h) at low polymer concentration
(1%).

Table 2
Viscosities of prepared polymer solutions at minimum and maximum rates of shear.

Formula codea Viscosity at minimum shear rate (Cp) ± S.D Viscosity at maximum shear rate (Cp) ± S.D

LCh 1 1636 ± 449 19 ± 3

LCh 1.5 6250 ± 2173 77 ± 9
LCh 2 12,857 ± 1904 93 ± 40
HCh 1 4060 ± 1126 207 ± 8
HCh 1.5 8186 ± 1656 262 ± 31
HCh 2 14,190 ± 2086 328 ± 6
NaCMC 1 13,016 ± 4200 332 ± 10
NaCMC 1.5 21,333 ± 577 1481 ± 46
NaCMC 2 25,620 ± 1579 18,356 ± 134
G1 3964 ± 3342 40 ± 11
G 1.5 13,000 ± 1520 88 ± 11
G2 17,444 ± 1953 212 ± 70

Low MW Chitosan (LCh), high MW Chitosan (HCh), sodium carboxy methyl cellulose (NaCMC) and Gelrite (G).
a
Mean of three experiments.
 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34 27

Fig. 2. Effect of (a) polymer type and (b) polymer concentration on viscosity of polymer solution. “Lch: Low chitosan, Hch: High chitosan, NaCMC: Sodium carboxymethyl cellulose,
G: Gelrite”.

Fig. 3. In vitro release profile of levofloxacin hemihydrate from semi sponges formulated using (a) Low chitosan; (b) High chitosan; (c) NaCMC and (d) Gelrite in STF (pH 7.4) at 35  C
in comparison to lyophilized drug solution.

3.3.5. Kinetic analysis of the release data coefficient of determination (R2) deduced for the parameters
The preference of a certain mechanism was based on the studied. The highest coefficient of determination was preferred for
28 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34
Fig. 4. Effect of (i) polymer type and (ii) polymer concentration on (a) Q12 h, (b) T50%. “Lch: Low chitosan, Hch: High chitosan, NaCMC: Sodium carboxymethyl cellulose, G: Gelrite”.
the selection of the order of release. The data is presented in Table 3
showed that the release of levofloxacin hemihydrate from all
formulae followed diffusion controlled mechanism according to
Higuchi model except formula G 2 that followed zero order of
release. According to KorsemeyerePeppas model, all formulations
had n values between 0.45 and 0.89 indicating an anomalous (non
Fickian) transport.
3.3.6. Selection of optimal formula through the determination of the
desirability factor
The selection criteria of the best semi-sponge formulae were the
highest polymer solution viscosity, the lowest Q12h and the highest
T50%. Formula containing 2% w/w Gelrite (G 2) showed the highest
desirability value (0.894) as shown in Fig. 6. This formula was
selected for further testing.
3.3.7. Scanning electron microscope imaging (SEM)
Scanning electron micrographs of formula G 2 was conducted.
On top view (Fig. 7a); this semi-sponge had a uniform non-porous
surface. Side views (Fig. 7bed) showed that the investigated for-
mula possessed a porous nature.
3.3.8. Effect of g-sterilization
Both physical characteristics and drug content showed no
extraordinary changes after g-sterilization at (25 kGy). Average
drug content was 107.8 ± 0.021% and complied with pharmaco-
poeial limits (85%e115%). The release of levofloxacin hemihydrate
from formula G 2 before and after g-sterilization showed no sig-
nificant change in the release pattern, as similarity factor was found
to be 67, which was within the acceptable range (50e100) [33,40].
3.4. In vivo characterization of prepared levofloxacin hemihydrate
semi-sponges
3.4.1. Eye irritation test
Eye irritation test showed that the formula G 2 exhibited no
signs of irritation (Fig. 8) over our study period (24 h). Thus, it could
be concluded that the formula was safe and non-irritant to the eye.
3.4.2. Microbiological susceptibility testing
Table 4 shows the score values of inhibition zone at different
time intervals of either commercially available Levoxin® eye drops
or formula G 2. Results revealed that formula G 2 had longer resi-
dence time (12 h) in the fluids of the outer tissues of the eye after
application in comparison to Levoxin® eye drops (4 h only). Fig. 9
shows the pattern of inhibition zone diameter against time
following the application of the market product Levoxin® and G 2
semi-sponge. Statistical analysis of inhibition zone diameters at
 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34 29

Fig. 5. Combined effects of polymer type and concentration on (a) T50% and (b) Q12h.

Table 3
Kinetic analysis of the release data of all levofloxacin hemihydrate semi-sponges.

Formula R2a Mechanism Korsmeyer e Peppas model T50% (minutes)

Zero First Diffusion Diffusion exponent (n) R2 Mechanism

NaCMC 1 0.921 0.821 0.975 Diffusion 0.718 0.999 Anomalous 37.73

NaCMC 1.5 0.905 0.752 0.974 Diffusion 0.799 0.995 Anomalous 55.73
NaCMC 2 0.968 0.814 0.999 Diffusion 0.573 0.992 Anomalous 55.03
G1 0.877 0.729 0.967 Diffusion 0.734 0.990 Anomalous 122.24
G 1.5 0.913 0.796 0.981 Diffusion 0.743 0.996 Anomalous 184.14
G2 0.994 0.845 0.986 Zero 0.681 0.999 Anomalous 458.74
HCh 1 0.891 0.804 0.955 Diffusion 0.569 0.992 Anomalous 18.72
HCh 1.5 0.845 0.415 0.939 Diffusion 0.829 0.998 Anomalous 31.31
HCh 2 0.895 0.734 0.979 Diffusion 0.761 0.998 Anomalous 36.71
LCh 1 0.895 0.734 0.979 Diffusion 0.718 0.996 Anomalous 42.13
LCh 1.5 0.896 0.717 0.978 Diffusion 0.799 0.999 Anomalous 62.92
LCh 2 0.964 0.862 0.997 Diffusion 0.573 0.998 Anomalous 87.68

Low MW Chitosan (LCh), high MW Chitosan (HCh), sodium carboxy methyl cellulose (NaCMC) and Gelrite (G).
a
Underlined values indicate the highest R2 values in each formula.

different time intervals using (SPSS ver. 19®), showed that all di-
ameters were not significantly different from each other except at
12 h (P < 0.05).
3.4.3. In vitro e in vivo correlation (IVIVC)
Two in vitro e in vivo correlations were established for the
optimized formula G 2. The first relation constructed was between
log percentage drug released and square of average diameter of
inhibition zone using third order polynomial regression (Fig. 10a)
(R2 ¼ 0.951) [41]. Polynomial regression is a form of linear
regression that had been generally used to describe nonlinear
phenomena as the rate of growth of tissues [42] and epidemic
diseases progression [43]. Second relation (Fig. 10b) was adopted
between fraction released and fraction diameter of inhibition zone
using third order polynomial regression (R2 ¼ 0.947).
4. Discussion
Water absorption ability is associated with the presence of hy-
drophilic groups such aseOH, eCOOH, and eOSO3H. Water
Entrance into polymer network results in the hydration of these
functional groups, which leads to expansion and ordering of the
polymer chains. The maximum water uptake is achieved, when
balance occurs between osmotic forces of the functional groups and
restrictive forces of the ordered polymer chains [44]. NaCMC results
were similar to the results obtained by Refai & Tag [45] who found
that NaCMC being a charged polymer had a higher extent of water
uptake compared to neutral polymers. Bertram & Bodmeier [23]
also found a good correlation between water uptake of NaCMC
and its charge density. Chitosan and Gelrite are also charged
polymers. Yet, their water uptake is lower than NaCMC. In a study
made by Chu et al. [46] on chitosan, it was demonstrated that
chitosan has neither free negative nor positive charges in neutral
medium. This uncharged nature of chitosan in neutral media was
30 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34
Fig. 6. Desirability results of the prepared semi-sponges.
Fig. 7. Scanning electron micro-graphs of (G 2) formula (a) Top view (magnification 100), (b, c and d) Side views (magnifications 100,160 and 300 respectively).
responsible for the decrease in both the uptake of water and in its
swelling. Regarding Gelrite, Chang et al. [47] suggested that water
uptake behavior of Gelrite showed to be closely related to the
porosity. Certain studies showed that scaffolds having higher
porosity increased water storage space and hence increased the
water uptake [38]. Other studies have suggested that small pore
sizes may potentiate the capillary phenomenon leading to an in-
crease in water absorption. The water uptake profile in our case
showed an initial rapid water uptake that might be caused by
capillary forces. The following reduction in water uptake rate may
be resulted from the formation of a dense gel layer that restricted
water entrance and produced slower hydration [45]. The significant
effect of polymer concentration on water uptake may be explained
that upon increasing the polymer concentration, a highly viscous
gel layer was formed at the contact area between the semi-sponge
and the medium, hence decreasing water uptake. Another expla-
nation stated by Chang et al. [47] that upon increasing polymer
concentration, the porosity of the prepared sponges decreases,
which will consequently decrease the degree of water uptake.
Regarding rheological characterization, Ocular semi-sponges
were supposed to take up tear fluids and transform to a gel or a
viscous solution. The viscosity of polymer solution used for prep-
aration and accordingly the viscosity of the formed gel is of great
importance for the performance of semi-sponges with respect to
water uptake and drug release. The resulted order of viscosity can
be explained as there are many factors that may affect the solution
viscosities and their resistance for the shear applied. In our case,
secondary bonding (namely hydrogen bonds and Van der Waals
forces) was believed to play the major role [48,49]. NaCMC struc-
ture contains free C]O and OH groups that can form strong
intermolecular and intramolecular hydrogen bonds. Moreover, Van
der Waals forces are highly dependent on the substance molecular
size and mass [50]. Thus, the considerable high molecular weight
(reaching 700 KDa) of NaCMC may be responsible for significant
higher Van der Waals forces and higher viscosity compared with
other used polymers. Gelrite and high chitosan may both have
similar molecular mass (around 200 KDa) and so, they have rela-
tively the same Van der Waals forces. However, the number of OH
and C]O groups of the Gelrite molecule is greater and more
available for hydrogen bonding when compared to high chitosan.
This will allow a higher degree of cross linking and hence higher
viscosity. In our case, Gelrite showed higher viscosity than HCh at
minimum shear rate, while it was more easily destroyed than HCh
upon an increase in the shear rate as shown in the results of
 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34 31

Fig. 8. Rabbit's right eye after (a) 1 h and (b) 8 h of formula G 2 application.

Table 4 stated that viscoelastic substances with high viscosity at low shear
Score values of inhibition zone of levoxin and formula G2 according to the afore- rates and low viscosity at high shear rates are preferred, since the
mentioned score guide.
ocular shear rate is very high, ranging from 0.03 s1 during inter-
Time (hrs.) Score of inhibition zones blinking periods to 4250e28,500 s1 during blinking.
Levoxin® Semi-sponge (G2) Low chitosan has the least viscosity, because it has the lowest
molecular weight, and hence the lowest Van der Waals forces
0.5 þþþþþ þþþþ
1 þþþ þþþþ among all the used polymers. The significant effect of concentration
2 þþþ þþþ over viscosity may be attributed to the increase in the degree of
4 þþ þþ cross linking with the increase of polymer concentration.
6  þþ The slower drug release from semi-sponges than the drug so-
8  þþ
10  þþ
lution in all polymers may be a result of the influence of viscosity on
12  þþþ the diffusion of the drug. The results were in accordance with what
described by the StokeseEinstein equation [52], which demon-
Score guide: Inhibition zone diameter  25 mm ¼ “þþþþþ”, 20 mm ¼ “þþþþ”,
15 mm ¼ “þþþ”, 10 mm ¼ “þþ”, 6e10 ¼ “þ”, no inhibition zone ¼ ““. strates that an increased viscosity of the formulation causes slower
diffusion of the drug across the gel matrix.
The pattern of release seems to pass through three different
mechanisms: (1) drug release from the surface of sponges, (2)
diffusion through the swollen rubbery matrix or (3) drug release
due to polymer erosion [53]. The release profiles of all formulae
showed rapid drug release during the first 30 min. This result might
be attributed to the release of the drug adhering to the surface and
not entrapped in the inner matrix of the sponge. These results were
in agreement with results of Foda et al. [15] and Kassem et al. [53].
They reported that a considerable percentage of their drugs were
released within the first hour for both uncross-linked and cross-
linked lyophilized matrices, and they attributed this to the pres-
ence of surface drug. After that, the drug release was due to the
diffusion process, which was much slower when compared to the
initial release. The results of NaCMC and Gelrite can be augmented
by the study done by Michailova et al. [54] which correlated the
water uptake, rheological properties of the used polymers with
drug release rate. Moreover, Gelrite release results was confirmed
by the results obtained by Rupenthal et al. [49] who found that
release rates of carrageenan, xanthan gum and gellan gum were
Fig. 9. Average diameters of inhibition zones around discs indicating drug levels in the significantly lower than those of chitosan, alginate, and HPMC. They
external eye fluids of albino rabbits versus time in hours following the instillation of explained these result by the fact of slow diffusion of the drug
formula (G 2) and levoxin® eye drops.
through the gel matrix. They also assumed the presence of ionic
interactions between the anionic polymer chains of gellan gum,
viscosity at maximum shear rate (Table 1). This may be explained xanthan gum and carrageenan and the positively charged model
by Rupenthal et al. [49] when they demonstrated that there was a drug used, causing additional slowing down of the release rate.
high degree of elasticity (due to secondary bonding) in polymer They added that there might be a repulsion caused between chi-
samples at rest. However, these secondary bonds were easily tosan's positively charged backbone amino groups and the used
destroyed upon an increase in the shear stress. Hence, it is expected drug, facilitating the drug diffusion through the chitosan gel matrix.
that upon blinking there will be a decrease in viscosity, subsequent Gorle and Gattani [55] provided a similar explanation for the zero
reduction in reflex tearing, and consequently prolonged residence. order release from gellan gum films. They stated that the pro-
According to Srividya et al. [51], this would be beneficial. They grammed release of Gatifloxacin from the ocular film prepared may
32 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34
Fig. 10. IVIVC correlations between (a) log % released and square of average diameter of inhibition zone using 3rd order polynomial regression, and (b) fraction released and fraction
of average diameter of inhibition zone using 3rd order polynomial regression.
be due to the formation of hydrogen bonds between the drug and
the polymers used. Gatifloxacin is a fourth-generation fluo-
roquinolone and similar in structure to Levofloxacin. Hence, for-
mation of hydrogen bonds between the drug and the polymer is a
possible scenario in our case and might has contributed to the
controlled release rate of drug. On the other hand, HCh and LCh
results in our study did not comply with the results obtained by Ko
et al. [56] and Polk et al. [57] who both stated that the molecular
weight of chitosan was a fundamental variable in the release of
their drugs from chitosan. They reported that the release rate of
their drugs increased with the decrease in chitosan molecular
weight. However, in our study LCh showed more hindrance for the
drug release than HCh. This result may be attributed to the differ-
ence in the pore size between LCh and HCh sponges. LCh had
smaller pore size than HCh. This might cause a slower entry of the
dissolution medium inside the sponge and hence a slower diffusion
of the drug, resulting in more release retardation by LCh sponges.
This results in the present study are consistent with the previous
observations of Foda et al. [15] who demonstrated that release of
tramadol HCl from LCh sponges was slower than from HCh sponges
because of the smaller pore sizes in LCh sponges compared to
sponges prepared by higher chitosan grades.
The significant effect of polymer concentration over release
results might be explained by the assumption stated by Sankalia
et al. [58] assuming that increasing in the amount of the polymer in
the matrix would increase degree of hydration with simultaneous
swelling that would result in lengthening of the drug diffusion
pathway and reduction in drug release rate. Formula G 2 had the
highest (T50%) and the lowest (Q12h) as a result to the low water
uptake and relatively high viscosity resulted from the combined
effects of polymer type and this high concentration (2 w/w %) used
that consequently retarded the drug release from this formula. The
minor significance of polymer type on the values of (Q12h) at low
polymer concentration (1%) may be explained by the fact that, at
this low polymer concentration, there were rapid release rates of
the drug from the formulae regardless to the polymer type, due to
the low viscosity and high water uptake values at this concentra-
tion. The zero order kinetics of release followed by formula G 2
might also have the same previous explanation as the high viscosity
and the low water uptake, would make the passage of the drug
through the gel layer a rate-limiting step for the release.
The porous nature presented in the side views of the SEM were
probably the cause of water penetration, drug dissolution, and
release. The existence of only one site (straight side) for drug
release might also explain the in-vitro zero ordered drug release
from formula G 2.
 O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34
In vivo results showed to be in coherence with the in vitro drug
release results from formula G 2. This in vivo behavior might be a
result of a combination of the three release mechanisms that
appeared in vitro: drug release from the surface of the sponge,
diffusion through the swollen rubbery matrix and drug release due
to polymer erosion [59]. The large inhibition zones at the first 2 h
might indicate the rapid drug release from the semi-sponge owing
to the dissolution of the drug adhering to the surface and not
entrapped in the inner matrix of the sponge. After the first 2 h, the
diameters of the zones decreased, but still insignificant (P < 0.05)
and remained steady for 10 h. Drug release during this stage might
be due to a combination of both the diffusion process, which was
slower compared to initial rapid release, and the polymer erosion.
The significant increase (P < 0.05) of the inhibition zone diameter
after 10 h might be an indication for the beginning of polymer
erosion predominance over the diffusion process.
These close correlations appeared between in vitro dissolution
results and in vivo data suggested that the stated dissolution
method could be beneficial to predict the in vivo data of the sus-
tained release semi-sponge formula G 2.
5. Conclusion
Ocular semi-sponges containing levofloxacin hemihydrate can
be considered as a promising new drug delivery systems to treat
bacterial conjunctivitis with minimum patient discomfort. Ocular
semi-sponges resemble the shape of the lower cul-de-sac, and
provide sustainment of drug release in the tear film for a long
period (up to 12 h). As a result, the need for frequent instillation of
levofloxacin eye drops could be substituted by the insertion of
semi-sponge in the lower cul-de-sac of the infected eye two times
daily. This dosage regimen is expected to highly increase the
compliance of patients, leading to better treatment in a shorter
duration.
Declaration of interest
The author reports no conflicts of interest. The author alone is
responsible for the content and writing of the paper.
Acknowledgment
The authors would like to thank Dr Heba attia, Microbiology and
immunology department, Faculty of Pharmacy, Cairo University, for
her help in the microbiological susceptibility testing.
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