European Journal of Pharmaceutical Sciences 167 (2021) 106041

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European Journal of Pharmaceutical Sciences

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

Chitosan/β-glycerophosphate in situ forming thermo-sensitive hydrogel for

improved ocular delivery of moxifloxacin hydrochloride
Marwa Hasanein Asfour a, Sameh Hosam Abd El-Alim a, *, Ghada Elsayed Ahmed Awad b,
Ahmed Alaa Kassem a
a
Pharmaceutical Technology Department, National Research Centre, El-Buhouth St., Dokki, Cairo 12622, Egypt
b
Chemistry of Natural and Microbial Products Department, National Research Centre, El-Buhouth St., Dokki, Cairo 12622, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of the current work is to develop a thermo-sensitive hydrogel system of moxifloxacin hydrochloride
Chitosan (MOX) for improved ocular delivery. Fifteen formulations were prepared at different concentrations of β-glyc­
β-glycerophosphate erophosphate disodium salt (β-GP) 12–20% (w/v) and chitosan (CS) 1.7–1.9% (w/v). The optimized MOX loaded
Thermo-sensitive hydrogel
thermo-sensitive hydrogel system (F8), consisting of CS (1.8%, w/v) and β-GP (16%, w/v), showed optimum
Ocular delivery
gelation temperature (35 ◦ C) and gelation time (2 min), thus was selected for further investigations. It showed a
Moxifloxacin HCL
Staphylococcus aureus significant decrease (p < 0.05) in the zeta potential value compared to CS solution with a favorable pH value
(7.1) and confirmed thermoreversible behavior. MOX loaded F8 displayed a porous structure under scanning
electron microscopy. Rheological investigation of MOX loaded F8 revealed the presence of a strong hydrogel
network with high elasticity along with a small loss factor of 0.08 indicating a great ease of gel formation. The
release of MOX from F8 was found to be governed by a combined mechanism of diffusion and relaxation. Bio­
logical assessment of two concentrations of MOX loaded F8 (0.25 and 0.5%) was conducted using healthy and
infected male albino New Zealand rabbits, where an improved and prolonged antibacterial activity against
Staphylococcus aureus compared to plain MOX (0.5%), marketed MOX eye drops (0.5%), was shown. Moreover,
histopathological examination of ocular tissues confirmed the antibacterial efficacy of the optimized formulation
eight days post topical therapy. Consequently, the developed CS/β-GP thermo-sensitive hydrogel system (F8)
reveals a promising potential for enhancing the ocular delivery of MOX for treatment of bacterial infections.

1. Introduction important in drug delivery (Liu et al., 2016). Numerous mechanisms can
possibly result in the formation of in situ hydrogel, e.g. temperature
Traditional ocular drug delivery systems, e.g. eye drops, normally modulation, pH alteration and ionic cross-linking (Ruel-Gariepy and
have a bioavailability of no more than 10% (Sieg and Robinson, 1977). Leroux, 2004). Among these in situ forming hydrogel systems,
Ophthalmic solutions are eliminated rapidly owing to the presence of an thermo-sensitive hydrogels have gained increasing interest recently.
efficient drainage apparatus and fast turnover of lacrimal fluid, which They may be easily instilled into the eye and distribute vastly on eye
leads to limited transcorneal absorption as well as a short precorneal surface in solution form owing to their low viscosity at room tempera­
residence time (Makoid et al., 1976). Accordingly, attention has been ture. Afterwards, the solution is converted to a viscous hydrogel due to
focused on the development of ocular delivery systems that extend elevated temperature on the conjunctiva. This would eventually
contact time at the ocular surface, thus increasing corneal penetration e. decrease the clearance rate of the tear fluid and drainage system (Chen
g. ointments, inserts, suspensions, collagen shields and pre-formed et al., 2012; Kolawole et al., 2019).
hydrogels (Edsman et al., 1998). A typical ophthalmic formulation Chitosan (CS) is a commonly used natural polymer, which is ob­
should have suitable strength and mucoadhesive force to withstand tained through partial depolymerization and deacetylation of chitin
lacrimal dilution. This would enhance the precorneal retention of the present in shells of crustacean. It has gained increasing interest in drug
drug and thus increase bioavailability (Qi et al., 2007). delivery applications owing to its biodegradability, biocompatibility,
In recent years, in situ forming hydrogels have become increasingly besides its low toxicity and immunogenicity (Dutta et al., 2004; Lee

* Corresponding author.
E-mail addresses: [email protected], [email protected] (S.H. Abd El-Alim).

https://doi.org/10.1016/j.ejps.2021.106041
Received 10 August 2021; Received in revised form 29 September 2021; Accepted 12 October 2021
Available online 14 October 2021
0928-0987/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
M.H. Asfour et al. European Journal of Pharmaceutical Sciences 167 (2021) 106041

et al., 1995). In physiological environments, various enzymes degrade Table 1

CS and form harmless products (Giri et al., 2012). CS can be biodegraded Composition and characterization parameters of MOX loaded CS/β-GP thermo-
mainly by the lysozyme in vivo since lysozyme is highly concentrated, sensitive hydrogel systems.
particularly, in the ocular mucosa (Das et al., 2020). CS is a suitable Formulation CS β-GP Gelation Gelation time
candidate in ophthalmic formulations due to its mucoadhesive proper­ code (%) (%) temperature (◦ C) (min)
ties, permeation enhancement, corneal wound healing effects, besides F1 1.7 12 – –
F2 1.7 14
its antimicrobial and antifungal properties (Irimia et al., 2018). It also
– –
F3 1.7 16 – –
has pseudoplastic and viscoelastic characteristics that do not disturb the F4 1.7 18 – –
tear film (Alonso and Sánchez, 2003; Krtalić et al., 2018). F5 1.7 20 – –
Lately, CS-based thermo-sensitive gels with several polyols e.g. F6 1.8 12 39 7
F7 1.8 14 36 4
glycerol, sorbitol and ethylene glycol have earned special consideration
F8 1.8 16 35 2
(El-Feky et al., 2018; Kolawole et al., 2019; Wu et al., 2007). These F9 1.8 18 35 2
polyols form a water shield surrounding CS chains (pKa 6.5), thus F10 1.8 20 35 2
maintaining CS as a liquid at lower temperatures, even at pH values > F11 1.9 12 – –
6.5, and as a gel at higher temperatures. β-glycerophosphate disodium F12 1.9 14 39 12
F13 1.9 16 ambient spontaneous
salt (β-GP) is one of these polyols, which is reported to form
F14 1.9 18 ambient spontaneous
thermo-sensitive CS gel (Kolawole et al., 2019; Li et al., 2010). Glycer­ F15 1.9 20 ambient spontaneous
ophosphate is an organic compound naturally found in the body. It is
usually used as a source of phosphate in the treatment of unbalance of
phosphate metabolism and its intravenous administration has been Germany. All other chemicals were of analytical grade.
approved by FDA (Zhou et al., 2015). The formed gel exists in the so­
lution state and can be transformed to the gel state at body temperature
(Kolawole et al., 2019; Supper et al., 2014; Szymaǹska et al., 2015). 2.2. Methods
CS/β-GP thermo-sensitive hydrogel was reported to be safe and biode­
gradable with the ease of drug loading, which allows its release at the 2.2.1. Preparation of MOX loaded CS/β-GP thermo-sensitive hydrogel
administration site (Hastings et al., 2012). systems
Moxifloxacin hydrochloride (MOX) is a novel fluoroquinolone anti­ MOX loaded CS/β-GP thermo-sensitive hydrogel systems (0.5%)
bacterial agent that has a broad spectrum of activity against gram- were prepared employing the method previously reported by Chenite
positive and gram-negative bacteria. Compared to ciprofloxacin, it ex­ et al. (Chenite et al., 2001). Briefly, predetermined concentrations of CS
hibits enhanced activity against anaerobes and gram-positive species were dissolved in 0.1 N HCl, while predetermined concentrations of
(such as streptococci, staphylococci and enterococci) (Balfour and Wise­ β-GP were dissolved in double distilled water along with MOX. After­
man, 1999). The mechanism of MOX bactericidal activity is the inhibi­ wards, 1 ml of chilled β-GP/MOX solution (4 ◦ C) was gradually added to
tion of DNA gyrase (topoisomerase II) and topoisomerase IV. These 4 ml of CS solution. The resulting mixture was mixed using a vortex
enzymes are essential members of bacterial DNA replication, repair, mixer at room temperature until a clear and homogenous solution was
transcription and recombination (Pestova et al., 2000). MOX is used for obtained. Various concentrations of CS and β-GP were investigated and
the treatment of respiratory tract bacterial infections such as are listed in table 1.
community-acquired pneumonia, sinusitis and acute exacerbations of
chronic bronchitis (Muijsers and Jarvis, 2002). MOX ophthalmic solu­ 2.2.2. Characterization of MOX loaded CS/β-GP thermo-sensitive hydrogel
tion was approved by the FDA in April 2003 for the treatment of bac­ systems
terial conjunctivitis caused by designate susceptible organisms (Al
Omari et al., 2014). 2.2.2.1. Determination of gelation temperature. The temperature-
The biochemical properties of CS/β-GP thermo-sensitive hydrogel dependent sol-gel phase transition behavior of MOX loaded CS/β-GP
systems offer them wide applications in biomedical field including local thermo-sensitive hydrogel systems was studied by the test tube inverting
drug delivery and tissue engineering (Zhou et al., 2015). Despite the method (Chen et al., 2012; Ghasemi Tahrir et al., 2016). The systems
potential of CS/β-GP systems to provide sustained drug release (Vigani were incubated in a water bath at ambient temperature and the gelation
et al., 2020), their full potential for use as ocular delivery sytems has not temperature was examined with increments of 1 ◦ C every 10 min.
been extensively explored, especially to counteract bacterial infections. Flowability of the systems was checked every 1 min via tilting the tubes.
Thus, the current study presents the first report on the formulation and The gelation temperature was determined as the temperature at which
characterization of a thermo-sensitive in situ forming CS/β-GP hydrogel no fluidity was observed when inverting the tubes.
for ocular delivery of MOX to increase its residence time in the eye, thus
achieving better therapeutic efficacy and improved patient compliance. 2.2.2.2. Determination of gelation time. The gelation time of MOX
Biological evaluation of the optimized MOX loaded hydrogel was stud­ loaded CS/β-GP thermo-sensitive hydrogel systems was determined by
ied on healthy as well as infected male albino rabbit eyes. Histopatho­ using the test tube inverting method. Briefly, 2 ml of each system was
logical examination of eye tissues was also performed to support the added into a test tube immersed in a water bath at the determined
results. gelation temperature. Flowability of the systems was checked every 30 s
via tilting the tubes. The gelation time is determined as the time at which
2. Materials and methods flow of the systems stopped (Chen et al., 2012).

2.1. Materials 2.2.2.3. Determination of zeta potential. The zeta potential (ZP) of CS
solution, drug-free optimized CS/β-GP thermo-sensitive hydrogel system
MOX (99%) was kindly donated by the Medical Union Pharmaceu­ and optimized MOX loaded CS/β-GP thermo-sensitive hydrogel system,
ticals (MUP), Abou Sultan, Ismailia, Egypt. CS (molecular weight at the solution state, was estimated employing Zetasizer (Nano Series
400,000, 85% deacetylated) was purchased from Alpha Aesar, Germany. ZS90, Malvern Instruments Ltd., Worcestershire, UK). Hydrogel systems
β-GP and dialysis tubing cellulose membrane, molecular weight cut-off were appropriately diluted employing double distilled water before ZP
12,000–14,000, were bought from Sigma-Aldrich Co., St. Louis, USA. determination. Measurements were repeated in triplicate at room
Hydrochloric acid 37% (HCl) was procured from Riedel-de Haën, Seelze, temperature.

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M.H. Asfour et al. European Journal of Pharmaceutical Sciences 167 (2021) 106041

2.2.2.4. pH measurement. The pH of the optimized MOX loaded CS/ 2.2.4. In vivo evaluation studies
β-GP thermo-sensitive hydrogel system was measured, at the solution
state, using a digital pH meter (Vstar 92, Orion Versa Star™, Thermo 2.2.4.1. Animals. In the present study, male adult New Zealand albino
Fisher Scientific Inc., USA) at room temperature. rabbits (2–2.5 kg) were obtained from the animal house of the National
Research centre (NRC), Cairo, Egypt. Rabbits were kept in individual
2.2.2.5. Evaluation of the thermoreversible behavior. To investigate the cages and fed with a standard dry food and water ad libitum. Animals
thermoreversible behavior, the optimized MOX loaded CS/β-GP thermo- were treated humanely and the investigation protocols were performed
sensitive hydrogel system was stored at 4–8 ◦ C for 24 h before being following the ethical guidelines concerning care and use of experimental
observed for flowability (Ghasemi Tahrir et al., 2016). animals approved by the Medical Research Ethics Committee (MREC) at
the NRC (Reg. No. 16/144). All eyes were firstly checked using a hand-
2.2.2.6. Rheological study. The viscoelastic properties of the optimized held slit lamp and those with no inflammatory signs of the eyes were
MOX loaded CS/β-GP thermo-sensitive hydrogel system were evaluated involved in the investigation (Di Colo and Zambito, 2002; Tamilvanan
utilizing parallel-plate rheometer (Physica MCR 301, Anton Paar, Ger­ and Benita, 2004).
many) run at the oscillatory mode employing 40 mm parallel plate with
a trim gap of 0.4 mm. The sample (gel state) was gently placed onto the 2.2.4.2. Susceptibility testing. Rabbits were randomly allocated into five
rheometer’s lower plate and the selected trim gap was adjusted to groups (n = 6). Group 1 received drug-free optimized CS/β-GP thermo-
decrease sample shearing. A “strain sweep” was performed to determine sensitive hydrogel system. Groups 2 and 3 received two concentrations
the linear viscoelastic region of the sample at 25 ± 0.5 ◦ C. The strain of optimized MOX loaded CS/β-GP thermo-sensitive hydrogel system
magnitude applied on the samples was gradually elevated from 0.05 to (0.25 and 0.5%, respectively). Group 4 received marketed MOX eye
10%, at a steady frequency of 1 Hz. The strain, where the storage/elastic drops (0.5%), whereas group 5 received plain MOX solution (0.5%).
modulus (G’) and loss/viscous modulus (G”) was constant and inde­ Sterile filter paper discs (6 mm diameter) were inserted under the eyelid
pendent of the prevailing frequency, was selected for the frequency of each rabbit for 30 s at predetermined time intervals after a single
sweep investigations (Kolawole et al., 2019). Gel strength was then installation (50 μl) of the studied formulation, each in the conjunctival
determined by frequency sweep analysis at 34 ± 0.5 ◦ C applying a fre­ sac of both rabbit eyes. Afterwards, discs were transferred into nutrient
quency ranging from 0.01 to 10 Hz. The gel strength of the sample was broth tubes inoculated with 100 µl of Staphylococcus aureus (S. aureus,
evaluated based on the ratio G’ to G” at a frequency of 1 Hz. (Kolawole ATCC 29,213) bacterial suspension followed by incubation for 24 h at
et al., 2019). Loss factor (tan δ) was calculated as tan G’’/G’. 37 ± 0.5 ◦ C. The optical density of cultures was measured at 600 nm
employing UV–VIS spectrophotometer in order to evaluate the growth
2.2.2.7. Scanning electron microscopy. Scanning electron microscopy inhibition of the inoculated bacteria. Inhibition% was estimated
(SEM) was performed to reveal the morphological structure of the considering the medium inoculated with S. aureus as control. In order to
optimized MOX loaded CS/β-GP thermo-sensitive hydrogel system. The assess and compare the antibacterial effect of the examined formula­
system was examined utilizing SEM (Quanta FEG 250, ThermoFisher tions, the area under the inhibition curve (AUC0–6h) was calculated via
Scientific Co., Czech Republic). Dried hydrogel sample was fixed on the linear trapezoidal method (Khalil et al., 2017).
aluminum stubs with double-sided tape, sputter-coated with a thin-layer
of gold and investigated at 20 kV and a distance of 10 mm. 2.2.4.3. Induction of ocular bacterial infection. Induction of bacterial
ocular infection was performed as previously reported by Khalil et al.
2.2.3. In vitro drug release study (Khalil et al., 2017). A 24 h old bacterial suspension of S. aureus was
The dialysis bag method, using cellulose membrane, was employed adjusted to contain 1 × 106 CFU/ml using sterile physiological saline.
to assess MOX release profile from the optimized CS/β-GP thermo- Each rabbit’s eye was infected with 100 µl of bacterial suspension. Forty
sensitive hydrogel system. One ml from the optimized MOX loaded eight hours post infection, bacterial infection was assured through the
CS/β-GP thermo-sensitive hydrogel system as well as plain MOX solu­ appearance of the following inflammatory signs, namely, redness,
tion, containing the equivalent amount of drug (5 mg), were placed in conjunctivitis and excessive tearing.
the dialysis bag followed by sealing at both ends to avoid leakage. The 2.2.4.3.1. Antibacterial therapy. A group of healthy rabbits was
bags were inserted in a 100 ml screw-capped glass container filled with assigned as a negative control (group 1). Infected rabbits were divided
phosphate buffered saline (PBS) pH 7.4 (El-Feky et al., 2018). The into six groups (n = 6) at random. Group 2 served as a positive control.
experiment was conducted in a thermo-stated shaking water bath (SV Group 3 received drug-free optimized CS/β-GP thermo-sensitive
1422, Memmert GmbH, Germany) adjusted to 100 rpm and 34 ± 0.5 ◦ C, hydrogel system. Groups 4 and 5 received optimized MOX loaded CS/
mimicking the physiological temperature of the eye surface (Morrison β-GP thermo-sensitive hydrogel system at two concentrations (0.25 and
et al., 2017). At predetermined time intervals, samples were withdrawn 0.5%, respectively). Group 6 received marketed MOX eye drops (0.5%),
and replenished with an equal volume of fresh medium. MOX concen­ whereas group 7 received plain MOX solution (0.5%). Topical treatment
trations in the samples were determined by spectrophotometric analysis took place by a single instillation (50 µl) every 12 h for 8 consecutive
at 287.8 nm (Motwani et al., 2007) based on the regression equation of a days, while the positive control group was left untreated. Swabs were
calibration curve carried out in the same medium. The cumulative taken daily from the infected eyes into tubes containing 1 ml of sterile
release percentages were calculated as the ratio of the quantity of MOX distilled water until the end of the study. Serial dilutions were performed
released to the initial quantity of MOX in the dialysis bag. Three inde­ for the samples and 100 µl of each dilution was poured into a sterile
pendent samples were included for all measurements. Petrie dish containing sterile Mueller-Hinton agar medium and incu­
bated at 37 ± 0.5 ◦ C for 24 h. Afterwards, colonies were counted and %
2.2.3.1. Kinetics studies of the release data. The release data were fitted of bacterial infection inhibition was calculated according to the
to various mathematical models to assess the release mechanism of MOX following equation:
from the optimized hydrogel system, namely, Zero order (% cumulative Inhibition (%)=(Ac-AS\AC)×100
drug released against time), first order (log% cumulative drug retained Where Ac is the colony count of the positive control and As is the
against time), Higuchi model (% cumulative drug released against colony count of the sample.
square root of time) and Peppas exponential equation (log% cumulative In order to assess and compare the antibacterial effect of the exam­
drug released against log time). In Peppas model, the release exponent ined formulations, the area under the inhibition curve (AUC0–8days) was
“n” was computed, which indicates the mechanism of drug release. calculated via the linear trapezoidal method (Khalil et al., 2017).

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M.H. Asfour et al. European Journal of Pharmaceutical Sciences 167 (2021) 106041

Table 2 employing predetermined concentrations of CS and β-GP (Table 1). The

Modified Draize grading scale for clinical evaluation of ocular irritation. thermo-sensitive systems investigated in this work were based upon the
Parameter Observation Score interaction of CS and β-GP. Polyol salts having only one anionic head, e.
Conjunctival Normal 0 g. polyol-phosphate salts, were reported as perfect candidates for con­
discharge verting actually pH-gelling CS solutions into temperature controlled
Slight discharge 1
hydrogel systems (Chenite et al., 2001). The main molecular mecha­
Severe discharge covering a small area around the 2
cornea nisms involved in gel formation were described by chenite et al.
Severe discharge covering a large area around the 3 (Chenite et al., 2000) as following: (1) electrostatic attraction between
cornea negatively charged phosphate moieties of β-GP and positively charged
Conjunctival Normal 0 ammonium groups of CS, (2) the increase of hydrogen bonding between
chemosis
CS chains due to the basic action of β-GP, which neutralizes the
Slight chemosis including nictitating membrane 1
Severe chemosis with eye partially closed 2 ammonium group of CS chains, thus reducing their electrostatic repul­
Severe chemosis with eye closed 3 sion and (3) the CS-CS hydrophobic interactions enhanced by the glyc­
Conjunctival redness Normal blood vessels 0 erol action in water. At decreased temperatures, powerful CS-water
Some blood vessels definitely hyperaemic 1
interactions prevent CS chains from agglomeration. Rising the temper­
Diffuse color, individual vessels not easily 2
discernible
ature leads to the removal of sheaths of water molecules via the glycerol
Diffuse beefy red 3 moiety, allowing CS chains to come closer, and hydrophobic interaction
between the polymer chains predominates, resulting in association of CS
macromolecules. This explains the temperature dependence of the
2.2.4.3.2. Histopathological examination. Twenty four hours after gelation process. Therefore, the gelation of CS/β-GP solution is antici­
the last treatment dose, 3 rabbits from each group were sacrificed. Eyes pated to be governed mainly by hydrophobic interaction. It is worthy to
were surgically removed, washed smoothly with normal saline and mention that such gelation process would not take place devoid of the
processed for microscopic inspection. In brief, eyes were fixed in 10% attractions via mechanisms (1) and (2), which exist inside the CS/β-GP
buffered formalin solution for 24 h followed by embedding in paraffin. solution (Chenite et al., 2001, 2000).
Sections of about 4 μm thickness were cut from the paraffin blocks and
stained with hematoxylin and eosin (HE) for histopathological 3.2. Characterization of MOX loaded CS/β-GP thermo-sensitive hydrogel
examination. systems

2.2.4.4. Ocular irritation assessment. The ocular irritation assessment 3.2.1. Determination of gelation temperature
was performed utilizing a slightly modified version of Draize test The optimum ocular in situ thermo-sensitive hydrogels should exhibit
(Baeyens et al., 2002; Draize et al., 1944; Mishra and Jain, 2014). Fifteen a gelation temperature greater than room temperature and transform to
healthy rabbits were randomly allocated into 5 groups (n = 3). Group 1 gel at the temperature of the conjunctival sac (34 ± 0.5 ◦ C) (El-Feky
received drug-free optimized CS/β-GP thermo-sensitive hydrogel sys­ et al., 2018). Table 1 shows the gelation temperature for the prepared
tem. Groups 2 and 3 received optimized MOX loaded CS/β-GP CS/β-GP systems. Only nine formulations had the ability to form
thermo-sensitive hydrogel system at two concentrations (0.25 and 0.5%, hydrogel within the investigated temperature range. Results reveal that
respectively). Group 4 received marketed MOX eye drops (0.5%), gelation is influenced by the concentration of both CS and β-GP. No
whereas group 5 received plain MOX solution (0.5%). Fifty microlitres hydrogel formation was observed at CS concentration of 1.7% (w/v) for
of each formulation was instilled into the lower conjunctival cul-de-sac all investigated CS/β-GP ratios (F1-F5). However, increasing CS con­
of the right eye of each rabbit following gently pulling the lower lid centration to 1.8% (w/v) allowed gelation as a result of the enhanced
away from the eyeball. The un-treated contra lateral eye served as a CS-CS physical interactions (Cho et al., 2006; Zhou et al., 2008). The
control. The eyelids were gently held together for about 10 s in order to gelation temperature of F6-F10 ranged from 35 to 39 ◦ C. Increasing the
avoid any loss of the instilled solution (Mishra and Jain, 2014). Eyelids, concentration of β-GP led to a decrease in gelation temperature. This is
conjunctiva, cornea and iris were examined by external observation at due to the fact that increasing the concentration of β-GP results in an
0.5, 1, 2, 4, 6 and 24 h post instillation. The assessment of ocular irri­ increase of the system pH. This would reduce CS protonation and
tation was carried out by providing 0 (absence) to 3 (highest) grades on intermolecular electrostatic repulsion, thus endorsing the sol-gel tran­
clinical evaluation scale (Draize et al., 1944; Singh et al., 2014) as sition by enhancing hydrophobic interactions between CS chains upon
illustrated in table 2. Overall ocular irritation index (Iirr) was computed slight increase of temperature (Supper et al., 2014). Nevertheless, at
by summing up the total clinical evaluation scores over the observation higher concentrations of β-GP (starting from 16% w/v), almost no
time points. A score of 2 or 3 in any category or Iirr greater than 4 was change in gelation temperature was observed. Further increase of CS
considered as an indicator of clinically significant irritation (Mishra and concentration to 1.9% (w/v) resulted in a solution of higher viscosity,
Jain, 2014; Singh et al., 2014). thus hindering the diffusion of CS and β-GP, which hampered or delayed
the gelation process (F11 and F12, respectively) (Cho et al., 2006). On
2.2.5. Statistical analysis the other hand, spontaneous gelation was observed at higher concen­
Results were presented as mean ± standard deviation (SD). Com­ trations of β-GP 16–20% (w/v). This might be attributed to a synergetic
parisons between different groups were evaluated using one-way anal­ effect at higher concentrations of both CS and β-GP, which led to a
ysis of variance (ANOVA) followed by Fisher’s LSD post-hoc test for sudden drop of the gelation temperature (Cho et al., 2006).
multiple comparisons utilizing SPSS software (version 22.0; IBM Co.,
USA). The difference was considered significant at p < 0.05. 3.2.2. Determination of gelation time
The results reveal that the gelation time decreased as the β-GP con­
3. Results and discussion centration increased (Table 1). Shorter gelation time was observed with
the increase in β-GP concentration up to 16% (w/v). This could be
3.1. Preparation of MOX loaded CS/β-GP thermo-sensitive hydrogel attributed to the increase in the medium pH by the incorporated β-GP, as
systems lower energy is required to induce gelation by promoting hydrophobic
interactions between CS chains (Cho et al., 2005; Supper et al., 2014).
In order to reach an optimum MOX loaded CS/β-GP thermo-sensitive Based on the results of gelation temperature and gelation time ex­
hydrogel system, a total of fifteen formulations were prepared periments, F8-F10 exhibited a gelation temperature of 35 ◦ C, which is

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M.H. Asfour et al. European Journal of Pharmaceutical Sciences 167 (2021) 106041

Table 3 coating of CS chains, thus preventing their aggregation in solution and

Zeta potential values of CS solution, drug-free and MOX maintaining CS solubility at low temperature (Chenite et al., 2001).
loaded F8 (n = 3).
Formulation ZP (mV) ± SD 3.2.5. Evaluation of the thermoreversible behavior
CS solution 50.7 ± 2.5 MOX loaded F8 exhibited a noticeable liquefaction after storage at
Drug-free F8 2.6 ± 0.2 4–8 ◦ C for 24 h. It has been reported that cooling after heat-induced
MOX loaded F8 2.8 ± 0.2
gelation weakens CS-CS hydrophobic interaction, which is the main
force of gelation. On the other hand, interchain hydrogen bonding is not
affected by the temperature change (Supper et al., 2014). This is
dependent on the pH of CS/β-GP solution before heating; systems with
pH values that ranged from 6.9 to 7.2 prior to heating were found to be
partially thermoreversible. However, CS/β-GP solutions having pH
values between 6.5 and 6.9 revealed complete thermoreversibility
(Chenite et al., 2000). In light of this finding, the pH value of MOX
loaded F8 (7.1) explains its liquefaction after storage at 4–8 ◦ C, indi­
cating a partial thermoreversibility of the prepared hydrogel.

3.2.6. Rheological study

Frequency dependence of storage (G’) and loss (G’’) moduli of MOX
loaded F8 is revealed in Fig. 1. Ideally, investigating the rheological
properties of the hydrogel should be carried out in linear viscoelastic
region, which is determined by a preliminary “strain sweep” analysis. In
non-linear viscoelastic region (i.e. region higher than the critical strain),
extreme oscillatory strain damages the hydrogel mechanical structure
leading to the disability to attain the consistent information concerning
inter-particle and inter-molecular forces in the matter. Hence, frequency
sweep analysis of the hydrogel systems should be conducted at an
oscillatory strain lower than the critical strain (i.e. in the linear visco­
elastic region) to guarantee that the hydrogel structure is not destructed
Fig. 1. Storage modulus (G’) and loss modulus (G”) as a function of frequency (Kolawole et al., 2019).
(Hz) on MOX loaded F8 at 34 ± 0.5 ◦ C. Based on the strain sweep analysis, a strain of 1% was chosen for the
present rheological investigation. This was to ensure that the storage
the closest to that of the conjunctival sac, as well as the lowest gelation (G’) and loss (G”) moduli of the system were independent of the utilized
time (2 min). However, F8, consisting of the least concentration of β-GP strain. Fig. 1 reveals that the storage modulus (G’) is much higher than
16% (w/v) along with CS 1.8% (w/v), was selected for further the loss modulus (G”) over the assessed frequency range at 34 ± 0.5 ◦ C,
investigations. where G’/G’’ ratio at a frequency of 1 Hz was 12.2 ± 0.8. This finding
indicates the presence of a strong hydrogel network with high elasticity
3.2.3. Determination of zeta potential (Kempe et al., 2008; Kolawole et al., 2019; Strom et al., 2014). This
The ZP of CS solution displayed a high positive value of 50.7 mV behavior is preferable as it promotes the rapid gelation of hydrogel at
(Table 3). This is attributed to the cationic nature of CS having pro­ the investigated physiological temperature. The loss factor (tan δ) was
tonated amino groups (NH3+) on its chain at acidic pH (< 6.5) (Asfour calculated and found to be 0.08 ± 0.01 indicating greater ease of gela­
et al., 2017). On the other hand, ZP values of drug-free F8 as well as tion for the formulation (Kolawole et al., 2019).
MOX loaded F8 showed a significant decrease (p < 0.05) compared to CS
solution (Table 3). This could be explained by the partial neutralization 3.2.7. Scanning electron microscopy
of cationic CS macromolecules with the anionic β-GP. This finding is in Fig. 2 depicts the SEM micrographs of MOX loaded F8. The micro­
good agreement with previous reports (Kim et al., 2012; Kolawole et al., graphs demonstrated that CS/β-GP hydrogel exhibited interconnection
2019; Owczarz et al., 2018). pore structure forming a three dimensional network as previously re­
ported for similar CS-based hydrogel systems (Chang et al., 2009). The
3.2.4. pH measurement porous structure has the advantage of providing void space that allows
pH is an important factor regarding tolerance of ophthalmic for­ oxygen and nutrient transportation (Rahman et al., 2019). Furthermore,
mulations by the eye. MOX loaded F8 exhibited a pH value of 7.1 ± 0.1, the displayed high porosity of CS/β-GP hydrogel could enhance its water
which is suitable for ocular administration (Kouchak et al., 2019). uptake ability, which assures a prolonged profile for the drug release
Although the initial CS solution is acidic in nature (0.1 N HCl), the (Szymaǹska et al., 2015).
incorporation of β-GP, at decreased temperature, led to an increase of
the CS solution pH to the physiologically suitable range (6.8–7.2) devoid 3.3. In vitro drug release study
of spontaneous precipitation. Normally, increasing the pH of CS solution
above its pKa (6.5) using a strong base, results in its precipitation. This is The in vitro release profile of MOX from F8 was compared with that of
due to deprotonation of the positively charged ammonium groups MOX from aqueous solution, in order to assess the ability of CS/β-GP
(NH3+) to neutral amino groups (NH2), leading to a decrease in the hydrogel to effectively deliver MOX in a sustained pattern. Fig. 3 dis­
charge density of CS backbone. This increases the intermolecular plays that the release of MOX from F8 was slower than that from its
attraction and enhances the hydrophobic and hydrogen bonding solution. MOX solution revealed 75.6% release after 1 h compared to
attraction forces to predominate and accordingly, precipitates CS 53% from F8. After 3 h, MOX release from solution reached a plateau,
(Asfour et al., 2017). On the contrary, addition of β-GP maintains CS signifying an almost complete MOX release (>95%). On the other hand,
solubility even at neutral pH owing to the attraction of negatively the release from F8 continued up to 8 h where 83.3% of the drug was
charged phosphate groups of β-GP with positively charged ammonium released, indicating a sustained release of the drug and potential pro­
groups of CS. The consequent exposure of the glycerol moiety results in longation in dosing interval. These results might be ascribed to the

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M.H. Asfour et al. European Journal of Pharmaceutical Sciences 167 (2021) 106041

Fig. 2. SEM micrographs of MOX loaded F8 at a magnification of (A) 5000 x and (B)10,000 x.

polymeric network of the hydrogel, which hinders the drug diffusion governing the drug release. Peppas equation has been applied, where
(El-Badry et al., 2015; Kassem et al., 2017). Drug release from swellable 60% of release data were incorporated (Ritger and Peppas, 1987), and
matrices is generally complex in nature. It was previously reported that good linearity (R2=0.97) was obtained (Table 4). According to Peppas
the drug release from the hydrogel takes place by two major mecha­ theory, release of a drug follows Fickian diffusion mechanism when n ≤
nisms: Firstly, in the early phase of release, drug molecules diffuse from 0.5, an anomalous (non-Fickian) diffusion when n ranges from 0.5 to
the hydrogel. Secondly, in the late phase of release, relaxation of the 1.0, case II transport for n = 1.0 and super-case II transport for n > 1.0
hydrogel matrix occurs leading to release of the drug. Although some (Ritger and Peppas, 1987). As presented, the value of the release expo­
processes may be clearly classified as either controlled by diffusion or nent “n” was 0.52, indicating a non-Fickian release mechanism where
relaxation, drug release from the gel is mostly governed by both the drug release was governed by a combination of diffusion and
mechanisms (Cai et al., 2012). relaxation mechanism signifying an “anomalous” diffusion. This sig­
nifies that the drug release is controlled by more than a single mecha­
3.3.1. Kinetics studies of the release data nism (Ritger and Peppas, 1987).
The analysis of MOX release kinetics from F8 is presented in table 4.
The release data didn’t demonstrate appropriate fitting to zero order,
first order or Higuchi model (R2=0.69–0.85). Thus, applying Peppas 3.4. In vivo evaluation studies
equation for analysis of release data, as well as elucidation of the release
exponent “n”, provides a better explanation of the mechanisms 3.4.1. Susceptibility testing
Fig. 4 displays the percentage inhibition of S. aureus growth plotted

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M.H. Asfour et al. European Journal of Pharmaceutical Sciences 167 (2021) 106041

Fig. 5. Percentage inhibition of S. aureus growth produced by different treat­

ment groups in the eyes of infected New Zealand albino rabbits up to 8 days (n
= 6).
Fig. 3. In vitro release profile of MOX from solution and F8 at 34 ± 0.5 ◦ C up to
8 h (n = 3).
marketed MOX eye drops revealed a rapid decrease up to 6 h. Notice­
ably, at the end of the experiment, both groups exhibited a
non-significantly different antibacterial efficacy (p > 0.05) compared to
Table 4
the drug-free F8 group (5.7% and 3.7%, respectively). On the other
The calculated correlation coefficients and Peppas release exponent of MOX
hand, the two investigated MOX loaded F8 (0.25 and 0.5%) exhibited a
release profile from F8 (n = 3).
non-significantly different antibacterial effect (p > 0.05) after 0.5 h
Formulation Zero order First order Higuchi model Peppas
compared to the marketed product and the plain drug. Obviously, both
model
R2
R2
R2
R2 n
concentrations were able to maintain a significantly higher (p < 0.05)
MOX loaded F8 0.69 0.83 0.85 0.97 0.52 antibacterial activity until the end of the experiment (33.6 and 37.9%,
respectively).
Furthermore, the calculated AUC0–6h of the two investigated MOX
loaded F8 (0.25 and 0.5%) was found to be 410.2 ± 5 and 442 ± 0.5%.h,
respectively. Both values are significantly higher (p < 0.05) compared to
the plain drug solution (182.7 ± 0.2%.h) and the marketed product
(259.1 ± 1.4%.h). This indicates the superiority of the optimized CS/
β-GP thermo-sensitive hydrogel system even at half the marketed MOX
concentration. This could be explained by the fact that after application
of the ocular thermo-sensitive hydrogel, it undergoes phase-transition
and forms viscoelastic gel in response to the ocular temperature. The
formed in situ gel can withstand the shear forces in the conjunctival cul-
de-sac, providing sustained release of the loaded drug (Kumar and
Himmelstein, 1995; Vigani et al., 2020).

3.4.2. Antibacterial therapy

The percentage inhibition of S. aureus growth in the eyes of infected
rabbits treated twice daily with drug-free F8, plain MOX solution
(0.5%), marketed MOX eye drops (0.5%) as well as MOX loaded F8 (0.25
and 0.5%) is presented in Fig. 5. The results revealed that only a very
low percentage of inhibition was observed for the drug-free F8 group.
On the other hand, all other treatment groups displayed a gradual in­
crease in the percentage inhibition of S. aureus up to 8 days post treat­
ment. The superiority of MOX loaded F8 (0.25 and 0.5%) was observed
Fig. 4. Percentage inhibition of S. aureus growth produced by different
starting from day 2 of treatment, where a significantly higher (p < 0.05)
experimental groups in the eyes of healthy New Zealand albino rabbits up to 6 h
percentage inhibition was observed compared to the plain drug and the
(n = 6).
marketed MOX eye drops.
Moreover, the calculated AUC0–8days showed significantly lower
against time for drug-free F8, plain MOX solution (0.5%), marketed
values (p < 0.05) for the plain MOX solution (456 ± 23.2%.days) and the
MOX eye drops (0.5%) and two concentrations of MOX loaded F8 (0.25
marketed MOX eye drops (520.4 ± 13.3%.days) compared to MOX
and 0.5%). The results showed that a mild antibacterial activity was
loaded F8, where AUC0–8days values of 576.9 ± 10.2 and 628 ± 8%.days
observed for the drug-free F8, which could be attributed to the reported
(0.25% and 0.5%, respectively) were revealed. These findings confirm
antibacterial activity of CS (Dutta et al., 2004). The antibacterial activity
the results obtained from the susceptibility study and come as a further
of all other groups exhibited the highest antibacterial activity after 0.5 h
proof of the superiority of the CS/β-GP thermo-sensitive hydrogel
of administration with inhibition percentage ranging from 83.2% to
system.
87.6%. The antibacterial activity of plain MOX solution and the

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M.H. Asfour et al. European Journal of Pharmaceutical Sciences 167 (2021) 106041

Fig. 6. Microscopic observations of sections of rabbit eye tissues with bacterial infection, 24 h after last treatment dose stained with HE with scale bar 500 μm: (A)
negative control group; (B & B’) positive control group; (C) rabbits treated with drug-free F8; (D) rabbits treated with MOX loaded F8 (0.25%); (E) rabbits treated
with MOX loaded F8 (0.5%); (F) rabbits treated with marketed MOX eye drops (0.5%); (G) rabbits treated with plain MOX solution (0.5%).

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M.H. Asfour et al. European Journal of Pharmaceutical Sciences 167 (2021) 106041

In view of the abovementioned results, MOX loaded F8 (0.25 and by both concentrations of CS and β-GP. Based on the results of gelation
0.5%) exhibited a more sustained antibacterial activity throughout the time and temperature, F8, consisting of CS 1.8% (w/v) and β-GP 16%
period of the study. This could be attributed to the longer ocular resi­ (w/v), was selected for further investigations. It showed a significant
dence time provided by the in situ gelling system, thus providing decrease (p < 0.05) in the zeta potential value compared to CS solution
extended release compared to the aqueous solution (Chen et al., 2012). with physiologically acceptable pH (7.1) and confirmed partial ther­
Another interesting finding is the superior performance of the hydrogel moreversible behavior. Scanning electron microscopy micrographs of F8
at half the concentration of the marketed product, which offers the showed a three dimensional porous network structure. Rheological
possibility of dose reduction. Accordingly, it can be concluded that the investigation revealed the presence of a strong hydrogel network with
novel developed MOX loaded CS/β-GP thermo-sensitive hydrogel sys­ high elasticity along with a small loss factor, thus indicating a great ease
tem revealed an improved efficacy for counteracting the bacterial of gel formation. The release profile of MOX from the selected hydrogel
infection of the eye. This could be attributed to the increase of corneal was found to be governed by a combined mechanism of diffusion and
residence time through decreasing the nasolacrimal drainage, tears relaxation indicating an “anomalous” diffusion pattern. In vivo evalua­
turnover and loss of drug on eyelids (Vandamme and Brobeck, 2005). tion of two concentrations of MOX loaded F8 (0.25 and 0.5%) was
performed using healthy and infected male albino New Zealand rabbits.
3.4.2.1. Histopathological examination. Fig. 6 shows sections of rabbit The results revealed an enhanced and sustained antibacterial activity
eye tissue from different experimental groups. Fig. 6A reveals tissue against Staphylococcus aureus compared to plain MOX (0.5%), marketed
sections from rabbit eyes of negative control group representing normal MOX eye drops (0.5%). This is due to the longer ocular residence time
histopathological findings of ocular tissue with intact retinal structure. provided by the in situ gelling system. Furthermore, histopathological
The inner retina composed of multi-layered structure, the outermost assessment of ocular tissues confirmed the antibacterial efficacy of the
retina consisted of pigmented epithelial cells and the layer of loose optimized formulation eight days post topical therapy. Accordingly, the
vascular supporting tissue lying between retina internally and the sclera developed CS/β-GP thermo-sensitive hydrogel system has potential for
externally. Figs. 6B and 6B’ display tissue sections of positive control improving the ocular delivery of MOX for management of bacterial
group showing significant inflammatory findings. Erosion of pigmented infections.
layer, choroid layer, sclera and retina were found. Thickness of the
supporting tissue and area of inflammation were observed (Fig. 6B). A Credit authorship contribution statement
significant increase of goblet cells and degeneration of pigmented layer
along with marked decrease in the corneal thickness was seen (Fig. 6B’). Marwa Hasanein Asfour: Conceptualization, Methodology, Re­
Examination of tissue sections of eyes treated with drug-free F8 showed sources, Writing - original draft, Writing - review & editing,
degeneration of the multi-layered structure of the inner retina and se­ Visualization.
vere necrotic area in the layer of loose vascular supporting tissue Sameh Hosam Abd El-Alim: Conceptualization, Methodology,
(Fig. 6C). Ocular tissue photomicrographs of rabbits treated with MOX Investigation, Writing - original draft, Writing - review & editing,
loaded F8 (0.25 and 0.5%) depicted normal histopathological findings Funding acquisition.
(Fig. 6D and 6E, respectively). The eye wall appeared intact with no Ghada Elsayed Ahmed Awad: Methodology, Investigation, Re­
inflammation or necrotic areas. Same findings were reported in tissue sources, Writing - original draft.
sections of rabbit eyes treated with the marketed MOX eye drops and Ahmed Alaa Kassem: Conceptualization, Methodology, Formal
plain MOX solution (Fig. 6F and 6G, respectively). analysis, Data curation, Writing - original draft, Writing - review &
editing.
3.4.3. Ocular irritation assessment
A drug delivery system intended for ocular use should not induce any Declaration of Competing Interest
ocular adverse effects e.g. burning, irritation, tearing and stinging
(Bourlais et al., 1998; Gaafar et al., 2014). The modified Draize test The authors report no declarations of interest.
revealed a slight redness of the conjunctiva and a slight reflex lachry­
mation with no chemosis during the first hour after instillation for all Acknowledgments
tested formulations. Same observation continued up to the second hour
in groups treated with drug-free optimized CS/β-GP thermo-sensitive This work was supported by the project’s sector at the National
hydrogel system and both MOX loaded optimized CS/β-GP Research Centre, Egypt through the research group project fund No.
thermo-sensitive hydrogel (0.25 and 0.5%). The Iirr score did not exceed 11010303.
4 at any time interval for all treatment groups, demonstrating that the
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