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ISSN: 0265-2048 (print), 1464-5246 (electronic)

J Microencapsul, Early Online: 1–9

! 2015 Informa UK Ltd. DOI: 10.3109/02652048.2015.1065915

RESEARCH ARTICLE

Preparation and in vivo evaluation of in situ gel system as dual

thermo-/pH-responsive nanocarriers for sustained ocular drug delivery
Soodabeh Davaran1,2, Farzaneh Lotfipour2, Naghmeh Sedghipour2, Mohamad Reza Sedghipour2,
Somayeh Alimohammadi1, and Roya Salehi1,3
1
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, 2Faculty of Pharmacy, Tabriz University of Medical Sciences,
Journal of Microencapsulation Downloaded from informahealthcare.com by University of Otago on 07/22/15

Tabriz, Iran, and 3School of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran

Abstract Keywords
Objective: Ciprofloxacin (CIP) was effective in treating bacterial keratitis. The purpose of this In situ nanogel, dual stimuli responsive,
study was to prepare an effective prolonged-release of CIP by both temperature and ocular drug delivery, ciprofloxacin,
pH-triggered in situ nanogels for the treatment of keratitis. Materials and methods: Poly(N- antimicrobial activity, keratitis
isopropylacrylamide-methacrylicacide-vinylpyrrolidone) [P (NIPAAm-MAA-VP)] nanoparticles
was synthesised and used for preparation of CIP-loaded nanogels. Antimicrobial and in vivo History
animal studies of the CIP-loaded nanoformulation were performed. Results: Nanoformulation
with a mean particle size between 10 and 50 nm and higher than 95% encapsulation efficiency Received 11 October 2014
was obtained. Ciprofloxacin released from the nanoparticles showed an enhanced antibacterial Revised 6 May 2015
effect as determined by minimal inhibitory concentrations. In vivo studies demonstrated Accepted 8 June 2015
reasonable efficacy in severe keratitis using the developed nanoformulation. Conclusions: Published online 20 July 2015
For personal use only.

Nanoformulation had acceptable efficacy in treating bacterial keratitis in an animal model.

Therefore, the developed system has the potential to be used in localised application for the
treatment of keratitis.

Introduction therapeutic effects and diminish the side effects. Over the last
decade, researchers are continuously motivated to outwit the
Keratitis is a corneal disease that may be caused by viral,
drawbacks associated with the conventional ophthalmic formula-
bacterial, fungal and parasitic infections. Moreover, in cases such
tions (Gao et al., 2010; Gupta et al., 2010; Khurana et al., 2014;
as sterile keratitis in Lyme disease, it may also be induced due to
Üstündağ-Okur et al., 2014). Some efforts has been made by
immune-related complications (Weyenberg et al., 2004; Marquart,
scientists to design CIP-loaded carrier as an effective ocular drug
2011). Bacterial infection of the cornea can vary from mild to
delivery (Law et al., 2000; Kaur and Smitha, 2002; Mortazavi
severe keratitis and if left untreated often leads to progressive
et al., 2010; Jain and Shastri, 2011; Pawar et al., 2011). Smart
tissue destruction, with corneal perforation (Üstündağ-Okur et al.,
polymeric in situ gel systems that show phase transition from sol
2014). Pseudomonas is one of the most common causes of
to gel upon getting biological stimulus can be designated as
bacterial keratitis in humans especially in patients wearing soft
system of choice in ocular drug delivery. Three types of biological
contact lenses (Stapleton et al., 2007, 2012). Ciprofloxacin (CIP)
stimulus such as temperature, pH and ions are presented by ocular
hydrochloride is an extremely effective antibacterial agent with
route. Some efforts has been done to design different types of
potent activity against most gram positive and gram negative
polymeric in situ gel systems for ocular drug delivery systems
bacteria. Ciprofloxacin used as topical eye drop is usually
(Gurny et al., 1993; Gupta et al., 2007; Agrawal et al., 2012;
effective against mild and moderate corneal ulcer caused by
Vadlapudi and Mitra, 2013). Tears have an average pH of 7.4 and
pseudomonas. However, in severe forms, it may not be effective
lack a strong buffering system. Therefore, the pH of the
and may progress leading to perforation and loss of vision
administered eye drop will determine the eye’s current pH. If
(Mundada and Shrikhande, 2006; Mortazavi et al., 2010).
the administered eye drop is acidic then it may cause the
In addition to virulence factors, drug wash out and dilution by
formation of insoluble complexes from the denatured proteins.
tear film are important contributors in low ocular bioavailability
Also, strong alkaline eye drops will damage of the eye’s cell
of the drug.
membrane integrity. Therefore, the ideal ocular drug should have
Topical drug delivery into eyes is the most accepted and
a pH between 6.8 and 7.4 (Pawar et al., 2013). Soluble pH- and
accessible way of administration for the treatment of various eye
T-responsive polymers that overcome transition at physiological
diseases. One of the major challenges in ophthalmic drug delivery
conditions (37  C and/or physiological pH) have been proposed as
systems is to design new soluble ocular carriers without causing
minimally invasive injectable systems (Hatefi and Amsden, 2002;
blurred vision get the drug into the target site to enhance the
Packhaeuser et al., 2004). The aim of this study was to develop a
novel CIP-loaded dual stimuli responsive nanogels formulation
Address for correspondence: Roya Salehi, School of Advanced Medical
with in situ gel-forming behaviour for topical ocular treatment of
Science, Tabriz University of Medical Sciences, Tabriz, Iran. Tel: bacterial keratitis. The physico-chemical characterisation, in vitro
+984113363161. Fax: +984113363132. E-mail: salehiro@tbzmed.ac.ir release, microbiological efficacy and in vivo efficiency of these
 2 S. Davaran et al.
nanoformulations were evaluated and compared with a commer-
cial CIP eye drop formulation.
Materials and methods
Chemicals preparations
N-Isopropylacrylamide (NIPAAm) purchased from Fluka
(Deisenhofen, Germany) was purified by re-crystallisation in
hexane and dried under vacuum at 25  C. Vinylpyrrolidone (VP;
Merck, Hohenbrunn, Germany) was freed from stabiliser by twice
vacuum distillation with continuous bubbling argon. Methacrylic
acid (MAA; Fluka, Deisenhofen, Germany) was used as supplied.
N,N-Methylene bisacrylamide (MBA; Sigma-Aldrich Co.,
Steinheim, Germany) was used directly without further purifica-
tion. Benzoyl peroxide (BPO; Sigma-Aldrich Co., Steinem,
Germany) was used as supplied as an initiator. All other reagents
were of analytical grade and used without further purification.
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Preparation of crosslinked poly(NIPAAM-MAA-VP)
Crosslinked poly(NIPAAM-MAA-VP) was synthesised through
free radical polymerisation (Rafie et al., 2010; Salehi et al., 2014).
Purified NIPAAm, MAA and VP were dissolved in 20 mL 1,4-
dioxan, with molar feed ratio of 75, 15 and 10, respectively, in to
which 0.3 mol% BP with respect to all the monomers was added.
To crosslink the polymer chains, 300 ml of MBA (0.049 g/ml) was
added to aqueous solution of monomers. The dissolved oxygen
was removed by passing nitrogen gas for 30 min. The polymer-
isation was carried out at 70  C for 24 h in nitrogen atmosphere.
The obtained polymer solution was precipitated in cold n-hexane
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and purified by dialysis for 5 days using dialysis membrane tubes
(Cellu SepH1) with MWCO of 2000. The external aqueous
solution was removed twice a day and displaced with fresh
distilled water. The polymer solutions were precipitated by
heating them at temperatures above lower critical solution
temperature (LCST). Then obtained gels were dried in vacuum
at 40  C for 24 h and frozen in liquid nitrogen and were
lyophilised immediately to obtain dry powder and dried again
under vacuum at 40  C for 24 h. The lyophilised powder may
again be dispersed in aqueous buffer or distilled water.
Instrumentation
Fourier transforms infrared spectroscopy
The chemical structures of the blank and CIP-loaded
poly(NIPAAM-MAA-VP) were studied by Fourier transform
infrared (FTIR) spectroscopy. Samples were mixed with KBr
and were pressed to disk. Infrared (IR) spectra of the samples
were scanned in the range from 400 to 4000 cm1 and recorded on
a Fourier transform infrared spectrometer (Equniox 55 LS 101
Bruker, German). FTIR spectra were obtained at a resolution of
4 cm1 with a minimum of 256 scan per spectrum. All
measurements were taken at room temperature. The spectra of
water, CO2 and KBr were subtracted from the sample spectrum
and the procedure was done under nitrogen gas to prevent
humidity interference.
Hydrogen nuclear magnetic resonance spectroscopy
The chemical composition of the synthesised poly(NIPAAM-
MAA-VP) was determined by hydrogen nuclear magnetic reson-
ance (1H NMR) using Bruker spectra spin 400 MHz.
Scanning electron microscopy studies
The surface morphology and size of the poly(NIPAAM-
MAA-VP) nanogels were assessed by a field emission scanning
J Microencapsul, Early Online: 1–9
electron microscope-energy dispersive using X-ray (FESEM-
EDX, S4160 Hitachi, Japan). The powder sample was spread on a
SEM stub and sputtered with gold. Particle size was obtained by
measuring the diameters of at least 300 particles shown in SEM
using image analysis software (Image-Pro Plus 4.5; Media
Cybernetics, Silver Spring, MD).
Study of thermal properties
To measure thermosensitivity of polymers, the cloud point
measurement (turbidimetry) method was employed. Optical
transmittance of aqueous polymer solution at various tempera-
tures was measured at 500 nm wavelength using UV–Vis spec-
trometer (UV-160, Shimadzu Corporation, Tokyo, Japan). Sample
and reference cells were thermostated with a circular water jacket
from 10 to 50  C. Heating rate was 1  C/min. At each temperature,
the samples were stabilised for 10 min before measurements
(Salehi et al., 2009a,b, 2013). Aqueous polymer solutions selected
for this study have variable pH (3, 7 and 8.5).
Preparation of CIP-loaded nanoparticles
Ciprofloxacin-loaded hydrogel nanogels were prepared by 24 h
incubation of pre-determined amount of dried nanogel (150 and
300 mg) in drug solution at 25  C for 24 h and were stirred well
to load drug in to the nanoparticles. The nanoparticle/drug ratio
was 10/1 (high nanogel concentration formulation) and 20/1
(low nanogel concentration formulation), respectively. The
solution was heated above the gelation temperature. After the
removal of immersed gel at the end of 24 h, the solution was
used to determine the concentration of unloaded CIP. The
obtained CIP-loaded P(NIPAAm-MAA-VP) nanogels was
freeze-dried and used for in vitro drug release study, antimicro-
bial and in vivo tests.
Determination of drug content of the nanoparticles
To determine drug entrapment within the nanoparticles, 10 mg
dried powder of CIP-loaded P(NIPAAm-MAA-VP) nanogels were
dissolved in 10 ml water. After complete dissolution of the
polymer, the designated amount of drug was quantified using a
high performance liquid chromatography (HPLC) method. The
HPLC system consisted of a Waters 515 pump, automatic injector
(7plus Waters autosampler), and Waters 2487 dual k absorbance
detector. Chromatographic separation was achieved with a
Nucleosil (Phenomenex, Torrance, CA) ODS 5 lm (25 cm3,
4.6 mm, i.d.) column. The mobile phase of 0.05 M potassium
hydrogen phosphate/acetonitrile (70/30 v/v) which its pH adjusted
to 3 with phosphoric acid solution was pumped. The flow-rate and
injection volume were 1 mL/min and 20 mL, respectively. The
pressure was adjusted up to 1400 psi. The UV detection was at
271 nm. The run time for the assay was 12 min and the retention
time for CIP was 7.6 min. Triplicate samples were used.
Ciprofloxacin solutions of known concentrations (1–100 mg/ml)
were used to generate calibration curves.
Ciprofloxacin encapsulation and loading efficiencies of
nanogels were measured using following equations.
Drug encapsulation efficiency ð%, w=wÞ
Mass of drug in nanogels
¼  100
Mass of feed drug
Drug loading efficiency ð%, w=wÞ
Mass of drug in nanogels
¼  100
Mass of nanogels
 DOI: 10.3109/02652048.2015.1065915
In vitro drug release
The powdered CIP-loaded P(NIPAAm-MAA-VP) nanogels
(10 mg) were re-dispersed in 3 mL of desired buffer solutions.
The dispersion was then transferred into a dialysis bag (cut-off
molecular weight 10 000 g/mol). The bag was subsequently
immersed in 10 mL buffer solutions with pH value of 7.4 with
stirring at a rate of 100 rpm for more than days at different
temperatures 37  C). At the required incubation time, the sample
was transferred to 10 ml of fresh buffer solution, and the released
CIP in the original buffer solution was determined. The detected
absorbance of CIP was converted to its concentration according to
the calibration curve of CIP in the same buffer. Then, the relative
percentage of the released CIP were calculated as a function of
incubation time and based on the amount of the drug existed in
the nanogels. Samples were measured using HPLC-UV. Percent
of drug released from nanogels was calculated by the following
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equation.
Amount of drug in release medium
Drug released % ¼  100
Amount of drug loaded in nanogels
Preparation of inoculum
The standard strain of Pseudomonas aeruginosa (ATCC: 9027)
used in this project was obtained in lyophilised form from
Institute of Pasture, Iran. They were activated by culturing in
sterile nutrient agar (Liofilchem, Italy) for 48 h at 37  C. A single
colony from grown plate was transferred into nutrient broth and
incubated over night at 37  C. Consequently, after incubation
For personal use only.
time, the cells were harvested by centrifugation at 3000 rpm for
15 min and washed twice and re-suspended in Ringer solution to
provide an optical density of around 0.1 at 540 nm with a
spectrophotometer (Coleman Electric Company, Maywood, IL) or
bacterial concentration around 108 CFU/ml.
Evaluation of the antimicrobial effect of CIP-loaded NPs
against P. aeruginosa
The antimicrobial activities of prepared nanoformulations were
compared to untreated CIP solution by Minimum Inhibitory
Concentration (MIC) determination against standard strain of
P. aeruginosa according to clinical and laboratory standards
institute (CLSI). Briefly bacterial inoculum in Muller–Hinton
Broth medium in the concentrations equal to 0.5 of McFarland
standard, added to serially diluted NPs suspension as well as drug
solutions (0.58–75 mg/ml). After 24 h incubation at 37  C, from
the content of the tubes streak cultures were made onto the
Muller–Hinton agar plates. The first concentration with no sign of
bacterial growth on plates considered as MIC. All experiments
were performed in three separate occasions.
Animal study
In an experimental study, 10 white female rabbits weighting
between 1.5 and 2.0 kg were investigated. This study followed the
recommendations of the Association for Research in Vision and
Ophthalmology (ARVO) regarding animal use in ophthalmic
research. Rabbits were selected as appropriate animal model of
interest to induce keratitis and have it experimentally treated by
the two trial treatment modalities. Two rabbits were used as
controls for preliminary assays followed by the experiments done
on eight rabbits through the main experimental phase of the study.
The eight rabbits were divided into two groups named A&B
which later formed our keratitis animal models and were assigned
to receive each of the two different treatment investigation
modalities. To guarantee the pathogenicity of the bacterium and
Preparation and in vivo evaluation of in situ gel system 3
to ensure applying the right method of keratitis ulcer develop-
ment, the procedures were carried out under general anaesthesia
by IM ketamine (Rotexmedica, Germany) 50 mg/kg and zylazine
(Alfasan, Holland) 10 mg/kg. Corneal epithelium was marked
using a trephine 6.0 mm in diameter at the central area. About
70% isopropyl alcohol drops were instilled inside the trephine;
after 30 min, it was dried using Merocele sponges. The trephine
was removed and the eye surface was rinsed with 0.9% sodium
chloride solution followed by mechanical de-epithelialisation
using No. 15 scalpel blade. Immediately, two perpendicular
incisions (4 mm long by 0.2 mm deep) were made on the stroma
using a diamond knife in one of the control rabbits and 20 ml
suspension of pseudomonas was instilled over the stroma and
inside the scratches. In another control rabbit, the same method
was used without making scratches. The latter one died after
about 30 min, probably due to anaesthetic drug overdose. Corneal
ulcer occurred in the first rabbit 36 h after inoculation. Therefore,
inducing the corneal ulcer on both eyes of the rabbits of the two
study groups was performed by de-epithelialisation and stromal
incisions. The dosage of anaesthetic drugs was decreased to
(35 mg/kg) ketamine and 10 mg/kg zylazine. Rabbit B4 died
2 days after inoculation due to an unknown aetiology.
Nanoformulation and conventional eye drops were instilled
every 1 h in both eyes of the remaining rabbits. The left eye in
each group was assigned as the test eye, while the right eye was
used as positive control. In group A, nanoformulations with low
polymer amount of (150 mg) and in group B, nanoformulations in
a high polymer amount (300 mg) was used on the left eye. The
positive control (right eyes of the rabbits) in both groups received
aqueous solution of CIP 0.3% W/V made in laboratory. After
fluorescein staining, the rabbit eyes were photographed over the
study period on day 2 and weeks 1 and 2 using photo slit
biomicroscope (Topcon, SL-D7, Tokyo, Japan). Ulcer surface
areas on rabbit eyes were measured using Scion Image analyzer
software (Scion Image Corporation, Frederick, MD). Data were
collected from the different photos in each rabbit and mean
surface areas (mm2) was reported. Regardless of the numeric
assessment of keratitis surface area, the ophthalmologists of the
research team made clinical classifications of the disease status
over the course of disease. Three parameters were assessed in this
regard namely, stromal infiltration, conjunctival injection and
corneal oedema. The keratitis status in each of the three
assessments was classified as none (0), mild (1), moderate (2)
and severe (3). Details of comparisons are given in Table 3.
Clinical assessment grading was done in a masked manner
according to the criteria described by Kowalski et al. (2004).
Results
Preparation and physicochemical characterisation of
poly(NIPAAm-MAA-VP) nanoparticles
Poly(N-isopropylacrylamide) (NIPAAM) is a well-known ther-
mosensitive polymer (Salehi et al., 2013). This polymer and its
derivatives have been widely investigated and utilised in biomed-
ical, pharmaceutical and other fields, such as topical ocular drug
delivery. The potential of N-isopropylacrylamide for topical
ocular drug delivery is particularly promising due to its unique
biological properties including favourable biocompatibility,
mucoadhesiveness and reversible sol–gel transition state.
Poly(methacrylic acid) (PMAA) was chosen as a pH-responsive
block. In contrast, poly(N-vinyl-2-pyrolidone) (PVP) (a biocom-
patible polymer for biomedical applications) was grafted to
PMAA and PNIPAAm (Salehi et al., 2009a; He et al., 2010;
Rawas-Qalaji and Williams, 2012; Rasouli et al., 2014).
Therefore, dual thermo-/pH-responsive P(NIPAAm-MAA-VP)
nanogel was successfully synthesised from NIPAAM, MAA
 4 S. Davaran et al.
Figure 1. 1HNMR spectra of P(NIPAAM-
MAA-VP) block copolymers.
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and VP monomers by free radical polymerisation method.
The characteristic signals of synthesised copolymer are found
below: yield of polymerisation: 90%, 1H NMR (400 MHz, CDCl3)
dH of NIPAAm: [d (ppm) ¼ 1, 60 (CH2–CH), d (ppm) ¼
1.11((CH3)2CH), d(ppm) ¼ 3.97(N–CH–(CH3)2), d(ppm) ¼
2.22(CH–C¼O)], MAA: [d(ppm) ¼ 0.9(–C–CH3)] and VP: [d
(ppm) ¼ 1.60(CH2–CH), d(ppm) ¼ 2.3(N–CH2–CH2), d (ppm) ¼
2.02(N–CH2–CH2), d (ppm) ¼ 3.3(CH2–C¼O)] (Figure 1).
In the FTIR spectra of the polymer (Figure 2a), an intense
peak at 3300–3600 cm1 can be attributed to the –OH of
For personal use only.
–COOH group of MAA. The C–H stretching vibration of the
polymer backbone is manifested through strong peak at
2928 cm1. Absorbance of amide carbonyl groups in
PNIPAAm occurs at 1650 cm1, bending frequency of amide
N–H appears at 1550 cm1. The peak at 1720 cm1 can be
attributed to the carbonyl groups in MAA moiety of polymer
(C¼O of COOH). In FTIR spectra of CIP (Figure 2b), one
prominent characteristic peak was found between 3500 and
3450 cm1, which was assigned to stretching vibration of –OH
groups and intermolecular hydrogen bonding. Another band at
3000–2950 cm1 represented alkenes and aromatic C–H stretch-
ing, mainly u ¼ C–H. The peak at 2900 cm1 was assigned to
CH stretching vibration of cyclopropyl group. The 1950–
1450 cm1 region exhibited FTIR absorption from a wide
variety of double bonded functional groups. The band at
1750–1700 cm1 represented the carbonyl C¼O stretching i.e.
C¼O. The peak at 1650–1600 cm1 was assigned to quinolones.
The band from 1450–1400 cm1 represented C–O at 1300–
1250 cm1 suggested bending vibration of O–H group which
proved the presence of carboxylic acid. A strong absorption peak
between 1050 and 1000 cm1 was assigned to C–F group. In
FTIR spectra of the CIP-loaded P(NIPAAm-MAA-VP) nanogels
(Figure 2c), the characteristic peaks of drug appeared between at
1100 and 1750 cm1 that represented C–F groups and carbonyl
C¼O stretching, respectively. The peak at 1650 cm1 can be
attributing to overlapping peak of quinolones and polymer amide
carbonyl groups. The morphology of the resulted particles was
studied by SEM (Figure 3). The prepared CIP-loaded
poly(NIPAAm-MAA-VP) nanogels were uniform spherical
nanogels with a mean particle size was ranged between 10
and 50 nm without obvious agglomeration.
Turbidimetry measurements
The transmittance of UV–visible light was measured as a
function of temperature. At low temperature, the solution was
transparent and transmittance of the light was high, while
J Microencapsul, Early Online: 1–9
temperature rose, the polymer started to aggregate and phase
separation occurred (Gao et al., 2010). The solution become
cloudy and the transmittance decreased rapidly as the solution
become opaque. The transition resulted in the precipitation and/
or sol to gel transformation of PNIPAAm or related copolymers
were determined with turbidimetry (Salehi et al., 2009a). The
effect of pH on the phase transition behaviour of temperature
sensitive P(NIPAAm-MAA-VP) nanogel were investigated by
cloud point measurements as shown in Table 1, developed
nanoformulation was clear solution that converted into nanogels
at temperatures above 36  C with natural and acidic medium (pH
3 and 7). The normal pH of tears ranges from 6.5 to 7.6. The
average pH of tears for most people is 7.0 (Abelson et al., 1981;
Rawas-Qalaji and Williams, 2012). Therefore, the developed
CIP-loaded poly(NIPMAAm-MAA-VP) nanoparticles by admin-
istration at targeted location (physiological condition of eye
37  C and pH of 7) became nanogels and resist from rapid
drainage by tears and could release CIP in smart and sustained
release manner (Table 1).
Drug loading and in vitro drug release study
In order to increase the EE%, the polymer solution was prepared
in water below LCST of the thermosensitive polymer (25  C).
Then it was mixed with a solution of CIP and the temperature was
raised above the LCST of the copolymer (above 36  C), which led
to direct loading of the drug into the gel. This method led to high
encapsulation efficiency. EE% of CIP for the poly(NIPMAAm-
VP-MAA) is high (above 95%).
In vitro CIP release from the poly(NIPMAAm-
VP-MAA) nanogels was studied in PBS (pH 7.4) at 37  C.
As illustrated in Figure 4, the CIP-loaded nanogels showed
well-developed sustained drug release patterns. Polymeric
nanogels yielded the initial burst release of CIP, reaching
50% in first two days at 37  C, these values reached up to
100% after 5 days.
In vitro antibacterial efficiencies of CIP-loaded
nanoparticles
First, the in vitro antimicrobial activity of prepared nanoformula-
tion in comparison with CIP solution against standard strain of
P. aeruginosa was studied. MIC value for drug solution was
measured to be 18.78 mg/ml compared to that for the prepared
nanoformulation that was decreased to 4.687 mg/ml. Indeed, the
MIC value of the CIP in nanoformulation was four times lower
than the CIP solution. Figure 5 depicts the results of streak
 DOI: 10.3109/02652048.2015.1065915
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Figure 2. The FTIR spectra of P(NIPAAM-MAA-VP) nanogels (a), ciprofloxacin (b) and ciprofloxacin-loaded P(NIPAAM-MAA-VP) nanogels (c).
Figure 3. Scanning electron micrographs of Ciprofloxacin-loaded
poly(NIPAAm-MAA-VP) nanoparticles.
cultures on the surfaces of Muller–Hinton agar plates, the first
concentration with no sign of bacterial growth on plates
considered as MIC.
Animal study
The keratitis surface area over the course of disease varied
according to the treatment types. The keratitis in starting control
rabbits developed total wound extension over the study period.
Keratitis wound area increased from a mean of 3.8 mm2 at the
second day up to 5.7 mm2 at day 7. After extension of the keratitis
Preparation and in vivo evaluation of in situ gel system 5
Table 1. Gelation temperature of P(NIPAAM-MAA-VP) nanogel at
different pH.
pH ¼ 3 pH ¼ 7 pH ¼ 8.5
Gelation temperature of 29  C 36  C 42  C
P(NIPAAm-MAA-VP)a
Note: aThe polymer concentration was 10% wt.
wound up to the 7th day of infection the reduction of wound
surface area from day 7 to day 14 was compared for the three trial
modalities, details of which is given in Table 2. Overall, different
degrees of reduction in keratitis surface area were observed in all
test groups (nanoformulation versus CIP solution). As shown in
Table 2, the efficacy of nanoformulation in its two concentration
modalities to reduce the keratitis wound area did not differ in mild
or moderate keratitis cases, whereas in severe cases as in rabbit
B1, the nanoformulation (high concentration) appeared to be more
effective than CIP solution. Figure 6 shows the clinical status and
eye ulcer areas of rabbit A1 and rabbit B1 in day 7 and day 14,
representative of severity and treatment variations. Results of
clinical assessment regarding stromal infiltration, conjunctival
injection and corneal oedema are given in Table 3. In this study,
paired corneas were compared. Methodologically, this is con-
sidered as an asset because it minimises the effect of biological
variation (Gupta et al., 2000; Dilbaghi et al., 2013).
Discussion
The cornea is a highly vulnerable organ tissue to immunological
inflammation and microorganisms. This is probably due to the
 6 S. Davaran et al. J Microencapsul, Early Online: 1–9

characteristics needed to maintain its valuable visual mission by
features as (1) ocular barriers, (2) the absence of lymphatic
drainage pathways, (3) the presence of immune modulatory
factors and (4) the presence of antigen presenting cells. Bacterial
infections are a common source of keratitis a major cause of
which is stated to be P. aeruginosa. P. aeruginosa is the aetiology
of a bacterial corneal ulcer that develops quite quickly and widely
involves the corneal area. It is most often seen in a weakened,
elderly patient and among those wearing contact lenses for longer
times. The condition usually starts from the centre with a gray
infiltrate underlying an epithelial defect. The infection, if not
treated, will quickly spread in all directions. It may affect the
sclera, leading to some grave consequences, or give rise to
destruction and perforation of the cornea. Although alpha
haemolytic streptococci have shown very low sensitivity to CIP
in treating keratitis, CIP has been shown to be generally effective
in treating bacterial keratitis (Hajoui et al., 2011; Shalchi et al.,
Journal of Microencapsulation Downloaded from informahealthcare.com by University of Otago on 07/22/15
cacy and in vivo efficiency of these nanoformulations were
evaluated and compared with a commercial CIP eye drop
formulation. As reported in the ‘‘Result’’ section, the in situ
CIP-loaded poly(NIPAAm-MAA-VP) nanogels with a mean
particle size between 10 and 50 nm and higher than 95%
encapsulation efficiency were obtained. The turbidimetry experi-
ment results indicated that sol–gel temperature of our developed
nanogel was 36  C. This is well-matched with the application of
nanogels for ocular drug delivery. The temperature of outer
surface of cornea is a bit lower than physiologic temperature. The
previous studies reported that it is around 34.8–36.6 (Mapstone,
1968; Purslow and Wolffsohn, 2005; Purslow et al., 2005; Ng and
Ooi, 2006; Purslow and Wolffsohn, 2007; Kawasaki et al., 2009;
Kessel et al., 2010; Gokul et al., 2014; Meister et al., 2014) which
meets the specifications of our developed nanogels.

2011; Prokosch et al., 2012). Ciprofloxacin and ofloxacin have Table 2. Keratitis surface wound area for both eyes (CIP nanoformulation
been found to be as effective as 80% in treating for Gram negative for the left ¼ test eyes versus CIP solution used in the right ¼ control eyes)
bacterial keratitis (Dhakwa et al., 2012). Improvement of the drug and both dosages of CIP-loaded nanoformulation (low concentrated for
delivery in treating keratitis has been the focus of a wide range of group A versus high concentrated in group B rabbits).
research in field of ocular pharmacology (Mannis, 2002;
Weyenberg et al., 2004; Ebrahim et al., 2005; Lv et al., 2005; keratitis wound area (mm2)
Mouly et al., 2006; Wu et al., 2010; Refai and Tag, 2011). In this Measurement
study, we developed a novel CIP-loaded dual stimuli responsive Group ID intervals OD OS
nanogels formulation with in situ gel-forming behaviour for
topical ocular treatment of bacterial keratitis. The physico- A1 2 days 6.67 5.97
7 days 9.70 6.52
chemical characterisation, in vitro release, microbiological effi- 14 days 1.68 1.92
A2 2 days 2.45 2.43
For personal use only.

7 days 3.04 2.95

14 days 1.03 1.37
A3 2 days 2.46 1.88
7 days 2.98 2.46
14 days 0 0
A4 2 days 1.45 1.85
7 days 1.92 2.2
14 days 0 0
B1 2 days 8.78 8.83
7 days 11.43 12.00
14 days 4.34 2.36
B2 2 days 2.81 2.84
7 days 5.36 6.78
14 days 1.57 1.45
B3 2 days 2.38 2.43
7 days 5.38 7.34
14 days 0 0
B4 Expired at day 2 – –
C 2 days 4.87 3.93
Figure 4. Cumulative release of ciprofloxacin from pH and thermo- 7 days 9.82 7.87
responsive P(NIPAAm-MAA-VP) nanogels under physiological environ- 14 days Whole cornea Whole cornea
ment (pH 7.4 and 37  C).

Figure 5. Streak cultures on the surfaces of

Muller–Hinton agar plates, the first concen-
tration with no sign of bacterial growth on
plates considered as MIC. (a) MIC value for
CIP solution was measured to be 18.78 mg/ml.
(b) MIC value for the prepared nanoformu-
lation was 4.687 mg/ml.
 DOI: 10.3109/02652048.2015.1065915 Preparation and in vivo evaluation of in situ gel system 7
Table 3. Results of clinical assessment of rabbit eyes regarding stromal
infiltration, conjunctival injection and corneal oedema (right
eye ¼ control eye, left eye ¼ test eye; A rabbits receiving low
concentrated nanogel and B rabbits receiving high concentrated nanogel).

Stromal Conjunctival Corneal

Rabbit eye Time infiltration injection oedema
A1 right eye Day 2 Moderate Severe Mild
Day 6 Severe Severe Severe
Day 14 Mild Mild Mild
A1 left eye Day 2 Moderate Severe Moderate
Day 6 Severe Severe Severe
Day14 Mild Mild Mild
A2 right eye Day 2 Mild Moderate Mild
Day 6 Moderate Mild Mild
Day 14 Mild None None
A2 left eye Day 2 Mild Moderate Moderate
Day 6 Moderate Mild Mild
Day 14 Mild None None
Journal of Microencapsulation Downloaded from informahealthcare.com by University of Otago on 07/22/15

A3 right eye Day 2 Mild Moderate None

Day 6 Mild Mild None
Day14 None None None
A3 left eye Day 2 Mild Mild None
Day 6 Moderate Mild Mild
Day 14 None None None
A4 right eye Day 2 Mild Mild None
Day 6 Mild Mild None
Day 14 None None None
A4 left eye Day 2 Mild Mild None
Day 6 Mild Mild Mild
Day 14 None None None
B1 right eye Day 2 Moderate Severe Mild
Day 6 Severe Severe Severe
For personal use only.

Day14 Moderate Mild Mild

B1 left eye Day 2 Moderate Severe Moderate
Day 6 Severe Severe Severe
Day 14 Mild Mild None
B2 right eye Day 2 Mild Moderate Mild
Day 6 Severe Severe Severe
Figure 6. The clinical status and eye ulcer areas of rabbit A1 (receiving Day 14 Mild Mild Mild
low concentrated nanogel) and rabbit B1 (receiving high concentrated B2 left eye Day 2 Mild Moderate Mild
nanogel in day 7 and day 14 (*: Corneal cyst with drug deposition that Day 6 Moderate Moderate Moderate
was later dissolved). Day 14 Mild Mild None
B3 right eye Day 2 Mild Moderate None
Day 6 Moderate Mild Mild
Day 14 None Mild None
B3 left eye Day 2 Mild Severe Mild
Ciprofloxacin released from the nanoparticles showed an Day 6 Moderate Mild Mild
enhanced antibacterial effect as determined by minimal inhibitory Day 14 None Mild None
concentrations. Since MIC value has declined from 18.78 to
4.687 mg/ml when tested against P. aeruginosa. In vivo studies
demonstrated reasonable efficacy in severe keratitis using the
developed nanoformulation. cell wall or membrane; (b) adsorbing to cell wall and serve as a
As it is known, the pKa value of the antimicrobial drug, CIP are antibiotic depot to continuously release drug molecules, which
6.09 for carboxylic acid group and 8.74 for the nitrogen on the will diffuse into the interior of the microorganisms (Zhang et al.,
piperazinyl ring (Torniainen et al., 1996). At pHs greater than 6.09, 2010).
the acid will be primarily dissociated and at pHs less than 8.74, the Our results are in good agreement with other studies in this
nitrogen will be primarily protonated. Therefore, at pH 7.4, CIP is regard. We have witnessed rapid development in the field of
positively charged. On the other hand, at pH 7.4, carboxylic acid ocular drug delivery in the last few years. One of the recent
groups of grafted PMAA in the carrier are deprotonated and reports is about a corneal targeted nanoformulation designed for
consequently negatively charged. The positively charged CIP binds sustained natamycin delivery in the treatment of mycotic keratitis
with the negatively charged P(NIPAAm-MAA-VP) to form the (Chandasana et al., 2014). It has reduced its dosage to 1/5 that
CIP-loaded P(NIPAAm-MAA-VP) by electrostatic interaction. means delivering less drug concentration for a longer period of
Also, the hydrogen bonding are present between the amino groups time. Hsiue et al. (2002) evaluated the intraocular pressure (IOP)
and hydroxyl groups in CIP molecules and the ionised –COO lowering effect of PNIAAm-loaded adrenalin on rabbit eyes. The
groups in PMAA and amine groups in VP of P(NIPAAm-MAA- effect of nanoformulations lasted 6–8 times longer than conven-
VP) nanogels at pH 7.4. this could be a possible reason for depot or tional eye drops. In their study, in vitro cytotoxicity tests showed
reservoir effect of our newly prepared formulation in eye environ- that the number of dead cells was identical to PBS control
ment to release the antibiotic in longer time intervals. solution. In our study, we did not notice any additional irritation in
Nanoparticle-based antimicrobial delivery systems could inter- the eyes treated with commercial or nano-ciprofloxacin eye drops.
act with microorganisms via two mechanisms: (a) fusing with A study by Dilbaghi et al. (2013) revealed that the optimised
microbial cell wall or membrane and release the cargo within the formulation of tropicamide-loaded TSX nano-aggregates have a
 8 S. Davaran et al.
significantly higher corneal permeation of tropicamide across the
isolated goat cornea compared to commercial conventional
aqueous formulation. In this study, paired corneas were compared.
Methodologically, this is considered as an asset because it
minimises the effect of biological variation (Gupta et al., 2000;
Dilbaghi et al., 2013). In a study by Calvo et al., three different
nanoparticles and nanocapsules were developed, and their
efficacy to increase the corneal penetration of drugs was
examined. They found that the in vitro corneal penetration of
the encapsulated indomethacin was more than 3-fold that of the
currently available ordinary eye drops. The results were consistent
with the three studied formulations (Calvo et al., 1996).
Controlled release of ophthalmic CIP using dual thermo-/pH-
sensitive poly(NIPAAm-MAA-VP) as in situ forming gel
nanocarrier has been the focus of this study. It revealed a
tendency in possible higher efficacy of CIP-loaded
poly(NIPAAm-MAA-VP) nanoparticles in treating bacterial kera-
Journal of Microencapsulation Downloaded from informahealthcare.com by University of Otago on 07/22/15
titis when compared to control treatment in severe forms of
keratitis in an animal model. However, in milder cases, the new
treatment did not appear to be more effective. Although, no case-
specific study was found to be comparable, similar drug delivery
application have also provided subtle evidence at least to foster
wider range of research to be done in this field.
Conclusion
The developed CIP-loaded poly(NIPAAm-MAA-VP) nanoformu-
lation used as an ocular delivery system represented both
temperature and pH-triggered in situ gelation property.
PNIPAAm (a thermosensitive polymer) in combination with
For personal use only.
MAA (pH-sensitive polymer) was used as gelling agent.
Developed formulation was clear solution, which converted into
gel at temperatures above 36  C and pH 7. Reasonable efficacy
was observed using the developed formulation. The developed
system is suitable for sustained topical drug delivery and has the
potential to be a viable alternative to conventional eye drops for
the treatment of ocular diseases.
Declaration of interest
The authors report no declarations of interest. The authors alone are
responsible for the content and writing of the paper.
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