162 Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2

Department of
Pharmaceutics, Faculty
of Pharmacy, Jamia
Hamdard,
1
Department
of Nuclear Medicine,
Institute of Nuclear
Medicine and Allied
Sciences, Ministry of
Defence, New Delhi, India
Address for correspondence:
Dr. Mohammed Aqil,
E-mail: aqilmalik@yahoo.com;
dr.hgupta@yahoo.com
H
yperacute bacterial conjunctivitis is a severe, sight
threatening ocular infection that warrants immediate
ophthalmic work-up and management. Bacterial conjunctivitis
requires treatment with antibiotics for 5-7 days that may result
in poor patient compliance with conventional dosage forms due
to greater frequency of drug administration, that is, 2-3 drops
every 2-3 h.
[1]
This is because in ocular delivery, the physiological
constraints imposed by the protective mechanisms of the eye
lead to low absorption of drugs, resulting in short duration of the
therapeutic effect. When a drug solution is dropped into the eye,
the effective tear drainage and blinking action of the eye result in
a 10-fold reduction in the drug concentration within 4-20 min.
[1]

The availability of medicament at corneal surface would be
significantly improved if the precorneal residence time of drugs
could be increased. Several new preparations such as inserts,
[2]

collagen shields,
[3]
and colloidal systems, such as liposomes,
[4,5]

nanoparticles,
[6,7]
and nanocapsules
[8,9]
have been developed for
ophthalmic use, not only to prolong the ocular contact time of
the vehicle but also to slow down drug elimination.
[10]
The use of
nanotechnology-based drug delivery systems like microemulsions,
nanosuspensions, nanoparticles, solid lipid nanoparticles,
niosomes, dendrimers, and liposomes has led to the solution of
various solubility-related problems of poorly soluble drugs, like
dexamethasone, budesonide, ganciclovir, and so on.
[11]
However,
these novel preparations have some disadvantages, such as poor
compliance. Out of these novel formulations; in situ gel and
nanoparticles have been established as the promising one. In our
previous work, we found that these formulations have their own
disadvantages. For example, in situ gel remains for 12-15 h,
[12]

whereas poly lactic co glycolic acid (PLGA) nanoparticles are
nonmucoadhesive, so are drained out of eyes quickly.
[13]
In our
present work, we try to combine both these formulation strategies
as a nanoparticles suspended in liquid dosage form suitable to be
administered by instillation into the eye which, upon exposure
to physiological conditions, changes to the gel phase, thus
increasing the precorneal residence time of the nanoparticles and
enhancing ocular bioavailability. Our group has coined the term
nanoparticle laden in situ gel for this formulation.
[14]
Nanoparticles laden in situ gel for sustained
ocular drug delivery
Himanshu Gupta, Mohammed Aqil, Roop K. Khar, Asgar Ali, Aseem Bhatnagar
1
,
Gaurav Mittal
1
ABSTRACT
Proper availability of drug on to corneal surface is a challenging task. However, due to ocular physiological
barriers, conventional eye drops display poor ocular bioavailability of drugs (1%). To improve precorneal
residence time and ocular penetration, earlier our group developed and evaluated in situ gel and nanoparticles
for ocular delivery. In interest to evaluate the combined effect of in situ gel and nanoparticles on ocular
retention, we combined them. We are the first to term this combination as nanoparticle laden in situ gel,
that is, poly lactic co glycolic acid nanoparticle incorporated in chitosan in situ gel for sparfloxacin ophthalmic
delivery. The formulation was tested for various physicochemical properties. It showed gelation pH near pH 7.2.
The observation of acquired gamma camera images showed good retention over the entire precorneal area
for sparfloxacin nanoparticle laden in situ gel (SNG) as compared to marketed formulation. SNG formulation
cleared at a very slow rate and remained at corneal surface for longer duration as no radioactivity was
observed in systemic circulation. The developed formulation was found to be better in combination and can
go up to the clinical evaluation and application.
KEY WORDS: Gamma scintigraphy, in situ gel, nanoparticle, nanoparticle laden in situ gel, ocular, PLGA,
sparfloxacin
Received : 18-03-13
Review completed : 22-03-13
Accepted : 27-03-13
How to cite this article: Gupta H, Aqil M, Khar RK, Ali A, Bhatnagar A, Mittal G. Nanoparticles laden in situ gel for sustained ocular drug delivery. J Pharm
Bioall Sci 2013;5:162-5.
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DOI:
10.4103/0975-7406.111824
Short Communication
Gupta, et al.: Nanoparticle laden in situ gel
Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2 163
In our study, we used third generation flouroquinolone
sparfloxacin for formulation evaluation. Hence, in the present
work we develop and evaluate new system, that is, sparfloxacin
PLGA nanoparticle laden in situ gel for ophthalmic delivery
to improve precorneal residence time and thus ocular
bioavailability.
Materials and Methods
Materials
Sparfl oxaci n was recei ved as ki nd gi ft from Mi cro
Labs Lt d. ( Chandi gar h, I ndi a) ; PLGA ( 50: 50) ,
intravenous0.2 dL/g, was obtained from Purac Biomaterial,
Singapore), polyvinyl alcohol (PVA, MW 95,000) was obtained
from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
Chitosan (practical grade, 75%-85% deacetylated, and molecular
weight 150 kDa) was obtained as kind gift from M/s India Sea
Foods, India. All other chemicals were of analytical grade.
Preparation of nanoparticle laden in situ gel
Nanoparticles were prepared as per our previous published
work.
[13]
Briefly, nanoprecipitation technique was applied to
prepare sparfloxacin nanoparticles. Drug and polymer in ratio
of 1:10 (keeping drug 10 mg and PLGA 100 mg) were dissolved
in acetone (5 mL) at room temperature. The resulted
solution was slowly dropped with speed manually (approx.
0.5 mL/min) into water (20 mL) containing PVA (1.5% w/v)
with continuous magnetic stirring at 1800 rpm. Acetone
and some water were evaporated, and the final volume of
the aqueous suspension was collected. The nanosuspension
was then centrifuged at 18,000 rpm, 20C for 1 h (Remi,
India). Nanoparticles were collected and washed (3 times)
with distilled water using previously described centrifugation
approach and then lyophilized by means of Christ Alpha
1-4 lyophilizator (Christ, Germany) using 1% w/v mannitol
as lyoprotectant. Whereas, in situ gel base is prepared by
dissolving 0.5% w/v chitosan in 1% v/v acetic acid, pH adjusted
to 5.5-6.0 with buffer saline.
[12]
Weighed quantity of drug-loaded freeze-dried nanoparticles
(equivalent to the prescribed dose of sparfloxacin 0.3% w/v) were
taken and dispersed in the respective 1 ml of placebo in situ gel
base (0.5%w/v chitosan solution) to form nanoparticle laden
in situ gel. Freeze-dried nanoparticles were weighed accurately
on the basis of its drug loading and release pattern [Table 1].
[13]
Physicochemical characterization
Physicochemical characterization of the developed nanoparticles
laden in situ gel were carried out and compiled in Table 2.
Clarity
The clarity of the formulations after and before gelling was
determined by visual examination of the formulations under
light alternatively against white and black backgrounds.
Gelation pH
Gelation pH is the pH at which the solution form of the
formulation was changed to gel. Formulation was taken in a
beaker and 1M NaOH was added dropwise with continuous
stirring. pH was checked using pH meter. The pH at which
sudden change in viscosity was observed and noted is recorded
as gelation pH.
Viscosity
Viscosity of formulations were determined by using Brookfields
viscometer (model DV II, spindle no. 02, at 20 rpm), on
formulation pH (6.0) and gelation pH near 7.0.
In vivo ocular retention study-Gamma scintigraphy
Gamma scintigraphy is used to assess in vivo precorneal drainage
of the developed formulation. Male New Zealand albino rabbits
of either sex weighing 1.8-2.5 kg and free of any signs of ocular
inflammation or gross abnormality were used in the study.
Animals were procured from the animal house of Institute of
Nuclear Medicine and Allied Sciences Delhi, India and all study
protocols were approved by local institutional animal ethics
committee. Sparfloxacin was radiolabeled with Tc-99m by direct
labeling method using stannous chloride as reducing agent as
per previous reported method.
[13]
Gamma camera (Millenium
VG, USA), autotuned to detect the 140 KeV radiation of Tc
99m

was used for scintigraphy study. Rabbits were anesthetized
using ketamine HCl injection given intramuscularly in a dose
of 15 mg/kg body weight. The rabbits were positioned 5 cm in
front of the probe and 50 PL of the radiolabeled formulation was
instilled onto the left corneal surface of the rabbits. Recording
was started 5 s after instillation and continued for 30 min using
128u128 pixel matrix. Individual 60 frames (60u30 s) were
captured by dynamic imaging process. Region of interest was
selected on one frame of the image and time-activity curve was
plotted to calculate the rate of drainage from eye. A single whole
body static image was also taken after 6 h of instillation of drug/
formulation. Each formulation was tested on three rabbits.
Table 1: Final composition of sparfloxacin nanoparticles
laden in situ gel
Ingredients Concentration (w/v) (%)
Sparfloxacin nanoparticles 0.4
Chitosan 0.5
Sodium chloride (NaCl) 0.45
Methylparaben 0.1
Water/base (q.s.) 100
Table 2: Physicochemical characterization of nanoparticles
laden in situ gel
Parameter Nanoparticle laden in situ gel
Clarity Very slight turbid solution
pH 6.08r0.07
Gelation pH 7.1r0.109
Viscosity (at pH 6.0) 42.83r2.041 cps
Viscosity (at pH 7.2) 248.33r4.84 cps
Gupta, et al.: Nanoparticle laden in situ gel
164 Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2
Results and Discussion
In situ gel and nanoparticles have their own merits and demerits.
In situ gel stays only for 12 h, whereas PLGA and conjunctiva is
anionic in nature and hence PLGA nanoparticles are not able
to be retained for the longer time. To increase the duration,
we entrapped PLGA nanoparticles in cationic chitosan in situ
gel system. Chitosan is a cationic polymer and hence will make
link with both PLGA nanoparticles and conjunctiva and thus
increases retention. As chitosan act both as pH sensitive and
permeation enhancer, the viscosity of the chitosan increases
when it reaches to eye pH at 7.4. This combination will allow
nanoparticles to stay for longer duration and provide optimum
drug release. To achieve most out of these two delivery systems,
we have combined both the system to develop nanoparticles
laden in situ gel. The nanoparticle size was near 180 nm in
concordant with our previously reported results.
[13]
The quantity
of nanoparticles was calculated on the basis of drug loading and
release, we have taken 40 mg of SN to prepare 1 ml of respective
nanoparticles laden in situ gel (SNG). Final composition of SNG
is given in Table 1. The developed nanoparticles laden in situ gels
were then evaluated for various physicochemical characteristics
as given in Table 2. The preparation is well-dispersed with slight
turbidity. After mixing, no difference is found in gelation pH
and viscosity of the formulation.
Gamma scintigraphy
Pharmacoscintigraphy is a tool to analyze the retention time of
the formulation in cul-de-sac. For gamma scintigraphic studies,
sparfloxacin was labeled with radionuclide Tc-99m using stannous
chloride as reducing agent. Tc-99m was chosen for the purpose
because of its moderate half life (6 h). Further it emits gamma
rays, which have relatively low energy as compared to D and E rays,
so leads to no serious health hazards to the workers. Sparfloxacin
was instantaneously labelled with Tc-99m. Labelling efficiency
was checked by instant thin layer chromatography (ITLC)
using 100% acetone as mobile phase. The Rf value of free Tc
is ~0.9, so it reaches to the top of the ITLC strip while the
complexed Tc (drug-Tc complex) cannot travel much due to
difference in molecular weight and is retained at the base of
ITLC strip. Thus, from the difference in the top and bottom
counts, labelling efficiency was calculated. After prelabeling
efficiency studies which include labelling parameters like
stannous chloride concentration and pH were optimized and a 50
Pg stannous chloride

concentration at pH 7.0 was found to give
the maximum labelling efficiency (95.2%). In these conditions,
minimum colloids (1%) were produced. The observation of the
acquired gamma camera images showed a good spreading over
the entire precorneal area for developed ocular formulations of
sparfloxacin [in situ gel (SG), nanosuspension (SN), nanoparticle
laden in situ gel (SNG)], immediately after administration as
compared to marketed formulation. Marketed formulation
cleared very rapidly from the corneal region and reached in
to systemic circulation via nasolacrimal drainage system as
significant activity was recorded in kidney and bladder after 6 h
of ocular administration [Figure 1a], whereas formulations SG,
SN, and SNG were retained for longer duration at corneal surface.
No significant radioactivity was observed in kidney and bladder
after 6 h of administration of these formulations [Figure 1b-d].
To differentiate between these formulations, the curves of the
% radioactivity remained on the corneal surface as a function of
time (30 min dynamic imaging) was also plotted [Figure 2]. The
retention on cornea follows the following sequence:
Figure 2: Gamma scintigraphy dynamic study
Figure 1: Static images of rabbit after 6 h of administering sparoxacin
formulations (a) marketed, (b) in situ gel, (c) nanosuspension, and
(d) nanoparticles laden in situ gel
d c
b a
Gupta, et al.: Nanoparticle laden in situ gel
Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2 165
Marketed formulation nanosuspension in situ
gelnanoparticle laden in situ gel
It shows that the nanoparticles laden in situ gel retained
for the longer duration in the eye giving extended release
than nanosuspension and in situ gel alone, whereas the
marketed formulation drained out of the eye with in
30 min. Due to mucoadhesive property of chitosan, in situ
gel-based formulation cleared at slowest rate as compare to
nanosuspension and retained at corneal surface for longest
time duration. Further, the viscosity of chitosan is raised due
to change in physiological conditions of pH (!7) and ionic
concentration of the formulation upon instillation in to eye as
a result of buffering action of the tear fluid. Chitosan also acts
as penetration enhancer that increases the transport of drug
across cornea.
Conclusion
Our previous study shows that the PLGA nanoparticles, due to
smaller size, are able to provide prolong retention on the eye
than the marketed formulation. PLGA is an anionic polymer and
hence it is nonmucoadhesive in nature. To enhance the efficacy
of the nanoparticulate formulation, we incorporate them in
cationic chitosan in situ gel to make nanoparticle laden in situ
gel, which helps in retention of nanoparticles on the eye. A good
spreading and retention of formulation was observed in gamma
scintigraphy studies as compared to marketed formulation.
In time activity curve, there is a minimal falls in counts/s of
formulation as compared to rapid fall of marketed formulation
further shows that the nanoparticles laden in situ gel stay for
longer time on the eye.
Acknowledgment
Authors are thankful to Council of Scientific and Industrial
Research (CSIR), New Delhi to provide Senior Research Fellowship
to Himanshu Gupta for conducting this work.
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Source of Support: Dr. Himanshu Gupta was recipient of Senior Research
Fellowship of Council of Scientifc and ndustrial Research (Government of
ndia), New Delhi, ndia, Conict of Interest: None declared.