Journal of Colloid and Interface Science 483 (2016) 132–138

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Journal of Colloid and Interface Science

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

Regular Article

Moxifloxacin loaded gelatin nanoparticles for ocular delivery:

Formulation and in-vitro, in-vivo evaluation
Alok Mahor a,⇑, Sunil Kumar Prajapati a, Amita Verma b,⇑, Rishikesh Gupta a, Arun K. Iyer c,d,
Prashant Kesharwani c,⇑
a
Institute of Pharmacy, Bundelkhand University, Jhansi, India
b
Department of Pharmaceutical Sciences, Faculty of Health Sciences, Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad, India
c
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, 259 Mack Ave, Wayne State University, Detroit, MI 48201, United States
d
Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48202, United States

g r a p h i c a l a b s t r a c t

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

Article history: The current research focuses on developing positively charged gelatin nanoparticles loaded with moxi-
Received 3 July 2016 floxacin for its effective ocular delivery and controlled release in corneal eye layer. We selected type A
Revised 6 August 2016 gelatin because of its biodegradable and non-toxic nature as the polymer of choice for fabricating the
Accepted 10 August 2016
nanoparticles by a modified two step desolvation technique. The produced nanoparticles were positively
Available online 10 August 2016
charged (+24 ± 0.12 mV) with a narrow particle size of 175 ± 1.11 nm as measured by dynamic light scat-
tering (DLS). The in-vitro drug release from the nanoformulations exhibited a burst effect in the first hour
Keywords:
followed by a controlled release of the drug for the subsequent 12 h. The Korsmeyer-Peppas model
Moxifloxacin
Gelatin
showed better linearity and the formulations displayed non-Fickian drug release pattern. The optimized
Ocular tissues formulation was assessed for its utility as an anti-bacterial agent and its effectiveness was tested on the
In-vivo tolerance corneal eye surface of rabbits. The in-vivo tolerance tests revealed that the drug loaded nano-
Staphylococcus aureus formulations was non-irritant to the ocular tissues indicating its safety. The in-vivo anti-bacterial activity
Bacillus subtilus of the nanosuspension was more effective against S. aureus than the commercially market product,
MoxiGramÒ. Microbiological efficacy assessed against B. subtilus using cup-plate method suggested that

⇑ Corresponding authors at: Institute of Pharmacy, Bundelkhand University, Kanpur Road, Jhansi 284128, India (A. Mahor). Department of Pharmaceutical Sciences, Faculty
of Health Sciences, Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad 211007, India (A. Verma). Department of Pharmaceutical Sciences, Eugene
Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, USA (P. Kesharwani).
E-mail addresses: alokmpharma@gmail.com (A. Mahor), amitaverma.dr@gmail.com (A. Verma), prashantdops@gmail.com (P. Kesharwani).

http://dx.doi.org/10.1016/j.jcis.2016.08.018
0021-9797/Ó 2016 Elsevier Inc. All rights reserved.
 A. Mahor et al. / Journal of Colloid and Interface Science 483 (2016) 132–138
our fabricated nanosuspension possess better anti-microbial activity as compared to the commercial
agent, MoxiGramÒ revealing promising potentials for the currently developed gelatin based
nanoformualtions.
1. Introduction
Drug delivery to the ocular tissues has been a formidable chal-
lenge because of the complex anatomy and physiological barriers
of the eye which does not permit the entry of drug molecules at
the site of action [1–3]. The eye is characterized by complex biolog-
ical barriers such as tear film barrier and the corneal barrier to
restrict the entry of any foreign materials [4]. The eye prevents
the entry of foreing objects and formulations including ophthalmic
formulation by washing out the materials as a defence mechanism
to external agents such as excessive lacrimation, reflex blinking,
high tear production causes short residence time [5] leading to
low availability of the drug at the corneal surfaces [6].
Penetration of lipophilic and hydrophilic drugs across the cor-
neal epithelium occurs via transcellular pathway or paracellular
pathway respectively but this transfer is hindered by drug binding
to the corneal tissues [7]. Corneal surface being lipidic is more per-
meable to non-ionized drugs, thus entry of ionized drugs depends
on the chemical equilibrium between the ionic and non-ionic form
of the drug in the ophthalmic dosage form and finally in the lacri-
mal fluid. The total charge of the drug thus plays an important role
in the drug permeation. Since, cornea and conjunctiva possess neg-
ative surface charges, positively charged hydrophilic cationic com-
pounds can permeate more easily through the cornea than anionic
forms [8–11].
Until recently, researchers have focussed mainly on improving
corneal retention/permeation of the drugs by employing colloidal
drug delivery systems like liposomes [12], nanoparticles [13–16]
and so forth with tangible success. In this regard, nanoparticles
with effective drug encapsulation, increased corneal uptake,
enhanced intra-ocular half-lives that are tolerable at corneal sur-
face [17] could be a promising for drug delivery for ophthalmic
applications.
For the fabrication of the nanoparticles, type A gelatin was cho-
sen because of its well-known biodegradable [18], biocompatible
and non-toxic nature. Indeed, collagen, the native protein, from
which gelatin is derived is present in the corneal stroma of the
eye and has been widely employed in ophthalmic drug delivery
systems [19,20]. Moxifloxacin, a hydrophobic drug, is a fourth gen-
eration fluoroquinolone antibiotic and is effective against anaero-
bic as well as gram positive microorganisms [21], its effective
dose is 1 drop t.i.d. for seven days for the treatment of acute
bacterial conjunctivitis. Thus, with an objective of increasing drug
solubility and reducing dosing frequency, the study was designed
to encapsulate moxifloxacin in gelatin matrix for controlled deliv-
ery at the ocular surface.
2. Materials and methods
2.1. Materials
Moxifloxacin was purchased from Yarrow Chem Products,
Mumbai, India, Gelatin Type A (Bloom175), glutaraldehyde, ace-
tone, sodium chloride, potassium chloride, calcium chloride dihy-
drate, magnesium chloride hexahydrate, sodium bicarbonate and
dialysis membrane (molecular weight cut-off 12,000–14,000 Da)
were purchased from Himedia, Mumbai, India. All other chemicals
133
Ó 2016 Elsevier Inc. All rights reserved.
used were purchased from Sigma Aldrich (St. Louis, MO) and were
of highest analytical reagent (AR) grade.
2.2. Animals
The experiments were performed using male New Zealand
albino rabbits, weighing 2.0–2.5 kg free devoid of any oddity of
ocular inflammation and uncultured malformations. Before com-
mencement of the experiments, the animals were familiarized to
the customary laboratory conditions in cross aerated animal hous-
ing at temperatures 25 ± 2 °C and 75% relative humidity with light
and dark cycles of 12:12 h. The rabbits were fed with regular pallet
nutrition and had free access to drinking water. The research
experiments were approved by the Institutional Ethics Committee
and were accomplished as per CPCSEA guidelines (approval no. BU/
Pharm/IAEC/11/035).
2.3. Fabrication of nanoparticles
Moxifloxacin loaded gelatin nanoparticles were prepared by a
two-step desolvation technique following the method as defined
by Coester et al. [22]. For this purpose, a fixed amount of type A
gelatin (Bloom strength 175) was dissolved in 25 ml of distilled
water under gentle heating (40 °C). The first desolvation step is ini-
tiated by adding 25 ml acetone (desolvating agent) and the drug
together. The supernatant containing desolvated gelatin was dis-
carded following subsequent sedimentation of high molecular
weight gelatin. The residue was dissolved again by adding distilled
water under heating and the pH was accustomed to 2.5 by 0.1 N
HCl. Gelatin nanoparticles were formed in situ when acetone was
added dropwise during the second desolvation step under stirring
(500 rpm). Subsequently, 200 ll of glutaraldehyde (25% aqueous
solution) was added after 10 min, to crosslink the nanoparticles.
Gelatin nanoparticles were further subjected to purification by a
3-fold centrifugation (16,000g for 20 min) and re-dispersion in
acetone/water mixture (30/70).
2.4. Characterization of the nanoparticles
2.4.1. Particle size and morphology
The size and polydispersity index of the particles were analyzed
by dynamic light scattering (DLS) also referred as photon correla-
tion spectroscopy (PCS). The samples were diluted in sterile
filtered, highly purified water and analyzed employing Nanosizer
ZS (Malvern Instruments, UK). Zeta potential was determined
using Nanosizer ZS (Malvern Instruments, UK) using specific
electrode containing cuvettes at concentrations of 20 lg/ml.
Surface morphology of the particles was assessed using Scan-
ning Electron Microscopy (SEM) (JSM-6500 F, Germany). Samples
were prepared by dispersing the lyophilized nanoparticles on a
double adhesive tape which was stuck on gold coated aluminium
stubs of 300 Å thickness and photomicrographs were taken at suit-
able thickness. Lyophilized nanoparticles were suspended in simu-
lated tear fluid (pH 7.4) and examined using transmission electron
microscope (TEM, Philips Morgagni 268, Netherlands) at an accel-
eration voltage of 100 kV to obtain TEM photomicrographs at
suitable magnification.
134 A. Mahor et al. / Journal of Colloid and Interface Science 483 (2016) 132–138

2.4.2. % Entrapment efficiency
The% entrapment efficiency of moxifloxacin loaded gelatin
nanoparticles was determined by centrifuging the nanoparticles
at 10,000 rpm for 30 min (REMI, Gwalior, India) and the super-
natant containing free drug was analyzed by UV spectrometer
(Shimadzu-1700, Japan) at 230 nm [23]. The % entrapment effi-
ciency can be determined by using the formula:
%Entrapment Efficiency ¼ 100  ðMass of drug in nanoparticles=
Mass of drug used in formulationÞ
2.5. Physicochemical characterization
Fourier Transform Infrared Spectroscopy (FTIR) of moxifloxacin,
gelatin, their physical mixture and fabricated nanosuspension was
obtained by preparing KBr pellet of the samples and analyzing
them using FTIR spectrometer (Shimadzu, 1800, Japan). The %
Transmittance (%T) was recorded in the spectral region of 4000–
500 cm1.
Differential Scanning Calorimetry (DSC) thermograms of drug,
polymer, physical mixture and the drug’s thermal behaviour in
nanoparticles was obtained using a Differential Scanning Calorime-
ter (DSC-60, Shimadzu, Japan) which was connected to a comput-
erized thermal analyzer. Endothermal transition of the drug was
analyzed using thermogravimetry.
Melting point of the drug and glass transition temperature of
gelatin was analyzed in the DSC thermogram of the physical mix-
ture. Thermal characteristics of the drug in the nanoparticles were
studied by heating the sample in a covered sample pan under
nitrogen gas flow. The investigations were carried out over the
temperature range of 50–250 °C with a heating flow rate of
10 °C/min.
2.6. In-vitro drug release
To assess the drug release from the nanosuspension, a Franz dif-
fusion cell was used. For this experiment, 1 ml of sample contain-
ing drug equivalent to 5 mg was added to the donor chamber and
the acceptor chamber was filled with 50 ml of simulated tear fluid.
The chamber was mildly agitated using a magnetic stirrer. At fixed
time intervals (0–12 h), twelve aliquots (1 ml) were withdrawn
t, k0 is the zero-order release constant, k1 is the first-order release
constant, kH is the Higuchi release constant, kHC is the Hixson-
Crowell rate constant, K is the Peppas release constant, and n is
the release exponent respectively.
2.8. In-vivo studies
2.8.1. In-vivo tolerance studies
In order to assess the biocompatibility and safety of the
nanoformulation at the corneal surface, an ocular irritation test
was performed. For this study, a single instillation of 50 lL of opti-
mized nanoparticulate formulation MGP1, marketed formulation
(MoxiGramÒ, Micro Labs, Bengaluru, India) and control (simulated
tear fluid {STF, pH 7.4}) was administered to the selected animal
models (three groups, comprising of three animals in each group)
respectively. The test was performed employing a clinical evalua-
tion scale [26,27] of 0 (absence) to 3 (highest) and animals were
observed for alteration in normal discharge, discomfort, alteration
in normal morphology of cornea, conjunctiva, eye lids. Each animal
was observed at 0.5, 2, 4, and 16 h after instillation. An index of
overall irritation (Table 2) was calculated by summing up the total
clinical evaluation scores over the observation time.
2.8.2. In-vivo anti-bacterial studies
A bacterial culture of Staphylococcus aureus (NCIM 2079),
sourced from IMTECH, Chandigarh, India was grown in nutrient
agar medium. The bacterial culture maintained on nutrient agar
media on petri dish was sub-cultured in liquid nutrient medium,
kept on rotary flask shaker at 37 °C and allowed to grow overnight.
The bacterial inoculum of S. aureus was inoculated into the right
eye of the animal model in group I, II and III respectively
with the help of transfer loop to harvest contamination. The
infection was produced after 2 days. To assess the anti-bacterial
effectiveness, three different preparations of moxifloxacin was
Table 1
Formulation, % entrapment efficiency, size, polydispersity index and zeta potential of
the moxifloxacin loaded gelatin nanoparticles.
Formulation NPX:GEL:GTA %EE Size (nm) PDI f (mV)

from the acceptor chamber and restored by the same volume of MGP1 1:1:3 57.21 ± 1.32 175 ± 1.11 0.067 ± 0.013 24 ± 0.12
fresh STF solution to retain sink conditions during the whole study. MGP2 1:1:4 44.32 ± 1.31 179 ± 0.12 0.071 ± 0.019 21 ± 0.04
MGP3 1:1:5 42.17 ± 1.41 162 ± 1.14 0.073 ± 0.021 19 ± 0.17
For the quantitative analysis UV–Vis spectrophotometry was uti-
MGP4 1:1:6 39.45 ± 1.75 151 ± 1.19 0.075 ± 0.023 18 ± 0.45
lized (Shimadzu UV-1700, Japan) at 245 nm wavelength. All MGP5 1:1:7 34.58 ± 1.63 149 ± 0.26 0.081 ± 0.024 15 ± 0.63
assessments were executed in triplicate and the (±SD) was calcu- MGP6 1:1:8 31.39 ± 1.45 138 ± 0.81 0.085 ± 0.028 14 ± 0.71
lated [24]. MGP7 1:1:9 33.73 ± 1.36 132 ± 1.69 0.090 ± 0.027 11 ± 0.78
MGP8 1:1:10 28.81 ± 1.26 130 ± 1.01 0.091 ± 0.029 09 ± 0.76

2.7. Drug release kinetics All values are mean (±SD), MOX: GEL: GTA refers to moxifloxacin, gelatin and
glutaraldehyde respectively, PDI refers to polydispersity index and f refers to zeta
To understand the kinetics and drug release mechanism, the potential.

data of in-vitro drug release from drug loaded gelatin nanoparticles

was fitted to five primarily applied mathematical models [25] (sta-
ted below) for the estimation of release kinetics of moxifloxacin
from the prepared gelatin nanoparticles. Table 2
Grading system of the macroscopic signs in the in-vivo tolerance studies of
moxifloxacin loaded gelatin nanoparticles.
Zero order model Dt = D0 + k0t
First-order model Dt = In D0 + k1t Grade Time
Higuchi square root model Dt = D0 + kHt1/2 Control Marketed formulation MGP1
Korsmeyer-Peppas model Dt/D1 = D0 + kPtn
0.5 h 4.0 h 0.5 h 4.0 h 16.0 h 0.5 h 4.0 h 16.0 h
Hixon-Crowell model Dt1/3 = D01/  kHCt
Discomfort 0 0 0 0 0 0 0 0
Discharge 0 0 0 1 0 1 0 0
Cornea 0 0 0 0 0 0 0 0
Conjunctiva 0 0 0 0 0 0 0 1
where Dt is the amount of drug released at time t, D0 is the initial Lids 0 0 0 0 0 0 0 0
amount of drug released, Dt/D1 is fraction of drug released at time
 A. Mahor et al. / Journal of Colloid and Interface Science 483 (2016) 132–138 135

Table 3 3. Results and discussion

Observation for physical parameter during in-vivo antibacterial evaluation of
formulations.
3.1. Particle size and morphology
Parameters Time Observation
(days)
Group I Group II Group III For the fabrication of nano-formulations and arriving at the best
(control) (MGP1) (moxifloxacin formulation, various batches were used and selection of the opti-
eye drop)
mized batch was done using varying ratios of crosslinking agent
Secretion 0 ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ (glutaraldehyde) keeping amount of drug and polymer constant
(discharge) 2 ⁄⁄⁄ ⁄⁄ ⁄⁄⁄ [29]. The particle size decreased upon increasing the amount of
4 ⁄⁄⁄ ⁄ ⁄⁄
6 ⁄⁄⁄ – ⁄
glutaraldehyde but the rate of reduction in size decelerated as
the ratio was increased [30]. A moxifloxacin:gelatin:glutaralde
Redness 0 ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄
2 ⁄⁄⁄ ⁄⁄ ⁄⁄⁄
hyde ratio (1:1:3) yielded particles of 175 ± 1.11 nm with highest
4 ⁄⁄⁄ ⁄ ⁄⁄ drug entrapment efficiency of 57.21 ± 1.32% amongst all developed
6 ⁄⁄⁄ – ⁄ batches (MGP1-MGP8). All fabricated batches were of nano-size
Swelling 0 ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ range, displaying monodisperse size distribution with polydisper-
2 ⁄⁄⁄ ⁄⁄ ⁄⁄⁄ sity index in the range of 0.067–0.091. The batches from
4 ⁄⁄⁄ ⁄ ⁄⁄ MGP1-MGP8 had a positive charge on the surface {since commercial
6 ⁄⁄⁄ – ⁄
gelatin is available in both cationic (type A gelatin with isoelectric
Infection 0 ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ point of 7–9), or anionic (type B gelatin with isoelectric point of
2 ⁄⁄⁄ ⁄ ⁄
4.8–5) [31]} exhibiting uniform distribution of the particles
4 ⁄⁄⁄ – –
6 ⁄⁄⁄ – – because of the repulsion leading to homogeneity (Table 1).
SEM micrographs showed spherical and smooth surface of the
Symbols: ⁄⁄⁄: Intense; ⁄⁄: Less; ⁄: Very less; –: absence, 0 day.
particles with slight perforations visible may be due to evaporation
of the solvating agent. TEM micrographs revealed a fairly solid and
consistent morphology of the prepared nanoparticles (Figs. 1 and
used: Group I (control), Group II (optimized formulation MGP1) 2). Variation in size from DLS and SEM can be attributed to the
and Group III (market product of moxifloxacin (MoxiGramÒ, Micro wet and dry states of the samples used for size determination using
Labs, Bengaluru, India). The treatment was initiated by instilling the two techniques respectively (Figs. 1 and 2).
MGP1, twice a day, while the MoxiGramÒ preparation was instilled
four times a day during the course of the experiment). The rabbit’s 3.2. Physicochemical characterization
eyes were monitored for discharge, redness, swelling and
suppression of infection up to 6 days (Table 3). FTIR spectrum of moxifloxacin, gelatin, their physical mixture
and related nanoparticles are shown in Fig. 3. The interaction of
drug with polymer leads to identifiable changes in the IR pattern.
2.8.3. Microbiological studies Notably, the FTIR spectra of the physical mixture of drug and poly-
The anti-microbial potential of the moxifloxacin loaded MGP1 mer suggested no possible interaction of moxifloxacin with gelatin
was assessed by comparing the results of the antibacterial effect as there were no distinctive changes in the spectra. A reduction in
of the marketed formulation (MoxiGramÒ) against Bacillus subtilus the intensity of the peak at 1264.00 cm1 of CAH bending and
(NCIM 2063, sourced from IMTECH, Chandigarh, India) using cup emergence of two pronounced peaks at 2938.56 cm1 and
plate method. 2939.22 cm1 of CAH aliphatic stretching was observed possibly
Nutrient agar medium was prepared and sterilized by autoclav- due to hydrogen bonding between drug and polymer in the
ing at 121 °C/15 psi pressure for 20 min. Then, 20 ml of medium nanoparticles.
previously inoculated with the B. subtilus (NCIM 2063) (0.2 ml of DSC thermogram of moxifloxacin displayed a prominent
stock) was transferred to the petri plate and was allowed to solid- endothermic peak at 213.31 °C suggesting anhydrous crystalline
ify. Subsequent to solidification, employing a sterile bore, three form of the drug. DSC thermogram of gelatin exhibited a fairly flat
cups of 4 mm in diameter were fabricated on the solidified agar thermal silhouette suggestive of the amorphous nature of the
layer. A fixed volume of the selected batch of MGP1 and
MoxiGramÒ eye drop (containing equivalent amount of drug)
was transferred into the cups. Positive control (petri dish with
microorganism, without drug) was also prepared. The petri plates
were allowed to stand at room temperature for 4 h and then were
subjected for incubation at 37 °C for 24 h. The zones of inhibition
were obtained. The diameter of the zone of inhibition was
measured by an antibiotic zone finder. Readings were taken in
triplicate [28].

2.9. Stability studies

Stability studies were conducted following WHO guidelines

using optimized batch MGP1. The sample batches were stored at
room temperature (25 ± 2 °C) and under accelerated storage condi-
tions i.e. 37 ± 2 °C/75 ± 5% RH in humidity control ovens. After six
months, all samples were analyzed for particle size, %entrapment
efficiency and in-vitro drug release was compared with the release
from initial batch prepared. Fig. 1. Scanning electron micrograph of MGP1.
136 A. Mahor et al. / Journal of Colloid and Interface Science 483 (2016) 132–138

Fig. 4. DSC thermogram of Moxifloxacin (MOX), Gelatin (GEL), physical mixture of

moxifloxacin and gelatin (MOX + GEL PM) and moxifloxacin loaded gelatin
nanoparticles (MOX + GEL NP).
Fig. 2. Transmission electron micrograph of MGP1.
hydrophilic nature of the polymer which might have absorbed
water from the aqueous release media. Controlled release of the
drug might have occurred due to drug entrapment in the core of
the particles (Fig. 5).
The values of n of 0.43 < n < 0.85 displays Fickian anomalous
model and case II transport respectively [32]. The drug release
from the prepared nanoparticles displayed non-Fickian release pat-
tern. Indeed, the release of the drug was governed by more than
one process, i.e. diffusion and erosion (anomalous diffusion) [33].
It was seen that the Korsmeyer-Peppas model showed a better lin-
earity (R2 = 0.993).

3.5. In-vivo studies

Fig. 3. FTIR spectra of moxifloxacin, gelatin, physical mixture of moxifloxacin and
gelatin (PM) and moxifloxacin loaded gelatin nanoparticles (NP). 3.5.1. In-vivo tolerance studies
In the ocular irritation test, visual examination showed that
conjunctival hyperemia and corneal opacity scores were zero at
polymer. A reduction in the peak intensity was observed of the
all annotations for MGP1. The in-vivo results for MGP1 batch
physical mixture of the drug and polymer at 211.06 °C. This might
showed no sign of irritation or damaging effects to ocular tissues
be due to the solubilization of the drug in the polymer or solid state
in rabbit eyes indicating its safety and biocompatibility. The tallies
interaction induced by heating. No distinctive peaks were observed
for conjunctival and eyelid swelling were constantly zero. The
for nanoparticles suggesting decrease in crystallinity of the drug in
absence of any in-vivo irritant action encourages the ophthalmic
the nanoparticles or the drug might have solvated in the polymer
use of the optimized gelatin nanoparticles (Table 2).
matrix. DSC thermograms of drug, polymer, physical mixture and
nanoparticles are shown in Fig. 4.
3.5.2. In-vivo anti-bacterial studies
The optimized batch (MGP1) was tested for the anti-bacterial
3.3. %Entrapment efficiency
activity in-vivo. The antibacterial activity of MGP1 was compared
with a marketed preparation of moxifloxacin (MoxiGramÒ eye
The MGP1 batch displayed highest entrapment efficiency in
comparison to other batches (Table 1). MGP1 showed 57% drug
entrapment while MGP8 showed 29%, this lowering in entrapment
of the can be attributed to the concentration of glutaraldehyde. As
the ratio is increased, the particles get dense because of effective
crosslinking of the nanoparticles as glutaraldehyde forms multiple
hydrogen bonds with single nanoparticle, leaving less space for
drug entrapment.

3.4. In-vitro drug release

The batches from MGP1-MGP8 showed a burst release pattern

in the first hour and subsequently followed controlled release
behaviour for 12 h. MGP1 and MGP8 showed drug release of 18%
and 13% respectively in the first hour and exhibited a controlled
drug release pattern till 12th hour (81% and 60% for MGP1 and
MGP8 respectively).
The burst release of the drug can be attributed to the adsorption
of the drug on the surface of the nanoparticles and to the Fig. 5. In-vitro release profile of batches MGP1-MGP8.
 A. Mahor et al. / Journal of Colloid and Interface Science 483 (2016) 132–138
Fig. 6. Comparative study between market product and MGP1.
drop, Micro Labs, Bengaluru, India) to assess their effectiveness as
an ophthalmic drug delivery system. Succeeding, inoculation of S.
aureus into the right eye of rabbits of the animal groups, the rab-
bit’s eye was observed. The results demonstrated that MGP1 exhib-
ited prominent effects in comparison to marketed preparation in
terms of suppression of discharge, redness, swelling and infection.
MGP1 provided symptomatic relief within four days at a dose
regimen of twice a day whereas marketed preparation (Moxi-
GramÒ) failed to show considerable effects in 6 days at a dose
regimen of four times a day (Table 3).
3.5.3. Microbiological studies
Optimized formulation gave clear zones of inhibition in com-
parison to commercial preparation (MoxiGramÒ). The diameter of
the zone of inhibition by MoxiGramÒ eye drop formulation was
found to be 10.49 ± 0.19 mm at 12 h and 12.52 ± 0.23 mm at 24 h
whereas the diameter of the zone of inhibition by MGP1 was found
to be 13.36 ± 0.11 nm at 12 h and 15.46 ± 0.17 nm at 24 h respec-
tively. From the results, it can be concluded that the fabricated
nanosuspension possess better anti-microbial activity as compared
to market product, MoxiGramÒ (Fig. 6).
3.6. Stability studies
Under normal and accelerated stability conditions, MGP1 was
selected for stability testing, following WHO guidelines. A slight
increase in particle size was noticed after six months of storage
(175 ± 1.11–185 ± 1.94 nm) possibly due to aggregation of the par-
Fig. 7. Comparative in-vitro drug release profile of MGP1 before and after stability
studies.
137
ticles. Drug entrapment decrease from 58% to 49% after stipulated
storage duration. The stored MGP1 displayed similar drug release
behaviour as that of the freshly prepared MGP1 batch with an
increased burst release in the first hour followed by a controlled
release of the drug. The increased burst release can be attributed
to the availability of higher amounts of the drug in the suspension
due to drug leaching from the surface of the nanoparticles during
storage, even though, release of the drug was controlled for the
next 12 h (Fig. 7). Thus, from the results obtained, the nanoformu-
lations can be considered stable under the tested conditions.
4. Conclusions
Cationic (type A) gelatin nanoparticles for topical administra-
tion were successfully prepared using a modified two step desolva-
tion method. Glutaraldehyde provided excellent crosslinking of the
nanoparticles resulting in particles with a mean size range of
175–130 nm for all the prepared batches with high drug entrap-
ment efficiency of 57%. The MGP1 nanoparticles had narrow PDI
of 0.067 with zeta potential of 24 ± 0.12 mV. FTIR studies showed
no drug-polymer interaction. DSC thermograms exhibited molecu-
lar level of distribution of moxifloxacin in the gelatin matrix.
An initial burst release followed by controlled release of the
drug was observed in the in-vitro drug release studies. All the
batches displayed non-Fickian drug transport. No irritational con-
sequences were witnessed in the in-vivo tolerance test. In-vivo
anti-bacterial studies showed that the optimized batch was able
to suppress discharge, swelling and infection in comparison to
market product, MoxiGramÒ. Antimicrobial testing revealed that
MGP1 considerably inhibited microbial growth (13.36 ± 0.11 nm
at 12 h and 15.46 ± 0.17 nm at 24 h) in comparison to commercial
MoxiGramÒeye drop solution. From the stability studies, moxi-
floxacin loaded gelatin nanoparticles can be rendered stable. The
current results are very promising and the future perspective can
include in-vivo pharmacokinetics and pharmacodynamics studies
to translate the nanoformulation for bench to the bedside.
Conflict of interest
The authors declare no conflict of interest.
Disclosures
There is no conflict of interest and disclosures associated with
the manuscript.
Acknowledgment
The authors would like to appreciate the kind support of Dr.
Arvind Jain, Post-Doctoral scientist, Weatherall Institute of Molec-
ular Medicine, University of Oxford, UK, Dr. Kuldeep Bansal, GLA
University, Mathura, Dr. Himanshu Pandey of SHIATS, Allahabad,
Dr. Upendra Sharma, Dr. Vijay Singh, Dr. Prem Prakash Singh, Dr.
Peeyush Bhardwaj and Dr. Devendra Singh of Institute of Phar-
macy, Bundelkhand University, Jhansi, in the compilation of this
manuscript.
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