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Colloids and Surfaces B: Biointerfaces 114 (2014) 36–44
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
j ournal homepage: www.elsevier .com/ locate /colsur fb
Carbopol gel containing chitosan-egg albumin nanoparticles for
transdermal aceclofenac delivery
Sougata Jana a,∗, Sreejan Manna a, Amit Kumar Nayak b, Kalyan Kumar Sen a,Sanat Kumar Basu a
a Division of Pharmaceutics, Department of Pharmaceutical Tecnology,Kolkata-700032,W.B., Indiab Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj 757086, Odisha, India
a r t i c l e i n f o
Article history:
Received 3 June 2013
Received in revised form
18 September 2013
Accepted 20 September 2013
Available online 30 September 2013
Keywords:
Carbopol 940
Chitosan
Egg albumin
Nanoparticles
Transdermal drug delivery
a b s t r a c t
In the present work, various aceclofenac-loaded chitosan-egg albumin nanoparticles were prepared
through heat coagulation method. These aceclofenac-loaded nanoparticles were characterized by FE-
SEM, FTIR, DSC and P-XRD analyses. The in vitro drug release from nanoparticles showed sustained drug
release over 8 h. Aceclofenac-loaded nanoparticles (prepared using 200 mg chitosan, 500 mg egg albu-
min and 2% (w/v) NaTPP) showed highest drug entrapment (96.32±1.52%), 352.90 nm average particle
diameter and −22.10 mV zeta potential, which was used for further preparation of Carbopol 940 gel for
transdermal application. The prepared gel exhibited sustainedex vivo permeation of aceclofenac over 8 h
through excised mouse skin. The in vivoanti-inflammatoryactivity in carrageenean-inducedratsdemon-
strated comparative higher inhibition of swelling of rat paw edema by the prepared gel compared with
that of the marketed aceclofenac gel over 4 h.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Transdermal drug delivery systems are prepared to deliver
drugs through skin at predetermined rate escaping the first-pass
effect by liver [1]. The most difficult aspect of transdermal drug
delivery system is to overcome the skin barrier. There is evidence
that the rate-limiting step in transdermal transport occurred at the
outermostlayer of the skin,stratum corneum [2]. Many approaches
have been investigated to enhance the drug permeation through
the barrier of skin for the use in transdermal drug delivery [1,3–7].
Currently, nanoparticles have shown great potential as novel
drug carriers for transdermal drug delivery [7–9]. The smaller
size of nanoparticles could ensure close contact with the stratum
corneum and increases the encapsulated drug amount penetrating
into the skin. The advantages of the use of these kinds of col-
loidal carriers are protection of unstable drugs from degradation
and control of drug release rate from these colloidal carriers [9,10].
Nanoparticlesare solidparticles ranging in size,1–1000nm [11,12].
Presently, polymeric nanoparticles have received lots of attention
due to their stability and ease of surface modification specificity
[13].
∗ Corresponding author. Tel.: +91 9434896683.
E-mail address: [email protected] (S. Jana).
Chitosan is a cationic biocompatible and biodegradable nat-
ural polysaccharide obtained by alkaline deacetylation of chitin,
which is composed of -1,4-linked 2-amino 2-deoxy -d-glucose
(N-acetyl glucosamine) [14,15]. Although chitosan is used in drug
delivery, it has a limited capacity for controlling drug release. So
researchers investigated various chemical modifications of chi-
tosan to develop chitosan-based formulations for controlled drug
release applications [16–18].
Egg albumin has recently received attention for its use in food
and pharmaceutical applications [19,20]. It behaves as anionic
polymer above its isoelectric point (pH 4.8) [21]. Therefore, it
is hypothesized that anionic egg albumin and cationic chitosan
might form a polyelectrolyte complex, if processed with each
other. Additionally, this approach could be beneficial to control
the drug release. Carbopols are polymers of acrylic acid cross-
linked with polyalkenyl ethers or divinyl glycol [22]. Because of
their hydrophilic nature, the cross-linked structures of Carbopols
make them potential candidates for the use as gel-type formula-
tions fortopicaluse [23,24]. In the previous literature, Carbopol gels
containing drug-loaded nanoparticles were investigated for trans-
dermal delivery [7,22]. In the present investigation, we attempted
to develop a transdermal Carbopol gel containing aceclofenac-
loaded chitosan-egg albumin nanoparticles.
Aceclofenac is a non-steroidal anti-inflammatory drug (NSAID)
withshort half-life(4 h) indicatedfor the symptomatic treatmentof
painand inflammation [15,25]. It is reported to produce side-effects
0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2013.09.045
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S. Jana et al. / Colloids andSurfaces B: Biointerfaces114 (2014) 36–44 37
like gastric irritation, ulcer, abdominal pain and flatulence, as a
result of prolonged treatment [26]. In view of the side-effects asso-
ciated with the oral administration of aceclofenac, it is increasingly
administered through topical route [27]. Furthermore, the trans-
dermal route of administration eliminates side-effects, increases
patient compliances, avoids first-pass metabolism and maintains
theplasma-drug levelfor a long period. Inthe current study,we pre-
pared aceclofenac-loaded chitosan-egg albumin nanoparticles by
heat coagulation method, incorporated these nanoparticles within
Carbopol 940 geland evaluatedthis newly formulated gelfor trans-
dermal delivery of acecalofenac, in vitro and in vivo.
2. Materials and method
2.1. Materials
Aceclofenac was received as a gift sample from Drakt Phar-
maceutical Pvt. Ltd., India. Chitosan (85% deacetylated) was
commercially purchased from Indian Sea Foods, Cochin, India. Egg
albumin,Carbopol 940,crospovidoneand sodium tripolyphosphate
(NaTPP) were purchasedfromLoba ChemiePvt.Ltd.,India. Allother
chemicals, and reagents used were of analytical grade.
2.2. Preparation of aceclofenac-loaded chitosan-egg albumin
nanoparticles
Aceclofenac-loaded chitosan-egg albumin nanoparticles were
prepared by heat coagulation method [28]. Different strength of
chitosan solutions were prepared by dissolving required amount
of chitosan in 1% acetic acid solution and egg albumin solutions
were prepared dissolving them in distilled water. Then, accu-
rately weighed amount of aceclofenac (100 mg) was dispersed into
the chitosan solutions and mixed thoroughly using a homoge-
nizer (Remi Motors, India). Chitosan-aceclofenac mixtures were
added drop-wise into egg albumin solutions under continuous
stirring (500 rpm) for30 min. Theresultant pHof these aceclofenac-
polymeric disperpersions was observed in between 4.00 and 4.30,
and was finally adjusted to 5.4 with the help of 0.1 N sodium
hydroxide. These adjusted dispersions were heated at 80◦C for
30min to aid in developing nanoparticles due to heat coagula-
tion. The prepared nanoparticles were collected by centrifugation
and were lyophilized (Eyela FDU 1200) to obtain dried samples.
Different chitosan-egg albumin nanoparticles formulations con-
taining aceclofenac along with amounts of chitosan, egg albumin,
and NaTPP were enlisted in Table 1.
2.3. Characterization of aceclofenac-loaded chitosan-egg albumin
nanoparticles
2.3.1. Estimation of drug entrapment efficiency10mg of lyophilized nanoparticles containing aceclofenac from
each formulation batch were accurately weighed and were taken
separately in 50ml phosphate buffer, pH 7.4 to keep for 48h under
continuous stirring followed by centrifugation at 2000rpm for
10min. Then, the supernatant fraction was further filtered through
Whatman® filter paper (No. 40). The aceclofeanc content in the
filtrate was determined using a UV-VIS spectrophotometer (Shi-
madzu, Japan) by measuring absorbance at Max of 274nm. The
drug entrapment efficiency of nanoparticles was calculated using
the following formula:
Drugentrapment efficiency (%) =theorticaldrug content in nanoparticles− amount of drug present in thefiltrate
theoretical drugcontent in nanoparticles (1)
2.3.2. Particle size and zeta potential determination
The nanoparticles were dispersed into 10ml phosphate buffer,
pH 7.4 and sonicated for 5 m in before size measurement. The
obtained homogeneous suspensions were examined for particle
size andzeta potentialusinga laser scattering particle size analyzer
(MAL500999, UK).
2.3.3. Field emission-scanning electron microscopy (FE-SEM)
Thelyophilizedparticles were spreadonto metal stubs andplat-inum coating was applied by using an ion-sputtering device. The
coated particles were then examined under FE-SEM (JSM 6701;
JEOL, Japan).
2.3.4. Fourier transform-infrared (FTIR) spectroscopy
Samples were powdered and analyzed as KBr pellets by
using a FTIR spectrophotometer (Perkin Elmer Spectrum RX I,
USA). The pellet was placed in the sample holder. Spectral
scanning was taken in the wavelength region between 4000
and 600 c m−1 at a resolution of 4 cm−1 with scan speed of
2 mm/s.
2.3.5. Differential scanning calorimetry (DSC)
Moisture free nanoparticles (7 mg) were placed into a platinumcrucible 40-l aluminum pan in hermetically sealed condition,
where alumina powder was used as a reference. Thermograms
were recorded from 29.80 to 351.90 ◦C at the heating rate of
5 ◦C/min under a constant flow of an inert nitrogen gas atmosphere
with the flow rate of 20ml/min. These analyses were done using
a Differential Scanning Calorimeter (Perkin Elmer® Instrument,
Japan).
2.3.6. Powder X-ray diffraction (P-XRD)
Samples were exposed to CuK radiation (40 kV × 20mA) in a
wide-angle X-ray diffractometer (Siemens D5000, Germany). The
instrument was operated in the step-scan mode in increments of
0.050◦ 2 . The angular range was 10◦–60◦ 2 , and counts were
accumulated for 1 s at each step.
2.4. In vitro release study of aceclofenac-loaded chitosan-egg
albumin nanoparticles
Invitrorelease of aceclofenacfrom thesepreparednanoparticles
was measured using dialysis bag diffusion technique. Accurately
weighed quantities of nanoparticles containing drug equivalent
to 50 m g aceclofenac were placed in one end of the dialysis bag
(Cellophane membrane, molecular cut off 10,000–12,000 Da, Hi-
Media, India) containing 5 ml of phosphate buffer, pH 7.4. After
that, other side of the dialysis bag was tied and immersed in
phosphate buffer (pH 7.4) contained in the USP type II dissolu-
tion apparatus (Veego VDA-6D, Veego Instruments Co-operation,
India). The system was maintained at 37±1◦
C under 100 rpmspeed. The dialysis bag acts as a donor compartment, and the ves-
sel of dissolution apparatus acts as the receptor compartment. 5 ml
of aliquots was collected at regular time intervals, and the same
amount of fresh dissolution medium was replaced into dissolution
vessel to maintain the sink condition throughout the experi-
ment. The collected aliquots were filtered, and suitably diluted
to determine the absorbance using a UV-VIS spectrophotometer
(Thermo Spectronic UV-1, USA) by measuring absorbance at Max
of 274 nm.
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38 S. Jana et al. / Colloids andSurfaces B: Biointerfaces114 (2014) 36–44
Table 1
The composition chart for the preparation of different aceclofenac-loaded chitosan-egg albumin nanoparticles with their drug entrapment efficiencies, average particle
diameters and zeta potentials.
Formulation code Composition Drug entrapment
efficiency (%)
(mean±S.D.; n= 3)
Average particle
diameter (nm)
Zeta potential
(mV)
Chitosan (mg) Egg albumin (mg) NaTPP (% w/v)
F-1 350 350 – 69.37 ± 0.93 553.40 −1.16
F-2 250 450 – 74.30 ± 0.95 501.25 −3.10
F-3 300 400 – 81.26 ± 1.38 489.52 −3.32
F-4 200 500 – 90.54 ± 1.03 446.30 −5.77
F-5 200 500 1 93.68 ± 1.08 388.94 −12.21
F-6 200 500 2 96.32 ± 1.52 352.90 −22.10
2.5. Analysis of in vitro drug release kinetics and mechanism
The in vitro drug release data were evaluated kinetically using
various important mathematical models like zero-order, first-
order, Higuchi, and Korsmeyer–Peppas models [26].
Zero-order model: Q = k0t +Q 0; where Q represents the drug
remaining to be released at time t , and Q 0 is the amount of drug
present in the formulation initially; k0 is the rate constant.
First-order model: Q =Q 0 ek1t ; where Q represents the drug
remaining to be released at time t , and Q 0 is the amount of drugpresent in the formulation initially; k1 is the rate constant.
Higuchi model:Q =kHt 0.5; whereQ represents the drug released
amount per unit area at time t , and kH is the rate constant.
Hixson–Crowell model:Q 1/3 = kt +Q 01/3; whereQ represents the
drugreleasedamountattime t ,andQ 0 isthe amountof drug present
in the formulation initially; k is the rate constant.
Korsmeyer–Peppas model:Q = kt n; whereQ represents the frac-
tion of drug released at time t , k is the rate constant and n is the
diffusional exponent, indicative of drug release mechanism.
Again, The Korsmeyer–Peppas model was employed in the in
vitro drug release behavior analysis of these formulations to dis-
tinguish between competing release mechanisms: Fickian release
(diffusion-controlled release), non-Fickian release (anomalous
transport), and case-II transport (relaxation-controlled release).When n is ≤0.43, it is Fickian release. The n value between 0.43
and 0.85 is defined as non-Fickian release. When n is ≥0.85, it is
case-II transport [26].
2.6. Preparation of Carbopol 940 gel containing
aceclofenac-loaded chitosan-egg albumin nanoparticles
In brief, 100mg of Carbopol 940 was dissolved in 6.50ml of
deionized water through a continuous stirring at 500 rpm by mag-
netic stirrer (Remi Motors, India) for 1 h to prepare Carbopol
940 solution. Then, accurately weighed quantities of aceclofenac-
loaded nanoparticles equivalent to 50mg aceclofenac were mixed
thoroughly with the above mentioned Carbopol 940 solution.Finally, weighed quantity of triethanolamine was added as a neu-
tralizer to increase the pH of the prepared Carbopol 940 mixture
and formation of gel occurred.
2.7. Characterization of Carbopol 940 gel containing
aceclofenac-loaded chitosan-egg albumin nanoparticles
2.7.1. pH determination
pH of the prepared gel was measured using a digital pH
meter (Systronics Instruments, India) by placing the glass elec-
trode completely into the gel systemand compared with marketed
formulation.
2.7.2. Viscosity measurement
The viscosity of gel was determined by using a Brookfield DV
III ultra V6.0 RV cone and plate viscometer (Middle-boro, MA) at
25±0.3 ◦C the software used for calculation was Rheocalc V2.6.
2.8. Ex vivo permeation study
2.8.1. Preparation of the skin
The ex vivo permeation of aceclofenac from Carbopol 940 gel
containing aceclofenac-loadedchitosan-egg albumin nanoparticles
were measured using excised skin of Swiss albino mice (weight
170–192 g). The experiment was approved by Institutional Animal
Ethical Committee, under registration number 955/A/06/CPCSEA.
The animals were sacrificed using anesthetic ether. The hair of
abdominal skin was removed by using an animal hair clipper. A full
thickness of skin was taken out and the fat adhering to the dermis
side was removed by using a surgical scalpel. Finally, the skin was
rinsed with phosphate buffer of pH 7.4and was kept in refrigerator
at temperature of −20 ◦C in aluminum foil packing. The stored skin
sample was used within 24h.
2.8.2. Ex vivo permeation by Franz diffusion cells
The ex vivo permeation through excised mouse skin was per-
formed by Franz diffusion cells. The cells consist of two chambers,
the donor and the receptor compartment with an available dif-
fusion area of 0.949cm2. The donor compartment was open at
the top and was exposed to atmosphere. The excised mouse skin
was mounted between the compartments of the diffusion cell with
stratum corneum facing the donor compartment and clamped into
position. Magnetic stirrer bars were added to the receptor cham-
bers and filled with the receptor phase. Phosphate buffer saline,
pH 7.4 was used as the receptor medium. The small concentration
of sodium azide (0.0025%, w/v) was added to prevent any micro-
bial growth [29]. The entire setup wasplaced over magnetic stirrer,
and the temperature was maintained at 37±0.5 ◦C. The skin sec-
tions were initially left in the Franz cells for 2 h in order to facilitate
hydration of the skin samples. After that 1 g of gel was applied
onto the excised mouse skin fitted on the Franz diffusion cell. 1 ml
of medium was collected from the receptor compartment at pre-
determined intervals over study period and was replaced with the
same amount of the fresh buffer. The amount of permeated drug
was analyzed by UV-VIS spectrophotometer (Thermo Spectronic
UV-1,USA) at 274nm wavelengthusing appropriate blank solution.
2.8.3. Permeation data analysis
The amount of aceclofenac permeated from gel through excised
mouse skin was plotted against the function of time. The slope and
intercept of the linear portion of plot were derived by regression.
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The permeation flux for gel was calculated as the slope divided by
the skin surface area [30]:
J SS =
dQ
dt
ss×
1
A
where J ss is the steady-state permeation flux (g/cm2/h), A is the
area of skin tissue (cm2) through which drug permeation takes
place, and (dQ /dt )ss is the amount of drug passing through the skin
per unit time at a steady state (g/h).
2.9. In vivo evaluation
The animal experiment was approved by Institutional Animal
Ethical Committee (Registration number 955/A/06/CPCSEA). The
carrageenean-induced rat-paw edema model [31] was performed
to assessthe anti-inflammatory activityevaluation of the optimized
gel. Male Sprague Dawley rats weighing 200–250 g were used for
the experiment. The acclimatized rats were kept fasting for 24h
with water ad libitum. The anti-inflammatory effect was evaluated
by applying 1 g of Carbopol 940gels containing aceclofenac-loaded
chitosan-egg albumin nanoparticles (F-6) on the rat paw. The con-
trol group was treated with the normal saline. After 3h interval,
0.1ml of a 1% carrageenan solution in physiological saline as thecontrol was injected intradermally in the right hind paw of the
rat. The edema volumes are measured using plethysmometer (Ugo
Bacile, model 7150) after 3 h of carrageenan injection. The extents
of swelling (%) were calculated from the difference in the volume
between immediately and 3 h after the carrageenan injection (6
rats in each group) using the following formula:
Swelling (%) =V − V 1V 1
× 100
whereV is thevolume 3 h after thecarrageenaninjectionin thesole
of the foot and V 1 is the volume immediately after the injection.
2.10. Statistical analysis
The in vivo data was tested for significant differences ( p< 0.05)
by paired samples t-test. All other data was analyzed with simple
statistics. The simple statistical analysis and paired samples t-test
were conducted using MedCalc software version 11.6.1.0.
3. Results and discussion
3.1. Preparation of aceclofenac-loaded chitosan-egg albumin
nanoparticles
Various aceclofenac-loaded chitosan-egg albumin nanoparti-
cles were prepared based on heat coagulation method at pH 5.4
and 80◦C. Chitosan is a polycation having pK a of about 6.3 and egg
albumin is a polyanion above its isoelectric point, pH 4.8 [21,22].Therefore, an insoluble polyelectrolyte complex could be formed
in the form of nanoparticles due to electrostatic attraction of these
two oppositely charged polymers at pH above the isoelectric point
of albumin.
3.2. Characterization of aceclofenac-loaded chitosan-egg albumin
nanoparticles
3.2.1. Drug entrapment efficiency
The results described that the drug entrapment efficiency
of these nanoparticles was within the range, 69.37±0.93 to
96.32±1.52% (Table 1) and formulation F-6 had the highest
drug entrapment efficiency (96.32±1.52%). The results of drug
entrapment efficiency of these formulations also described that,
increasing drug entrapment was found with the decrease of chi-
tosan amount and the increment of egg albumin amount in their
formulations. The increasing drug entrapment was also found with
the increase addition of NaTPP in the preparation of aceclofenac-
loaded chitosan-egg albumin nanoparticles. This could be due to
ionic cross-linking NaTPP with free chitosan in the dispersion.
3.2.2. Particle size and zeta potential
Average particle diameters of aceclofenac-loaded chitosan-
egg albumin nanoparticles were ranged within 352.90–553.40nm
(Table 1). Particle size distribution of 2% (w/v) NaTPP cross-linked
aceclofenac-loaded chitosan-egg albumin nanoparticles (F-6) pre-
pared using 200mg chitosan and 500mg egg albumin is presented
in Fig. 1a and average particle diameter was measured 352.00 nm.
Decreasing average particle diameter of these nanoparticles was
found with the decrease of chitosan amount and the increment
of egg albumin amount in their formulation. However, decreasing
average particle diameter of nanoparticles was also observed with
the addition and concentration of NaTPP used in their preparation,
which could be due to ionic cross-linking of chitosan by the NaTPP.
This phenomenon of decreased particle size with the addition of
NaTPP could be due to the shrinkage of polymeric gel by higher
degree of cross-linking with the high concentration of cross-linker
(i.e. NaTPP).
The measurement of the zeta potentialallows predictions about
the storage stabilityof various colloidaldispersions. In general, par-
ticle aggregation is decreased for charged particles with high zeta
potential due to electric repulsion. The zeta potentials of various
aceclofenac-loaded chitosan-egg albumin nanoparticles are pre-
sented in Table 1 and found within the range between 1.16 and
22.10 mV. From these results, it was clearly evidenced that the
zeta potential of these nanoparticles decreased with the increase
in addition of NaTPP.
3.2.3. FE-SEM analysis
FE-SEM of aceclofenac-loaded chitosan-egg albumin nanoparti-
cles (F-6) was performed. In the FE-SEM photograph at 65000×
magnification (Fig. 1b), these particles were appeared denselypacked to each other. Particles were mostly in the nanoscale range
and spherical in shape.
3.2.4. FTIR spectroscopy
FTIR spectrum of aceclofenac, chitosan, egg albumin and TPP
cross-linked aceclofenac-loaded chitosan-egg albumin nanoparti-
cles (F-6) is shown in Fig. 2. The FTIR spectra of aceclofenac showed
that principal peaks at 3028cm−1 due to aromatic C H stretch-
ing vibrations and 2937cm−1 due to aliphatic C H stretching
vibrations, a band at 1717cm−1 due to C O stretching, a sharp
band at 1772cm−1 due to C O stretching of carboxylic acid, band
at 3320cm−1 due to secondary N H rocking vibrations, and two
sharp peaks at 716 cm−1 due to 1,2 di-substituted C Cl stretching.
In case of FTIR spectra of chitosan, peaks at 3411.86 cm−
1 for O Hstretch, 2972.72 cm−1 for aliphatic C H stretch, 1650.00cm−1 due
to amide,1560.37cm−1 due toN H bending vibration ofsecondary
amine, 1076.08cm−1 for C O stretch of ether, 560.37cm−1 for 1◦
N H groups and bands at 1100–1000cm−1 are due to the saccha-
ride structure were observed. In addition, characteristic peaks at
1464.72cm−1 and 1431.03 cm−1 were seen due to bending vibra-
tion of CH2 group and OH group, respectively. The FTIR spectra
of egg albumin showed characteristic peaks at 3300.78 cm−1 for
N substituted amide, 2969.92 cm−1 for aliphatic C H stretching,
1645.34cm−1 due to amide band, 1560.63cm−1 for N H bend-
ing vibration, 1403.22 cm−1 for aliphatic C H bending vibration
and 1157.99 cm−1 due to C N stretching vibrations. Characteris-
tic peaks appeared in the FTIR spectra of aceclofenac were also
appeared clearly in the aceclofenac-loaded chitosan-egg albumin
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40 S. Jana et al. / Colloids andSurfaces B: Biointerfaces114 (2014) 36–44
Fig. 1. (a) Particle sizedistributionof 2% (w/v) NaTPP cross-linked aceclofenac-loadedchitosan-eggalbuminnanoparticles (F-6) preparedusing 200mg chitosan and 500mg
egg albumin; (b) FE-SEM image of 2% (w/v) NaTPP cross-linked aceclofenac-loaded chitosan-egg albumin-nanoparticles (F-6) at 65000× magnification.
nanoparticles (F-6). A small but distinct change or shifting of the
amide band was observed in these spectra. Thus, it can be said
that aceclofenac was successfully entrapped into the nanoparticleformulation and no significant changes occurred in drug proper-
ties.However,the spectrumof theaceclofenac-loadedchitosan-egg
albumin nanoparticles (F-6) exhibited the characteristic absorp-
tion bands, which were appeared in both egg albumin and chitosan
spectrum with only intensity differences except the appearance
of two additional peaks at 1080.88 cm−1 and 1028.23 cm−1. These
peaks were not present in either chitosan or egg albumin spec-
trum. This phenomenon indicates the interaction between anionic
egg albumin and cationic chitosan.
3.2.5. DSC analysis
DSC thermograms of pure aceclofenac and aceclofenac-loaded
chitosan-egg albumin nanoparticles (F-6) were presented in Fig. 3.
The thermal graph of pure aceclofenac showed sharp endother-
mic peaks at 152.5 ◦C, indicating the melting point of aceclofenac.
In case of aceclofenac-loaded chitosan-egg albumin nanoparticles
(F-6) two sharp endotherms were observed at 156◦C and 280 ◦C.
The first peak indicates that the drug remains in the polymer
matrix as a separate entry as well as in stable condition and the
second peak at 280◦C denotes the melting point of polymers of nanoparticles.
3.2.6. P-XRD analysis
P-XRDpatternsof aceclofenacand aceclofenac-loaded chitosan-
egg albumin nanoparticles (F-6) are presented in Fig. 4. P-XRD
of aceclofenac showed diffraction peaks at about 18.50◦, 19.11◦,
22.18◦, 23.89◦, 25.92◦ (2 ) with different signal intensities, indi-
cating important crystallographic characteristics of aceclofenac.
In the P-XRD pattern of aceclofenac-loaded chitosan-egg albumin
nanoparticles (F-6), slightly less intense peak were observed at
23.4◦ and 24.2◦ (2 ) indicating that the drug (aceclofenac) main-
tain little bit of its crystalinity in the nanoparticle formulation. The
above result suggests that most of the aceclofenac was convertedfrom crystalline to amorphous form in the aceclofenac-loaded
chitosan-egg albumin nanoparticles (F-6), which could be due to
the effect of polymers or formulation process.
Fig. 2. FTIRspectrum of aceclofenac, chitosan, egg albumin and aceclofenac-loaded chitosan-egg albumin nanoparticles (F-6).
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S. Jana et al. / Colloids andSurfaces B: Biointerfaces114 (2014) 36–44 41
Fig. 3. DSC thermograms of aceclofenac and aceclofenac-loaded chitosan-egg albumin nanoparticles (F-6).
3.3. In vitro drug release from aceclofenac-loaded chitosan-egg
albumin nanoparticles
In vitro drug ( aceclofenac) release from these prepared
aceclofenac-loaded chitosan-egg albumin nanoparticles was eval-uated using dialysis bag diffusion technique in phosphate buffer,
pH 7.4. The cumulative percentage of drug release from these
aceclofenac-loaded nanoparticles was found sustained over a
period of 8 h (Fig. 5). The in vitro drug release from all the for-
mulation batches of nanoparticles showed an initial burst release
of drugs, which might be attributed due to the presence of free
drug crystals and/or weakly bound drug on the surface of the
nanoparticles. The cumulative percentage of drug released from
these aceclofenac-loaded chitosan-egg albumin nanoparticles (F-1
to F-6) in 8h was within the range of 54.10±1.61–88.02 ±1.33%.
The drug release from these nanoparticles was mostly dependent
on theamount of drug-load. Thedrug release wasfoundfaster from
the nanoparticles of lower drug entrapment efficiency. The com-
paratively sustained release of drug was evidenced in case of TPP
cross-linked aceclofenac-loaded chitosan-egg albumin nanoparti-
cles (F-5 and F-6). However, slower drug release was found for TPP
cross-linked aceclofenac-loaded chitosan-egg albumin nanoparti-
cles prepared using higher concentration of ionic cross-linker, TPP,
indicating that the delayed drug release was due to presence of
higher degree of cross-linking.
The in vitro drug release data from various aceclofenac-loaded
chitosan-egg albumin nanoparticles were evaluated kinetically
using various important mathematical models like zero order, first
order, Higuchi, and Korsmeyer–Peppas models. The correlation (R2)
values of these models were determined for evaluation of accuracyfor the best fitting of models and values nearer to 1 considered as
best fitting of models. The result of the curve fitting into various
mathematical models was given in Table 2. When the respectiveR2
were compared, it was found that all these nanoparticles followed
the Korsmeyer–Peppas model over a period of 8h. The value of
release exponent (n) determined from in vitro aceclofenac release
data of various aceclofenac-loaded chitosan-egg albumin nanopar-
ticles rangedfrom 0.64 to 0.82, indicating anomalous (non-Fickian)
diffusion mechanism of drug release. The anomalous diffusion
mechanism of drug release demonstrates bothdiffusion controlled,
and swelling controlled drug release.
3.4. Preparation of Carbopol 940 gel containing
aceclofenac-loaded chitosan-egg albumin nanoparticles
In the current investigation, we attempted to develop Car-
bopol 940 gel containing aceclofenac-loaded chitosan-egg albumin
nanoparticles for transdermal use using 2% (w/v) NaTPP cross-
linked aceclofenac-loaded chitosan-egg albumin nanoparticles
(F-6). We have selected that nanoparticles formulation for the
Fig. 4. P-XRD patterns of aceclofenac and aceclofenac-loaded chitosan-egg albumin nanoparticles(F-6).
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42 S. Jana et al. / Colloids andSurfaces B: Biointerfaces114 (2014) 36–44
Fig. 5. The in vitrodrug release from various aceclofenac-loaded chitosan-egg albumin nanoparticles in phosphate buffer, pH 7.4 (mean±S.D.; n =3).
Table 2
Results of curve fitting of the in vitrodrug release profile of various aceclofenac-loaded chitosan-egg albumin nanoparticles.
Formulation code Correlation coefficient (R2) Release exponent (n)
Zero order First order Higuchi Korsmeyer–Peppas
F-1 0.9733 0.9744 0.9886 0.9994 0.82
F-2 0.9693 0.9166 0.9042 0.9736 0.81
F-3 0.9422 0.8681 0.8589 0.9674 0.64
F-4 0.9492 0.9307 0.9483 0.9860 0.76
F-5 0.9312 0.8846 0.8454 0.9736 0.79
F-6 0.9811 0.9577 0.9203 0.9792 0.80
Fig. 6. (a) The comparative ex vivo drug permeation from Carbopol 940 gel containing aceclofenac-loaded chitosan-egg albumin nanoparticles and a marketed aceclofenac
gel through excised mouse skin (mean±S.D.; n = 3); (b) comparative percentage inhibition profile of paw edema edema for Carbopol 940 gel containing aceclofenac-loaded
nanoparticles and marketed aceclofenac gel at various time intervals in carrageenan-induced rat model for anti-inflammatory activity evaluation.
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S. Jana et al. / Colloids andSurfaces B: Biointerfaces114 (2014) 36–44 43
preparation transdermal Carbopol940 gel due to its maximum drug
entrapment than other nanoparticles formulated in the study.
3.5. Characterization of Carbopol 940 gel containing
aceclofenac-loaded chitosan-egg albumin nanoparticles
3.5.1. pH
In the development of transdermal formulation, the pH of the
formulation isimportant.The more acidicor more basicpH oftrans-
dermal formulations may change the skin environment. This can
produce skin irritation upon application. The pH of the prepared
Carbopol 940gel containing aceclofenac-loaded chitosan-egg albu-
min nanoparticles was found 7.10, which is close to normal pH of
the human skin.
3.5.2. Viscosity
The viscosity of prepared Carbopol 940 gel containing
aceclofenac-loaded chitosan-egg albumin nanoparticles was com-
pared with a marketed aceclofenac gel. The prepared Carbopol 940
gel containing aceclofenac-loaded chitosan-egg albumin nanopar-
ticles showedviscosityof 523.26±2.08 cps, which washigher than
that of the marketed aceclofenac gel (303.47±1.44cps).
3.6. Ex vivo permeation
The prepared Carbopol 940 gel containing aceclofenac-loaded
chitosan-egg albumin nanoparticles and a marketed aceclofenac
gel were studied for ex vivo skin permeation through excised
mouse skin. The drug permeation from both the gels was found
sustained over a period of 8 h, which was evidenced in ex vivo
skin permeation study results (Fig. 6a). The permeation flux for
preparedCarbopol940 gel containingaceclofenac-loadedchitosan-
egg albumin nanoparticles was found 0.0681±0.0008g/cm2/h,
which was significantly higher ( p< 0.05) than that of the marketed
aceclofenac gel (0.0316±0.0004g/cm2/h).
3.7. In vivo evaluation
The in vivo anti-inflammatory activity of the prepared Car-
bopol 940 gel containing aceclofenac-loaded chitosan-egg albumin
nanoparticles and a marketed aceclofenac gel were evaluated in
male Sprague Dawley rats using carrageenean-induced rat-paw
edema model. The percent inhibition of swelling of rat paw edema
for Carbopol 940 gel containing aceclofenac-loaded nanoparticles
and marketed aceclofenac gel at various time intervals were pre-
sented in Fig. 6b. The prepared Carbopol 940 gel containing F-6
formulation showedhigherinhibition of swelling of ratpaw edema
compared with that of the marketed aceclofenac gel over a period
of 4 h, indicating improved permeation profileby thenewly formu-
lated Carbopol 940 gel containing F-6 formulation
4. Conclusion
Various aceclofenac-loaded chitosan-egg albumin nanoparti-
cles were prepared based on heat coagulation method at pH 5.4
and 80 ◦C. The drug entrapment efficiency of these nanoparticles
was within the range, 69.37±0.93 to 96.32±1.52%. Highest drug
entrapment efficiency (96.32±1.52%) was found in case of formu-
lation F-6, prepared using 200mg chitosan, 500 mg egg albumin
and2% (w/v) NaTPP.Averageparticle diameter andzetapotential of
2% (w/v)NaTPP cross-linked aceclofenac-loaded chitosan-egg albu-
min nanoparticles were found 352.90nm and −22.10 mV, respec-
tively. The stabilityand physical conditionof the loaded aceclofenac
within the nanoparticles were confirmed using FE-SEM, FTIR, DSC
and P-XRD analyses. The in vitro drug release from all aceclofenac-
loaded nanoparticles showed sustained drug release over a period
of 8h, which followed Korsmeyer–Peppas model and anoma-
lous (non-Fickian) diffusion mechanism of drug release. Carbopol
940 gel containing 2% (w/v) TPP cross-linked aceclofenac-loaded
chitosan-egg albumin nanoparticles was prepared for transdermal
delivery of aceclofenac. The prepared gel was characterized by pH
and viscosity. The Carbopol 940 gel containing aceclofenac-loaded
nanoparticles showed sustained permeation of aceclofenac over
8 h in ex vivo skin permeation study using excised mouse skin.
However, this Carbopol 940 gel formulation showed faster per-
meation of aceclofenac than that of the marketed aceclofenac gel.
The permeation flux for prepared Carbopol 940 gel was found sig-
nificantly higher ( p< 0.05) (0.0681±0.0008g/cm2/h) than that
of the marketed aceclofenac gel (0.0316±0.0004g/cm2/h). The
in vivo anti-inflammatory activity in male Sprague Dawley rats
using carrageenean-induced rat-paw edema model demonstrated
comparative higher inhibition of swelling of rat paw edema by
Carbopol 940 gel containing aceclofenac-loaded nanoparticles
compared with that of the marketed aceclofenac gel over a period
of 4h. Overall, these results indicated the promise of Carbopol
940 gel containing 2% (w/v) NaTPP cross-linked aceclofenac-loaded
chitosan-egg albumin nanoparticles for transdermal delivery of
aceclofenac with improved permeation profile and thus, improved
patient compliance.
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