Design and Characterization of Albumin-Chitosan Microspheres of Aceclofenac for Sustained Release
Sachin R Patil*1, Swati Patil4 ,Ravi Kumar1, MB Patil2 and Mahesh S Paschapur3
1Dept. of Pharmaceutics, K.L.E.S’s College of Pharmacy, Ankola-581314, Karnataka, India.
2Dept. of Pharmacognosy, K.L.E.S’s College of Pharmacy, Ankola-581314, Karnataka, India.
3Dept. of Pharmacology, K.L.E.S’s College of Pharmacy, Ankola-581314, Karnataka, India.
ABSTRACT
The
present study deals with the formulation and characterization of cross linked chitosan/ albumin microspheres containing an NSAID drug
Aceclofenac. The microspheres were prepared by suspension cross linking method
using gluteraldehyde as a cross linking agent of the
polymer matrix. Total eight formulation batches (F1 to F8) were formulated
using chitosan/albumin alone and in combinations. The
formulations were subjected to various evaluation parameters like % practical
yield, entrapment efficiency, particle size distribution, swelling ratio, in vitro
release and stability studies. Perfectly spherical cross linked microspheres
loaded with aceclofenac were obtained in the size
range of 50 – 500 µm. The % practical yield, entrapment efficiency, particle
size, swelling ratio were increased with increased concentration of polymer
used. The release of aceclofenac was influenced by
polymer concentration and size of microspheres. The stability studies of
formulation showed 4°C is suitable temperature for storage.
KEYWORDS:
Cross linked microspheres, Controlled release, Chitosan,
Albumin, aceclofenac.
INTRODUCTION
Controlled
release technology has rapidly emerged over the past three decades, as a new
interdisciplinary science that offers novel approaches to the bioactive agents.
Controlled drug delivery design involves the application of physical and
polymer chemistry to dosage form design, to produce a well characterized and
reproducible drug delivery profile. By achieving predictable and reproducible
release rates. Environment bioactive agents to the target environment for an
extended time controlled release delivery systems can achieve optimum
therapeutic responses, prolong efficacy and decreased toxicity1.
The
goal of any drug delivery system is to provide a therapeutic amount of drug to
the proper site in the body to achieve promptly and then maintain the desired
drug concentration that is the drug delivery system should deliver drug at a
rate detected by the needs of the body over the entire period of treatment.
This is possible through administration of conventional dosage form in a
particular dose and particular frequency to provide a prompt release of drug.
Therefore to achieve as well as to maintain the drug concentration within the
therapeutically effective range needed for treatment by repeated administration
a day. This results in a significant fluctuation in plasma-drug level, leads to
several undesirable toxic effects, and poor patient compliance2,3.
Aceclofenac is a novel NSAID known to exhibit
multifactor mechanism of action. Aceclofenac was developed in order to provide
a highly effective pain relieving therapy with a reduced side effect profile,
especially GI events that are frequently experienced with NSAID therapy. It is
used in the management of osteoarthritis, rheumatoid arthritis and ankylosing spondylitis. The mean
plasma elimination half-life is 4 - 4.3 hours. Clearance is estimated to 5 litres per hour. Approximately two-thirds of the
administered dose is excreted via the urine, mainly as conjugated
hydroxymetabolites4.
Microparticles
are polymeric particles ranging in size from 1 – 1000 mm. The mechanism of
drug release is either dissolution or diffusion of drug and the formulations
are either as encapsulated (microcapsule) or matrix (microsphere). The numbers
of methods are described for encapsulating medicaments with different coat
materials. The properties especially drug release characteristic of the
microspheres depends on the coat material employed in preparation5.
MATERIALS AND METHODS
Aceclofenac was obtained as a gift sample
from Amoli organics Pvt. Ltd; Mumbai. Chitosan was a gift sample from Central institute fisheries
technology; Cochin. Albumin, glutaraldehyde (25%),
liquid paraffin and petroleum ether were obtained from S.D. Fine Chemicals;
Mumbai. All other chemicals used were of A R grade.
Preparation
of chitosan microspheres6,7
The required amount
of chitosan was dissolved in 2% v/v acetic acid
solution. The drug (100 mg) was added in it and this dispersion was extruded
through syringe in 100ml of light liquid paraffin containing in a 500 ml beaker
and stirred on Remi-three blade stirrer at high
speed. The w/o emulsion formed was stabilized by adding 1% Tween-80. After 20
min of stirring, 1ml of glutaraldehyde (25% solution,
as cross-linking agent) was added and stirring was continued for 3 hours.
Microspheres thus formed were separated by filtration, washed repeatedly with
hexane/ cyclohexane to remove oil, and finally washed
with water to remove excess of glutaraldehyde.
Microspheres were then air dried at room temperature.
Preparation of albumin microspheres8,9
Albumin
microspheres were prepared by using the same technique with little
modification. The required amount of BSA was dissolved in little quantity of
distilled water at room temperature and the drug (100 mg) was added in it. This
dispersion was heated to 60°
for 2 min and then homogenized for 5 min. This dispersion was transferred to
500 ml beaker containing 100ml of light liquid paraffin which contain 1% of Tween 80. After 20 min of stirring, 1ml of glutaraldehyde (25% solution, as cross linking agent) was
added and stirring was continued for 3 hours. Microspheres thus formed were
separated by filtration, washed with hexane/ cyclohexane
to remove oil, and finally washed with water to remove excess of glutaraldehyde. Microspheres were then air dried at room
temperature.
Percentage Practical yield10
Microspheres were
collected and weighed to determine production yield (PY) from the following
equation.
Practical Mass (Microspheres)
PY
(%) =
x 100
Theoretical Mass (Polymer +
Drug)
Particle size analysis
Particle size of
the microspheres was determined by optical microscopy. Average of 100
microspheres were used for study and the mean particle size was considered to
be the deciding factor in selecting optimum formulation condition for each
variable parameter studied.
Scanning electron microscopy (SEM) of microspheres:
SEM of microspheres
was recorded using scanning electron microscope (Jeol.Jsm
T-330, Japan) with 75X magnification.
Drug content and encapsulation efficiency10
A microsphere
sample (10 mg) was dissolved in 10 ml of 0.1N HCl /
methanol (1:1 v/v) mixture with ultrasonication for 4
h at 30°C.
The samples were filtered using 0.2 mm membrane filter and absorbance of sample
was determined at 275 nm using spectrophotometer.
Actual drug content
and encapsulation efficiency were calculated in duplicate for all batches using
the equation as follows
Mact
Drug content % = x 100
MmS
Mact
Encapsulation efficiency = x 100
Mthe
Where Mact is the actual Aceclofenac content in
weighted quantity of microspheres, MmS
is the weighted quantity of sample microsphere and Mthe
is the theoretical amount of aceclofenac in
microspheres.
Swelling ratio11
The swelling
characteristics of microspheres were determined in order to check hydrophilic
affinity of spherical microspheres. The equilibrium water content (swelling
ratio) of the cross-linked microspheres, expressed as the weight fraction of
water in the equilibrated microspheres, was measured gravimetrically by
weighing the particles prior to and after swelling. The dried microspheres were
first weighed and then equilibrated in distilled water for ~72 h. Subsequently,
they were removed from water, carefully blotted with tissue paper and then they
were re-weighed. The equilibrium weight degree of swelling (QW) was calculated
by the following expression.
Ws - Wd
QW%
= x 100
Ws
Where, Ws and Wd are weights of swollen and dried microspheres,
respectively.
In vitro release kinetics12,13
In vitro release profile of aceclofenac microspheres was examined in pH 1.2 buffer from
0 to 2 h, pH 6.0 buffer from 2 to 3 h, and in phosphate buffer of pH 7.2 from 3
to 8 h as a dissolution medium with 10% v/v methanol using rotating basket
method specified in USP XXIII at 100 rpm. Microspheres equivalent to 50 mg of aceclofenac were taken in the dialysis bags in the basket
and rotated at a constant speed of 100 rpm.
The medium was
maintained at 37°C±0.5°C. Aliquots of
samples were withdrawn after predetermined periods of time and the same volume
of fresh medium was added immediately to the test medium. The concentration of
the drug release at different time intervals was then determined by measuring
the absorbance at 275 nm spectrophotometrically using Shimadzu 1201 UV-visible
spectrophotometer.
Stability studies
From the 8 batches
of aceclofenac loaded microspheres, formulations F2,
F5 & F7 were tested for stability studies. The microspheres were placed in
a screw capped glass container and stored at ambient humidity conditions at
room temperature (27±20 C), oven temperature (40±20 C)
and refrigerator (4-60 C) for a period of 45 d. The samples were
assayed for drug content.
RESULT
AND DISCUSSION
The prepared
microspheres exhibit good morphological characteristics, spherical and without
aggregation with medium size range from 50-500µm. SEM of microspheres at
magnification of 75X is presented in Fig. 1and 2, which revealed that the
microspheres were almost spherical in nature with slight smooth surface
morphology.
Fig. 1: SEM Photograph of aceclofenac-loaded
microspheres containing chitosan (F1)
With increase in chitosan/ albumin concentration in the microspheres from F1
to F8, the particle size of microspheres increases, which may be due to the
fact that increase in the concentration of polymer increases the cross linking,
and hence the matrix density of the microsphere increased, and that may result
in the increase in particle size of the microspheres.
The compiled thermograms of DSC of pure drug, drug loaded microspheres
and plain microspheres indicate that the pure sample of aceclofenac
showed endothermic peak at 1630C. It was observed that absence of
the endothermic peak of the drug at 163°C in the drug loaded microspheres indicated,
that there is no interaction between Aceclofenac and chitosan
and drug is molecularly distributed in the microspheres.
The effect of
concentration of polymer that is polymer drug ratio on particle size, %
practical yield, encapsulation efficiency, and swelling ratio is presented in
Table 1.
Among all drug to
carrier ratio used, the ratio 1:2:1 showed maximum % yield of 89.74% and the
ratio 1:4:0, 1:0:4, and 1:2:1 showed 76%, 70%, and 80% encapsulation efficiency
respectively. It was observed that entrapment efficiency increases with an
increase in polymer concentration, which may be due to the increase in
viscosity of the chitosan / albumin solution with
increase in concentration prevents drug crystals from leaving the droplets.
It has been seen
that as there is increase in concentration of polymer there is increase in
swelling ratio. It has also seen that, as the degree of polymer cross-linking
increased the equilibrium weight degree of swelling decreased. It may be due to
the increase of the degree of cross-linking of the microspheres results in a
significant decrease of the molecular weight between cross-links and, as a
consequence, the water uptake by the hydrogel
microspheres also decreases.
In vitro drug release
profiles of all batches are showed in Table 2. The release pattern of the drug
from the microspheres was observed to follow biphasic pattern, characterized by
initial burst effect followed by slow release over a period of 8 hr.
Fig. 2: SEM Photograph of aceclofenac-loaded
microspheres containing albumin (F4)
As shown in Fig. 2,
when the release of pure drug and the microspheres were compared, pure drug was
entirely released within 2 h where as in case of microspheres showed sustained
release. In the first 1 h drug release was 27.68%, 30.25%, 28.96%, 26.39%,
27.04%, 27.68%, 26.39%, and 27.68% for F1 to F8 respectively as shown in Table
2. The mechanism for the burst release can be attributed to the drug loaded on
the microspheres or imperfect entrapment of drug. The release of the drug is
dependent on the microsphere size, as expected. Drug release is faster from
spheres of smaller size owing to the decreased diffusional
path length and the increased surface area in contact with the dissolution
medium. With increased load of the drug in the microspheres matrix, there is an
increased release. At higher loadings, drug diffusion from the matrix produces
more pores and channels through which the release occurs at a faster rate.
Fig. 3: In vitro
release profile of Aceclofenac from microspheres
In vitro dissolution
profiles of Aceclofenac from microspheres formulations F1 (-¸-),
F2 (-p-),
F3 (-r-),
F4 (-¢-),
F5 (-£-),
F6 (-˜-),
F7 (-™-),
F8 (-Ü-)
and Pure Drug (-u-)
No appreciable
difference was observed in the extent of degradation of product during 45 d in
the microspheres. It was observed that there was slight decrease in drug
content when formulation stored in 40C but there was significant
difference in drug content when formulation was stored at 400C.
The method of
preparation of albumin-chitosan microspheres of aceclofenac was found to be simple and reproducible. The
carriers used, albumin and chitosan, are easily
available, biocompatible, and biodegradable. From the above data, it may be
concluded that the drug loaded microspheres appears to be suitable delivery
system for aceclofenac and may help to reduce dose of
a drug and frequency of administration.
ACKNOWLEDGEMENT
The authors would
like to acknowledge M/S Amoli organics Pvt. Ltd;
Mumbai, and Central institute of fisheries technology; Cochin for generous gift
sample of aceclofenac and chitosan
respectively.
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