Sangeetha et al
Tropical Journal of Pharmaceutical Research, March 2007; 6 (1): 653-659
© Pharmacotherapy Group,
Faculty of Pharmacy, University of Benin,
Benin City, Nigeria.
.
All rights reserved
Available online at http://www.tjpr.org
Research Article
Formulation of Sodium Alginate Nanospheres
Containing Amphotericin B for the Treatment of
Systemic Candidiasis
Shanmugasundaram Sangeetha*, Dhandapani Nagasamy
Venkatesh, Rajendran Adhiyaman, Kumaraswamy Santhi, and
Bhojraj Suresh
Centre for Research and Post-graduate studies JSS College of Pharmacy Ooty-643 001. The Nilgiris, India.
Abstract
Purpose: The aim of this work was to formulate sodium alginate nanospheres of amphotericin B by
controlled gellification method and to evaluate the role of the nanospheres as a “passive carrier” in
targeted antifungal therapy.
Methods: Sodium alginate nanospheres of amphotericin B were prepared by controlled gellification
method, and the particle size analysis was carried out by scanning electron microscopy. The carrier
capacity of sodium alginate was evaluated in terms of drug to polymer ratio. In vitro release study was
carried out on all drug loaded nanospheres by the dialysis method. Release kinetics of drug from
different drug loaded nanospheres was also determined. The in vivo antifungal efficacy of nanospheres
bound drug vis-à-vis the free drug was evaluated in candidiasis- induced mice models.
Results: Preparation of nanospheres through controlled gellification method yielded particles with a size
range of 419.6 ± 0.28 nm. Studies on drug to polymer ratio showed a linear relationship between
concentration of drug and drug loading capacity. In vitro release kinetic study revealed that the release
of drug from the nanospheres followed Fickian diffusion. In vivo studies showed that the nanospherebound drug produced a higher antifungal efficacy than the free drug.
Conclusion: The formulated sodium alginate nanospheres containing amphotericin B was found to
have better antifungal activity when compared to the free drug and also yielded sustained in vitro
release.
Keywords: Nanospheres, sodium alginate, amphotericin B, controlled gellification method, in vitro & in
vivo release.
*Correspondence E-mail : sangeethamadhesh@rediffmail.com, Tel: +91-423-2443393. Fax: +91-423-2442937.
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INTRODUCTION
Infections due to fungi of the genera Candida
and Aspergillus have become major nosocomial
causes of morbidity and mortality in
immunocompromised
individuals.
Their
incidence has increased during the last three
decades in parallel with the number of such
patients and it is now a frequent complication1,2.
More recently there has been a substantial
increase in the frequency of candidaemia
caused by species other than Candida albicans.3
Systemic candidiasis is associated with high
mortality rate and prolonged hospitalization.4
The treatment options are extremely limited.
Rapid treatment with amphotericin B or
fluconazole is required to reduce the mortality
observed in these patients.5 Amphotericin B is a
very potent antifungal agent, but it has multiple
severe toxic effects and about 36% rate of failure
in the treatment of immunocompromised hosts.6
Thus several lipid formulations (Ambisome®,
Amphocil®, Abelcet®) have been developed and
commercialized.7,8 Although they have been
proven to reduce amphotericin B toxicity, their
toxic effects and pharmacokinetic properties all
differ and their use has been limited by high
cost.
One of the suitable methods to overcome these
problems
could
be
association
with
biodegradable polymeric carriers such as
nanoparticles. Nanoparticles may become one of
the successful carriers by overcoming problems
caused by infections that are refractory to
conventional treatment and also widening the
therapeutic margin of antibiotics currently used
in clinical practice.9,10,11,12 Hence, the objective of
the work was to formulate sodium alginate
nanospheres of amphotericin B by controlled
gellification
method,
evaluate
its
physicochemical characteristics such as particle
size, drug loading capacity, in vitro release
characteristics, and also the in vivo efficacy in
candidiasis-induced
animal
models.
An
important requisite in the realization of an
injectable nanoparticle delivery system is the
proper choice of the polymeric substrate. The
polymeric bulk must fulfil many requirements as
well as have suitable mechanical properties and
biodegradability. Therefore, a suitable polymer
such as sodium alginate was selected for the
study as it met these requirements.
EXPERIMENTAL
Materials
Amphotericin B was generously donated by
Synbiotics Ltd, Baroda, India. Other materials
used include sodium alginate, calcium chloride,
poly-l-lysine, dialysis bag (Sigma Inc USA). SEM
(JSM-6400 scanning electron microscope,
Tokyo, Japan), research centrifuge R24 (Remi
instruments), UV spectrophotometer (UV 160 A
Shimadzu, Japan) were the equipments used for
the study.
METHODS
Preparation of sodium alginate nanospheres
by controlled gellification method
Sodium alginate nanospheres were prepared by
the method reported by Rajaonarivony et.al,10.
2ml of calcium chloride (18mM) was added to
38ml of sodium alginate solution (0.1%w/v) to
induce gellification. Then 16ml of poly-l-lysine
(0.1%w/v) was added to form a polyelectrolyte
complex. The nanospheres suspension obtained
was stirred for 2 h and kept overnight for
stabilization. The nanospheres were separated
by ultra centrifugation at 20000 rpm for 45
minutes and dried under vacuum to form a flaky
mass, which on redispersion in sterile water for
injection, produced discrete particles.
Determination of particle size
The nanospheres were spread over a glass slide
and dried under vacuum at room temperature
(25ºC). The sample was shadowed in a cathodic
evaporator with a gold layer 20nm thick. The
diameters of all the spheres in each field were
calculated using a JSM-6400 scanning electron
microscope (Tokyo, Japan).11
Study on drug to polymer ratio
To determine the drug: polymer ratio12, five
batches of nanospheres containing various
concentrations of drug were prepared. In each
batch the concentration of drug was varied, while
other processing variables were kept constant.
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Sangeetha et al
Amphotericin B equivalent to 10µg/ml was
dissolved in 38ml of sodium alginate solution
(0.1%). Then 2 ml of calcium chloride (18mM)
and 16ml of poly-l-lysine (0.1%) was added,
stirred for 2 h and kept overnight. The resulting
nanospheres were separated at 20000 rpm by
ultracentrifugation. The nanospheres were dried
under vacuum. This batch coded ASA-I.
Similarly ASA-II, ASA-III, ASA-IV and ASA-V
were prepared, containing 20µg/ml, 30µg/ml,
40µg/ml and 50µg/ml of amphotericin B,
respectively. The batches were subjected to
particle size analysis by SEM.
Determinations of drug content
An ultracentrifugation technique13 was used to
separate the free drug from nanospheres and to
estimate the drug loading of the nanospheres.
The final colloidal suspensions were ultra
centrifuged at 10000 rpm at 22 ± 2 ° C for 0.5 h.
The supernatant was analyzed for drug content
by measuring the absorbance at 345 nm using
UV spectrophotometer.
In vitro release studies
The drug release assessment was carried out
following the procedure reported earlier15, which
employed a diffusion cell. A Sigma dialysis
membrane was fixed to one end of a permeation
cell. The nanospheres were placed in the cell
donor compartment and the cell was immersed
in a beaker containing 50ml of phosphate buffer
(pH 7.4) which serve as a receptor compartment.
The cell was immersed to a depth of 1cm below
the surface of the receptor solvent. The medium
in the receptor compartment was agitated using
a magnetic stirrer and temperature of 37 ± 10C.
5ml of the sample from the receptor
compartment was taken at various intervals of
time over a period of 24 h and each time
replaced with fresh buffer. The samples
withdrawn
were
estimated
spectrophotometrically at 345nm.
In vivo studies in candidiasis induced mice
models
The institutional animal ethics committee of JSS
College of pharmacy approved the protocol
study. The in vivo study was carried out in
candidiasis induced mice models as reported
earlier14. A total of 30 animals were taken, and
divided into 3 groups. All 3 groups were induced
with candidiasis by intravenous administration of
0.1ml of 105 to 106 cells/ml of cell culture
suspension. After 24 h of candidiasis induction,
the Group I was treated with nanospheres bound
amphotericin B, Group II with free amphotericin
B. In each case, bound drug and free drugs were
dispersed in sterile water for injections. The dose
of the drug was 100mg/kg of animal weight and
the volume of injection was 1ml/100g of mouse.
The doses were given once a day for 7
consecutive days. Group III (control) was treated
with solvent only. The animals were observed for
mortality for 16 consecutive days. The mice were
sacrificed at the end of 16th day by cervical
dislocation and dissected immediately. Organs
like kidneys, lungs and liver were removed and
homogenized in 5ml of sterile saline with Tween
80. The number of colony forming units (CFU) in
each homogenate was determined by plate
counts on Sabourands dextrose agar media
(SDA) containing 0.05mg/ml of chloramphenicol.
RESULTS AND DISCUSSION
Preparation of nanospheres by controlled
gellification involves formation of calcium
alginate complexes. The interaction between the
calcium ions and the alginate polymer occurred
at the level of the oligopolyglucuronic
sequences. Furthermore, calcium ions induced a
parallel packing of the oligopolyglucuronic
sequences to give egg-box structures and the
addition
of
poly-l-lysine
allowed
only
strengthening of this system to obtain small and
well
defined
particles.
The
obtained
nanospheres were spherical and discrete with an
average particle size of 419.6 ± 0.28 nm.
Drug-loading capacity
The drug carrier capacity of sodium alginate with
respect to amphotericin B was determined
assessing the drug: polymer ratio. As Table 1
shows, the drug loading capacity was observed
to have a direct linear relationship, with the drug
loading capacity increasing as the concentration
of drug increased. Thus, it can be said that the
saturation capacity of the polymer with respect to
the selected drug occurred at a relatively lower
concentration and at a faster rate.
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Sangeetha et al
the drug was released in a slow manner over 24
h. In order to predict and correlate the release
Release kinetic analysis
The in vitro study of all the five batches showed
an initial burst release within 30 min and 60% of
Table 1: Amphotericin B - loading efficiency of sodium alginate nanospheres
Formulation code
Drug concentration
(µg/ml)
Drug loading
(%)
ASA I
10
10.7 ± 0.2
ASA II
20
13.5 ± 0.6
ASA III
30
17.2 ± 0.8
ASA IV
40
22.6 ± 0.4
ASA V
50
27.3 ± 0.7
Table 2: In vitro release kinetics of amphotericin B- loaded nanospheres
Formulation
code
ASA I
Drug
concentration
(µg/ml)
10
First order plot
Peppa’s
K×10-3
2.6357
r
0.9951
n
0.5199
r
0.9447
ASA II
20
2.8368
0.9956
0.5132
0.9265
ASA III
30
2.8735
0.9708
0.5223
0.9131
ASA IV
40
3.5046
0.9926
0.5437
0.9404
ASA V
50
4.2236
0.9957
0.5583
0.9325
Table 3: Percentage mortality and colony forming units of nanospheres bound amphotericin
B and free amphotericin B in organs of RES.
Drug treatment
Number of colony forming units (CFU)
Mortality (%)
Kidney
Amphotericin
free drug
Amphotericin
nanospheres
bound drug
Control
Liver
Lungs
B
40
5x103
4.2x103
9.5x103
B
20
5.6x103
3.9x102*
4.5x102*
100
1.8x104
7.5x103
1.9x104
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Sangeetha et al
Fig 1: Scanning electron micrograph of sodium alginate nanospheres containing amphotericin B.
100
90
80
Cumulative release (%)
70
60
50
40
30
20
10
0
0
4
8
12
16
20
24
Time (h)
Fig 2: In vitro release profiles of amphotericin B from different batches of drug loaded sodium
alginate nanospheres. (♦) ASA I-10µg/ml, (■) ASA II-20µg/ml, (▲) ASA III-30µg/ml, (×) ASA IV40µg/ml and (●) ASA- V-50µg/ml.
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behavior of the drug from the hydrophilic matrix it
is necessary to fit it to a suitable model. Hence,
the release data were fitted according to the well
known exponential equation16, often used to
describe the drug release behavior from
polymeric system:
mt/m∞= ktn ………………………… 1
Where mt/m∞ is the fractional release of the
drug, ’t’ is the release time, ‘k’ is a constant
which indicates the properties of the
macromolecular polymeric system and n is the
release exponent indicative of the mechanism of
release. The ‘n’ value used for analysis of the
drug release mechanism from the amphotericin
B nanospheres were determined from log
(mt/m∞) vs log(t) plots. To calculate the release
constant, k, the logarithm of the remaining
amphotericin B in nanospheres plotted versus
time. The release of drug from nanospheres
followed first order kinetics over a 24 h period.
Drug loading is another factor that influenced the
drug release rate from the nanospheres.
Generally, increasing the drug content in the
nanospheres increased. The values of ‘k’, ‘n’ and
‘r’ for five different batches are reported in Table
2, and the ‘n’ value was within 0.5132 to 0.5583.
The results of kinetic analysis reveal that the
release of amphotericin B from sodium alginate
nanospheres followed Fickian diffusion.
In vivo studies
An effective treatment of systemic candidiasis
with amphotericin B requires comparatively
higher concentrations of the antifungal drug in
major target organs, such as the like lung and
liver, which act as reservoirs of microbes and
may lead to reoccurrence of infection. As Table
3 indicates, the nanosphere-bound drug is
relatively more effective than the free drug
against the candidiasis in terms of significant
reduction in the colony forming units (CFU) in
liver and lungs (p<0.05 by SNK test). The
significant reduction in colony forming units
(CFU) in the liver and lung could be to enhanced
drug localization of nanosphere- bound drug as
a result of macrophage uptake. The results
obtained in this study showed a marked
reduction in the CFU in organs of liver and lungs
when compared with the CFU for groups treated
with the free drug. The reduction in CFU as well
as the fall in % mortality may facilitate a
reduction in the total dose required for therapy.
This, in turn, means that dose-related systemic
toxicities could also be reduced.
CONCLUSION
The formulated sodium alginate nanospheres of
amphotericin B were found to be an effective
and natural carrier in terms of discrete particle
size, optimum drug loading capacity, satisfactory
in vitro release characteristics, and in vivo
antifungal activity for the passive delivery of
amphotericin B. Furthermore, the nanospherebound drug may enhance drug localization in the
RES. A possible beneficial fall-out is a reduction
in total dose as well as dose-related systemic
side effects.
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