This document summarizes a study that developed and optimized chitosan-alginate nanoparticles for oral delivery of the drug rosuvastatin calcium. The nanoparticles were prepared using ionotropic gelation and characterized for properties such as size, surface charge, drug loading efficiency, and in vitro drug release. The optimized nanoparticles had an average size of 349.3 nm, a zeta potential of +29.1 mV, high drug loading and entrapment efficiencies, and showed a fast initial release followed by more gradual release over 24 hours. The study demonstrated that the chitosan-alginate nanoparticle system improved rosuvastatin solubility and has potential to enhance its oral bioavailability and therapeutic efficacy.

1. _____________________________________________________________________________________________________
*Corresponding author: E-mail: sajanqa@gmail.com, mmujtaba@nbu.edu.sa;
Journal of Pharmaceutical Research International
32(1): 50-56, 2020; Article no.JPRI.54773
ISSN: 2456-9119
(Past name: British Journal of Pharmaceutical Research, Past ISSN: 2231-2919,
NLM ID: 101631759)
Chitosan-sodium Alginate Nanoparticle as a
Promising Approach for Oral Delivery of
Rosuvastatin Calcium: Formulation,
Optimization and In vitro Characterization
Md. Ali Mujtaba1*
and Nawaf M. Alotaibi1
1
Department of Pharmaceutics, Faculty of Pharmacy, Northern Border University, Rafha,
Kingdom of Saudi Arabia.
Authors’ contributions
This work was carried out in collaboration between both authors. Author MAM designed the study,
performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Author
NMA managed the analyses of the study and literature searches. Both authors read and approved the
final manuscript.
Article Information
DOI: 10.9734/JPRI/2020/v32i130394
Editor(s):
(1) Rahul S. Khupse, Assistant Professor, Pharmaceutical Sciences, University of Findlay, USA.
Reviewers:
(1) Preksha Barot, India.
(2) Arthur Chuemere, University of Port Harcourt, Nigeria.
(3) Bekkouch Oussama, Mohamed First University, Morocco.
Complete Peer review History: http://www.sdiarticle4.com/review-history/54773
Received 02 February 2020
Accepted 17 February 2020
Published 27 February 2020
ABSTRACT
Rosuvastatin calcium is the most effective antilipidemic drug and is called "super-statin" but it
exhibits low aqueous solubility and poor oral bioavailability of about 20%. The present work aimed
to develop and optimize chitosan-alginate nanoparticulate formulation of rosuvastatin which can
improve its solubility, dissolution and therapeutic efficacy. Chitosan-alginate nanoparticles were
prepared by ionotropic pre-gelation of an alginate core followed by chitosan polyelectrolyte
complexation and optimization was done in terms of two biopolymers, crosslinker concentrations.
The chitosan-alginate nanoparticles were characterized by various techniques such as particle
properties such as size; size distribution (polydispersity index); Zeta-potential measurements and
Fourier transform infrared spectra respectively. The designed rosuvastatin loaded chitosan-alginate
nanoparticle had the average particle size of 349.3 nm with the zeta potential of +29.1 mV, and had
Original Research Article

2. Mujtaba and Alotaibi; JPRI, 32(1): 50-56, 2020; Article no.JPRI.54773
51
high drug loading and entrapment efficiencies. Fourier transform infrared spectra showed no
chemical interaction between the rosuvastatin and chitosan-alginate nanoparticle upon the
encapsulation of rosuvastatin within the nanoparticles. Nanoparticles revealed a fast release during
the first two hours followed by a more gradual drug release during a 24 h period. Hence, our
studies demonstrated that the new chitosan-alginate nanoparticle system of rosuvastatin is a
promising strategy for improving solubility and in turn, the therapeutic efficacy of rosuvastatin.
Therefore, the rosuvastatin-loaded chitosan-alginate nanoparticles are a promising approach for
the oral delivery of rosuvastatin.
Keywords: Chitosan; alginate; nanoparticles; rosuvastatin; drug release.
1. INTRODUCTION
The prevalence of dyslipidemia is high and
increasing in many developing countries,
including Saudi Arabia because of the
westernization of diet and other lifestyle
changes [1]. Rosuvastatin calcium (ROC) is a
widely used antihyperlipidemic drug use for the
treatment of atherosclerosis and is called
"superstatin" [2]. It competitively inhibits the
enzyme HMG CoA reductase, prevents the
conversion of 3-hydroxy-3-methylglutaryl CoA
to mevalonate and the production of cholesterol
and its circulating blood derivatives, including
LDL [3]. ROC is a poorly water-soluble drug
with only 20% oral bioavailability. It is classified
by the biopharmaceutical classification system
as a class II drug [4]. The poor solubility of
ROC affects its dissolution rate which in turn,
its bioavailability. Thus, enhancing the dissolution
of ROC can lead to improving its oral
bioavailability.
Drugs with poor solubility possess difficulty in the
formulation by applying conventional approaches
as they present problems such as the slow onset
of action, poor oral bioavailability, lack of dose
proportionality, failure to achieve steady-state
plasma concentration, and undesirable side
effects. Nanotechnology is a promising strategy
in the development of drug delivery systems
especially for those potent drugs whose clinical
development failed due to their poor solubility,
low permeability, inadequate bioavailability,
and other poor biopharmaceutical properties
[5,6]. Nanoparticles consisting of synthetic
biodegradable polymers, natural biopolymers,
lipids, and polysaccharides have been developed
and tested over the past decades. Nowadays,
polymeric nanoparticles prepared by natural
biopolymers such as chitosan and alginate are of
interest in the drug delivery system due to their
biodegradability, biocompatibility, and safe [7,8].
Alginate is a natural anionic polymer derived
from brown algae and its chemical structure is
composed of α-L-guluronic acid and β-D-
mannuronic acid. It can form nanoparticles by
ionotropic gelation with divalent cations
or cationic polymers [9] , but these nanoparticles
are not stable at room temperature [10] and
encapsulated active compounds easily leak from
the nanoparticles [11]. Mujtaba et al. [8] and
Lertsutthiwong et al. [12] overcame these
limitations by coating the alginate nanoparticles
with chitosan, a cationic polysaccharide
produced by deacetylation of chitin. Additionally,
chitosan has been reported to possess the
property of reducing cholesterol levels,
which may make chitosan as an attractive
polymer in the development of drug delivery
systems for the treatment of cardiac diseases
[13,14].
Recently, nanoparticle-based drug delivery
systems have emerged as a solution for
increasing the bioavailability of potential
therapeutic agents. Therefore, the aim of present
work is to develop and optimize the chitosan-
alginate nanoparticulate formulation of ROC
which can improve its solubility, dissolution and
therapeutic efficacy.
2. MATERIALS AND METHODS
2.1 Materials
Rosuvastatin calcium (ROC) was obtained from
Jamjoom Pharmaceutical Co. Ltd. (Jeddah,
Kingdom of Saudi Arabia) as a gift sample.
Chitosan with medium molecular weight and
degree of deacetylation about 85% were
purchased from Sigma–Aldrich, St. Louis, MO,
USA. The medium viscosity sodium alginate
isolated from Macrocystis pyrifera, having a
molecular weight between 75 and 100 kDa, and
mannuronic to guluronic acid ratio of 1.5 (60:40),
was purchased from CDH Labs., India. All other
reagents and chemicals used were of analytical
reagent grade.

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2.2 Preparation of Blank Chitosan-
alginate Nanoparticles (CANPs)
Accurately weight 20 mg sodium alginate and
dissolved in 10 mL distilled water to form a 0.2%
(w/v) sodium alginate solution. This solution was
adjusted to a pH of 5, and then it was added
dropwise to 10 mL of 0.2% (w/v) calcium chloride
solution under magnetic stirring at room
temperature. 50 mg of chitosan was dissolved in
50 mL of 1% (v/v) acetic acid solution to obtain a
0.1% (w/v) chitosan solution. After the pH of the
chitosan solution was adjusted to 5, it was
incorporated into the resultant calcium alginate
pre-gel and the mixture solution was further
stirred for 1 h. The mixture was then subjected
to probe sonication (Vibra Cell, Sonics &
Materials, Inc., USA) for 15 min at 20°C of probe
temperature in a pulsatile manner (50 s
sonication with 10 s pause) with 30% amplitude.
After probe sonication, the resulting opalescent
suspension was equilibrated overnight to allow
nanoparticles to form uniform particle size.
2.3 Preparation of ROC-loaded CANPs
Accurately, 2 mL of 1 mg/mL ROC solution was
added dropwise to 10 mL of 2 mg/mL alginate
and stir for 30 min. The above solution was then
added dropwise to 10 mL of 2 mg/mL calcium
chloride solution followed by a continuous stirring
of 30 min. Afterward 10 mL of 1 mg/mL chitosan
stock solution was added to the resultant alginate
solution and stir for another 60 min. The mixture
was then subjected to probe sonication for 15
min at 20°C of probe temperature in a pulsatile
manner (50 s sonication with 10 s pause) with
30% amplitude. After probe sonication, the
resulting opalescent suspension was equilibrated
overnight to allow nanoparticles to form uniform
particle size.
2.4 Characterization of CANPs
2.4.1 Nanoparticles size and surface charge
The diameter of the nanoparticles (z-average),
polydispersity index (PDI) and zeta potential was
measured for the formulation using dynamic light
scattering zetasizer (PSS NICOMP Z3000, Port
Richey, FL). The nanoparticle dispersions were
diluted with distilled water before the
measurement to avoid multi scattering
phenomenon. All the measurements were
performed in triplicates. Dynamic light scattering
(DLS) is used to measure the average
nanoparticle size and size distribution. This
technique measures time-dependent fluctuations
in the intensity of scattered light as a result of
Brownian motion exhibited by the particles. The
zeta potential is considered as one of the highest
measured parameters which denotes the overall
charges acquired by the particles in a particular
medium and it is considered as one of the
important factors for the stability of the
nanoparticles [15].
2.4.2 Fourier transform infrared
spectroscopy (FT-IR)
CANPs separated from nanoparticulate
suspensions were dried by a freeze dryer, and
their FT-IR transmission spectra was obtained
using a FT-IR spectrophotometer (FTIR-7600,
Angstrom Advanced Inc. USA). A total of
2% (w/w) of the sample, with respect to the KBr
disc, were mixed with dry KBr. The mixture
was ground into a fine powder using an
agate mortar before compressing into KBr
disc under a hydraulic press at 10,000 psi. Each
KBr disc was scanned at 4 mm/s at a resolution
of 2 cm over a wavenumber region of 400–4000
cm
-1
using IR solution software. The
characteristic peaks were recorded for different
samples. The interaction between ROC and
formulation excipients was investigated by FT-IR
spectrometry.
2.4.3 Determination of encapsulation
efficiency of CANPs
The encapsulation efficiency (EE) of
nanoparticles was determined by the separation
of drug-loaded nanoparticles from the aqueous
medium containing non-associated ROC by
ultracentrifugation at 18,000 rpm at 4°C for 30
min. The amount of ROC loaded into the
nanoparticles was calculated as the difference
between the total amount used to prepare
the nanoparticles and the amount that was
found in the supernatant. The amount of free
ROC in the supernatant was measured by a
validated UV-vis spectrophotometric method.
The ROC encapsulation efficiency (EE) of the
nanoparticles was determined in triplicate and
calculated as follows [16]:
Encapsulation efficiency =
–
(1)

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2.4.4 In vitro drug release characteristics
The release profiles of ROC from the ROC-
loaded CANPs were determined by dialysis bag
diffusion techniques [17]. The release media
were taken as simulated body fluid (SBF, pH 7.4)
was taken as release media under sink
conditions for ROC. ROC-loaded CANPs
suspension (20 mL) prepared under optimal
conditions and pure drug ROC at a concentration
equivalent to that in the nanoparticles will be
loaded into dialysis bags with a molecular weight
of 35 kDa (Fisher Scientific, USA) and
suspended in 150 mL of release medium at
37°C with a stirring rate of 150 rpm. At time
intervals, 0.2 mL of the release medium was
taken and immediately replaced by a fresh
release medium. The amount of drug in the
samples was determined using a UV-VIS
spectrophotometer. All experiments were
performed in triplicate.
3. RESULTS AND DISCUSSION
The main effort of this work was to develop
a nano delivery system for the drug ROC
using natural, biodegradable, biocompatible
polysaccharides especially chitosan and sodium
alginate. These nanoparticles are obtained
spontaneously under very mild conditions. The
preparation of CANPs systems, based on an
ionotropic gelation process, involves mixing the
two aqueous phases at room temperature.
The ionotropic gelation process did not include
the emulsification step and stage of organic
solvents, thereby minimizing the inactivation of
encapsulated drugs [7]. In aqueous solutions, at
pH around 4 to 5.5 the amine groups on chitosan
became easily positively charged. On the other
hand, alginate dissolved in a neutral pH solution
where the carboxylate groups were negatively
charged. In aqueous solutions with pH around
5.2 chitosan amino groups interacted with
alginate carboxylate groups to form the hydrogel.
The formulation, which gave an opalescent
suspension with a positive zeta potential, was
selected as the optimum ratio for the ROC
encapsulation.
The DLS analysis revealed that the blank CANPs
and ROC loaded CANPs have a mean
hydrodynamic diameter of 299.1 nm and 349.3
nm as shown in Fig. 1(A) and (B), respectively.
DLS measures the apparent size (hydrodynamic
radius) of a particle, including hydrodynamic
layers that are formed around hydrophilic
particles, leading to an overestimation of
nanoparticles size. Hence, the discrepancy in the
size of nanoparticles may be due to the fact that
the DLS method gives the hydrodynamic
diameter rather than the actual diameter of
hydrophilic nanoparticles. The PDI value of blank
CANPs was 0.493 while that of ROC loaded
CANPs was 0.446, thus indicating a narrow and
favorable particle size distribution [18]. Zeta
potential is quite important for colloids and
nanoparticles in suspension. Its value is closely
related to suspension stability and particle
surface morphology. Zeta potential of the
optimized ROC loaded CANPs was found to be
+29.1 mV. The zeta potentials of ROC loaded
CANPs show large positive values reflecting
the stability of the colloidal suspensions.
Having positively charged surfaces is an added
advantage when using nanoparticles in drug
delivery since they are able to transfer easily
through negative channels in the cell membrane
[19].
FT-IR was adopted to characterize the potential
interactions in the NPs and successful loading of
ROC into the NPs. FT-IR spectra of alginate,
chitosan, ROC and ROC loaded CANPs are
shown in Fig. 2. In the spectra of pure chitosan,
characteristic peaks at 3449 cm
−1
for –NH2 and –
OH stretching, 1659 cm−1
for –CO stretching, and
1596 cm
−1
for –NH2 bending vibrations were
observed [20]. The bands around 1079 cm−1
(C-
O-C stretching) presenting in the FT-IR spectrum
of sodium alginate are attributed to its saccharide
structure. In addition, the bands at 1662 and
1400 cm
−1
are assigned to asymmetric and
symmetric stretching peaks of carboxylate salt
groups [21]. The characteristic IR absorption
peaks of free ROC were present at 3341.5 cm
−1
,
2972.2 cm−1
and 1425.8 cm−1
correspondings to
cyclic amines, CH stretching, C=O stretching, O-
H bending. These were similar to what has been
previously reported in the monograph for
rosuvastatin [22]. The characteristic absorption
band of ROC appeared in the ROC loaded
CANPs, which probably indicate that the ROC
molecule was filled in the polymeric network.
These results indicate that the carboxylic groups
of alginate associated with ammonium groups of
chitosan through electrostatic interactions to form
the polyelectrolyte complex. In other words, the
characteristic peaks of ROC were not altered in
their position after successful encapsulation in
the CANPs. Thus, it can be concluded that there
is no chemical interaction between the ROC and
CANPs upon the encapsulation of ROC within
the NPs. The % EE of CANPs was 83.65% ±
1.53 (n = 3). The % EE acts as an important
factor influencing the drug release, as well as the

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54
overall efficacy of the production process. The
CANPs protect the encapsulant, have
biocompatible and biodegradable characteristics,
and limit the release of encapsulated materials
more effectively than either alginate or chitosan
alone [8].
Fig. 1. Particle size distribution of (A) blank chitosan-alginate nanoparticles (B) ROC loaded
chitosan-alginate nanoparticles
Fig. 2. FTIR spectra of chitosan, alginate, drug loaded chitosan-alginate nanoparticles and
rosuvastatin

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Fig. 3. Percentage of cumulative release of
free ROC and ROC loaded Chitosan-alginate
NPs
The in vitro release profiles of optimized ROC
loaded CANPs formulations in phosphate-
buffered saline (PBS) solution (pH 7.4) are
shown in Fig. 3. It shows the initial burst release
of ROC from NPs in PBS solution which can be
observed up to 2 h. This accounts for about 45%
of ROC from the total encapsulated amount.
Then, it followed a more gradual and sustained
release phase for the next 24 h. Drug release
from CANPs was significantly rapid as well as
complete than the pure ROC drug. The initial
fast release of ROC may be due to the rapid
hydration of nanoparticles because of the
hydrophilic nature of chitosan and alginate. The
release medium penetrates into the particles and
dissolves the entrapped drug; therefore, it can be
proposed that the major factor determining the
drug release from nanoparticle is its solubilization
or dissolution rate in the release medium.
4. CONCLUSION
Preparation of ROC loaded CANPs was
successfully carried out through the ionotropic
gelation and polyelectrolyte complexation
method to obtain particles with the highest
payload potential (83.65%) at the smallest size
(349.3 nm). The dispersion of ROC in aqueous
alginate with calcium chloride as a crosslinking
agent created hydrogel nanoparticles with
chitosan added for structural support. This
occurs due to electrostatic interactions among
carboxylate groups on alginate and positively
charged calcium ions and protonated amine
groups on chitosan. Finally, the in vitro release
profile observed for these nanoparticles was
characterized by an initial fast release followed
by a controlled release phase. In conclusion, this
new nanosystem also offers an interesting
potential for the delivery of hydrophobic
compounds.
CONSENT
It is not applicable.
ETHICAL APPROVAL
It is not applicable.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the approval
and the support of this research study by the
grant no. 7681-PHM-2018-3-9-F from the
Deanship of Scientific Research at Northern
Border University, Arar, K.S.A.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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