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IJPT| March-2014 | Vol. 5 | Issue No.4 | 6131-6150 Page 6131
ISSN: 0975-766X
CODEN: IJPTFI
Available Online through Research Article
www.ijptonline.com DESIGN OF DOCETAXEL- LOADED CHITOSAN NANOPARTICLES: COMPARISON OF
TWO PREPARATION METHODS
Preeti Kusha*, KiranjeetKaur
b, AbhiniThakur
b
Chandigarh College of Pharmacy Landran Mohali(Punjab) India 140307.
Email: [email protected] Received on 18-01-2014 Accepted on 20-02-2014
Abstract
The aim of the present work is to investigate the best method for thepreparation of nanoparticles (NPs) as a potential
drug carrier and system for the treatment of cancer disease. Docetaxel (Dtx) was chosen as the model drug to be
incorporated within nanoparticles. Two different preparation method is use to design the nanoparticles- ionotropic
gelation method and emulsion crosslinking method. Nanoparticles prepared by ionotropic gelation method have a
smaller particle size of 171.4 nm, better entrapment efficiency (78%) and loading capacity as compared to nanoparticles
prepared by emulsion cross linking method, resulted in larger particle size of 330 nm with less entrapment efficiency of
64.38%.
Keywords:Chitosan, Docetaxel, Emulsion crosslinking method,Ionotropic gelation method, Nanoparticles.
Introduction
Chitosan, derived from chitin by deacetylation, is the second most abundant naturally occurring biopolymer (after
cellulose) and a major structural polysaccharide found in the exoskeleton of crustaceans such as crab and shrimp (Juan
Xu et al. 2012).It is consisting of β-(1,4)-2-acetamido-2-deoxy-d-glucose and β-(1,4)-2-anaino-2-deoxy-d-glucose units.
Thus, it comprises of copolymers of glucosamine and N-acetyl glucosamine. The molecular formula is C6H11O4N
(Kaloti and Bohidar 2010). It is considered to be the most widespread polycationic biopolymer, as well as having non-
toxic, biocompatible, biodegradable characteristics. CS can be applied in food processing, agriculture, biomedicine,
biochemistry, wastewater treatment, membranes, microcapsules, nanoparticles, liquid crystalline material, etc. (Min-
Lang Tsai et al. 2011, Chang, Chang and Tsai 2007, Rinaudo 2006, Tsai, Bai and Chen 2008). The CS nanoparticle has
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attracted great attention in pharmaceutical applications including being targeted for colon or mucosal delivery, cancer
therapy, or delivery of vaccines, genes, antioxidants, etc. because the primary amine groups render a positive charge and
mucoadhesive properties that make CS very useful in drug delivery applications (Hu et al. 2008, Jang and Lee 2008,
Sarmento, Ferreira, Veiga and Ribeiro 2006, Vila et al. 2004, Yuan, Li and Yuan, 2006).
Docetaxel is a semisynthetic derivative of taxoid family of antineoplastic agents. It is an analog of paclitaxel which is
extracted from the needles of the European yew tree (Taxusbaccata L.) in 1986 (AfrouzYousefi et al. 2009). Docetaxel
has been effective against breast, ovarian, lung and head and neck cancers. Being a microtubule stabilizing agent, it
inhibits microtubule disassembly and consequently inhibits cell proliferation (Horwitz SB 1992). Due to the poor
solubility of docetaxel in water, tween80 (polysorbate 80) and ethanol (50:50, v/v) are used for the formulations
currently available in the market. Both tween80 and ethanol are responsible for hypersensitivities that occur after
docetaxel administration and make premedication of the patients with corticosteroids and antihistamines a necessity. To
overcome this problem and to improve efficacy, novel formulations of docetaxel have been attempted, such as
liposomes, cyclodextrins, mixed micelles, submicron emulsion and nanoparticles. Among them, the nanoparticle
formulation holds greatest promise for this purpose. The nanoparticles showed advantages such as more stable during
storage over others (8).
In this study, different parameters were investigated for formulation of docetaxel nanoparticles by two different methods
in order to reach to the best nanoparticle size, encapsulation efficiency, drug loading, in-vitro release, and stability.
Materials and methods
Materials
Docetaxel was obtained from Sigma-Aldrich (USA). Chitosan was obtained from Himedia Pvt. Ltd. Sodium
tripolyphosphate (TPP), glutaraldehyde (GLU), cyclohexane, n-hexanol, acetonitrile and acetic acid glacial was obtained
from Lobachemie.
Methods
Ionotropic gelation method
Docetaxel chitosan nanoparticles were obtained based on ionic gelation of TPP with chitosan.Based on an optimization
procedure design by us, a number of parameters were investigated by changing one parameter while keeping the others
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constants. These varying parameters including concentration of chitosan solution (0.2 to 1.0% w/v), concentration of
TPP solutions (0.25 to 1.25% w/v) and concentration of drug (0 to 0.8 mg/ml).
The preparation of chitosan NPs involves the mixture of two aqueous phases at room temperature. One phase contains a
solution of polycation chitosan and the other contains a solution of polyanion TPP.For this purpose chitosan solution
(0.2% w/v) was obtained by dissolving chitosan in 1% v/v acetic acid. The chitosan solution was stirred overnight at
room temperature using a magnet stirrer. The pH of the resulting solution was around 3.6 and this was adjusted to 5.5
using 20 wt% aqueous sodium hydroxide solution. The chitosan solution was then passed through a syringe filter (pore
size 0.45 um) to remove residues of insoluble particles. TPP was dissolved in distilled water and also passed through a
syringe filter (pore size 0.22 um). The chitosan nanoparticles formed spontaneously upon addition of various
concentration of TPP (0.25 to 1.25% w/v) to chitosan solution by dropwise. The selected volume ratio of CS to TPP was
5:1.For preparation of Dtx nanoparticles, various concentrations of Dtx (0 to 0.8 mg/ml) in TPP solution (firstly drug
was dissolved in 1-2 ml of acetonitrile) were prepared. Nanoparticles were formed by adding this solution into chitosan
solution. The nanoparticle suspensions were continuously stirred for 1 h and centrifuged at 16,000rpm for 30 min. The
resulting nanoparticle products were lyophilized and stored.
Emulsion crosslinking method
Docetaxel chitosan nanoparticles were obtained based on water in oil microemulsion system.Based on an optimisation
procedure design by us, a number of parameters were investigated by changing one parameter while keeping the others
constants. These varying parameters including concentration of chitosan solution (0.2 to 1.0% w/v), concentration of
glutaraldehyde solutions (10 to 50% v/v) and concentration of drug (0 to 0.8 mg/ml).The preparation of chitosan NPs
involves the mixture of two phases i.e. oil phase and aqueous phase.Chitosan solution was prepared by dissolving
calculated amount of chitosan powder in 1% (w/w) acetic acid solution. The cyclohexane, hexanol and chitosan solution
were mixed in a flask at the volume ratio of 11:6:6. Triton X-100 was dropped into the mixture while stirring until the
mixed emulsion became transparent or semitransparent, indicating that the nanoemulsion was formed. The w/o
nanoemulsion containing glutaraldehyde but without chitosan was prepared with the same procedure. The w/o
nanoemulsion containing glutaraldehyde was added drop wise into the w/o nanoemulsion with chitosan under stirring,
and kept the mixed nanoemulsion at 40 0C for 4 h to allow the water pools containing glutaraldehyde to collide with the
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water pool containing chitosan. The chitosan was cross linked and the chitosan nanoparticles (CS-GLU) were formed.
For preparation of Dtx nanoparticles, various concentrations of Dtx (0 to 0.8 mg/ml) in cyclohexane (firstly drug was
dissolved in 1-2 ml of acetonitrile) were prepared. After the reaction, acetone was added into the system to break the
nanoemulsion, the nanoparticles were then precipitated with centrifugation (4000 rpm for 20 min) at room temperature,
and rinsed with acetone. Finally, the nanoparticles were dried in air at room temperature for 48 h.
Characterization of Docetaxel-loaded Chitosan nanoparticles
Particle size, size distribution and zeta potential of nanoparticles
The mean diameter and size distribution of the nanoparticles were measured by dynamic light scattering using Zetasizer
(Malvern Instruments, Malvern, UK). The analysis was performed at a scattering angle of 90º and a temperature of
25ºC. The mean particle size and polydispersity index and zeta potential of each sample was determined three times and
the average values are calculated.
Encapsulation efficiency and loading capacity
Lyophilized Dtx loaded nanoparticles (equivalent to 10 mg Dtx) was dissolved in 2 ml acetonitrile to extract docetaxel
into acetonitrile for determining the encapsulation efficiency. The samples in acetonitrile were gently shaken on a shaker
for 4 h at room temperature to completely extract out docetaxel from the nanoparticles into acetonitrile. These solutions
were centrifuged at 14,000 rpm (Centrifuge Remi equipment, Mumbai) and supernatant was collected. The docetaxel
concentrations were measured spectrophotometrically(Shimadzu UV spectrophotometer, Japan) at 230 nm and each
sample was determined three times. And the percentage encapsulation efficiency and loading capacity was calculated by
using the equations.
�� �%� =������� ����� �� ���������
������� ��× 100 (1)
���%� =���������
������������������� × 100 (2)
Morphological characterization of nanoparticles
The surface morphology of the optimized nanoparticles was measured by field emission scanning electron microscopy
(Hitachi, Japan MSW-301). The lyophilized samples were carefully mounted on an aluminium stub using a double stick
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carbon tape. Samples were then introduced into an automated sputter coater and coated with a very thin film of gold
before scanning the samples under FESEM.
Percentage yield: The lyophilized nanoparticles from each formulation were weighed and the respective percentage
yield of production was calculated as the ratio between the amount of NPs weight obtained and the total weight of solid
materials used for the preparation multiplied by 100.
%��� =������������������� ��������(�)
������������ ������������ (�)× 100 (3)
In-vitro drug release studies
The in-vitro release profile of docetaxel from polymeric nanoparticles was evaluated according to the protocol. For the
in-vitro release studies, about 20 mg of DTX-NPs were suspended in 3.0 ml of release medium (phosphate buffer saline
solution of pH 7.4).The mixture was introduced into a cellophane membrane dialysis bag. The bag was closed and
transferred to dissolution rate test apparatus containing200 ml of the same solution maintained with rotating speed 50
rpm at 37 0C. Theexternal solution was continuously stirred, and 5 ml sampleswere removed at selected intervals and
equal volume of fresh medium was replaced immediately. Triplicate samples were used. After a suitable dilution,
sample was analyzed by UV spectrophotometer. Results are expressed as the cumulative percent released drug as a
function of time.
Table 1: Equations for different model*
Release models Equations
Zero order (�� = �� + �� )
First order ���� = ��� + �� �
Higuchi’s square root (����� + �� �/�)
Korsmeyer-Peppas �� �∞
= � �⁄
*Qt is the initial drug amount (100% when represented as percentage); �� the amount of drug remaining at a specific
time (calculated as % of��); k is the rate constant; t is the time.
Stability studies
Docetaxel loaded chitosan NPs (20 mg) was kept in sealed glass vials and maintained at 4°C for a period of 6 months.
Nanoparticles were characterized for change in particle size, encapsulation efficiency and percent drug loading
according to the above mentioned protocols.
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Release models and kinetics
To determine the drug release mechanism and to compare the release profile amongst various nanoparticles
formulations, the in-vitro release data was fitted to various kinetic equations.The plots were drawn as per the following
details:Cumulative percent drug released as a function of time (zero order kinetic plots), Log cumulative percent drug
retained as a function of time (first order kinetics plots),Log cumulative percent drug released as a function of log time
(Korsmeyer-Peppas plots), Cumulative percent drug released versus square root of time (Higuchi’s square root plots).
Results and discussions
Docetaxel loaded chitosan nanoparticles were prepared by ionotropic gelation and emulsion crosslinking method using
TPP and glutaraldehyde as the cross linking agents respectively. The effect of various processing variables like polymer
concentration, cross linker concentration and drug concentration on particle size, PDI, zeta potential, %EE and %LC
were studied. Further optimized batches of nanoparticles prepared by both the methods were characterized by surface
morphology, in-vitro drug release, and modeling of drug release.
Effect of processing variables on particle size, PDI, zeta potential.
Ionotropic gelation method
The nanoparticles were prepared by ionic gelation upon addition of TPP to Chitosan solution under mechanical stirring
at room temperature. TPP has five negative ionic charge points that interact with the positive amino groups of Chitosan
in acetic acid solution (Hosniyeh H et al. 2012). Different parameters influence the characters of the nanoparticles.
These include pH (Ajun W et al. 2009), molecular weight of Chitosan (Zheng Y et al. 2006), chitosan and TPP
concentration and addition of an active compound (Calvo P et al. 1997, Papadimitriou S et al. 2008).Chitosan and TPP
can form nanoparticles in specific moderate concentrations.Nanoparticles with smaller size have valuable characteristics
such as improved drug delivery, longer circulation in blood, and lower toxicity (Papadimitriou S et al. 2008, Ferrari M
2005). The effect of each of these variables is expressed as follows
Effect of TPP concentration
It was observed that application of TPP with higher concentration can significantly increase the size of the particles
(Wen Fana et al. 2012). This could be due to the increase in the amount of anionic groups in the preparation medium,
which causes more electrostatic interaction with positive amino sites on Chitosan, reduction of the positive surface
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charge, and increments in nanoparticles size. Zeta potential influences the stability of the nanoparticles through
electrostatic repulsion (YangchaoLuo et al. 2010, Ajun W et al. 2009, Gan Q and Wang T 2007). The positive zeta
potential was due to the residual amine groups (JingouJi et al. 2011). TPP concentrations higher than 1.25% w/v form
aggregated solutions. The prepared nanoparticles by ionotropic gelation method have a polydispersity index below 0.5
indicating the uniformity of particle size. Fig.1 represents bar graphs showing a comparative effect of TPP concentration
on particle size, polydispersity index and zeta potential.
Fig.1: Bar graphs showing a comparative effect of TPP concentration on particle size, polydispersity index and
zeta potential of nanoparticles prepared by ionotropic gelation method.
Effect of chitosan concentration
Fig.2 represents bar graphs showing a comparative effect of chitosan concentration on particle size, polydispersity index
and zeta potential. In all cases, TPP concentration was kept constant (0.75% w/v). The increased viscosity of higher
chitosan concentrations prevents effective ionic interaction between TPP and Chitosan solution, which increases
nanoparticle size with increase in zeta potential (Ajun W et al. 2009, Hosniyeh H et al. 2012). It is known that under
acidic conditions, there is electrostatic repulsion between chitosan molecules due to the protonated amino groups of
chitosan meanwhile, there also exist interchain hydrogen bonding interactions between chitosan molecules. Below a
certain concentration chitosan (2.0 mg/mL as reported), the intermolecular hydrogen bonding attraction and the
intermolecular electrostatic repulsion are in equilibrium (Qun G and Ajun W 2006). Therefore, in this concentration
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range, as chitosan concentration increases, chitosan molecules approach each other with a limit, leading to a limited
increase in intermolecular cross-linking, thus larger but still nanoscale particles are formed (Wen Fana et al. 2012). PDI
of nanoparticles were favourable i.e. below 0.5.
The higher positive surface charge of CS/TPP nanoparticles were formed by the interaction between protonized -NH3+
in CS and the polyanionic phosphate groups in TPP, the zeta potential of nanoparticles increased linearly due to a more
available protonized –NH3+ on the surface of nanoparticles formed with higher CS concentration (YangchaoLuo et al.
2010).
Fig.2: Bar graphs showing a comparative effect of Chitosan concentration on particle size, polydispersity index
and zeta potential of nanoparticles prepared by ionotropic gelation method.
Effect of drug concentration
Dtxloaded nanoparticles were prepared upon addition ofDtx in 0.75% w/v TPP into 0.2% w/v Chitosan solution. To
determine the effect of Dtxconcentrationon particle size, various concentrations of Dtx in 0.75%w/v TPP solution were
applied. Table 2 shows that generally addition of Dtx increases the size of Chitosan nanoparticles.
In general, Dtx loaded Chitosan nanoparticles size didnot grow significantly at concentrations up to 0.4 mg/mL, but there
was a change in size when the concentration of Dtx was increased from 0.4 to 0.6 mg/mLand the size remains constant at
concentrationsabove 0.6 mg/mL of Dtx in TPP solution. Therefore it may notbe possible to severely increase the Dtx
particle diameter until it reaches its maximum capacity inside the nanoparticles. Dtx concentration did not influence the
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zeta potential of the prepared nanoparticles.Fig.3 represents bar graphs showing a comparative effect of drug
concentration on particle size, polydispersity index and zeta potential.
Table 2: Results of Mean Particle size and Poly dispersity index,Zeta Potential, Encapsulation Efficiency and
Loading Capacity of Various Formulations prepared by Ionotropic Gelation method
Formulation
Code
Dtxconcn
(mg/ml)
Particle size
(nm)
PDI Zetapotential
(mV)
EE (%) LC (%)
F1 - 159.2 ± 3.31 0.217 ±0.029 31.2 ± 1.66 - -
F2 0.2 167.2 ± 3.43 0.205 ± 0.034 31.7 ± 1.12 69.37 ± 0.74 9.94 ± 0.81
F3 0.4 171.4 ± 3.22 0.211 ±0.027 30.3 ± 1.67 78.28 ± 0.91 11.26 ± 0.79
F4 0.6 198.9 ± 4.58 0.298 ± 0.041 29.1 ± 2.11 75.11 ± 1.02 12.01 ± 0.92
F5 0.8 204.1 ± 6.09 0.332 ± 0.043 29.8 ± 1.96 70.41 ± 1.35 10.56 ± 0.99
Note: Chitosan = 0.2% w/v, TPP = 0.75% w/v
Fig.3: Bar graphs showing a comparative effect of Drug concentration on particle size, polydispersity index and
zeta potential of nanoparticles prepared by ionotropic gelation method.
Emulsion cross linking method
Docetaxel loaded Chitosan nanoparticles were prepared by water-in-oil nanoemulsion system or prepared by emulsion
crosslinking method (ZhiJia et al. 2005). To achieve this, emulsification of aqueous chitosan solution in the oil phase
was carried out. The formed micro-droplets were crosslinked with glutaraldehyde to obtain more or less solid spherical
particles. A new w/o nanoemulsion system with glutaraldehyde as a cross linking agent, cyclohexane as the oil phase, n-
hexanol as a cosurfactant and dilute acetic acid solution containing chitosan as the aqueous phase.By adding
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glutaraldehyde into the nanoemulsion system, the chitosan polymer was solidified and the nanoparticles could form
from the nanoemulsion system. The effects of different processing variables were studied.
Effect of Glutaraldehyde (cross linking) concentration
Glutaraldehyde is mainly used as a cross linking in emulsion cross linking method. The chitosan polymer is solidified on
addition of glutaraldehyde and the nanoparticles could form from the nanoemulsion. Glutaraldehyde is a dipolar anionic
linear molecule and binds to 2 amino groups. Chitosan cross links by nucleophilic interaction between amino group of
chitosan and aldehyde group of glutaraldehyde (Xiao ying et al. 2011). It is observed that an initial increase in
concentration of the glutaraldehyde decreases the particle size upto a concentration of 40%. The higher concentration of
glutaraldehyde (>40%) has no significant influence on particle size. This may be attributed to the fact that at higher
(upto 40%) concentration of cross linking agent, pore networks get partially filled with excess of glutaraldehyde and size
of particles appears to be smaller (Moralesa MA et al. 2013). Zeta potential influences the stability of the nanoparticles
through electrostatic repulsion. The results showed to be no significant effect of cross linking agent on zeta potential, a
small decrease of zeta potential due to decrease of residual amine group into the solution. The prepared nanoparticles
have a polydispersity index below 0.5 indicating that nanoparticles by this method have a favourable PDI.
Fig.4represents bar graphs showing a comparative effect of glutaraldehyde concentration on particle size, polydispersity
index and zeta potential.
Fig.4: Bar graphs showing a comparative effect ofGlutaraldehyde concentration on particle size, polydispersity
index and zeta potential of nanoparticles prepared by emulsion cross linking method.
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Fig.5:Bar graphs showing a comparative effect of Chitosan concentration on particle size, polydispersity index
and zeta potential of nanoparticles prepared by emulsion cross linking method.
Effect of chitosan concentration
Chitosan undergoes nucleophilic interaction with the cross linking agent leading to the formation of nanoparticles.
Glutaraldehyde interacts with 2 amino groups of chitosan (Moralesa MA et al. 2013). Further values of PDI are less than
0.5 indicating uniformity of particle size. Also zeta potential increases with an increase in chitosan concentration due to
increase of amine group.
Effect of drug concentration
Table 3 shows that generally addition of Dtx increases the size of Chitosan nanoparticles. The size of nanoparticles
didnot grow significantly at concentrations up to 0.4 mg/ml. A significant increase in size was observed at concentration
0.6 mg/ml andthe size remains constant at concentrationsabove 0.6 mg/mL of Dtx.Fig.6 represents bar graphs showing a
comparative effect of drug concentration on particle size, polydispersity index and zeta potential.
Table 3: Results of Mean Particle size and Poly dispersityindex,Zeta Potential, Encapsulation Efficiency and
Loading Capacity of Various Formulations prepared by emulsion cross linking method
Formulation
code
Dtxconcn
(mg/mL)
Particle size
(nm)
PDI Zetapotential
(mV)
EE (%) LC (%)
S1 - 306.4 ± 3.21 0.220 ± 0.023 19.9 ± 1.23 - -
S2 0.2 318.1 ± 5.42 0.215 ± 0.058 21.2 ± 1.02 56.47 ± 0.94 7.94 ± 0.71
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S3 0.4 329.9 ± 4.23 0.227 ±0.027 22.4 ± 1.47 64.38 ± 1.12 9.23 ± 0.89
S4 0.6 367.3 ± 7.59 0.297 ± 0.051 21.6 ± 2.01 60.41 ± 1.52 10.01 ± 0.95
S5 0.8 376.1 ± 7.02 0.321 ± 0.046 20.3 ± 1.94 58.44 ± 1.75 8.56 ± 0.97
Note: Chitosan = 1% w/v, GLU = 40%v/v.
Fig.6:Bar graphs showing a comparative effect of Drug concentration on particle size, polydispersity index and
zeta potential of nanoparticles prepared by emulsion cross linking method
Encapsulation efficiency and loading capacity of nanoparticles
Docetaxel loaded chitosan nanoparticles were prepared by two methods namely ionotropic gelation method and
emulsion cross linking method in order to make a comparative evaluation. Encapsulation efficiency and drug loading
capacity is an important parameter to be considered while choosing the appropriate method. Fig.7represents the effect of
different concentrations of Docetaxel on encapsulation efficiency and loading capacity of nanoparticles prepared by
ionotropic gelation and emulsion cross linking method.
Ionotropic Gelation method
A maximum EE (78%) was achieved at 0.4 mg/ml of Dtx concentration (Batch F3). Initially, %EE showed an increase
with increase in Docetaxel concentration up to 0.4 mg/ml. From 0.4 - 0.8 mg/ml, the %EE decreased from 78% to 70%.
While maximum %LC was observed at 0.6 mg/ml of Dtx in TPP solution, the increase in drug concentration from 0.4
mg/ml to 0.8 mg/ml did not significantly effect on the %LC.
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Emulsion cross linking method
Fig.7 represents the effect of varying Docetaxel concentration on % EE and %LC. A maximum EE (64.38%) was
achieved at 0.4 mg/ml of Dtx concentration (Batch S3). The encapsulation efficiencies were obtained in the range of 56-
64%. While maximum %LC was observed at 0.6 mg/ml of Dtx.
Fig.7: Comparative Evaluation of Encapsulation efficiency and Loading capacity of nanoparticles by Ionotropic
gelation method and emulsion cross linking method.
Characterization of Optimized batches of nanoparticles
The effect of different variables and their concentration was studied in both the methods. The main parameters studied
are concentration of chitosan, TPP and drug in ionotropic gelation method while in emulsion cross linking method these
were concentration of glutaraldehyde, chitosan and drug. On the basis of the above studies, an optimized batch was
selected having suitable particle size, encapsulation efficiency, zeta potential, loading capacity and polydispersity index.
Batch F3 was the optimized formulation of ionotropic method and S3 of emulsion cross linking method. The optimized
batch of nanoparticles was further characterized.
Morphological characterization of nanoparticles
Docetaxel and chitosan nanoparticles prepared by both the methods were subjected to field emission scanning electron
microscopy to determine their surface morphology. The images indicate that nanoparticles were found to be spherical
present with smoother surfaces. Fig.8 (a-b) represents the FESEM images of F3 and S3 prepared by ionotropic gelation
method and emulsion cross linking method respectively.
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(a) (b)
Fig.8: Field emission scanning electron microscope images of nanoparticles (a) Batch F3 (b) Batch S3
The yield of optimized batch of nanoparticles was determined by weighing the batch of nanoparticles after
lyophilisation. The yield of nanoparticles was found to be 86% (F3) in ionotropic gelation method and 72% (S3) in
microemulsion method.
In-vitro drug release studies
The in vitro drug release data of docetaxel loaded chitosan nanoparticles in PBS 7.4 as a release medium after 24 hours
presented in Fig. 9.
The following figures display the in vitro release profile of docetaxel from both ionotropic gelation and emulsion cross
linking method. F3 represents the batch prepared by ionotropic gelation and S3 by emulsion cross linking method. F3
showed a greater release of drug up to 80% in 24 h whereas that of S3 was 58% during 24 h time duration. The release
of drug from nanoparticles showed a sustained drug release. This can be attributed to the drug release occurring in three
phases. Firstly, F3 showed an initial burst release due to the release of drug from surface of nanoparticles. The second
phase includes a sustained release of drug due to release of drug from matrix. Lastly, slow release of drug is obtained
due to polymer degradation (Das S et al. 2012, JingouJi et al. 2011). Fig.9 shows that F3 shows a sustained release of
the drug over 24 h. The release of the drug from S3 is less as compared to F3 releasing only about 58% of drug in 24 h.
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Fig 9: In vitro release rate profiles of docetaxel chitosan nanoparticles F3(Ionotropic gelation method), S3
(emulsion cross linking) in phosphate buffer saline (pH 7.4) over24 hours of the study
(a) (b)
Fig. 10: The particle size, encapsulation efficiency and drug loading of NPs (a) F3 (b) S3 against storage time at 4ºC
Storage stability of Dtx loaded chitosan nanoparticles
The long term storage stability of the Dtx chitosan NPs is an important parameter. Nanoparticles formulations increase
the surface area by many folds and this may lead to very high aggregation after long periods of storage. This poor long
term stability may be due to different physical and chemical factors that may destabilize the system. Lyophilization is a
promising approach for the stabilization of Dtx chitosan nanoparticles. For lyophilized nanoparticles, cryoprotectant
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serves as stabilizers during the freeze drying process. For our study, mannitol (2% w/v) was chosen as the
cryoprotectants to prevent the hydrolytic instability, aggregation between nanoparticles, protection during processing
and storage. Fig. 10 represents the effect of storage time on particle size, % encapsulation efficiency and % drug loading
of nanoparticles prepared by ionotropic (F3) and emulsion cross linking method (S3) respectively.
After 6 months of storage with cryoprotectant at 4°C, the nanoparticles appear to be stable without any collapse or
aggregation. We saw no major changes in both batches (F3 and S3) besides a slight increase in particle size and a slight
decrease in encapsulation efficiency and drug loading. Therefore, Dtx chitosan NPs formulated by both ionotropic
gelation methodand emulsion crosslinking method were found to be stable for a long period of time.
Modeling and Release kinetics of nanoparticles
To determine the release kinetics, the release data was fitted into various kinetic models: zero order, first order,
Higuchi’s square root and Korsmeyer-Peppasmodels. Table 4 shows the correlation coefficient (r2) used as an indicator
of the best fitting models consider for optimized NPs (batch F3 and S3). The r2
values for Higuchi kinetics of optimized
NPs were greater than that of zero order and first order (Sanna V et al. 2011, Costa P and Sousa LJM 2001). Higuchi
kinetic model states that diffusion is the one of major method drug release best described the controlled release phase (r2
of F3= 0.851 and r2 of S3= 0.848). During later part of release which may be controlled by a combination of slow and
gradual erosion and diffusion. Beside to understand the drug release mechanism, the drug release 60% was fitted to
korsmeyerpeppas exponential model Mt/M∞= Ktn , where Mt/M∞ is fraction of drug released after time ‘t’ and ‘K’ is
kinetic constant and ‘n’ is release exponent, which characterizes the different drug release mechanism.Based on various
mathematical models, the magnitude of the release exponent ‘n’ indicates the release mechanism.The limits considered
were n ≤ 0.43 (for a classical Fickian diffusion-controlled drug release) and n = 0.85 (indicates a case II relaxational
release transport; non-Fickian, zero order release). Values of n between 0.43 and 0.85 can be regarded as an indicator of
both phenomena (drug diffusion in the hydrated matrix and the polymer relaxation) usually called anomalous transport
(Siepmann J and Peppas NA 2001, Higuchi T 1963). In optimized F3 and S3 formulations, n values is 0.50 and 0.51
respectively, suggesting an anomalous or non-Fickian diffusion, which is related to combination of both diffusion of the
drug and dissolution of the polymer.
PreetiKush* et al. International Journal Of Pharmacy & Technology
IJPT| March-2014 | Vol. 5 | Issue No.4 | 6131-6150 Page 6147
Table 4: In Vitro Release Kinetics of DocetaxelLoaded Chitosan nanoparticles.
(Correlation Coefficient), K0 (Zero Order Rate Constant), K1 (First Order Rate Constant), KH (Higuchi
Dissolution Rate Constant), h (Hour Unit of Time).
Conclusion
In this study, Chitosan nanoparticles containing anticancer agent Docetaxel were successfully prepared by using two
preparation methods (i) Ionotropic Gelation method (ii) Emulsion cross linking method. The study suggests the
importance of controlling the process parameters during formulation as they greatly influenced the final product, such as
particle size, polydispersity index, zeta potential, drug encapsulation efficiency. FESEM images indicate nano-sized
spherical particles with smooth surface.
Nanoparticles prepared by ionotropic gelation method (F3) gave a smaller particle size of 171.4 nm, better entrapment
efficiency (78%) and Loading capacity as compared to batch S3 prepared by emulsion cross linking method, resulted in
larger particle size of 330 nm with less Entrapment efficiency of 64.38%. Moreover, in vitro release studies showed
almost complete release of drug from F3 releasing about 80% (In 24 hr) of drug in a sustained manner following
Higuchi’s square root kinetics and as compare to Batch S3 showed incomplete release of drug releasing 54% drug in 24
h. The above results clearly confirm the superiority of Ionotropic gelation method over emulsion cross slinking method.
Thus, Preparation method by ionically cross-linking cationic chitosan with TPP was particularly successful as, aside
from its complexation with negatively charged polymers, chitosan formed gel spontaneously on contact with TPP due to
the formation of inter and intramolecular cross-linkage. Chitosan nanoparticles produced by ionic crosslinking with TPP
increased the drug loading efficiency in the chitosan nanoparticles and also prolonged the drug release period.
Release kinetics
Zero order First order Higuchi’s
squareroot
KorsemeyerPeppas
r2 K0
( h-1)
r2 K1
(h-1)
r2 KH
(h-0.5)
r2 K
(h-1)
N
F3 0.5834 0.043
5
0.7643 0.00069
1
0.851
4
1.998 0.791
8
0.3702 0.5010
S3 0.5949 0.030
2
0.7068 0.00046
1
0.848
1
1.369 0.804
7
0.3036 0.5124
PreetiKush* et al. International Journal Of Pharmacy & Technology
IJPT| March-2014 | Vol. 5 | Issue No.4 | 6131-6150 Page 6148
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Corresponding Author:
PreetiKusha*,
Email: [email protected]