coden: ijptfi available online through research article ...chandigarh college of pharmacy landran...

PreetiKush* et al. International Journal Of Pharmacy & Technology

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



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



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



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



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.


IJPT| March-2014 | Vol. 5 | Issue No.4 | 6131-6150 136

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



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



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



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



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.



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



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.



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.



(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.



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



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.



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



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Corresponding Author:

PreetiKusha*,

Email: [email protected]

coden: ijptfi available online through research article ...chandigarh college of pharmacy landran...

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