chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

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Chemical Engineering Research and Design

journal homepage: www.elsevier.com/locate/cherd

Preparation and statistical optimization of Losartan

Potassium loaded nanoparticles using Box
Behnken factorial design: Microreactor
precipitation
Pritam Patil a , Gokul Khairnar b , Jitendra Naik a,
a

Department of Chemical Engineering, University Institute of Chemical Technology, North Maharashtra University,
Jalgaon, Maharashtra, India
b Department of Pharmaceutical Technology, University Institute of Chemical Technology, North Maharashtra
University, Jalgaon, Maharashtra, India

a r t i c l e

i n f o

a b s t r a c t

Article history:

The current study deals with the development and optimization of Losartan Potassium (LP)

Received 27 January 2015

loaded nanoparticles by continuous ow microreactor precipitation methodology for sus-

Received in revised form 4 June 2015

tained release. Formulation of LP loaded nanoparticles was conducted by using box Behnken

Accepted 21 July 2015

(33 ) experimental design to evaluate the effect of three independent process variables viz.,

Available online 29 July 2015

polymer concentration (Ethyl cellulose, X1 ), surfactant concentration (Tween 80, X2 ) and the

Keywords:

(Y1 ) and drug release (Y2 ) over 12 h. The average particle size and polydispersity index of LP

Losartan Potassium

loaded nanoparticles were characterized by particle size analyzer. The interaction between

inner diameter (X3 ) of the microreactor on the preferred responses: encapsulation efciency

Microreactor

ethyl cellulose and the LP was investigated by FTIR spectroscopy. The surface morphology

BoxBehnken design

and physical nature of LP loaded nanoparticle were characterized by FESEM and XRD anal-

Encapsulation efciency

ysis, respectively. The design formulations showed the encapsulation efciency within the

Drug release

range of 57.5 to 82.8% with sustained release prole of 77.2 to 100% over 12 h. The actual
and predicted values of both the responses were close to each other. It is veried that the
Box Behnken factorial design provides a useful platform for the optimization of LP loaded
nanoparticle by microreactor precipitation methodology.
2015 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1.

Introduction

Over the past two decades, development of nanoparticles has

been gaining more prominence from industries in the eld
of chemistry, microelectronics, sensors, energy, optic, life sciences and pharmaceutics (Capretto et al., 2012; Cheng et al.,
2012; Horn and Rieger, 2001; Zhao et al., 2010; Patil and Naik,
2014). In pharmaceutical industries, the size and size distribution of nanoparticles plays an important role in drug
delivery processes such as sustained release, targeted release
and the kinetics of taking away from the body (Rapoport, 2007).

Therefore, nanoparticles have prominent attribute because of

their physicochemical properties and better control of process
parameters, is vital in order to produce such nanomaterials with desired features (Carugo et al., 2011). In addition,
nanoparticles have a series of advantages, including control
drug delivery, a large amount of drug availability and increase
in the amount of drug uptake by the target cells (Capretto et al.,
2013).
Usually, nanoparticles or micro particles are prepared
by conventional methods such as solvent evaporation,
spray drying, ultrane mechanical milling and high speed

Corresponding author. Tel.: +91 257 2258441; fax: +91 257 2258403.
E-mail address: jbnaik@nmu.ac.in (J. Naik).
http://dx.doi.org/10.1016/j.cherd.2015.07.021
0263-8762/ 2015 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

homogenization, etc. However, this bottom up and top down

methods has some limitations, such as low yield, low thermal efciency and broad particle size distribution (Zhao et al.,
2007; Appalakutti et al., 2015). Owing to these disadvantages,
the progress in the eld of microreactor technology offer a
series of advantages over conventional methods in terms of
smaller particle size, shorter residence time and controllability of the process (Song et al., 2008; Dreher et al., 2008; Patil
et al., 2012). Result reported by Zhang et al. and Chen et al.
showed that the micro channel reactor having an excellent
micromixing property with narrow size distribution and large
surface area throughout the reactor (Carugo et al., 2011; Zhao
et al., 2007). In addition, microreactor are characterized by
their high surface area to volume ratio, it enables to reduce
heat and mass transfer resistance for the development of uniform nanoparticle in their smallest dimension (Dreher et al.,
2008; Wang et al., 2014; Horikoshi et al., 2013; Fletcher et al.,
2002). However, the small dimension of microreactor restricts
uids to low Reynolds number (<2000). This implies the ow
of miscible uid moving through a micro- channel are linear
and initiate the diffusion of drug molecule to the non solvent
across the interface, followed by the nucleation and growth of
the nanoparticles (Liu et al., 2014; Beebe et al., 2002; Weibel
and Whitesides, 2006).
Losartan Potassium (LP) is a potent and very much precise
antihypertensive drug. The use of the LP is to reduce the risk of
stroke in people who have high blood pressure and left ventricular hypertrophy condition (McMahon et al., 1990; Chobanian
et al., 2003). It belongs to group of angiotensin II receptor
antagonist (Singh and Deep, 2011). LP has an oral bioavailability of 33% and has a half life of 1.5 to 2.5 h with the terminal half
life of the metabolites being longer at 6 to 9 h (Chopra et al.,
2007; Ripley and Hirsch, 2010). Notably, LP drug is widely bound
to plasma proteins and also causes neutropenia, gastrointestinal disorders, acute pancreatitis and headache (Vidyadhara
et al., 2013). Ethyl cellulose (EC) is very popular biocompatible
polymeric material used for the development of drug delivery
system (Herrmann and Bodmeier, 1995; Polard et al., 1996). EC
is an inert to the core, brittleness, stable and hydrophobic polymer that have been widely used in developed release dosage
forms for targeted or sustained release purpose (Michael et al.,
2004; Rowe, 1992; Akbuga, 1991). Thus, in order to reduce the
dosing frequency, a dose form is designed to maintain a constant drug concentration for an extended period of time with
minimum side effects.
Response surface methodology is one of the most
prominent statistical modeling technique. This methodology
includes various types of experimental designs such as Box
Behnken, 3 level factorial, central composite and D-optimal
design, etc., which are widely useful for the development
and optimization of the relationship between measured variables and a number of independent variables in the form
of polynomial equations (Deshmukh and Naik, 2013; Roy
and Shahiwala, 2009; Tajber et al., 2009). The selection of
experimental design is depending on the objectives of the
experiment and the number of factors to be investigated.
Box Behnken design is one of the most popular Response
surface designs and advantage of such methodology is to
provide less experimental runs and time, thus provides more
efcient optimization (Chaudhary et al., 2013; Peng et al.,
2013). BoxBehnken statistical design is a type of independent
quadratic design that does not contain a fractional factorial
design (Sood et al., 2014). The present study, investigates the
inuence of variables on the nanoparticle properties.

99

In the present investigation LP loaded nanoparticles were

prepared by microreactor precipitation methodology. The aim
of this work was to develop sustained drug delivery formulation of LP for oral therapy of hypertension. A Computer-aided
optimization techniques using BoxBehnken design were
employed to investigate the effect of three factors viz., concentration of polymer, surfactant and inner diameter of
microreactor, on percentage encapsulation efciency (EE) and
drug release (DR) of nanoparticles. Furthermore, in vitro study
was carried to evaluate all physicochemical and drug release
parameters of developed LP loaded nanoparticles.

2.

Experimental

2.1.

Materials

Losartan Potassium was a generously gifted by Wockhardt

Research Centre (Aurangabad, India). Ethyl cellulose (20cp viscosity grade), Methanol (HPLC grade), Tween 80 was procured
from Merck Specialties Private Limited (Mumbai, India). HPLC
grade water was procured from Avantor performance material ltd. (Ankleswar, India) and all other chemicals were of
analytical grade and used as received.

2.2.

Experimental setup and procedure

Microreactor was fabricated using silicon tube with different

channel diameters (0.8 to 1.5 mm, ID) having a length of 183 cm
for each channel. Syringe pump (Infusor, universal medical
instruments, India) and syringes (20 and 50 ml, Dispovan) were
used for respective experiments.
Continuous-ow nano precipitation of LP loaded nanoparticles was carried out in a tubular microreactor shown in Fig. 1
(Capretto et al., 2013). In brief, 100 mg of the LP and required
amount of EC were dissolved in 20 ml methanol and sonicated
for 5 min to get transparent solution. In another beaker; the
required amount of Tween 80 was dissolved in 40 ml water
with constant stirring. Organic (LP, EC and methanol) and non
solvent (water and Tween 80) solutions were lled separately
with syringes and driven by syringe pump. Both solutions were
passed through spiral microreactor. The ow regime of the
solutions was usually laminar owing to the small dimension
of microreactor. At low Reynolds number solution streams
were alienated leading to diffusion controlled mixing throughout the microreactor. The collected nanosuspension from
the outow of microreactor was stirred continues till complete evaporation of methanol. The ow rate of organic and
non-solvent solution was set at 60 ml h1 and120 ml h1 and a
volumetric ratio of 1:2. The resultant nanosuspension was
lyophilized with addition of mannitol. Lyophilized nanoparticles were used for further studies.

2.3.

Experimental design

Various factors were evaluated in the formulation of LP

loaded nanoparticles using 33 (three-factor and three-level)
BoxBehnken factorial design. The selection of BoxBehnken
factorial design based on screening experiments in order to
identify minimum number of experimental runs and tting
surface model based on the second order polynomial equation
(Deshmukh and Naik, 2014; Cheng and Wu, 2001). The independent variables selected were the amount of EC (X1 , mg),
amount of Tween 80 as a surfactant (X2 , % v/v) and the inner

100

chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

Fig. 1 Schematic diagram of the experimental set-up for preparation of LP loaded nanoparticle by microreactor
methodology.
diameter of the microreactor (X3 , mm), which were varied at
three levels(1,0, +1). The drug EE (Y1 %) and DR over 12 h (Y2 %)
were selected as dependent factors. The statistical experimental data were analyzed using the Design-Expert Software
(Version-8.0.7.1, Stat-Ease Inc., Minneapolis). The matrix of
design, including dependent and independent variables is
shown in Table 1. The interaction of independent variables
and measured responses were modeled using the following
quadratic mathematical model (Chaudhary et al., 2013),
Y = b0 + b1 x1 + b2 x2 + b3 x3 + b12 x1 x2 + b13 x1 x3 + b23 x2 x3

U-2900, Tokyo, Japan). An accurately weighed 10 mg of prepared LP loaded nanoparticles were stirred with enough
quantity of methanol to dissolve the polymeric coat and
extracted in distilled water. The mixture was stirred continued
until complete evaporation of methanol followed by ltration.
The drug content within the ltrate was analyzed after suitable dilution using UVvis spectrophotometer by measuring
absorbance at 210 nm. The encapsulation efciency (EE) of the
LP loaded nanoparticles was calculated using the following
equation (Deshmukh and Naik, 2014), % EE = actual quantity
of drug determined/theoretical quantity of drug 100.

+ b11 x12 + b22 x22 + b33 x32

2.4.2.
Y is the response, b0 is the intercept and b1 to b33 are the regression coefcient. x1 , x2 , and x3 are individual effect, x1 x2 ,x1 x3 ,
and x2 x3 are interaction effect, and x1 2 ,x2 2 , and x3 2 are the
quadratic effect. ANOVA of the model was analyzed the signicance of the statistical parameters and model at the 95%
condence level (P < 0.05).

2.4.

Characterization of the nanoparticles

2.4.1.

Evaluation of Encapsulation Efciency (%)

The amount of LP drug encapsulated within the nanoparticles was determined by UVvis spectrophotometer (HITACHI

In vitro release study

In vitro dissolution studies of LP loaded nanoparticles were

performed using XXVIII apparatus, Type-I (rotating paddle
method) for 12 h. 5 ml of nanosuspension was introduced into
the pretreated dialysis membrane, tied at both ends and then
membrane was placed in a dissolution test apparatus containing distilled water. Stirring was performed at 100 RPM by
using a magnetic stirrer at 37 0.5 C (Khairnar et al., 2014a;
Khairnar et al., 2014). Samples (2 ml) were withdrawn at regular interval and replenished with contemporary dissolution
media to keep up sink condition. The LP content of each
sample was analyzed after suitable dilution using UVvis spectrophotometer by measuring absorbance at 210 nm.

Table 1 Variable and three levels.

Independent variable
X1 = Polymer conc. (mg)
X2 = Surfactant conc (%).
X3 = Inner diameter of micro reactor (mm)
Dependent variables
Y1 = %EE
Y2 = %DR (at 12 h)

Low level (1)

100
0.5
0.8

Medium level (0)

300
0.75
1

High level (+1)

500
1
1.5

chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

2.4.3. Field emission scanning electron microscopy

(FE-SEM) analysis

3.

The shape, size and surface characteristics of nanoparticles

were examined by Scanning electron microscopy (FESEM-S
4800, Hitachi, Japan) at a working distance of 8.68.8 mm and
accelerating voltage of 15.0 kV. For the morphological study,
the nanoparticles were gold coated by mounted on a brass
stub using double-sided adhesive tape under vacuum (5 pa)
in an ion sputter (Hitachi E1010) to make them electrically
conductive.

3.1.
Mechanism of formation of drug loaded
nanoparticles in a microreactor

2.4.4.

Fourier transform-infrared (FT-IR) spectroscopy

In order to examine the drug polymer interaction, the infrared

spectra of the LP, EC, and LP loaded nanoparticles were
obtained by using an FTIR spectrophotometer (FTIR-8400; Shimadzu, Asia Pacic Pvt. Ltd. Singapore) by the potassium
bromide pellet method. For that, sample (1 mg) was mixed
with KBr (40 mg) and compressed into a disc in a manual
press. Spectra were recorded in the wavelength region of
4000400 cm1 .

2.4.5.

X-ray diffraction analysis (XRD)

The physical nature of the LP, EC and LP loaded nanoparticles were examined by an X-ray diffractometer (Bruker, D8
The
Advanced, Germany) with Cu K radiation ( = 1.5406 A).
anode X-ray tube was operated at 40 kV and 40 mA. The samples were analyzed over the angle range of 080 on the 2
scale at a step size of 0.03/s.

2.4.6.
(PDI)

Particle size distribution and polydispersity index

The particle size and PDI of the LP loaded nanoparticles were

determined by laser scattering technique using nanozetasizer (ZS 90, Malvern Instruments, UK) at 25 C. The obtained
nanoparticles were suspended in distilled water as a dispersion medium and sonicated before analysis for 1 min.
The sample SOP was generated at a refractive index of
1.52, 243.8 of count rates (kcps) and 0.8872(mp s) of viscosity.

2.5.
Release kinetics and comparison of optimized
formulations with marketed formulation
There are various kinetic models viz., Zero order, rst order,
Hixson Crowell, Higuchi and Korsmeyer-Peppas were selected
as a model dependent approach to review the mechanism of
in vitro release of the optimized nanoparticles (Armin et al.,
2014; Misra and Mohanty, 2014; Malakar and Nayak, 2012).
These model dependent approaches are used to describe the
in vitro release behavior of LP from the formulation system
when the release mechanism is not known or when more
than one type of release phenomenon is concerned. Correlation coefcient (R2 ) was selected to dene the accuracy and
prediction ability of these models. The model with a highest
correlation coefcient of determination (R2 ) was considered
to be the most relevant kinetic model for describing the
in vitro release of an LP from the formulation. The optimized
nanoparticle formulations were compared with the marketed
formulation Losakind (25 mg).

101

Result and discussion

LP loaded nanoparticles are usually prepared by different conventional methods by taking into consideration of
the physiochemical properties of LP and surfactants. Thus,
Microreactor precipitation technology opens a novel way of
preparation of drug loaded nanoparticles in a controlled
way. Two solutions streams of organic and non solvent were
injected with the constant ow rate and the solutions are
mixed with the central channel. The ow regime of the solutions was usually laminar owing to the small dimension of
microreactor. At low Reynolds number solution streams were
alienated leading to diffusion controlled mixing throughout
the microreactor (Palanisamy and Paul, 2012). This may result
to the solution streams ows parallel to the side wall of the
microreactor and initiate the diffusion of drug molecule across
the interface. The dissolved drug molecules are coming in contact with supersaturated non-solvent solution formed drug
nuclei within the interface; provides nucleation sites, increase
the nucleation rate and starts the growth of the particle. However, the growth of particle was controlled by the addition of
surface active agent (Aghajani et al., 2013). It was found that
with increase in volume ratio of non solvent to organic solvent the nucleation rate will increase and reduces the particle
size, this may due to the nucleation and growth kinetics (Sinha
et al., 2013).
From the preliminary studies, it was observed that LP, EC
and inner diameter of the microreactor were found to have
signicant impact on the formulation of LP loaded nanoparticles. The effects of these independent variables were studied
by Box Behnken factorial design and are described as follows.

3.2.

Statistical analysis of EE and DR

A BoxBehnken statistical design with 3 independent variables

at 3 different levels was used to study the effects on selected
dependent variables (Khairnar et al., 2014b). The independent
variables were Polymer concentration (X1 ), Surfactant concentration (X2 ) and the inner diameter of the microreactor (X3 ),
while the dependent variables were EE (Y1 ) and DR over 12 h
(Y2 ). The EE was found in the range of 57.50 (F10) to 82.8% (F4)
and minimum DR among the all batches of the nanoparticles
was 77.2% (F11) are listed in Table 2. Transformed values of
EE and DR are represented in Table 3. The quadratic equation
obtained for the EE (Y1 ) was given by:
Y1 = 84.52 2.76X1 + 0.45X2 + 0.97X3 + 6.68 X1 X2 3.12X1 X3
0.19X2 X3 6.5X12 8.86X22 13.30X32
And for the DR (Y2 ) was given by:
Y2 = 93.84 3.44X1 + 0.72X2 + 4.77X3 7.08X1 X2
7.11X1 X3 + 2.39X2 X3 1.27X12 + 3.59X22 7.02X32

A positive value in the quadratic equation for a response

represents an effect that favors the optimization (synergistic
effect); while a negative value indicates an inverse relationship
(antagonistic effect) between the factor and the response. In
regression equations of Y1 and Y2 , the main effects of X1 , X2 ,

102

chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

Table 2 %EE and % DR over 12 h.

Experimental run

Polymer conc. (mg)

Surfactant conc. (%).

100.00
300.00
300.00
300.00
500.00
100.00
300.00
300.00
500.00
500.00
100.00
300.00
300.00
500.00
300.00
300.00
100.00

0.75
1.00
0.50
0.75
1.00
0.50
1.00
0.75
0.50
0.75
0.75
0.75
0.75
0.75
0.50
0.75
1.00

F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
F11
F12
F13
F14
F15
F16
F17

Inner diam. (mm)

1.50
0.80
1.50
1.00
1.00
1.00
1.50
1.00
1.00
1.50
0.80
1.00
1.00
0.80
0.80
1.00
1.00

% EE

% DR at t = 12 h

70.56
60.30
64.72
82.80
73.28
72.56
65.12
81.00
58.64
57.50
66.56
82.00
81.50
64.00
59.30
81.00
60.48

99.40
84.20
92.77
91.63
83.48
88.20
100.00
90.40
100.00
79.20
77.20
88.50
91.00
86.77
85.05
91.50
100.00

Table 3 Formulation of LP nanoparticle using BoxBehnken design.

Factor

Batches

F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
F11
F12
F13
F14
F15
F16
F17

Responses

X1

X2

X3

Y1

Y2

1
0
0
0
+1
1
0
0
+1
+1
1
0
0
+1
0
0
1

0
+1
1
0
+1
1
+1
0
1
0
0
0
0
0
1
0
+1

+1
1
+1
0
0
0
+1
0
0
+1
1
0
0
1
1
0
0

70.56
60.30
64.72
82.80
73.28
72.56
65.12
81.00
58.64
57.50
66.56
82.00
81.50
64.00
59.30
81.00
60.48

99.40
84.20
92.77
91.63
83.48
88.20
100.00
90.40
100.00
79.20
77.20
88.50
91.00
86.77
85.05
91.50
100.00

and X3 represents the average results of changing 1 variable at

a time from its low-level to high level. The interaction terms
X1 X2 , X1 X3 , and X2 X3 show how the EE and DR changes when
2 variables are simultaneously changed. The value of correlation coefcient (R2 ) was found to be 0.9752 for the quadratic
model of response Y1 , indicating good t, as shown in Table 4.
The results of ANOVA, as shown in Table 6, indicated that the
model F-Value was found to be signicant at P < 0.05. In this

case X1 , X1 X2 , X1 X3 , X1 2 , X2 2 , and X3 2 are signicant model

terms. Also for DR, The value of correlation coefcient (R2 ) was
found to be 0.9645 for the quadratic model of response Y2 , indicating good t, as shown in Table 4. Table 6 listed the results
of ANOVA; the model was found to be signicant at P < 0.05
(F-value = 21.10). In this case X1 , X3 , X1 X2 , X1 X3 , X2 X3 , X2 2 ,
and X3 2 are signicant model terms. Diagnostics case statistics for various response variables were also studied by using

Table 4 Summary of regression analysis for responses Y1 and Y2 .

Models
Response Y1
Linear
2FI
Quadratic
Cubic
Response Y2
Linear
2FI
Quadratic

R2

Adjusted R2

0.0403
0.2005
0.9752

0.1812
0.2791
0.9434

0.9983

0.9933

0.1804
0.7387
0.9645

0.0087
0.5819
0.9187

Predicted R2

0.4786
0.7348
0.5341

Std. dev

10.09
10.50
2.21

Press

2039.49
2392.80
642.66

7.10
4.57
2.01

. . .. . .. . .
. . .. . .. . .
suggested
. . .. . .. . .

0.76
0.7305
0.5079
0.4757

Remarks

1382.89
1205.00
419.01

Cubic
0.9919
0.9676
1.27
+
Regression equations of the tted models
Y1 = 84.52 2.76X1 + 0.45X2 + 0.97X3 + 6.68 X1 X2 3.12 X1 X3 0.19 X2 X3 6.56 X1 2 8.86 X2 2 13.30 X3 2
Y2 = 93.84 3.44X1 + 0.72X2 + 4.77X3 7.08 X1 X2 7.11 X1 X3 + 2.39 X2 X3 1.27 X1 2 + 3.59 X2 2 7.02 X3 2

. . .. . .. . .
. . .. . .. . .
suggested
. . .. . .. . .

chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

103

Table 5 Diagnostics case statistics for various response variables.

Batch no
F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
F11
F12
F13
F14
F15
F16
F17

Response variables
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2
Y1
Y2

Actual value
70.56
99.40
60.30
84.20
64.72
92.77
82.80
91.63
73.28
83.48
72.56
88.20
65.12
100.00
81.00
90.40
58.64
100.00
57.50
79.20
66.56
77.20
82.00
88.50
81.50
91.00
64.00
86.77
59.30
85.05
81.00
91.50
60.48
100.00

design expert software and it was represented in the Table 5

with actual value, predicted value and residual value for each
response. Thus, the optimized difference between actual and
predicted values was very less; it concludes that the model
was best tted. From the contour plot (Fig. 2A) it could be predicted that when polymer concentration was maintained at
(1) level the EE was between 60.48 to 72.56%. The EE was further enhanced when polymer concentration was maintained
at (0) level. In this case the EE was between 59.30 to 82.80%.
But at (+1) level, there was a decrease in EE, it was found to
be 57.50 to 73.28%. The EE was in the order of 0> +1 > 1. The

Predicted value
71.50
100.96
62.04
84.06
63.07
92.16
81.66
90.61
72.03
85.14
73.81
86.54
63.59
98.39
81.66
90.61
57.60
99.91
59.75
79.86
63.33
77.21
81.66
90.61
81.66
90.61
64.04
85.54
60.74
87.41
81.66
90.61
61.52
100.09

Residual
0.94
1.56
1.74
0.14
1.65
0.61
1.14
1.02
1.25
1.66
1.25
1.66
1.53
1.66
0.66
0.21
1.04
0.09
2.25
0.66
3.23
0.01
0.34
2.11
0.16
0.39
0.043
2.23
1.44
2.36
0.66
0.89
1.04
0.090

effect of polymer concentration on EE was found statistically

signicant with P < 0.0115. So it was clear that there was no
linear relationship between polymer concentration and EE. EE
was increased up to the certain level by increasing the polymer
concentration, but further increase in polymer concentration
there was a decrease in EE which was found at +1 level of
polymer. This may result to the higher quantity of polymer
utilized that taken more time for the precipitation (Deshmukh
and Naik, 2013). In addition, from the contour plot (Fig. 2B), it
can be predicted that when polymer concentration was maintained at (1) level the minimum DR was 77.20% over 12 h and

Fig. 2 Effect of individual variables (polymer and surfactant) on responses EE% and DR% presented by contour plots (A and
B).

104

chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

Table 6 ANOVA of models for Y1 and Y2 .

Source

DF

Sum of squares

Mean square

F value

P value

Model for Y1
X1
X2
X3
X1 2
X2 2
X3 2
X1 X2
X1 X3
X2 X3

9
1
1
1
1
1
1
1
1
1

1345.18
56.22
1.52
7.49
181.33
330.34
452.58
178.49
42.40
0.16

149.46
56.22
1.52
7.49
181.33
330.34
452.58
178.49
42.40
0.16

30.64
11.53
0.31
1.54
37.18
67.73
92.79
36.59
8.69
0.034

<0.0001
0.0115
0.5954
0.2552
0.0005
<0.0001
<0.0001
0.0005
0.0215
0.8593

Model for Y2
X1
X2
X3
X1 2
X2 2
X3 2
X1 X2
X1 X3
X2 X3

9
1
1
1
1
1
1
1
1
1

770.72
87.40
3.83
181.93
6.84
54.21
126.19
200.51
220.56
25.05

85.64
87.40
3.83
181.93
6.84
54.21
126.19
200.51
220.56
25.05

21.10
21.54
0.94
44.83
1.68
13.36
31.10
49.41
54.35
6.17

0.0003
0.0024
0.3635
0.0003
0.2354
0.0081
0.0008
0.0002
0.0002
0.0419

for (0) level it was found 84.20%. But when this concentration
was maintained at (+1) level, it was 79.20%. From this it can
be concluded that there was no linear relationship between
the polymer concentration and drug release. The inuence
of polymer concentration on drug release was found statistically signicant with P < 0.0024. DR was increased up to the
certain level by increasing the polymer concentration, but further increase in polymer concentration there was a decrease
in DR. This was due to hydrophobic nature of ethyl cellulose
which results to the increase in the length of the diffusion
pathways (Siepmann et al., 2007).
During the development of nanoparticles, tween 80 was
used as a surfactant due to its high water solubility. The major
role of the surfactant was to reduce the interfacial tension
between two phases and to stabilize the nal nanoparticles
by preventing the aggregation. In case of surfactant concentration when it was maintained at (1) level the maximum EE
was 72.56%, at (0) level it was 82.80% and at (+1) level it was
73.28% are shown in Fig. 3A. So, The EE was in the order of
0> +1 > 1. Also in case of the effect of surfactant on DR, it can
be predicted from the contour plot that (Fig. 3B) at (1) level
the minimum DR was 85.05% over 12 h and for (0) level, it was
77.20% but when this concentration was maintained at (+1)
level it was 83.48%. So from contour plot it was concluded
that the (0) level was found at an optimized concentration
level of surfactant to retard the DR from the nanoparticles.
In order to assess the effect of ID of microreactor on DR and
EE as shown in Fig. 4A and B, it was selected as an independent variable during the experiments. The maximum EE was
obtained 82.80% when the ID was used at (0) level. An in case of
DR the effect of ID was statistically signicant with P < 0.0003.
Minimum DR from nanoparticles was 77.20% over 12 h when
ID was maintained at (0) level.

3.3.

Characterization of nanoparticles

3.3.1. Field emission scanning electron microscopy

(FE-SEM) analysis
Field emission scanning electron microscopy was performed in order to examine the surface characteristic of LP

loaded nanoparticles. Fig. 5 shows uniform spherical shaped

nanoparticles in the size range of 200350 nm. The nanoparticles were found to be isolated, uniform surface and free
owing in nature. The sustained release of LP drug caused
due to the lack of pores and channels on the surface of the
nanoparticles.

3.3.2. Fourier transforms infrared spectroscopy (FT-IR)

analysis
The FTIR spectrum of pure LP, EC and LP loaded nanoparticles
are shown in Fig. 6. The IR spectrum of pure LP drug (Fig. 6A)
showed the principal peaks at 3003.26,1575.81,1052.20, 949.09,
and 764.8 cm1 corresponding to C H stretching, C N stretching, Aromatic C H in plane blend, Aromatic C H out plane
blend, and C Cl stretching of chlorine group, respectively.
The IR spectrum of EC (Fig. 6B) showed the principal peaks
at 3471.98, 2976.26, 2874.03, and 1375.29 cm1 conrmed the
OH stretching, symmetric C H stretching of ethoxy group,
C H stretching of ethoxy group, and bending of OH group,
respectively. The nal formulation of LP loaded nanoparticles
(Fig. 6C) showed the peaks at 3479.7, 3007.12, 2848.24, 1649.19,
1444.56, 1251.84, 1079.11, 951.90, and 760.94 cm1 which stayed
close in the FTIR spectrum of LP and EC, signifying there was
no interaction between the drug (LP) and polymer utilized
(EC).

3.3.3.

X-ray diffraction (XRD) analysis

XRD patterns of pure LP, ethyl cellulose and LP loaded nanoparticles are shown in the Fig. 7. Pure LP (Fig. 7A) showed
distinct peaks at 2  angle of 6.349 , 10.122 , 13.919 , 18.01 ,
20.06 , 23.03 , 25.66 , 28.82 , 32.31 , 36.36 , and 39.29 conrmed the crystalline (81.6%) nature of the LP drug. There
was no clear peak in the XRD pattern of ethyl cellulose
(Fig. 7B) indicates the amorphous nature of the polymer.
The absence of crystalline peaks of pure LP in the diffractogram of LP loaded nanoparticles (Fig. 7C) suggested the
amorphous (49.8%) nature of optimized formulation. Also,
the amorphous nature of the formulation reveals that the
majority of drug distributed homogeneously within the polymer.

chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

105

Fig. 3 Effect of individual variables (surfactant and Inner diameter) on responses EE% and DR% presented by contour plots
(A and B).

Fig. 4 Effect of individual variables (Inner diameter and polymer) on responses EE% and DR% presented by contour plots (A
and B).

Fig. 5 FE-SEM of optimized LP loaded nanoparticle formulation showing sphericity, smooth surface of nanoparticles.

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chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

Fig. 6 FTIR spectra of LP (A), Ethyl cellulose (B), and LP loaded nanoparticles (c).

3.3.4. Particle size distribution and polydispersity index

analysis
The particle size of LP loaded nanoparticles prepared by
microreactor precipitation method was found in a range of
141.8 to 342.09 nm with the average particle size of 357.8 nm
as shown in Fig. 8. The ow rate and volumetric ratio of organic
and non solvent during the microreactor precipitation process
was 1:2. Owing to this higher volume ow of non solvent, the
particle size of nanoparticles decreases. In addition, due to
high surface area to volume ratio of microreactor, the preliminary higher supersaturation level leading to increase the
nucleation rate followed by growth resulting uniform smaller
size of particles developed (Ali et al., 2009). The 0.469 PDI value

of LP loaded nanoparticle was close to zero indicates that the

monodispersed and narrow size distribution of particles.

3.4.
Release kinetics and comparison of optimized
formulations with marketed formulation
The comparative in vitro drug release prole of optimized
formulations of nanoparticles with the marketed formulation of the LP was shown in Fig. 9. A rapid burst release
was observed in case of Losakind (25 mg) tablet. Almost 100%
drug was released within the period of 2 h while the formulations 11 and 5 showed excellent sustained release behavior.
In case of Run 11(F11) the initial burst release was 23.95 and

Fig. 7 XRD spectra of LP (A), Ethyl cellulose (B), and LP loaded nanoparticles (c)

chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

107

Fig. 8 Particle size analysis of optimized LP loaded nanoparticle formulation.

Fig. 9 In vitro drug released prole of formulations Run 11, Run 5 and losakind (25 mg) tablet in distilled water.

for Run 5 (F 5) it was 27.35% in the rst hour, and drug

release was sustained over the period of 19 and 16 h, respectively. From this, the release pattern was biphasic consisting
initial burst release followed by slower sustained release of
drug. Due to such sustained release behavior of the nanoparticles, it would denitely reduce the dosing frequency of the
medication which is the major drawback of the conventional
drug delivery system for the LP. The mechanism of in vitro
release of the nanoparticles was evaluated by employing a
different model dependent approach viz., zero order, rst
order, Higuchi, KorsMeyerPeppas, HixonCrowell equation.
The kinetic model with higher values of coefcient correlation (R2 )was considered to be a best t model for the drug
release. It was observed from the data that both the formulation (Run 11 and 5) were best tted to the Higuchi model with
R2 = 0.9944 and R2 = 0.9817. This model is based on the hypothesis that (i) initial drug concentration in the matrix is much
higher than drug solubility; (ii) drug diffusion takes place only
in one dimension (edge effect must be negligible); (iii) drug

particles are much smaller than system thickness; (iv) matrix

swelling and dissolution are negligible; (v) drug diffusivity is
constant; and (vi) perfect sink conditions are always attained
in the release environment (Dash et al., 2010).
Also, both the formulations showed slope (n) higher than
0.89 (n = 1.0562 and n = 1.1046 for Run 11 and 5, respectively),
this clearly indicated the super caseII transports, i.e. drug
release by both diffusion and relaxation of the polymer chain.

4.

Conclusion

The Losartan Potassium loaded nanoparticles were successfully prepared by a microreactor precipitation method using
BoxBehnken design. The average particle size of nanoparticle was 359.4 nm with narrow size distribution. The prepared
nanoparticles were found to be uniform surface, free owing
in nature and without any channel and pores on the surface.
XRD and FTIR characteristics of optimized formulation indicated that the LP drug was present in an amorphous state

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chemical engineering research and design 1 0 4 ( 2 0 1 5 ) 98109

and there were no interaction between LP and EC polymer utilized. The independent variables having a signicant effect
on responses viz., encapsulation efciency and drug release.
The high drug encapsulation efciency (82.8%) of the formulation was achieved by run 4 using 300 mg of EC, 0.75% of Tween
80 and 1 mm inner diameter of the microreactor. The in vitro
drug release studies revealed that, the LP loaded nanoparticle
was found sustained as compare to the marketed formulation.
Thus, LP loaded nanoparticles prepared by the microreactor precipitation method represents a promising formulation
technique for encapsulation efciency and sustained drug
release of the LP.

Conict of interest statement

Authors do not have any conict of interest.

Acknowledgements
The authors are grateful to Technical Education Quality
Improvement Program (TEQIP-II) for providing nancial assistance to carry out this research work. Authors are also thankful
to Wockhardt research Centre (Aurangabad, India) for providing a gift sample of Losartan potassium.

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