Powder Technology 256 (2014) 436–449

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Powder Technology
journal homepage: www.elsevier.com/locate/powtec

A comparative study of top-down and bottom-up approaches for the

preparation of nanosuspensions of glipizide
Koneti Venkata Mahesh, Sachin Kumar Singh ⁎, Monica Gulati
School of Pharmaceutical Sciences, Lovely Professional University, Punjab 144411, India

a r t i c l e i n f o a b s t r a c t

Article history: Glipizide nanosuspensions were prepared by both liquid antisolvent precipitation and media milling technique
Received 11 June 2013 using bead mill. From this study it was concluded that ratio of polymer to drug, milling time and milling speed
Received in revised form 16 January 2014 played significant role in controlling the zeta potential of nanosuspension prepared by media milling whereas,
Accepted 1 February 2014
milling time and milling speed were considered to be significant factors for controlling particle size distribution
Available online 8 February 2014
d (90) of nanosuspension. In the bottom up process ratio of surfactant to drug and speed of mixing played a
Keywords:
significant role in controlling the zeta potential of glipizide whereas ratio of polymer to drug and speed of mixing
Glipizide were considered to be the significant factors that affect the particle size distribution d (90) of nanosuspension. An
Nanosuspensions increase in particle size distribution was observed with the optimized nanosuspension prepared by liquid
Liquid antisolvent precipitation antisolvent precipitation method when tested under accelerated conditions (40 °C at 75% RH for 6 months) as
Media milling compared to media milling method. The X-ray diffraction data shows no form conversion of the drug due to
Zeta potential processing parameters involved in the production of nanosuspension. The formulated nanosuspension has
Particle size distribution d (90) shown a faster dissolution profile (98.97 using bottom up and 96.44% using top down method in 10 min), relative
to that of pure glipizide (20.17% in 10 min), mainly due to the formation of nanosized particles.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction presence of a stabilizer. Various adaptations of this approach include:

Poorly soluble molecules have been successfully formulated by
employing a variety of techniques such as: (i) solubilization in surfactant
solutions; (ii) use of cosolvents; (iii) pH adjusted solutions;
(iv) emulsions; (v) liposomes; (vi) complexation with cyclodextrins;
and (vii) solid dispersions [1,2]. However, most of these techniques
require a large amount of additives limiting their use from the safety
perspective. Moreover, these techniques offer little or no help in the
formulation of molecules that are poorly soluble in both aqueous and
non-aqueous solvents [3]. Nanosuspensions by the virtue of their
large surface area to volume ratio provide an alternative method to
formulate poorly soluble compounds. These are sub-micron colloidal
dispersions of discrete particles that have been stabilized using surfac-
tants, polymers or a mixture of both. Extensive review has been done
on the various approaches for the development and characterization
of nanoparticles, nevertheless briefly it has been classified into two
basic approaches i.e. top down technology and bottom up technology
[4]. Top-down processes consist of particle size reduction of large drug
particles into smaller particles using various wet milling techniques
such as: media milling, microfluidization and high pressure homogeni-
zation. In the bottom-up approach the drug is dissolved in an organic
solvent and is then precipitated on addition of an anti-solvent in the
⁎ Corresponding author at: School of Pharmaceutical Sciences, Lovely Professional
University, Phagwara 144411, Punjab, India. Tel.: +91 9888720835; fax: +91 1824501900.
E-mail address: singhsachin23@gmail.com (S.K. Singh).
(i) solvent–anti-solvent method; (ii) supercritical fluid processes;
(iii) spray drying; and (iv) emulsion–solvent evaporation [3,5].
The method of manufacture can significantly impact the formation
and stability of nanosuspensions and hence their overall performance
[6–8]. In the top-down process considerable heat is generated which
may cause degradation of heat sensitive active pharmaceutical ingredi-
ents (APIs). Milling has been shown to cause mechanical activation
at drug particle surfaces [9]. Crystal defects due to disordering of the
crystal surface and generation of localized amorphous regions have
been implicated in increased surface energetics [10]. Reordering of
crystal defects and re-crystallization of amorphous regions have result-
ed in both physical and chemical instability of processed materials on
storage. Alternatively, the bottom-up process can adversely influence
nanosuspension formulations as well by the generation of various
unstable polymorphs, hydrates and solvates during processing. These
approaches involve the use of solvents which are usually difficult
to completely remove [2]. Any residual solvent can cause physical
and chemical instability of the formulation. Moreover, bottom-up
approaches usually result in needle shaped particles due to rapid
growth in one direction which influences the physical stability of the
nanosuspensions [3,11].
In order to overcome the problem of stability of nanosuspensions
various stabilizers are added. The stability and robustness of a
nanosuspension is mainly governed by various formulation and process
variables. Selection of proper steric and electrostatic stabilizers and its
optimum quantity plays a major role in formulating a nanosuspension.

0032-5910/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.powtec.2014.02.011
 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449
Commonly used steric stabilizer includes hydroxyl propyl methyl
cellulose (HPMC), hydroxyl propyl cellulose (HPC), polyvinylpyrroli-
done (PVP K 30), and pluronics (F68 and F127) whereas electrostatic
stabilizer includes polysorbate (Tween-80), sodium lauryl sulfate
(SLS) and docusate sodium (DOSS). A suitable working polymer to
drug ratio (steric stabilizer) is from 0.05:1 to 0.5:1 [1]. At high stabilizer
concentrations, well above of the plateau of the adsorption isotherm,
electrostatic stabilizers can cause a decrease in the diffuse layer leading
to a decreased zeta potential and a decreased physical stability. Electro-
lytes are present in the gastrointestinal tract and the contact of the
nanocrystals with these electrolytes cannot be avoided. Electrostatic
stabilization is reduced in its efficiency in an electrolyte containing
environment. Therefore it is important to find the optimal concentra-
tion for a stabilizer. For a physically stable nanosuspension solely
stabilized by electrostatic repulsion, a zeta potential of ± 30 mV is
required as a minimum. In the case of a combined electrostatic and
steric stabilization, a rough guide line of ±20 mV is sufficient [12,13].
The bottom up technology involving solvent antisolvent precipita-
tion technique requires dissolving the drug in a solvent which is then
added to a non-solvent to precipitate the crystals and the subsequent
growth of crystal is controlled by the addition of polymer and/or surfac-
tant to produce fine particles [14]. These methods give better control
over particle properties such as, size, morphology and crystallinity as
compared to top-down methods [15]. Other solvent removal methods
such as, evaporative precipitation into aqueous solution (EPAS) [16]
and microemulsions have also been reported, though these are one-
step processes they have certain disadvantages such as low yield
and degradation of heat sensitive materials [17]. The supercritical fluid
technology is another extensively researched bottom-up technology
for the preparation of nanoparticles [18], however, it has inherent
disadvantages of using extremely high pressures which require high
pressure pumps, temperatures, and specially designed fine nozzles
which may pose operational problems.
High-pressure homogenization relies on the forcing of a suspension
through a small gap which makes miniaturization of this technology
less straight forward. Media milling, on the other hand, can be performed
by agitation of devices containing the starting suspension and milling
media. Furthermore, nanosuspension production by media milling is
characterized by its ease of scale-up [5], making results generated on
nanosuspensions in downscaled designs valuable and was therefore
selected for this study. Apart from this several research articles are
available for better understanding of nanocrystals formulated by other
than media milling technology. Though most of the products existing
in market are developed based on the media milling technique, there is
no research work available in the public domain on the same considering
the industrial perspective of nanosuspension development, which
includes the cost and time of manufacturing [19].
The aims of this study were to compare top-down and bottom-up
approaches and evaluate the effect of the processing method and
stabilizer suitability on nanosuspension preparation and stability using
Box–Behnken design (BBD). The objective of this study also includes
optimization of polymer to drug ratio and surfactant to drug ratio that
could give nanosuspensions with ideal characteristic, to optimize
processing factors that could affect the manufacturing process on large
scale development.
In the present study glipizide was used as a model drug which is a
potent sulfonylurea and has established potential benefits such as
lower dose, rapid onset, lower insulin levels, increased sensitizing and
insulin mimetic effects. However, it is a poorly soluble drug with relatively
high permeability that warrants it to be classified under biopharmaceuti-
cal classification system (BCS) classification-II. It is observed that admin-
istration of sulfonylurea under fasting condition significantly increases
the area under curve for 24 h and increases the maximum concentration
of drug in blood compare to their administration under feeding condi-
tions. The presence of food and certain dietary supplements interfere
with its dissolution and in turn its absorption [20]. In view of the time
437
required to reach on optimal conditions in plasma, sulfonylureas may
be more effective if given 30 min prior to meal [21] conversely, this
might reduce patient compliance since after taking the drug if the patient
is not able to have the meal it would result in severe hyperglycemia and if
taken with meal, food would interfere sequentially with its absorption.
Hence, improving the dissolution characteristics of glipizide by preparing
nanosuspensions might allow its concomitant dosing with food.
2. Materials and methods
2.1. Materials
Glipizide was a gift sample from Micro Labs. Baddi, India. Sodium
lauryl sulfate (SLS) was purchased from purchased from Stepan Co.,
USA. Hydroxyl propyl methyl cellulose (HPMC 6 cps) was purchased
from Samsung fine chemicals Co., Ltd., Korea. Purified water USP was
used in this study. Pluronic F68, F-127 and polyvinylpyrrolidone (PVP)
K-30 were from BASF (Ludwigshafen, Germany); Tween-80 (polysorbate
80) was supplied by RANKEM, India. Other laboratory chemicals used,
such as sodium hydroxide and potassium dihydrogen phosphate,
were purchased from Sigma-Aldrich (Steinheim, Germany) and were
of analytical grade. A UV–visible double beam spectrophotometer
used throughout the study was manufactured by Shimadzu, Japan.
2.2. Preliminary studies to select the suitable stabilizers
Screening for an optimal stabilizer(s) and its amount is very impor-
tant for the product quality. A suitable stabilizer has to be added to
stabilize the nanosuspension system and prevent aggregation and
Ostwald ripening. A series of stabilizers were assorted in preparing the
nanoparticles and different formulations were prepared by varying
the type of stabilizer at a constant concentration of stabilizer. The
various stabilizers investigated were sodium lauryl sulfate (SLS), polyvi-
nylpyrrolidone (PVP K 30), pluronics F-68 and F-127, Tween-80 and
hydroxyl propyl methyl cellulose (HPMC 6 cps).
2.2.1. Bottom up process
2.2.1.1. Effect of the types of solvent and antisolvent. The preliminary study
was aimed to screen the optimal solvent and antisolvent systems that
together could aid the formation of nanoparticles. The solubility of drug
was found out in various organic solvents (acetone, dichloromethane,
ethyl acetate and isopropyl alcohol) to find out the most suitable solvent
which had a maximum drug loading providing high super saturation
which would aid in rapid nucleation and precipitation. It was seen that
amongst the various organic solvents assorted, acetone showed the
highest solubility for glipizide (drug loading up to ~ 40 mg/5 ml) and
was selected as the solvent phase. Owing to poor solubility of glipizide
in water and high miscibility of water with acetone, water was then
selected as the antisolvent phase to assist the precipitation process.
2.2.1.2. Design of experiments. A set of experiments with Box–Behnken
Design (BBD) was adopted to develop the nanoparticles of glipizide by
liquid antisolvent precipitation method. Initial screening trials were
carried out in evaluating the formation and processing aspects of
nanosuspension. Various factors like concentration of drug, amount of
polymer, amount of surfactant, solvent/antisolvent ratio and speed of
mixing were identified as critical to give a product in nanorange and
with required stability. The results from the initial screening trials sug-
gested that polymer to drug ratio, surfactant to drug ratio, solvent/
antisolvent ratio and speed of mixing are the main factors which affect
the particle size d (90) and zeta potential of nanosuspension. Based on
the number of factors and their level a Box–Behnken design was used
to evaluate the effect of formulation and processing parameters affecting
the physical properties of nanosuspension. The four independent factors
identified for this study were polymer to drug ratio, surfactant to drug
438 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449

Table 1 Table 2
Variables for Box–Behnken study for Bottom up technology. Formula composition of nanosuspension batches prepared by bottom up
technology.
Independent factors Design level
Ingredients Quantity/batch (g)
Uncoded Coded Uncoded Coded
Glipizide 4.0
Ratio of surfactant to drug (g) A 0.06–1.0 −1
PVP K 30 0.25–0.50
0.12–1.0 0
SLS 0.25–0.75
0.18–1.0 +1
Solvent/antisolvent 1:10–1:30
Ratio of surfactant to drug (g) B 0.06–1.0 −1
0.09–1.0 0
0.12–1.0 +1
Solvent/Antisolvent (ml) C 1:10 −1
1:20 0
1:30 +1 2.2.2.2. Nanosuspension preparation by top down media milling. Preparation
Speed of mixer (rpm) D 1500 −1 of nanosuspension by media milling involves two main steps. The first
3000 0 one is uniform dispersion of drug and stabilizer in dispersion media and
4500 +1
the second one is particle size reduction in milling chamber. The uniform
dispersion of drug (initial particle size d (90) 6.0 μm) and stabilizer in
dispersion media was prepared by using Heidolph mixer (Model:
RZR2051 control, rose scientific Ltd., Alberta, Canada) operated at
500 rpm. This suspension was loaded in milling chamber of bead mill
ratio, solvent/antisolvent ratio and speed of mixing. All these factors
(Model: Lab Star 1, Netzsch mill, Germany) for particle size reduction.
were operated at three levels (+1, 0, −1). The concentration of drug,
The milling media used for this study was 0.2-mm yttrium-stabilized
type of polymer, type of surfactant, and time of mixing was kept the
zirconium beads. The milling operation was performed in a recirculation
same for all the experiments. Design-Expert 8.0.7.1 software was used
mode with the suspension fed at a rate of 100 ml/min. The bead mill was
to conduct the study. A total of 29 experiments were designed by the
operated at specific speed and time as designed by the DoE. The temper-
software with 2 center points. Experiments were run in random order
ature of suspension was controlled during milling by circulating cold
to increase the predictability of the model. Table 1 shows the indepen-
water through the outer jacket.
dent factors and their design level used in this study. Table 2 list out
The nanosuspensions were dried using spray dryer (Spray Dryer
the formula composition of nanosuspension with respect to the amount
Model: LU 222, Labultima, Mumbai, India) under the following set of
as mentioned in Design of Experiment (DoE).
conditions: inlet temperature, 85 °C; outlet temperature, 60 °C; feed
rate, 2.5 ml/min; and atomization pressure, 2 kg/cm2.
2.2.1.3. Preparation of glipizide nanoparticles using antisolvent precipita-
tion technique. Glipizide nanoparticles were prepared using liquid 2.3. Characterization of glipizide nanoparticles
antisolvent precipitation technique. Glipizide was dissolved in acetone
at definite concentration and sonicated in a bath sonicator for 2 min. 2.3.1. Particle size measurement
The solution was filtrated through a 0.22 μ Whatman filter paper to The particle size of nanosuspension was measured using Malvern
remove possible particulate impurities. The prepared glipizide solution Zetasizer ZS200. Each sample was measured at least three times. The
was injected by syringe on to the tip of the antisolvent water containing average values were employed for the calculations of the response
each specific concentration of polymer and/or surfactant with stirring. surfaces.
Precipitation took place immediately upon mixing and formed a
suspension with bluish appearance. The consequences of the formulation 2.3.2. Zeta potential
and process parameters, such as the type of solvent, the solvent/ The zeta potential of nanosuspension was measured using Malvern
antisolvent ratio, the speed of mixing, and the concentration of glipizide Zetasizer ZS200 at 25 ± 0.5 °C. Each sample was measured at least
on the properties of the nanoparticles were investigated. The freshly three times. The average values were employed for the calculations of
formed suspension was centrifuged at 5000 rpm (REMI Centrifuge, the response surfaces.
REMI Instruments Pvt. Ltd) for 10 min and washed twice with 5 ml of
deionized water; the obtained nanoparticles were then dried by using a 2.3.3. Percentage yield and drug content
vacuum dryer at 50 °C for 8 h and stored in a desiccator till further use. The glipizide content was determined by dissolving accurately
weighed quantity of glipizide nanoparticles in methanol. The solution
2.2.2. Top down process

2.2.2.1. Experimental design. A set of experiments with Box–Behnken Table 3

Variables for Box–Behnken study for top down technology.
design was adopted to develop the nanoparticles of glipizide by media
milling method. The four independent factors identified for this study Independent factors Design level
were polymer to drug ratio, surfactant to drug ratio, milling time and mill- Uncoded Coded Uncoded Coded level
ing speed. All these factors were operated at three levels (+1, 0 and −1).
Ratio of polymer to drug (g) A 0.06–1.0 −1
The concentration of drug, type of polymer, type of surfactant, milling
0.12–1.0 0
media, volume of milling media, and solvent i.e. purified water were 0.18–1.0 +1
kept the same for all the experiments. Design-Expert® 8.0.7.1 software Ratio of surfactant to drug (g) B 0.06–1.0 −1
was used to conduct the study. A total of 29 experiments were designed 0.09–1.0 0
0.12–1.0 +1
by the software with 2 center points. Experiments were run in random
Milling time (h) C 3.0 −1
order to increase the predictability of the model. A batch size of 250 g 4.5 0
was kept constant for all experimental trials. Table 3 shows the indepen- 6.0 +1
dent factors and their design levels used in this study. Table 4 list out the Milling speed (rpm) D 1500 −1
formula composition of nanosuspension with respect to the ratios as 3000 0
4500 +1
mentioned in DoE.
 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449 439

Table 4 2.3.7. Statistical analysis

Formula composition of nanosuspension batches prepared by top All the data were statistically analyzed by analysis of variance or
down technology.
Turkey's multiple comparison test. Results are quoted as significant
Ingredients Quantity/batch (g) where P b 0.05.
Glipizide 4.0
PVP K 30 0.25–0.50
SLS 0.25–0.75
3. Results and discussions
Purified water qs
3.1. Effect of stabilizers on particle size distribution and zeta potential
qs = quantity sufficient to 250g.

A series of various stabilizers were used for preparing glipizide

was filtered, diluted appropriately in the samples, and measured
spectrophotometrically at 225 nm to determine drug content.
2.3.4. In vitro dissolution studies
In vitro dissolution studies were carried out in 900 ml 0.05 M
phosphate buffer (pH 7.5) at 37 ± 0.5 °C at 50 rpm (paddle method,
Lab India dissolution tester DS 8000, USP-II). 5 mg of pure glipizide
and its equivalent optimized batches of nanosuspensions prepared by
bottom up and top down technology were added to the dissolution
medium and 5 ml of sample was withdrawn at 5, 10, 15, 30, 45 and
60 min, respectively and replaced with fresh media. The solutions
were filtered with Whatman filter paper (0.22 μm) and assayed spectro-
photometrically for the dissolved drug at 225 nm.
2.3.5. Powder X-ray diffraction analysis
The powder X-ray diffraction (PXRD) patterns of pure glipizide,
glipizide nanoparticles prepared by liquid antisolvent precipitation
technique, nanosuspensions milled at high speed (4500 rpm) and milled
at slow speed (1500 rpm) were recorded using an X-ray diffractometer
(Bruker axs, D8 Advance) with a Cu line as the source of radiation.
Standard runs using a 40-kV voltage, a 40-mA current, at a scanning
rate of 0.010°min−1 over a 2θ range of 3–45° were used.
2.3.6. Stability studies
The potential for Ostwald ripening of the suspensions was evaluated
by conducting stability studies at 4 °C and 25 °C for three months. The
accelerated studies were also carried out at 40 °C and 75% relative
humidity (RH) for three months.
nanoparticles. Different formulations were prepared by varying the
type of stabilizers at a constant concentration of stabilizer using liquid
antisolvent precipitation technique and media milling technique
respectively. As can be seen from Fig. 1, amongst the polymeric group
PVP K 30 showed the highest particle size reduction (particle size d
(90) is 243 nm) while amongst surfactant groups SLS showed the
highest particle size reduction (particle size d (90) is 219 nm) using
liquid antisolvent precipitation method. The same types of results
were also observed while using milling method for nanosuspension
preparation. The highest particle size reduction was observed using
PVP K 30 as polymer (particle size d (90) is 264 nm) and using SLS as
surfactant (particle size d (90) is 196 nm) as shown in Fig. 2.
The same polymer (PVP K 30) and surfactant (SLS) have shown
the highest zeta potential using both technologies for preparation
of nanosuspension. PVP K 30 has provided zeta potential of − 20 mV
and − 18 mV using liquid antisolvent precipitation technique and
media milling respectively (Figs. 3 and 4). Also, SLS has provided zeta
potential of − 32 and − 31 mV using liquid antisolvent precipitation
technique and media milling respectively (Figs. 3 and 4). Hence it was
decided to employ SLS as well as PVP K 30 to prepare glipizide nanopar-
ticles using liquid antisolvent precipitation as well as media milling
technique.
3.2. Liquid antisolvent precipitation technique
3.2.1. Effect of independent factor on particle size distribution (Y1) and zeta
potential (Y2)
A total of 29 experiments were carried out to study the effect of
formulation and processing factors affecting the zeta potential and
particle size distribution of nanosuspension. Response data for all
experimental runs of Box–Behnken experimental design is presented
in Table 5.

Fig. 1. Particle size d (90) of glipizide suspension obtained with various stabilizers using liquid antisolvent precipitation method.
440 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449

Fig. 2. Particle size d (90) of glipizide suspension obtained with various stabilizers using media milling method.

The responses obtained for this study are well modeled by a linear
function of the independent variables; hence the first order polynomial
was used for approximating the function as shown in Eq. (1).
Y ¼ β þ β1 X1 þ β2 X2 þ β3 X3 þ β4 X4 þ €
where, € represents noise or error, X represents independent variable, Y
represents response and β represents coefficient. The values of response
Y1 (particle size d (90)) and Y2 (zeta potential) ranges from − 7.0 to
−32.0 mV and 219 to 453 nm, respectively. The ratio of maximum to
minimum for both the responses Y1 and Y2 is 2.14 and 2.20, respective-
ly; therefore power transformation was not applied to the obtained
values. Analysis of variance (ANOVA) was applied to determine the
significance and the magnitude of the effects of the main variables and
their interactions. The regression model obtained was used to generate
the counter plots for independent factors. The ANOVA table confirms
the adequacy of the model (i.e. F b 0.05) as shown in Table 6. It also
identifies the significant factors that affect the responses Y1 and Y2 of
nanosuspension. For zeta potential the ratio of surfactant to drug and
speed of mixing (factors A and D) were identified as significant model
terms. The particle size of the nanoparticles also depends on ratio of
surfactant to drug and speed of mixing, however, ratio of polymer to
drug and solvent/antisolvent ratio were also found to affect the particle
size.
The final mathematical model in terms of coded factors as determined
ð1Þ by Design-Expert software is shown below in Eqs. (2) and (3) for
responses Y1 and Y2, respectively.
Y1 ¼ þ360:75−13:65  A þ 17:63  B þ 2:68  C−48:25  D ð2Þ
Y2 ¼ þ24:31 þ 5:73  A−0:60  B þ 0:083  C þ 3:86  D: ð3Þ
A positive sign represents a synergistic effect, while a negative sign
indicates an antagonistic effect. In the case of Y1, negative coefficients
of A and D the model refers to decreased particle size at higher level
of surfactant to drug ratio and higher level of speed of mixing respec-
tively. Similarly, the positive coefficients of B and C indicate the increase
in particle size with increase in ratio of polymer to drug and solvent to
antisolvent ratio. Whereas for Y2 the negative coefficients of B refers
to decrease in the zeta potential as the concentration of polymer in-
creases. However, the positive coefficients of A, C and D refer to increase

Fig. 3. Zeta potential of glipizide suspension obtained with various stabilizers using liquid antisolvent precipitation method.
 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449 441

Table 6
Summary of ANOVA of Box–Behnken screening design batches using liquid antisolvent
precipitation technique.

Response variables Regression parameters P value

R2 Fcal

Particle size 0.8685 6.61 0.0006

Zeta potential 0.7200 11.23 b0.0001

and resist crystal growth. When two particles surrounded by an adsorbed

Fig. 4. Zeta potential of glipizide suspension obtained with various stabilizers using media
milling method.
in zeta potential with increase in surfactant concentration, solvent to
antisolvent ratio and speed of mixing.
A perturbation graph was plotted to find those factors that most
affect the response. A steep slope or curvature in a factor shows that
the response is sensitive to that factor. A relatively flat line shows insen-
sitivity to change in that particular factor. In the case of Y1 factors B and
D show a steep slope, and factors A and C also show a noticeable bend.
Whereas, in Y2 factors A and D show a steep slope, and factor B shows a
slight bend, whereas, factor C shows a flat line. Figs. 5 and 6 represents
perturbation plot for responses Y1 and Y2.
It was observed that the particle size d (90) decreases as the concen-
tration of sodium lauryl sulfate was increasing and at lower concentra-
tion of PVP K 30. PVP K 30 is vinyl pyrrolidone/vinyl acetate copolymer,
and is acknowledged to get adsorb on the surface of the drug particle
polymer layer approach each other, there will be a local increase in
polymer concentration in that region which results in an osmotic
pressure locally and leads to an increase in total potential energy which
might be responsible for prevention of agglomeration of the particles.
However, a further increase in the concentration of PVP K 30 could result
in a decrease in diffusion of the solvent towards the antisolvent caused by
the high viscosity of the solution, which in turn increases the mean
particle size.
At the same time the mean particle size was found to be decreased
because of the excellent dispersion property of SLS. SLS is an anionic
surfactant and due to its surface active property improves the wettabil-
ity of the drug powder in the dispersion medium which may contribute
to a reduction in mean particle size. It was also observed that when only
PVP K 30 was employed, the glipizide particles did not wet adequately
and particles floated on the aqueous vehicle while when SLS was
employed, it increased the wettability of glipizide particles and hence
showed better particle size reduction. When S/AS volume ratio was
increasing the mean particle size of glipizide was also increasing
irrespective of the fact that the concentration of surfactant is high.
This may be attributed to solvent mediated transformation during the
crystal growth that may lead to Ostwald ripening which in turn leads
to increase in particle size. At high surfactant concentration and low
speed of mixing, the larger particles were formed. At lower concentra-
tion of surfactant and high speed of mixing the mean particle size of

Table 5
Factor level and response data for BBD study (liquid antisolvent precipitation method).

Runs Factor-1 A. Ratio of Factor-2 B. Ratio of Factor-3 C. S/AS (ml) Factor-4 D. Speed Response-1 Response-2
surfactant to drug (g) polymer to drug (g) of mixing (rpm) PSD d (90) (nm) Zeta potential (mV)

1 5.0 5.0 60 4500 241 −30.0

2 7.5 3.75 90 3000 294 −29.3
3 2.5 2.5 60 3000 350 −15.2
4 2.5 3.75 60 4500 243 −23.1
5 2.5 3.75 30 3000 256 −20.3
6 5.0 2.5 90 3000 260 −26.0
7 5.0 3.75 90 1500 309 −28.9
8 5.0 3.75 60 3000 360 −27.5
9 5.0 2.5 60 4500 211 −26.8
10 2.5 3.75 90 3000 247 −13.0
11 2.5 5.0 60 3000 276 −18.1
12 7.5 3.75 60 4500 222 −31.6
13 7.5 5.0 60 3000 377 −29.9
14 5.0 3.75 60 3000 360 −27.9
15 5.0 2.5 60 1500 241 −19.3
16 5.0 5.0 60 1500 367 −18.2
17 7.5 3.75 30 3000 284 −28.2
18 5.0 5.0 30 3000 239 −24.1
19 5.0 3.75 60 3000 360 −27.9
20 5.0 2.5 30 3000 324 −23.2
21 2.50 3.75 60 1500 408 −15.1
22 5.0 3.75 30 4500 209 −27.1
23 5.0 2.75 30 1500 331 −18.5
24 5.0 5.0 90 3000 328 −16.3
25 5.0 3.75 90 4500 239 −28.9
26 7.50 3.75 60 1500 289 −21.2
27 5.0 3.75 60 3000 350 −27.9
28 5.0 3.75 60 3000 360 −27.9
29 7.50 2.50 60 3000 190 −33.3

g, gram; ml, milliliter; rpm, rotation per minute; nm, nanometer; mV, millivolts; PSD, particle size distribution; BBD, Box–Behnken design.
442 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449

Fig. 5. Perturbation graph for effect of individual factors on response Y1 using liquid antisolvent precipitation method.

glipizide is very less. The probable reason for this may be high
energy and shear force generated as a result of the impaction of
media with the drug which provides the energy inputs to break
the microparticulate drug into nanosized particulates. Thus a little
amount of surfactant at high speed of mixing was just sufficient to
reduce the particle size. The same statement is found true when it
was observed that the particle size at high speed of mixing was
significantly reduced irrespective of the concentration of PVP K 30.
It was also observed that at low S/AS volume ratio and high speed
of mixing the particle size reduction was more as compared to
high S/AS volume ratio and low speed of mixing.
Zeta potential is the potential at the hydrodynamic shear plane and
can be determined from particle mobility under an electric field; the
mobility will depend on surface charge and electrolyte concentration.
As can be seen the zeta potential value was increased at low level factor
B where the factor A was high. At low level of the factor B the particle of
drug was not covered so densely with PVP K 30. Due to which SLS
diffuses faster to the particle surfaces due to its excellent dispersion
properties. Adsorption of SLS on to the particulate surface leads to a
high zeta potential value. The zeta potential value of nanosuspension
decreased at high level of PVP K 30, irrespective of factor level A. At
this stage due to increased concentration of PVP K 30 in nanosuspension
its adsorption on drug particles increases which leads to a reduction of
the measured zeta potential. The adsorption layer of stabilizer shifts
the plane of shear, at which the zeta potential is measured, to a larger
distance from the particle surface. Consequently the measured zeta
potential is lowered. It was also observed that there is no much difference
in the zeta potential with increase or decrease in solvent/antisolvent
volume ratio and concentration of polymer. However, a slight decrease
in zeta potential is observed while increasing the polymer. An increase
in surfactant concentration and speed of mixing increased the zeta
potential. The probable reason for this may be that due to high speed of
mixing the adsorption of steric and electrostatic stabilizers was more
which increases the particle mobility and zeta potential value. However,
there was no much effect of solvent to antisolvent ratio on zeta potential
but the speed of mixing increased the zeta potential. Once again this may
be due to excellent adsorption of steric and electrostatic stabilizers on the
drug due to high speed of mixing.

Fig. 6. Perturbation graph for effect of individual factors on response Y2 using liquid antisolvent precipitation method.
 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449 443

Fig. 7. Overlay plot for optimized parameters of nanosuspension prepared by liquid antisolvent precipitation technique.

3.2.2. Optimization of formulation and processing parameters using
graphical optimization method
Optimization of nanosuspension was performed to find the levels of
factors A–D which gives particle size d (90) Y1 of 200–300 nm range
and Y2 of −4 to −32 mV range. Under this model predicted Y1 and Y2
in required range at A, B, C and D values of 0.25 g (ratio of surfactant to
drug), 0.50 g (ratio of polymer to drug), 300/3000 (solvent/antisolvent),
and 3000 rpm (speed of mixing), respectively for a batch size 4 g. By
using these values of factors three different batches of nanosuspensions
were prepared. Fig. 7 represents an overlay plot showing the optimized
parameters suggested by DoE software to get the responses in required
range.
3.3. Nanosuspension preparation by top down technology
3.3.1. Effect of independent factors on particle size distribution (Y1) and
zeta potential (Y2)
A total of 29 experiments were carried out to study the effect of
formulation and processing factors affecting the zeta potential and
particle size distribution of nanosuspension. Response data for all

Table 7
Factor level and response data for BBD study (media milling method).

Runs Factor-1 A. Ratio of Factor-2 B. Ratio of Factor-3 C. Milling Factor-4 D. milling Response-1 Response-2
surfactant to drug (g) polymer to drug (g) time (h) speed (rpm) PSD d (90) (nm) Zeta potential (mV)

1 5.0 3.75 6 4500 210 −22.5

2 7.5 5.0 4.5 3000 318 −16.6
3 5.0 3.75 3 4500 240 −4.32
4 5.0 2.5 4.5 4500 308 −28.1
5 5.0 3.75 4. 3000 292 −27.1
6 2.50 2.5 4.5 3000 352 −26.9
7 5.0 3.75 6 1500 243 −14.3
8 5.0 5.0 6 3000 292 −19.5
9 5.0 3.75 3 4500 240 −24.0
10 5.0 3.75 3 1500 432 −11.6
11 7.5 3.75 4.5 4500 251 −24.3
12 2.5 3.75 3 3000 271 −20.9
13 5.0 5.0 4.5 4500 220 −15.9
14 5.0 5.0 4.5 1500 330 −12.9
15 5.0 3.75 4.5 3000 292 −21.7
16 2.5 3.75 6 3000 235 −23.5
17 5.0 5.0 3 3000 410 −15.9
18 5.0 2.5 3 3000 268 −32.8
19 7.5 3.75 6 3000 232 −22.5
20 5.0 2.5 4.5 1500 415 −25.5
21 5.0 3.75 4.5 3000 292 −21.7
22 7.5 3.75 4.5 1500 302 −23.9
23 5.0 3.75 4.5 3000 283 −21.9
24 2.5 5.0 4.5 3000 366 −16.0
25 2.5 3.75 4.5 1500 341 −17.2
26 5.0 2.5 6 3000 209 −28.2
27 5.0 3.75 4.5 3000 292 −21.7
28 7.5 3.75 3 3000 245 −18.5
29 7.5 2.5 4.5 3000 302 −24.2

g, gram; h, hours; rpm, rotation per minute; nm, nanometer; mV, millivolts; PSD, particle size distribution; BBD, Box–Behnken design.
444 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449
Table 8
Summary of ANOVA of Box–Behnken screening design batches using media milling
technique.
Response variables Regression parameters P value
R2 Fcal
Particle size 0.4767 5.47 0.0028
Zeta potential 0.4984 5.96 0.0018
experimental runs of Box–Behnken experimental design is presented
in Table 7. The responses obtained in this study are also well modeled
by a linear function of the independent variables and thus first order
polynomial equation as shown in Eq. (1) was used for approximating
the function. The values of response was used for Y1 (particle size d
(90) and Y2 (zeta potential) ranges from 209 to 432 nm and −4.32 to
− 32.8 nm, respectively. The ratio of maximum to minimum for both
responses Y1 and Y2 is 2.06 and 7.59, respectively; therefore power
transformation was not applied to the obtained values. ANOVA was
applied to determine the significance and the magnitude of the effects
of the main variable and their interaction in the regression model
obtained was used to generate the counter plots for independent
factors. The details of ANOVA for responses Y1 and Y2 are mentioned
in Table 8 which confirms the adequacy of linear model (model
prob N F is less than 0.05). It also identifies the significant factors that
affect the responses Y1 and Y2 of nanosuspension. For particle size
distribution (Y1) milling time and milling speed (factors C and D)
were identified as significant model terms whereas for zeta potential
(Y2) ratio of polymer to drug and milling speed (factors B and D)
were identified as significant model terms. The final mathematical
model in terms of coded factors as determined by Design-Expert
software is shown below in Eqs. (4) and (5) for responses Y1 and Y2
respectively
Y1 ¼ þ292:69−12:50  A þ 6:42  B–37:08  C–49:50  D ð4Þ
Y2 ¼ þ20:65 þ 0:18  A–5:78  B þ 2:13  C þ 1:21  D: ð5Þ
Figs. 8 and 9 represent perturbation plot for responses Y1 and Y2. In
the case of response Y1 factors C and D show a steep slope and factors A
Fig. 8. Perturbation graph for effect of individual factors on response Y1 using media milling method.
and B show a noticeable bend. Whereas, in the case of response Y2
factor B shows a steep slope, factors C and D exhibit a slight slope or a
noticeable bend and factor A shows a flat line.
The particle size distribution (PSD) d (90) response surface varies in
linear pattern with increasing milling speed and milling time. PSD d
(90) of nanosuspension decreases as the milling time and milling speed
increases. The minimum value of PSD d (90) 209 nm is at the higher
milling time and milling speed. The probable reason for this may be
high energy and shear forces generated as a result of the impaction of
the milling media with the drug which provides the energy input to
break the microparticulate drug into nano-sized particles. As can be
seen, the PSD d (90) of nanosuspension decreases at the lower polymer
concentration. The probable reason for this may be, at lower polymer
concentration collision of drug particles due to high impaction of
milling media is increased which in turn decreases the PSD d (90)
of nanosuspension. Moreover, at higher polymer concentration
the viscosity of polymer increases drastically which hinders the
processing of nanosuspension on bead mill. Issues such as increases
in product temperature and increase in pressure on the bead mill
were also observed during manufacturing nanosuspension with
high polymer to drug ratio and high milling speed. These state that
nanosuspension with high polymer to drug ratio to be milled at
higher milling speed is not suitable for manufacturing and scale
up. In view of this an attempt was carried out to overcome this
issue by operating the bead mill of lower speed of initial 10 min
and further increasing the speed slowly. The milling time shows a
prominent effect on the PSD d (90) of nanosuspension even at high
polymer concentration. The particle size of nanosuspension decreases
with the increase in concentration of surfactant and milling time. The
same case was observed when the particle size decreases with increase
in concentration of surfactant and milling speed. Table 7 reveals that
the particle size decreases at low level of polymer and high level of
surfactant and increases at high level of polymer.
The zeta potential values increases at low level of factor A where the
level of factor B is high. PVP K 30 is a non-ionic polymer and SLS is an
anionic surfactant. At low level of factor B particle surface of drug is not
covered so densely with PVP K 30, due to which SLS diffuses faster to
the particle surfaces as it has excellent dispersion properties. Adsorption
of SLS onto the particle surface leads to high zeta potential value. The
zeta potential value of nanosuspension decreases at level of factor B,
 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449 445

Fig. 9. Perturbation graph for effect of individual factors on response Y2 using media milling method.

irrespective of factor level A. At this stage due to increased concentration
of PVP K 30 in nanosuspension its adsorption on drug particles increases
which leads to reduction of the measured zeta potential. The adsorption
layer of the stabilizer shifts the plain of shear at which the zeta potential
is measured to a larger distance from the particle surface. Consequently
the measured zeta potential is lower. In such cases zeta potentials
of about ± 20 mV are still sufficient to fully stabilize the system in
combination with steric stabilization [12]. It was also observed that
zeta potential value increases at high milling speed and lower polymer
concentration. The probable reason for this may be that due to high
milling speed the adsorption of steric and electrostatic stabilizers is
more which increases the particle mobility and zeta potential value.
The zeta potential value increases at low level of factor B (polymer
concentration) where the level of factor C is high. The zeta potential
value increases with increase in concentration of factor A and milling
speed (D). The same response was observed when the milling speed
and milling time are kept high.
3.3.2. Optimization of formulation and processing parameters using
graphical optimization method
Optimization of nanosuspension was performed to find the levels
of factors A–D which gives Y2 in − 20 to − 25 mV range and Y1
(d(90)) of 250–300 nm. Under this the model predicted Y1 and Y2 in
required range at A, B, C and D values of 0.50 g, 0.38 g, 6 h, 4500 rpm,
respectively for a batch size of 4 g. By using these values of factors
three different batches of nanosuspensions were prepared. Fig. 10
represents and overlay plot showing the optimized parameters
suggested by DoE software to get the responses in required range.

Overlay Plot
5.0

Particle Size: 246

4.5
B: ratio of polymer to drug

4.0

3.5

particle size: 292.69 Zeta Potential: 28.0

3.0
zeta potential: 20.649
X1 5.00
X2 3.75
2.5

2.5 3.5 4.5 5.5 6.5 7.0

A: ratio of surfactant to drug

Fig. 10. Overlay plot for optimized parameters of nanosuspension prepared by media milling technique.
446 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449

Fig. 11. Mean dissolution profiles of pure glipizide and glipizide nanosuspension prepared by liquid antisolvent precipitation method and media milling respectively.

3.4. In vitro dissolution studies
In order to ascertain whether the goal of improving the rate of
dissolution of glipizide is achieved, the results of in vitro dissolution of
different glipizide samples are shown in Fig. 11. The rate of dissolution
of pure glipizide was very low: only 20.17% of the drug was dissolved in
the first 10 min; only 68.33% of the glipizide was dissolved after 60 min.
The formulation of glipizide nanosuspensions significantly improved
the dissolution rate, since almost 100% of the drug was dissolved in the
first 10 min for both the nanosuspensions prepared by liquid antisolvent
precipitation method as well as by media milling method, respectively.
The surface-active agents may have contributed to the increase in disso-
lution rate due to the improved wettability and solubility of the drug.
Moreover, the zeta potential of optimized batch of nanosuspension
was found to be − 22 mV for liquid antisolvent precipitation method
and − 23.6 mV for media milling method, as they are stabilized by
steric and electrostatic stabilizers which suggests that no aggregation of
nanoparticles took place.
3.5. Powder X-ray diffraction studies
3.5.1. Powder X-ray diffraction studies of nanosuspensions prepared by
liquid antisolvent precipitation technique
As shown Fig. 12a, the diffraction pattern of glipizide powder
revealed several sharp high intensity peaks at diffraction angles (2θ)
of 7.5°, 20.5°, 21°, 22°, 23.5°, 29°, 32° and 33°, respectively, suggesting
that the drug existed as crystalline material. While in glipizide nanopar-
ticles of optimized batch the peaks are almost similar in intensity and
position, indicating that the crystallinity of the drug is intact (Fig. 12b)

3000 3000
a b

2000 2000
Counts
Counts

1000 1000

0 0
20 30 40 50 60 70 80 20 30 40 50 60 70
2000 2000
c d
1500 1600

1200
Counts

1000
Counts

800 800

400 400

0 0
20 30 40 50 60 70 80 20 30 40 50 60 70 80
Degrees 2-Theta Degrees 2-Theta

Fig. 12. X-RD pattern of a. pure glipizide b. nanosuspension prepared by liquid antisolvent precipitation technique c. nanosuspension prepared by media milling at high speed and d.
nanosuspension prepared by media milling at low speed.
 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449 447

Fig. 13. Mean dissolution profiles of fresh and aged glipizide nanosuspensions prepared by liquid antisolvent precipitation method and media milling respectively at different
stability conditions.

and increased dissolution as seen from dissolution studies could be
attributed to a decrease in particle size from micrometer to nanometer.
3.5.2. Powder X-ray diffraction studies of nanosuspensions prepared by top
down media milling technique
The powder X-ray diffraction study of spray-dried nanosuspension
operated at high milling speed and low milling speed showed no
significant shift in the main peaks when compared with pure drug
(Fig. 12a). The characteristic peaks for milled drug were observed at
same 2θ value as those of unmilled drug. A slight decrease in intensity
of peaks was observed with spray-dried nanosuspension operated at
higher milling speed. Fig. 12c and d shows the X-RD pattern of unmilled
drug, spray-dried nanosuspensions at low speed, and spray-dried
nanosuspensions at high speed.
3.6. Stability studies
In order to study the effect of aging on dissolution profile of
glipizide nanosuspension, the nanosuspension was kept at 4 °C,
25 °C and 40 °C/75% RH for 6 months. Then, dissolution rate was mea-
sured. The results revealed that the dissolution rates of nanosuspension
prepared by top down media milling method were not affected by the

Fig. 14. Effect of storage on glipizide nanosuspensions prepared by liquid antisolvent precipitation technique: a. 4 °C, b. 25 °C, and 40 °C/75% RH up to 180 days (6 months).
448 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449
Fig. 15. Effect of storage on glipizide nanosuspensions prepared by media milling technique: a. 4 °C, b. 25 °C, and 40 °C/75% RH up to 180 days (6 months).
effect of aging, as there was no significant difference (P N 0.05) in
dissolution rates of aged nanosuspension compared with the fresh
nanosuspension (P value 0.49 at 4 °C, 0.48 at 25 °C and 0.32 at
40 °C/75% RH respectively, which were greater than 0.05). Fig. 13
shows the dissolution profile of fresh and aged nanosuspensions.
Although the aged nanosuspension appears to have lower dissolu-
tion rate than the fresh nanosuspension in the graph, similarity
factor (F 2 ) of the two release profiles was 77.51 at 4 °C, 55.93 at
25 °C and 51.51 at 40 °C/75% RH respectively, indicating acceptably
similar profiles (if F2 ≥ 50 the data is considered similar).
Also, there was no much change observed in the dissolution profiles
of nanosuspensions prepared by bottom up liquid antisolvent precipita-
tion technique kept at 4 °C and 25 °C, but a drastic decrease in drug
release was observed with the nanosuspensions kept at 40 °C/75% RH.
There was a significant difference (P b 0.05) found in the dissolution
profile of fresh and aged nanosuspension prepared by bottom up liquid
antisolvent precipitation technique kept at 40 °C/75% RH (Fig. 13). The
decrease in dissolution profile may be attributed to increase in the
particle size of the nanosuspensions due to Ostwald ripening. The P
values for the release profiles of fresh and aged nanosuspensions
prepared by antisolvent precipitation technique kept at 4 °C, 25 °C
and 40 °C/75% RH are 0.49, 0.48 and 0.03 (P b 0.05). The F2 values for
the release profiles of fresh and aged nanosuspensions prepared by
antisolvent precipitation technique kept at 4 °C, 25 °C and 40 °C/75%
RH are 73.57, 62.67 and 3.66 (F2 value less than 50), respectively.
The above findings were further supported by the particle size
analysis of fresh and aged nanosuspensions kept at different stability
conditions. The change in the particle size of nanosuspensions was inves-
tigated as a function of time and temperature to determine the efficiency
of preparation process to prepare stable nanoparticles with no Ostwald
ripening. Fig. 14 shows the effect of storage on glipizide nanosuspensions
at 4 °C (Fig. 14a), 25 °C (Fig. 14b) and 40 °C/75% RH (Fig. 14c) prepared
by liquid antisolvent precipitation method. Fig. 15 shows the effect of
storage on glipizide nanosuspensions at 4 °C (Fig. 15a), 25 °C (Fig. 15b)
and 40 °C/75% RH (Fig. 15c) prepared by media milling method. Increase
in particle size d (90) was observed in all the formulations with the great-
er increase at 40 °C/75% RH as compared to 4 °C and 25 °C respectively.
Agglomeration, crystal growth due to Ostwald ripening or both may be
the contributing factors. It is important to note that there was negligible
increase in particle size of nanosuspensions prepared by either liquid
antisolvent precipitation method or media milling method at 4 °C and
25 °C up to 6 months (180 days). However, a drastic increase in particle
size was observed in nanosuspension prepared by liquid antisolvent
precipitation technique (Fig. 14c) at 40 °C/75% RH after 6 months. The
particle size of initial nanosuspension was 244.32 nm and changed to
826.35 nm after 6 months. This showed that liquid antisolvent precipita-
tion technique failed to prevent Ostwald ripening under accelerated
conditions. This increase in particle size could be the reason for decreased
% drug release of nanosuspensions prepared by liquid antisolvent
technique kept at 40 °C/75% RH (Fig. 13). However, there were no signif-
icant/drastic increase in particle size observed in nanosuspensions
prepared by top down media milling method at any of the aforemen-
tioned storage conditions. This shows the potential of media milling
method to prevent Ostwald ripening or particle agglomeration and thus
formation of stable nanosuspension.
4. Conclusion
Glipizide nanoparticles were prepared by both liquid antisolvent
precipitation and media milling technique. The Box–Behnken design
has been applied successfully to optimize formulation and process
parameters for nanosuspensions. From this study it was concluded
that polymer concentration (ratio of polymer to drug), milling time
and milling speed played significant role in controlling the zeta poten-
tial of nanosuspension prepared by media milling. On the other hand
milling time and milling speed were considered to be significant factors
for controlling particle size distribution d (90) of nanosuspension. In the
bottom up process surfactant concentration (ratio of surfactant to drug)
and speed of mixing played a significant role in controlling the zeta
potential of glipizide. On the other hand polymer concentration (ratio
 K.V. Mahesh et al. / Powder Technology 256 (2014) 436–449
of polymer to drug) and speed of mixing were considered to be the
significant factors that affect the particle size distribution d (90) of
nanosuspension. The graphical optimization method helped in finding
the sweet spot or design space to get nanosuspension with desired
physicochemical properties. Both the top down and bottom up process-
es give similar initial formulations with comparable stability at lower
temperatures (at 5 °C and 25 °C for 6 months). However, an increase
in particle size distribution (PSD d (90)) was observed with the
optimized nanosuspension prepared by liquid antisolvent precipitation
method when tested under accelerated conditions (40 °C at 75% RH for
6 months). This increase in particle size has also influenced the dissolu-
tion profile of the nanosuspension (i.e. drug release was observed 100%
in the first 10 min for fresh nanosuspension and 38.42% in the first 10
min for aged nanosuspension). The increase in particle size (PSD d
(90)) was also observed with media milling but there was very
less increase and it did not alter the dissolution profiles of the pre-
pared nanosuspensions. This revealed that the preparation of
nanosuspension of glipizide by wet media milling method may pro-
duce more stable nanoparticles as compared to liquid antisolvent
precipitation technique. The X-ray diffraction data shows no form
conversion of the drug due to processing parameters involved in
the production of nanosuspension and thus it can be concluded
that the enhancement of dissolution of optimized formulations
(100% in the first 10 min for both the process) as compared to pure
glipizide (20.17% in the first 10 min and 68.33% in 60 min) is only
due to reduction of particle size in nanorange. Thus this improved
formulation could offer an improved drug delivery strategy which
might allow the concomitant use of glipizide with food that still
needs to be correlated with in vivo studies.
Acknowledgments
We are grateful to shree Ashok Mittal, Honorable Chancellor, Lovely
Professional University, Punjab, India for providing facilities to carry
out the aforementioned research. We also owe our thanks to Micro
Laboratories, Baddi, India, for providing the gift samples of glipizide.
We also thank Dr. Gaurav Jain, assistant professor, Jamia Hamdard,
India and Dr. Surajpal Verma, assistant professor, Lovely Professional
University for helping us in carrying out zeta potential and particle size
distribution (PSD d (90)) studies.
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