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Formulation Development and Taste Masking

of Rifaximin Nanosuspension
ARTICLE JANUARY 2013

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RESEARCH ARTICLE

Formulation Development and Taste Masking of Rifaximin

Nanosuspension
M A M Danish1*, Azhar Shetsandi1, Kiran S Bhise1
Abstract: The aim of this study was to prepare and characterize rifaximin nanosuspensions to mask bitter taste and enhance the
solubility of this drug. Nanosuspensions were prepared by the precipitation ultrasonication method. The effects of two
important process parameters, i.e. the concentration of PVA in the anti-solvent and the time length of ultrasonication on the
particle size of nanosuspensions were investigated systematically and the optimal values were 0.15% and 15 min, respectively.
The particle size and zeta potential of nanocrystals were 129 nm and 23.9 mV, respectively. The morphology of nanocrystals
was found to be flaky and spherical in shape by scanning electron microscopy (SEM) observation. The X-ray powder diffraction
(XRPD) and differential scanning calorimetry (DSC) analysis indicated that there was no substantial crystalline change in the
nanocrystals compared with raw crystals. The taste of rifaximin was significantly masked and its solubility increased by
reducing the particle size.

size of the particles produced after precipitation, usually

with a rapid growth rate and, therefore, they are large and
have a broad particle size distribution (PSD). Recently, the
control of particle size and PSD has become relatively easy
by using a static mixer [6] and a confined impinging jet
reactor. These methods are all based on the classical theory
of nucleation and crystal growth and they have been
described in detail. [7] In the last decade, ultrasound has
received much attention and has been used as an effective
method of controlling the nucleation and crystallization
process Ultrasound irradiation has been proved to be a
feasible mixing method and it can intensify mass transfer
and accelerate molecular diffusion. [8] Rifaximin is a
Antibiotic, Antiinfective Agent, semi synthetic, rifamycinbased non-systemic antibiotic, meaning that the drug will
not pass the gastrointestinal wall into the circulation as is
common for other types of orally administered antibiotics.
It is used to treat diarrhea caused by E. coli. Rifaximin is
classified as a Class IV API (poorly soluble and highly
permeable) by the Biopharmaceutics Classification System
(BCS). Rifaximin have very bitter taste so having less
patient compliance. The absolute oral bioavailability of this
drug is reported to range from about 10% to 20%,
depending in part on the dosage form. The dissolution is
the rate-limiting factor for absorption. In this article, stable
nanosuspensions were prepared by the precipitation
ultrasonication method. The effects of the process
parameters, such as the concentration of PVA in antisolvent
and the time length of ultrasonication on the particle size of
the nanosuspension were investigated systematically. The
corresponding physical properties of the prepared
rifaximin nanocrystals were characterized by scanning
electronic microscopy (SEM), X-ray powder diffraction
(XRPD) and differential scanning calorimetry (DSC). The
nanosuspension was also evaluated by particle size
distribution and zeta potential distribution. [9]

INTRODUCTION
A surprisingly large proportion of solid active
pharmaceutical ingredients (API) have poor aqueous
solubility and therefore poor bioavailability. The
bioavailability of these so-called brick dust API can now is
improved by formulating them as nanosuspensions. [1] The
dissolution rate of API is proportional to the surface area
available for dissolution as described by the Noyes
Whitney equation and, in addition to the dissolution rate
enhancement, an increase in the solubility of nanosized API
is also expected as described by the OstwaldFreundlich
equation. [2] The nanocrystals can be obtained either by
particle size reduction of larger crystals (top-down
approach) such as high-pressure homogenization [3] and
media milling or by building up crystals by precipitation of
dissolved molecules (bottom-up approach). [4] The topdown approach, however, is more frequently used, and this
approach requires high energy and expensive equipment.
Regarding the bottom-up method, in the last decade,
supercritical fluid-based techniques have been widely
investigated to obtain nanosized drug particles, such as
supercritical anti-solvent precipitation (SAS)) or rapid
expansion of a supercritical solution into a liquid solvent
(RESOLV). [5] However, these methods are difficult to
control and scale up and are expensive. Anti-solvent
precipitation is an effective way to prepare micro or nanosize drug particles. In this method, briefly, the drug is first
dissolved in the solvent, then; the drug solution is quickly
introduced into the anti-solvent. Precipitation occurs
immediately by a rapid desolvation of the drug. [6] Aqueous
solutions containing some stabilizers, such as polymers and
surfactants, are commonly used as the anti-solvent.
Polymers, such as hydroxyl propyl methylcellulose (HPMC)
and methylcellulose (MC), [6] and polyvidone (PVP), can
form strong hydrogen bonds with the drugs, which can be
adsorbed on the hydrophobic particle surface, inhibiting
the crystal growth of the drugs. However, there are some
basic problems associated with common anti-solvent
precipitation techniques, i.e. it is difficult to maintain the

MATERIALS AND METHODS

Materials
RFX (RFX) was received as gift sample from Lupin
pharmaceutical (India), polyvinyl alcohol (PVA, Mw 3070
kDa, 88% alcoholysis) PEG 200 and acetone of analytical
grade were purchased from Luba chemicals (MH).

M. C. E. Societys Allana College of Pharmacy, Pune-411001, Maharashtra,

India.
E-mail: danishbagban@gmail.com
*Corresponding author
1

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RESEARCH ARTICLE
Methods
1. Preparation of RFX Nanosuspensions
RFX nanosuspensions were prepared by the precipitation
ultrasonication method. Briefly, RFX was dissolved in a
mixed solvent of PEG 200 and acetone (ratio of 1:1, v/v) to
form a series of organic solutions containing 200 mg/ml of
drug. PVA was dissolved in water to obtain a series of antisolvents with the concentrations of 0.15%, 0.25%, 0.50%,
1.0% and 1.5% (w/v). Both solutions were passed through
a 0.45 m millipore filter paper. The anti-solvent was
cooled to below 3C in an ice-water bath. Then, 2 ml of
organic solution was quickly introduced into 20 ml of the
pre cooled anti-solvent at a stirring speed of 400 rpm. After
thaximine anti-solvent precipitation, the samples were
immediately transferred to a test tube (2 cm in diameter
and 10 cm in length) and treated with an ultrasonic probe
at ultrasonic power inputs 150 W for different time lengths
( 10, 15 and 30 min).
The probe with a tip diameter of 8mm was immersed 10
mm in the liquid, resulting in the wave traveling
downwards and reflecting upwards. The period of
ultrasound burst was set to 3 sec. with a pause of 3 sec.
between two ultrasound bursts. During the process, the
temperature was controlled using an ice-water bath. The
obtained nanosuspensions were concentrated by
centrifugation at 16,000 rpm for 40 min using an
ultracentrifuge. After the centrifugation, the supernatant
was replaced with 2ml of 0.2% PVA solution. The solid
residue was redispersed using a bath sonicator and the
final drug content was adjusted to 20 mg/ml (drug
weight/volume of nanosuspension) using an appropriate
volume of 0.2% PVA solution. Different process parameters
were systematically investigated by 32 factor method to
clarify their effects on the particle size of nanocrystals. The
process parameters included the concentration of PVA in
the anti-solvent and time length of ultrasonication. For
long-term stability, RFX nanosuspensions were lyophilized.
The nanosuspensions were rapidly frozen in liquid nitrogen
and freeze-dried in a FD-1C-50 freeze-drier for 15 h.

rotating paddle. Accurately measured nanosuspension

liquid equivalent to 200 mg of drug dose was put into a
treated cellophane membrane which has been boiled and
equilibrated in 0.1 M Sodium Lauryl Sulphate The bag was
secured with two knots at each end and space was
minimized as far as possible and was immediately placed
into release apparatus containing 900 ml of the 6.8
phosphate buffer with addition of 1.5% SLS and kept at
370.5C with paddle stirring rate of 100 rpm. One ml
sample was pipette out from the release medium at the
time interval. 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 420
and 480min. and the volume of the samples were replaced
by the fresh buffer to maintain sink condition. The samples
were analyzed by UV-spectrophotometrically at 436 nm.

2. Charactrization of Rfx Nanosuspension

a. Entrapment Efficiency
The % entrapment efficiency (% EE) of RFX in the
nanosuspension was determined after sonication. For the
removing free RFX the nanosuspension was subjected to
centrifugation on a cooling ultracentrifuge at 1600 rpm for
45 min. The clear supernatant was siphoned off to separate
the unentrapped drug. One ml of supernatant was taken
and diluted with PEG-acetone system up to 10 ml and
absorbance was recorded at 436 nm using UV
spectrophotometer.

e. SEM Studies of Nanosuspension for Surface

Morphology
SEM studies were performed for the nanosuspension. The
lyophilized powder of nanosuspension was spread on a
double-sided adhesive plate one side of which was stuck to
a glass slide. Excess powder was removed and the slide
was kept on the sample holder and the scanning electron
micrographs were taken using an electron microscope
JEOL- JSM- 6360A. (Welsh and Rhodes, 2001 have reported
that these studies can be used to study the surface
morphology of the dry nanosuspension powders). The SEM
of nanosuspension at magnification 1000X has been
depicted in Result and Discussion.

c. Particle Size Analysis

Mean particle size and size distribution of optimized batch
of SLN was determined by dynamic light scattering using
Zetasizer Ver. 6.34 (Malvern instrument Ltd., Malvern, UK)
at room temperature. Before measurement, batches were
diluted with filtered double distilled water until the
appropriate concentration of particles was achieved to
avoid multi-scattering. The diluted sample was filled in the
Disposable transparent sizing cuvette. The size was
measured at 25C. The dispersant Refractive index and
Material refractive index was 1.330 and 1.59 respectively.
The analysis was performed to obtain the Z-average size
and the polydispersity index value. The width of the size
distribution was indicated by the polydispersity index (PI).
The particle size analysis data were evaluated using
volume distribution to detect even a few large particles.
d. Zeta Potential
Charge on drug loaded droplet surface was determined
using Zetasizer Ver. 6.34 (Malvern Instruments Ltd.,
Malvern, UK). Analysis time was kept for 60s and average
ZP, charge and mobility of optimized batches of polymeric
nanoparticles was determined. All measurements were
done at 25C.

DrugEntrapment%
Massofdruginnanoparticle

Massofdrugusedinformulation

f. Powder X-Ray Diffractometer

RFX and formulation were subjected to PXRD using X-Ray
Generator, D8 Advance, Germany. To study X-Ray
Diffraction pattern, the sample was placed into aluminum
holder and the instrument was operated between initial

b. Cumulative % Drug Release

The release of RFX from nanosuspension was determined
using an USP dissolution apparatus II equipped with

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RESEARCH ARTICLE

Figure 1: Cumulative % drug release study

Figure 2: Powder X-Ray Diffraction of RFX, nanosuspension

Figure 3: Scanning electron microscopy

Figure 4: Differential scanning calorimeter (DSC)

Figure 5: FT-IR spectroscopy

and final 2 angle of 5-50 respectively in an increment of

0.1 2.

whereas others over the range of 10-100C. Peak

transitions and enthalpy of fusion were determined for the
samples using TA 60 integration software.

g. Differential Scanning Calorimeter (DSC)

The thermal behavior of RFX was examined by DSC, using a
Shimadzu DSC-60 Differential Scanning Calorimeter. The
system was calibrated with high purity sample of Indium.
RFX and formulation were scanned at a heating rate of
10C/min over a temperature range of 50 to 300C

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h. FT-IR Spectroscopy
Fourier Transform Infrared Spectroscopy (FT-IR) of the
Nanosuspension was conducted using Jasco FTIR 4100
spectrophotometer (Jasco Tokyo Japan) and the spectrum
was recorded in the wavelength region of 4000-400cm-1

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RESEARCH ARTICLE

Figure 6: Size distribution by Intensity

Figure 7: Zeta potential distribution

Figure 8: 3D surface plot for drug release

Figure 9: 3D surface plot for entrapment efficiency

Table 1: % Entrapment Efficiency and Cumulative % Drug Release of Factorial Batches

S. No.
1
2
3
4
5
6
7
8
9

Batches
FA1
FA2
FA3
FB1
FB2
FB3
FC1
FC2
FC3

% Entrapment Efficiency*
330.22
450.28
570.21
710.14
830.17
840.22
400.91
500.97
360.55

the FTIR spectrum was taken by mixing very small amount

of the formulation with previously dried KBr at 160C for
30 min in the ratio of 1:3.

The dissolution rate was carried out same as that of trial

formula. The cumulative % drug release study indicated
that after 7 hrs the 81.083 release is obtained by
nanosuspension.

i. In-vitro Evaluation of Bitter Taste of Nanosuspension

Nanosuspension lyophilized powder (equivalent to 8 mg of
RFX) were placed in volumetric flask with 25 ml of
phosphate buffer pH 6.8 and stirred for 5 min. The mixture
was filtered and filtrate was analyzed for RFX
concentration at 430 nm by UV-visible spectrophotometer
and that was compared with the threshold value.

% Entrapment Efficiency
The % Entrapment efficiency is carried out same as of trial
formula. An obtained result is 850.37.
Powder X-Ray Diffraction of RFX Nanosuspension
PXRD of RFX that exhibited numerous peaks at 2 value of
11.2, 11.8, 12.1 and 22.3 confirming the RFX to be
Amorphous in nature. Whereas nanosuspension
lyophilized powder exhibited characteristic peaks at 2

RESULT AND DISCUSSION

Cumulative % Drug Release Study

Inventi Rapid: Pharm Tech Vol. 2013, Issue 4

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Cumulative % Drug Release*

53.6790.21
55.7600.46
65.1610.56
64.4890.54
72.8360.91
80.1240.40
51.3570.32
55.2360.21
59.5750.81

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RESEARCH ARTICLE
Table 2: Size Distribution by Intensity

Z-Average (r.nm):
PdI:

201.3
0.586

Size (r.nm)
124.2
533.9

Peak 1:
Peak 2:

% Intensity
60.3
35.9

Width (r.nm)
39.66
191.6

Table 3: Zeta Potential Distribution

Zeta Potential (mV):

Zeta Deviation (mV):
Conductivity (mS/cm):

-23.9
6.34
0.201

Mean (mV)
-23.9
0.00
0.00

Peak 1:
Peak 2:
Peak 3:

values of 15.42, 16.98, 18.9 and 20.94. X-ray diffraction has

been used to analyze potential changes in the inner
structure of RFX crystals. It was confirmed that no
crystalline change was found in the nanocrystals, because
their powder X-ray diffraction patterns were consistent
with the pattern of the raw crystals and spherical crystal.
However, the differences in the relative intensities of their
peaks might be attributed to the differences in the
crystallinity of the samples.

Width (mV)
6.34
0.00
0.00

CONCLUSION
From the present study it can be concluded that, the
nanosuspension of RFX can be formulated which can be
converted to lyophilized powder for long term stability.
Formulation of RFX into nanosuspension mask its bitter
taste, enhances the solubility and dissolution as well as
permeability. Concentration of nano sized particles has
marked effects on the dissolution of RFX from
nanosuspension.

Scanning Electron Microscopy

The morphology of nanocrystals was found to be spherical
and flaky in shape by scanning electron microscopy (SEM)
observation. The surface topography of spherical crystals
showed agglomerates of small crystal nuclei by SEM
observation.

REFERENCES AND NOTES

1. Barrett E Rabinow. Nanosuspensions in drug delivery. Nature
Reviews Drug Discovery, 3:785-796, 2004.
2. Raval Patel. Preparation and Characterization of Nanoparticles
for Solubility and Dissolution Rate Enhancement of
Meloxicam. Intl R J of Pharmaceuticals, 1(2):42-49, 2011.
3. RA Nash. Suspensions. Encyclopedia of pharmaceutical
technology, 2:2045-3032 ,2002,
4. Huabing Chen, Chalermchai Khemtong. Nanonization
strategies for poorly water-soluble drugs. Drug Discovery
Today, 2010.
5. Atul Pathak, Suresh P Vyas. Nano-vectors for efficient liver
specific gene transfer. Int J Nanomedicine, 3(1):3149, 2008.
6. Jianxin Zhanga, Matthew Bunkera. Nanoscale thermal analysis
of pharmaceutical solid dispersions. International Journal of
Pharmaceutics, 380:170173, 2009.
7. Y Sugimoto, P Pou, M Abe, P Jelinek, R Perez, S Morita and O
Custance. Nanosuspensions. Nature, 446:64-68, 2007.
8. Dengning Xiaa, Peng Quan. Preparation of stable nitrendipine
nanosuspensions using the precipitationultrasonication
method for enhancement of dissolution and oral
bioavailability. European Journal of Pharmaceutical Sciences,
40:325334, 2010.
9. Robert Steffen M D, David A Sack. Therapy of Travelers
Diarrhea With Rifaximin on Various Continents. The American
Journal of Gastroenterology, 98:5-9, 2003

Differential Scanning Calorimeter (DSC)

In order to further confirm the physical state, DSC was also
performed to analyze the different samples. In each case, a
DSC scan of each sample showed a single sharp
endothermic peak described to the melting of the drug
which also indicated that there was no substantial
crystalline change. However, the melting point of the
nanocrystals and spherical crystals was lower than that of
the raw crystals and this might be due to the size reduction
in the crystals.
FT-IR Spectroscopy
The FT-IR of RFX nanosuspension showed all the peaks of
drug and Excipients.
In-vitro Evaluation of Bitter Taste of Nanosuspension
For drug concentration analysis sample subjected to UV
and drug concentration in filtrate after 5 min of stirring
was found to be 5.8 g.

Acknowledgments
Authors are thankful to Lupin limited, Pune, for the gift sample
of RFX.

Size Distribution by Intensity

Mean particle size of optimized batch was found to be
124.2 nm.

Cite this article as: M A M Danish, Azhar Shetsandi,

Kiran S Bhise. Formulation Development and Taste
Masking of Rifaximin Nanosuspension. Inventi Rapid:
Pharm Tech, 2013(4):1-5, 2013.

Zeta Potential Distribution

Zeta potential of optimized batch was found to be -23.9
(mV).

Inventi Rapid: Pharm Tech Vol. 2013, Issue 4

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Area (%)
100.0
0.0
0.0

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