FORMULATION AND IN-VITRO CHARACTERIZATION OF RISPERIDONE

NANOSUSPENSIONS FOR THE ENHANCEMENT OF DRUG RELEASE RATE

Abstract
Poorly water-soluble compounds are difficult to develop as drug products using conventional
formulation techniques and are frequently abandoned early in discovery. In the present study,
nanosuspensions of risperidone were prepared by nanoprecipitation method for the improvement
of solubility and dissolution rate. Through this process the particle size of risperidone can be
obtained in the nano-size range, by adjusting the operation parameters, such as the stabilizer
concentration. The optimized formulation of nanosuspension obtained is with a stabilizer PVA.
The dissolution of nanosized risperidone is significantly enhanced compare with the pure drug.
The method was evaluated in comparison with the well known solvent diffusion process for the
efficiency. Control of the preparation variables (stabilizers, drug content, homogenization
procedure and cooling conditions) allowed formation of nanosuspensions with diameters less
than 300 nm. The major advantage of the nanoprecipitation method over the conventional
methods is the avoidance of organic solvents during production, although the mean particle size
is slightly greater. The formulations of PVA and Tween 80 as stabilizers yielded
nanosuspensions with the smallest average particle size. The formulation of risperidone as a
nanosuspension, either in the form of lyophilized powder or granules, was very successful in
enhancing dissolution rate, more than 95% of the drug being dissolved in the 60 min compared
to less than 15% of the micronized drug. The increase in in-vitro dissolution rate may favourably
affect bioavailability and improve safety for the patient by decreasing gastric irritancy.

Keywords: Dissolution, nanosuspension, precipitation, risperidone, solubility

Introduction
The design and formulation of a dosage form require consideration of the physical, chemical, and
biological characteristics of all the drug substances and pharmaceutical ingredients to be used in
its preparation. An important property of a drug substance is solubility, especially aqueous
system solubility1. One of the critical problems associated with poorly soluble drugs is too low
bioavailability and erratic absorption because of their low dissolution rates 2. The solubility
dissolution behavior of a drug is a key factor to its oral bioavailability. Nanotechnology can be
used to solve the problems associated with these conventional approaches for solubility
dissolution and bioavailability enhancement. In Nanosuspension technology, the drug is
maintained in the required crystalline state with reduced particle size (i.e. increase in the surface
area) leading to an increased dissolution rate and therefore improved bioavailability 3.Reduction
of drug particles to nanometer range leads to an enhanced dissolution rate not only because of
increased surface area but also because of saturation solubility.
Materials and Methods
Risperidone was obtained as gift from Panacea biotech Ltd. Chandigarh, India. Polyvinyl
alcohol, Polyvinyl Pyrrolodine K44, Hydroxymethyl propyl cellulose were procured from Drugs
India (Hyderabad, India). All other materials, reagents and solvents used were of analytical
grade.
Risperidone estimation:
Determination of absorption maxima

10mg of pure drug was dissolved in 10ml methanol (primary stock solution - 1000
µg/ml). From this primary stock solution 1 ml was pipette out into 10 ml volumetric flask and
made it up to 10ml with the media (Secondary stock solution – 100µg/ml). From secondary stock
solution again 1ml was taken it in to another volumetric flask and made it up to 10 ml with
media (working solution - 10µg/ml). The working solution was taken for determining the
wavelength.

Determination of Calibration Curve:

10mg of pure drug was dissolved in 10ml methanol (primary stock solution - 1000 µg/ml). From
this primary stock solution 1 ml was pipette out into 10 ml volumetric flask and made it up to
10ml with the media (Secondary stock solution – 100µg/ml). From secondary stock solution
required concentrations were prepared and those concentrations absorbance was found out at
required wavelength at 278nm and which is shown in table-1 and figure-1.

Preformulation studies:

Preformulation testing is the first step in the rationale development of dosage forms of a drug
substance, alone and when in combined with excipients. The overall objective of the pre
formulation testing is to generate information useful to the formulator in developing stability and
bioavailability of the product..

Characterisation of Drug:
Physicochemical Evaluation of Drugs:

1. Colour and Appearance

The Colour and Appearance of Drug was observed and recorded with the descriptive
terminology.

2. Melting Point

The Melting Point is determined by using melting point apparatus. The melting point is
determined according to the procedure given in Indian pharmacopoeia 2007. The
melting point of pure drug was determined by taking small amount of drug in a capillary tube
closed at one end. The capillary tube was placed in an electrically operated melting point
apparatus and the temperature at which the drug melts was recorded.

3. Solubility Studies

It is important to know about solubility characteristic of a drug in aqueous system, since they
must possess some limited aqueous solubility to elicit a therapeutic response. The solubility of
drug was recorded by using various descriptive terminology specified in Indian Pharmacopoeia,
2007.

4. Identification of drugs:

The obtained sample was examined by infrared absorption spectral analysis and was compared
with the standard IR spectrum of pure drug.
Drug and excipient compatability studies by FTIR

FTIR spectra were performed using Bruker FTIR spectrophotometer to find out any possible
drug-stabilizer interactions. Samples of drug and of each stabilizer used as well as samples of the
prepared nanosuspensions were grounded separately and mixed thoroughly with potassium
bromide. The ratio of sample and KBr was kept for all the preparations. The potassium bromide
discs were prepared by compressing the powders at a pressure of 5 tones for 5 min in a hydraulic
press. Scans were obtained from 4000 to 500 cm-1.

Preparation of Nano suspension by Microprecipitation:

Microprecipitation technique is for the preparation of Nano Suspension. The drug dissolved in
suitable solvent. In another beaker stabilizer in water. Drug and stabilizer different ratios were
taken as shown in Table.

 Drug was dissolved in Methanol

 Stabilizer dissolved in Water

Formulation optimization
The optimized formulation was selected on the basis mean particle size, particle dispersity index
and zeta potential and stability.
Preliminary experiments for nanosuspension formulation
Preliminary parameters were optimized by varying one parameter at a time, while keeping other
constant, so that effect of various parameters could be evaluated. For optimization of NS, various
parameters affecting the nano-suspension like concentration of drug, concentration of zirconium
beads, concentration of excipients (different stabilizers were used) and stirring time were studied.
The parameters were optimized to obtain nano-ranged size with narrow size distribution.4
Polymeric stabilizers such as PVA, Tween 80, PVP, and HPMC were taken for study. In order to
select the appropriate stabilizer, preliminary experiments with 0.75 %, 1.5 % and 2.0 % (w/v) of
various stabilizers were performed. Also, effect of combination of polymeric and surfactant
stabilizer (SLS) on mean particle size and zeta potential were investigated. Effect of different
concentrations of drug (0.75 %, 1.5 % and 2.0 % w/v) on particle size was investigated.
Nanosuspension formulations were prepared by micro precipitation method. The drug is
dissolved in suitable organic solvent acetonitrile in which the drug is soluble. This was poured
into different amount of water containing different amount of stabilizers and SLS at maintained
at room temperature and subsequently stirred on homogenizer to allow volatile solvents to
evaporate. Addition of organic solvents by means of a syringe positioned with the needle directly
into stabilizer containing water. Organic solvents were left to evaporate off under continuous
stirring of the nanosuspension at room temperature for 5 hours 5.formulation composition is
shown in table-2.

Evaluation of Nanosuspension:

Drug Content

Accurately weighed amount of each preparation dissolved in required amount of methanol and
diluted suitably in pH 6.8 phosphate buffer. The drug content was determined
spectrophotometrically at required wavelength. Calculation was done using following formula6

Determination of Nanosuspension Percentage Yield

The nanosuspension production yield was calculated by gravimetric method. Fixed volumes of
nanoparticles suspension were centrifuged (15,000×g, 30 min, 15ºC) and sediments were dried7.

The percentage yield was calculated as follows:

Particle size measurement

The particle size analysis and particle dispersity index of nanosuspensions were determined
using a Malvern Zeta Sizer Nano ZS (Malvern Instruments, Malvern, UK). The particle size
index indicates the width of a particle distribution. Prior to the measurement, the samples were
diluted with double distilled filtered water to a suitable scattering intensity and re-dispersed by
shaking before the measurement. All measurements were performed in triplicate. The results are
expressed as mean ± standard deviation (SD).

Analysis of Zeta Potential

The zeta potential is a measure of the electric charge at the surface of the particles indicating the
physical stability of colloidal systems. Zeta Potential was measured using a Zeta Sizer Nano ZS
(Malvern Instruments, Malvern, UK). Each sample was suitably diluted with double distilled
filtered water and placed in a disposable zeta cell. The Zeta potential values were assessed by
determining the particle electrophoretic mobility. The electrophoretic mobility was converted to
the zeta potential via the Helmholtz Smoluchowski equation. All measurements were performed
in triplicate. The results are expressed as mean ± SD.

Solid state evaluation

Changes in the crystalline state can affect the solubility, dissolution velocity, the oral
bioavailability as well as the stability of a pharmaceutical formulation. Therefore crystalline
structure of nasuspension was investigated by DSC.

Differential Scanning Calorimetry

DSC scans of prepared dried powdered drug sample, pure drug and physical mixture were
recorded using DSC Hitachi 7020 with muse software. All samples were weighed (8-10mg) and
heated at scanning rate of 10oC/min under dry nitrogen flow (50ml/min). Aluminum pans and
lids were used for all samples.

Scanning Electron Microscopy

Surface morphology of nanosuspensions was investigated using Scanning Electron Microscopy

(SEM). The samples for SEM study were prepared by lightly sprinkling the formulation on a
double-adhesive tape stuck to an aluminum stub. The stubs were then coated with gold to a
thickness of ~300 Å under an argon atmosphere using a gold sputter module in a high vacuum
evaporator. The coated samples were then randomly scanned and photomicrographs were taken
with a scanning electron microscope. SEM was operated at an acceleration voltage of 20 kV 9 and
shown in figure-4.
In-Vitro Drug Release Studies

In-vitro dissolution study of nanosuspensions formulations was carried out using USP
dissolution apparatus type II. Studies were carried out using phosphate buffer pH 6.8 as
dissolution medium (900ml) and 50 rpm was set throughout the study. Samples were withdrawn
at regular time interval of 10, 20, 30, 40, 50 and 60 minutes. Samples were replaced by
equivalent volume of fresh dissolution medium. The withdrawn samples were
spectrophotometrically analyzed at respective wavelength on UV Spectrophotometer8.

Results and Discussion

The nanosuspension formulations prepared with no stabilizer showed rapid agglomeration of
drug particles instantly after preparation. The agglomeration of particles is may be due to the
attractive forces between the particles in the absence of significant energy barrier, in addition to
above it might also due to so-called hydrophobic effect. The presence of hydrophobic particles or
molecules in water causes distortion and rearrangement of hydrogen bonding in the aqueous
medium, thus greatly increasing the free energy of the system. As a result, these hydrophobic
particles tend to agglomerate to reduce the system free energy.
Four stabilizers (PVA, Tween 80, HPMC and PVP) were tested for their stabilization potential.
Important function of stabilizer is that they can form a substantial mechanical and
thermodynamic barrier at the interface that retards the approach and coalescence of individual
nanoparticles. As per results obtained, it may be concluded that mean particle size varies with
stabilizer and with PVA it shows lowest size followed by then the other stabilizer. As shown in
results an appropriate amount of stabilizer is required to achieve smaller particle size. The crystal
growth was protected by the adsorbed stabilizers, and the quantity of stabilizer should be enough
to cover the crystal surface to provide enough steric repulsion between the crystals. Inadequate
surface coverage of stabilizer could result in rapid crystal growth and agglomeration, while high
concentration of stabilizer could result in enhanced viscosity of the solution which would
obstruct the diffusion between the solvent and anti-solvent during precipitation. The type of
compound and their amount employed for stabilization has a prominent effect on particle size. At
the low drug concentration, the particle size was smaller with a narrow size distribution.
However, at the higher drug concentration, due to greater supersaturation, a higher diffusion
controlled growth and agglomeration rate were achieved, resulting in larger crystals. Stirring
speed is obviously affecting the particle size, as increasing the stirring speed, decrease in mean
particle size because of high shear force.
Role of surfactant in formulation
In precipitation which is bottoms up technique nanosuspensions are formed by building particles
up from molecular stage. During this new surface area is formed, which produces high free-
energy, leading to agglomeration of particles and subsequent increase in particle size. This
tendency can be avoided by addition of surface active agents, which reduces the free surface
energy. Hence, during the formulation of nanosuspension the optimization of surface active
agents has prime importance.
Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy Figures 1(a)–1(h) revealed that there is neither appearance of new peaks nor
disappearance of existing peaks, which indicated that there is no interaction between the drug
and each of the stabilizers used. The characteristic ketone (C=O) stretching vibration at 1747.22
cm-1, C-H bending at 1412.57 cm-1 , C-O stretching at 1261.59 cm-1 , C-N vibration at 1313.29
cm-1 , aromatic C-H stretching at 747.69 cm -1 and the stretching band in the region of 3500-
3200 cm-1 assigned to the non bonded aromatic amino group were identified in all the spectrums
of the nanosuspensions and shown in figure-2 and figure-3.
Drug Content
During the preparation process there was no any drug loss step involved, so theoretically the
formulation was considered as being 100% drug content. It is obvious that the drug content of
nanosuspension can be considered as a function of both the nature of the stabilizer used as well
as the method of preparation and it can be seen that all the drug content were within acceptable
limit and shown in table-3.
Particle size analysis
The mean particle size varies 248±4.78 nm to 358±4.28 nm and showed good correlation
coefficient (0.8935. The particle size of different formulation, which clearly indicates the batch
F2 had less particle size as compare to other formulation. The batch F3 had a Z-average particle
size of 346 nm. Results of the mean particle size equation indicate that the all three independent
variable significantly affect the mean particle size. Insufficient surface coverage of stabilizer
could result in rapid crystal growth and agglomeration, while high concentration of stabilizer
could result in enhanced viscosity of the solution. However, drug concentration was more
significantly affect the mean particle size because at the higher drug concentration, due to greater
supersaturation, a higher diffusion controlled growth and agglomeration rate were achieved,
resulting in larger crystals.

Zeta Potential
It is generally acknolowdged that a zeta potential of approximately ± 20 mV is required. Zeta
potential analysis was performed to get information about the surface properties of
nanosuspensions. The zeta potential of the prepared nanosuspensions was found to be +21.29
mV. This value indicates the stability of the formulation and it is closer to the required zeta
potential value.
Scanning Electron Microscopy
Pure drug and nanosuspensions surface morphology and shape were analyzed by SEM,
representative examples are shown in figure 2 and 3. It can be seen that the raw drug particles
existed as irregular tabular and prismatic crystals with smooth surface. Micronized pure form of
drug powder showed irregular shapes with particle size generally larger than the prepared
nanosuspensions and with a broad particle size distribution. The prepared nanosuspensions were
more uniform in shape as compared to pure drug but with more or less rough surfaces. The
images revealed the presence of aggregates or particle assemblies which were composed of a
large number of individual nanoparticles. The weakened steric hindrance effects, derived from
the thinner stabilizer layer, would probably result in partial aggregation among nanosuspensions,
which would increase the particle size distribution of nanosuspensions and shown in figure-5 and
figure-6.
In-vitro dissolution studies
Dissolution studies were compared for pure drug, and optimized nanosuspension formulation.
The amount of drug released from the optimized nanosuspension formulation was 96.41% within
60 min compared to amount of 15.82 % of pure drug after 1 hour in phosphate buffer pH 6.8.
The increase in accessible surface area to the dissolution medium and hydrophilic surfactant
coating on the particle surfaces may be the reason for increase in dissolution rate. This enhanced
dissolution rate can be attributed to the higher surface area of nanocrystals available for
dissolution and the decreased diffusion layer thickness.
Conclusion
Risperidone nanosuspensions were prepared by nanoprecipitation. Nanoprecipitation technique
has been described as a simple method for drug nano-sizing at laboratory scale. Preliminary trails
helped in identifying the significant parameters that affected the response variables. All the
predetermined independent variables except drug concentration were found to affect the
dependent variables. Particle size is significantly influenced by concentration of drug,
concentration of stabilizer and starring speed. Nanosized risperidone dissolved significantly
faster than pure drug powder. The optimized formulation maintained the crystallinity of
risperidone and released almost 96.41 % drug within 60 minutes and shown in figure-7.

References
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 Table-1: Calibration curve of Risperidone
S.No Concentratio Absorbanc
n (µg/mL) e
1 0 0 Figure-1: Calibration curve
2 10 0.198 of Risperidone

3 20 0.396
4 30 0.601
5 40 0.804
6 50 0.998

Figure-2: FTIR graph for drug

 Figure-3: Risperidone Drug+ PVA FTIR

Table-2: Composition of risperidone nanosuspensions

Batch Code Stabilizer Drug (mg) Drug: Stabilizer

NS without 20 -
F1
Stabilizer
F2 PVA 20 1:0.4
F3 Tween 80 20 1:0.4
F4 PVP K44 20 1:0.4
F5 HPMC K4M 20 1:0.4

Evaluation parameters:

Table-3: Drug content, percentage yield, particle Size (P.S.), and size distribution of nano
suspensions prepared

Formulation Drug P.S. in nm Polydispersibility ZP±SD

Stabilizer % Yield
Code Content % Index (Mv)-
Risperidone - - 3548±25.15 0.51 -47.3±7.63
NS without -52.7±7.91
F1 62.38 99.6 1264±64.85 1.00
Stabilizer
F2 PVA 96.67 94.3 248±4.78 0.48 -56.2±7.67
F3 Tween 80 94.78 95.7 346±3.48 0.38 -48.5±5.60
F4 PVP K44 88.49 93.1 358±4.28 0.69 -49.9±7.62
F5 HPMC K4M 90.78 95.6 346±3.34 0.58 -55.1±9.73
Figure-4: Drug and PVA mixture DSC image

Figure-5: Risperidone Pure Drug

 Figure-6: Risperidone nano suspension formulation

In-vitro study:

120

100

80
% drug release

F1
60 F2
F3
F4
40 F5

20

0
0 10 20 30 40 50 60 70
Time in minutes

Figure-7: Percentage drug release profile for formulations in phosphate buffer