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ISSN: 0898-2104 (print), 1532-2394 (electronic)

J Liposome Res, Early Online: 1–11

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08982104.2014.974058

RESEARCH ARTICLE

Preparation and evaluation of a buflomedil hydrochloride niosomal

patch for transdermal delivery
Nida Akhtar1, Seema Arkvanshi1, Shiv Sankar Bhattacharya1, Anurag Verma1, and Kamla Pathak2
Journal of Liposome Research Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 11/18/14

1
School of Pharmaceutical Sciences, IFTM University, Lodhipur-Rajput, Moradabad, Uttar Pradesh, India, and 2Department of Pharmaceutics,
Rajiv Academy for Pharmacy, P.O. Chhattikara, Mathura, Uttar Pradesh, India

Abstract Keywords
Context: Niosomes are the non-ionic surfactant vesicles obtained on hydration of synthetic Cholesterol, drug, flux, permeability, systemic
non-ionic surfactants. These are the promising vehicles for effective transdermal drug delivery. circulation
Objective: The present research work was aimed to develop niosomal-based transdermal
buflomedil hydrochloride patch containing a stable formulation with improved drug History
permeation.
Materials and methods: Niosomes were prepared by solvent evaporation method using 32 Received 27 May 2014
factorial design. All the formulations were evaluated for vesicle size, zeta potential and percent Revised 30 August 2014
entrapment efficiency. Optimized niosomal and liposomal formulation were loaded into a Accepted 4 October 2014
patch system. All the patches were then characterized for drug–excipient interaction study, Published online 30 October 2014
scanning electron microscopy, pharmacotechnical properties and in vitro permeation studies.
For personal use only.

Result: F9 formulation having optimum vesicle size (10.09 ± 1.2 mm), highest zeta potential
(85.4 ± 0.56 mV) and maximum percent entrapment efficiency (97.09 ± 0.11%) was selected as
optimized formulation. In case of liposomes, formulation F12 was selected. Patches loaded
with niosomes showed 95.12 ± 1.19% cumulative amount of drug permeated as compared
to liposomal vesicle-loaded patches which showed 82.21 ± 1.24% and control patches
70.10 ± 1.33%. Discussion: Flux, permeation rate and permeability coefficient were found to
be higher in case of niosomal patches as compared to liposomal patches and control patches.
Surfactant present in niosomes act as a penetration enhancer which contribute in the
permeation enhancement of buflomedil hydrochloride from niosomes.
Conclusion: Thus, it was concluded that niosomal vesicles represented to be an efficient and
stable vesicular carrier for transdermal delivery of buflomedil hydrochloride.

Introduction investigated, niosomal vesicles have been found to be capable

enough as a transdermal drug carrier in enhancing the
Transdermal drug delivery system is considered to be an
permeation. These are the vesicular carriers prepared using
attractive alternative route to oral route of drug delivery
non-ionic surfactants and cholesterol (Prasanthi & Lakshmi,
(Prausnitz & Langer, 2008). This system uses skin for the
2012). Enhanced permeation through these vesicles is due
delivery of systemically acting drugs (Prasanthi & Lakshmi,
to the fact that these vesicles get interacted with stratum
2012). Despite of having the advantages of avoiding gastro-
corneum by aggregation, fusion and adhered to the cell surface
intestinal absorption of drugs, bypassing first-pass hepatic
causing a high-thermodynamic activity gradient of the drug
metabolism, controlling drug release (Gaikwad, 2013), non-
at the surface of vesicle and stratum corneum. This acts
invasive, self-administration (Prausnitz & Langer, 2008) and
as a driving force for the penetration of drugs across
improved patient compliance, this system has the limitation of
the stratum corneum. Niosomes also modify the structure of
slow diffusion of drugs due to lesser permeability across the
stratum corneum; loosen the intercellular lipid barrier of
skin. Thus, limiting the delivery of the drug through the skin
stratum corneum. Thereby, increasing the stratum corneum
(Gaikwad, 2013). Therefore, several approaches have been
permeability. Non-ionic surfactant present in niosomes works
used to weaken this barrier and to enhance the transdermal
as a permeation enhancer and plays a key role in enhancing the
delivery of the drug into the systemic circulation. One of these
permeation (Rahimpour & Hamishehkar, 2012). Buflomedil
approaches is the use of vesicle-based formulations as
hydrochloride [4-(1-pyrrolidinyl)-1-2,4,6-trimethoxyphenyl)-
transdermal system. Of the various vesicular systems
1-butanone hydrochloride] is a vasoactive drug employed
widely in the treatment of vascular diseases increasing the
Address for correspondence: Nida Akhtar, Assistant Professor, peripheral and arterial blood flow in ischaemic patients (El-
School of Pharmaceutical Sciences, IFTM University, Lodhipur-
Rajput, Moradabad 244102, Uttar Pradesh, India. E-mail:
Desoky et al., 2010). It is also employed in the treatment of
nidakhtr378@gmail.com dementia, disorientation, insomnia, intermittent claudication,
 2 S. Arkvanshi et al. J Liposome Res, Early Online: 1–11

ischaemic rest pain, loss of concentration, loss of memory, Selection of concentration of cholesterol
Raynaud’s syndrome, etc. The present investigation was
Blank niosomes were prepared using different concentration
undertaken to develop niosomal formulations that can improve
of cholesterol to select the optimum concentration required
the transdermal delivery of buflomedil hydrochloride. As
for the preparation of niosomes. For this, niosomes were
postulated by various authors, the oral and parenteral forms
prepared at concentrations 0.1–5.0% w/v. Niosomal suspen-
administered of buflomedil hydrochloride has risk of adverse
sions were then visualized by taking micrographs through
reactions and may cause premature inactivation of the drug by
optical photomicrograph (Photomicrograph, HICON, Delhi,
first pass hepatic metabolism. Liposomes were then formulated
India) at 100.
by Roesken et al. to overcome the limitations possessed by oral
and parenteral formulations. But, these vesicles were found to
Preparation of buflomedil hydrochloride niosomes
possessed limited permeability across the skin (Roesken et al.,
2000). Thus, in this research work, an attempt has been made to Niosomes loaded with buflomedil hydrochloride were for-
formulate niosomal-based transdermal delivery system encap- mulated by ether injection method based on 32 factorial design
(Table 1). 32 Factorial design is a three-level and two factors-
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sulating buflomedil hydrochloride, a vasoactive drug. This can

improve the therapeutic efficacy of drug due to its greater
concentration and longer time duration at the desired site of
action with improved permeability across the skin. It also
avoids inactivation of drug as it bypasses the first-pass hepatic
metabolism. Further, it can also reduce the side effects
associated with the drug usage (Muri et al., 2010). In vitro
permeability study was done to layout the comparison among
control formulation, niosomes and reference liposomes for
improved permeability across the skin.
Materials
Buflomedil hydrochloride was obtained as a gift sample from
Fresenius Kabi Oncology Ltd. (Kalyani, West Bengal, India).
For personal use only.
based factorial design as reported by Webb (1971). In total,
there are nine runs or combinations. In designing niosomes,
cholesterol and span 60 were selected as two independent
variables, both at three different levels (1, 0, +1). In the
design, the lower concentration of cholesterol, i.e. 1.0%w/v,
has been assigned at 1 level, whereas 2.0% w/v concentration
was assigned the level 0. The concentration at highest level +1
was 3.0% w/v. In case of span 60, concentrations at three levels
from lowest to highest (1, 0, +1) were 0.5, 1.0 and 3.0% w/v,
respectively. Based on this factorial design, nine niosomal
formulations were developed. Span 60, cholesterol and drug
were dissolved in a mixture of diethyl ether and methanol as
per the composition given in Table 1 and was stirred

continuously on a magnetic stirrer (Macro Scientific Works,

Cholesterol, Span 40, Span 60 and Span 80 were obtained
from Central Drug House (CDH) Pvt. Ltd. (New Delhi,
India). Methanol and sodium chloride extrapure LR were
procured from CDH Pvt. Ltd. (New Delhi, India). Potassium
dihydrogen orthophosphate was purchased from SD Fine
chemicals Ltd. (Mumbai, India). HIMEDIA Dialysis mem-
brane 150 was procured from Sigma Aldrich (Mumbai, India).
Diethyl ether, hydroxy propyl methyl cellulose (HPMC K4M)
and sodium chloride were purchased from Central Drug
House (CDH) Pvt. Ltd. (New Delhi, India). Sodium hydroxide
pellets LR were procured from RFCL Ltd. (New Delhi,
India). Glycerin was procured from SD Fine Chemicals Pvt.
Ltd. (Mumbai, India).
Delhi). Distilled water was heated to 55 ± 1  C. Organic phase
was then injected into an aqueous phase.
Purification of drug-loaded niosomes
Drug-loaded niosomes were purified by dialysis membrane
technique to remove the free drug from niosomal suspension.
For this, Hi-media dialysis membrane was kept in saline
solution for 2 h before dialysis to ensure complete wetting of
the membrane. Niosomal vesicles loaded with buflomedil
hydrochloride were placed in a dialysis bag, which was
transferred into 200 ml of phosphate buffer pH 7.4. The
receiver medium was stirred with a magnetic stirrer at 500 rpm

Methods Table 1. 32 Factorial design of liposomal and niosomal formulations of

buflomedil hydrochloride.
Preliminary studies on blank niosomes
Preliminary studies were performed to optimize the blank Formulation Drug Cholesterol Span 60 Diethyl ether Methanol
code (mg) (% w/v) (% w/v) (% w/v) (% w/v)
niosomes prepared by ether injection method described later.
Blank niosomes were prepared using different types of Span Niosomes
F1 10 1.0 () 0.5 () 1.0 1.0
(Span 40, 60 and 80). An appropriate type of span and its F2 10 1.0 () 1.0 (0) 1.0 1.0
optimum concentration were selected. Selection of required F3 10 1.0 () 3.0 (+) 1.0 1.0
concentration of cholesterol was also done by preparing the F4 10 2.0 (0) 0.5 () 1.0 1.0
blank niosomes. F5 10 2.0 (0) 1.0 (0) 1.0 1.0
F6 10 2.0 (0) 3.0 (+) 1.0 1.0
F7 10 3.0 (+) 0.5 () 1.0 1.0
Selection of type and concentration of span F8 10 3.0 (+) 1.0 (0) 1.0 1.0
F9 10 3.0 (+) 3.0 (+) 1.0 1.0
Blank niosomes containing span 40, 60 and 80 were prepared. Liposomes
For this, niosomal suspensions were visualized by taking F10 10 1.0 () – 1.0 1.0
micrographs through optical photomicrograph (Photomicro- F11 10 2.0 (0) – 1.0 1.0
graph, HICON, Delhi, India) at 100. Further, blank niosomes F12 10 3.0 (+) – 1.0 1.0
Fx* 10 1.5 1.5 1.0 1.0
were prepared by changing the concentration of span 60 in the
range of 0.1–3.0% w/v to optimize its concentration. *Extra design check point formulation.
 DOI: 10.3109/08982104.2014.974058 Buflomedil hydrochloride niosomal patch for transdermal delivery
(13.98  g). Five millilitres of sample was withdrawn at
appropriate time intervals and replaced with an equal volume
of fresh media and analyzed spectrophotometerically for the
amount of free drug. Purification time was optimized by
applying statistical paired t test at 5% level of significance.
Evaluation of niosomes
Vesicle size and zeta potential
Vesicle size, zeta potential and polydispersity index were
measured by Zetasizer ver. 6.01 (Malvern Instruments Ltd,
Malvern, Worcestershire) by diluting one drop of liposomal
and niosomal suspension with hydroethanolic solution at
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25  C in clear disposable zeta cells. All the measurements
were done in triplicate for each sample.
Entrapment efficiency
Purified vesicular suspension was transferred into the centri-
fuge tube and centrifuged (Macro Scientific Works,
New Delhi, India) for 1 h at 4000 rpm (894.0  g). The
sediment was lysed using methanol and filtered through nylon
filter disc (0.22 mm). The drug was assayed both in the
sediment and supernatant to determine the entrapment
efficiency by the following equation:
% Entrapment efficiency ¼ ½Wa  ðWs þ WP Þ=Wa , ð1Þ
For personal use only.
where Wa is the amount of drug added in the system, Ws is the
amount of drug in the supernatant after centrifugation and WP
is the amount of drug in the purification medium.
Validation of experimental design
The polynomial equation was generated for dependent
variable using Design Expert Software version 8.0.5 (Stat-
Ease Inc., Minneapolis, MN). An extra design check point
formulation (fx) was developed by selecting the levels as 1.5%
w/v of cholesterol and 1.5% w/v of span 60. This was used to
validate the obtained polynomial equation model. A statistical
model consisting of interactive and polynomial terms was
used to evaluate the responses. Polynomial and transformed
polynomial equations were obtained. Extra design check point
formulation was then evaluated for dependent variable, i.e.
percent entrapment efficiency to get the experimental value.
This value was compared with the predicted value obtained
from transformed polynomial equation and evaluated statis-
tically by pooled t test at 5% level of significance.
Evaluation of optimized niosomes
Scanning electron microscopy
Scanning electron microscope (Model JSM 6360, Jeol Make,
Tokyo, Japan) was used to characterize optimized niosomes
and reference liposomal formulation. The samples were
visualized under scanning electron microscope at 1500
magnification with accelerating voltage of 17 kV.
In vitro release study
For in vitro release study, dialysis membrane was washed and
soaked in saline solution. Optimized niosomal (F9) and
3
liposomal vesicles (F12) were placed in a dialysis bag. The
dialysis bag was then placed in 250 ml of phosphate buffer pH
7.4 in a beaker with constant stirring at 37  C for 8 h. Samples
withdrawn at appropriate time intervals were analyzed
spectrophotometerically. Percent cumulative amount of drug
released was then determined.
Stability study
Stability of the optimized (F9) and reference (F12) formula-
tion was determined by storing the vesicles at 4  C
(refrigerated temperature) and 25  C (room temperature) for
6 months. After appropriate time intervals, samples were
withdrawn and evaluated for their mean particle size, zeta
potential and percent entrapment efficiency.
Preparation of patches
Solvent evaporation method
Transdermal patches loaded with niosomal vesicles were
prepared using solvent evaporation method. HPMC K4M was
dissolved in a mixture of chloroform and methanol (6:4).
Optimized niosomes and reference liposomal formulation(s)
were centrifuged at 2000 rpm (447.0 g), and the pellets were
incorporated in the above polymer base. Glycerin was added
under continuous stirring on the magnetic stirrer (Macro
Scientific Works, for 5–6 h. The mixture was spread in the
petridishes. The petridishes were then placed in hot air oven
at 36  C temperature.
Evaluation of patches
Differential scanning calorimetry
Differential scanning calorimetric analysis of drug, span 60,
cholesterol, HPMC K4M (Hydroxy Propyl Methyl Cellulose),
niosomal patch, liposomal patch and control patch were
carried out. The analysis was conducted in the heating
range of 54–260  C in nitrogen atmosphere at the rate of
150 ml/min), using differential scanning calorimetry (DSC,
Pyris Diamond TG/DTA, PerkinElmer, Singapore).
Scanning electron microscopy
Scanning electron microscope (Model JSM 6360, Jeol Make,
United Kingdom) was used to characterize the loading of
niosomes in patches and reference liposomal patches as well
as control patches. The samples were visualized under
scanning electron microscope at 1500 magnification with
accelerating voltage of 17 kV.
Thickness
Thickness was measured using screw gauge (Sterling
Manufacturing Company, India). The study was done in
triplicate.
Weight uniformity
The patches were subjected to weight variation test by
weighing the selected patches individually. Three dried
patches were weighed on an electronic balance (Citizen
Scale CY220). The study was done in triplicate.
 4 S. Arkvanshi et al.
Tensile strength
In order to determine the elongation as a tensile strength, the
polymeric patch was pulled by means of a pulley system;
weights were gradually added to the pan to increase the
pulling force till the patch was broken. The elongation, i.e. the
distance travelled by the pointer before break of the patch,
was noted with the help of magnifying glass on the graph
paper. Equation 2 was used to calculate the tensile strength.
Tensile load at break
Tensile strength ¼ ð2Þ
Dis tan ce travelled by broken patch
Flatness
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Three longitudinal strips were cut out from each patch, i.e.
one from the centre, one from the left side and third one
from the right side. The length of each strip was measured.
The change in length because of non-uniformity in flat-
ness was measured by determining percent constriction,
with 0% constriction equivalent to 100% flatness (Patel et al.,
2009).
Folding endurance
This was determined by repeatedly folding one film at the
same place till it broke. The number of times the film could
be folded at the same place without breaking/cracking gave
the value of folding endurance.
For personal use only.
Percentage of moisture content
The patches were weighed individually and kept in desicca-
tors containing activated silica at room temperature for 24 h.
Individual patch was weighed repeatedly until they showed a
constant weight. The percentage of moisture content was
calculated as the difference between initial and final weight
with respect to final weight.
Determination of percent drug content
Drug content was determined by taking the patches of 1 cm2
surface area and dissolved in 5 ml methanol. Vortex shaking
(Vortex shaker, HICON, India) was done for 5 min, and
volume was made up to 10 ml with methanol and examined
spectrophotometrically (Shimadzu UV 1800, Japan) for drug
content.
In vitro release studies
In vitro release studies were performed for niosomal patches,
liposomal and control patches. For this study, Franz Diffusion
cell (Bhanu Scientific Instrument CO, Bangalore, India) was
used with a receptor compartment of 10 ml capacity and area
3.14 cm2. Hi-media dialysis membrane 150 (molecular weight
cut off (MWCO) 12–14 kDa; Prajapati et al., 2011a) was
soaked in phosphate buffer pH 7.4 and mounted on the Franz
diffusion cell. The transdermal patch of area 3.14 cm2 area
was placed in the donor compartment and it was separated
from the receptor compartment by dialysis membrane. The
donor and receptor compartment was joined together using
clamp (Prajapati et al., 2011b). The receptor compartment
containing 10 ml of the phosphate buffer pH 7.4 maintained at
J Liposome Res, Early Online: 1–11
32  C ± 0.5  C was stirred using magnetic stirrer at 60 rpm.
One-millilitre sample was withdrawn from the receptor
compartment at appropriate time intervals and analyzed
spectrophotometerically. An equal volume of fresh phosphate
buffer pH 7.4 was replaced after each sampling into the
compartment (Wahid et al., 2008). The study was performed
in triplicate, and average values were calculated. The percent
cumulative amount of drug permeated across the skin per
square surface area was evaluated and plotted against time to
calculate the transdermal flux.
Results and discussion
Preliminary studies on blank niosomes
Blank niosomes were formulated by ether injection method,
and several preliminary optimization studies were conducted
in order to optimize the methodology. From the micrographs
taken, it was observed that in case of span 40, vesicles formed
were non-uniform in size and had indefinite structure, while
span 80 did not accompanied the formation of vesicles.
However, niosomes formulated with span 60 were uniform
and spherical in shape with well defined and specific
boundary (Figure 1). The results obtained are in accordance
with the fact that span 60 is the better surfactant of all the
surfactants as it is having high-phase transition temperature
and possesses low-hydrophilic–lipophilic balance (HLB) so it
form the vesicles of appropriate size (Akhilesh et al., 2012).
Critical packing factor (CPF) is another parameter considered
behind the selection of span 60. It is the molecular charac-
teristic of surfactants that depicts whether the aggregate form
is spherical micelles, inverted micelles or vesicles
(Israelachvili, 1994). Surfactants with CPP value 0.5–1 can
only form vesicles. As span 60 was reported to have above
specified CPP value can lead to the formation of vesicles
(Akhilesh et al., 2012). Span 60 has relatively high hydro-
phobicity and less value of critical packing parameter. So, on
adding cholesterol, it has potential to form optimum mem-
brane curvature for lamellar (vesicular) structure (Manosroi
et al., 2003). Thus, span 60 was selected for preparing
niosomes. Further, an appropriate concentration of span 60
was selected, using varying concentrations of it. It was
observed that at 0.5% w/v concentration of span 60, niosomal
vesicles formed were firm, with well-defined boundary and
spherical in shape confirming uniformity. At concentration
below 0.5% w/v, vesicle could not be formed and on
increasing the concentration of span 60 beyond 0.5% w/v
till 5.0% w/v, the size of vesicle increased with loss in
uniformity in size. Thus, the optimum concentration of span
60 was identified to be 0.5, 1.0 and 3.0% w/v. After the
selection of span and its concentration, optimum concentra-
tion of cholesterol was selected. Among the concentration of
cholesterol taken (0.5–5.0% w/v), it was observed that
uniformity of vesicles was good at concentration 1.0–3.0%
w/v. This also guided the selection of levels of span 60 and
cholesterol for 32 experimental design.
Optimization of purification time
Selected formulation (F9) was subjected to purification, and
the amount of free drug released was observed for 60 min.
 DOI: 10.3109/08982104.2014.974058 Buflomedil hydrochloride niosomal patch for transdermal delivery 5
Journal of Liposome Research Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 11/18/14

Figure 1. Photomicrographs of niosomes prepared with (a) Span 40; (b) Span 60 and (c) Span 80 at 100.

Initially, the amount of free drug released increased till 20 min Table 2. Pharmacotechnical properties of niosomal vesicles (F1–F9) and
and reached plateau level by 30 min (data not shown). Paired t reference liposomal vesicles (F10–F12).
test was applied to determine any significant difference in the
amount of free drug released at different time intervals. It was Zeta Percent
For personal use only.

Formulation Particle size Polydispersity potential entrapment

observed that there was a significant difference (p50.05) in code range (mm) index (mV) efficiency
the amount of free drug released between 10 and 30 min, but
there was no significant difference (p40.05) in amount of F1 2.87 ± 1.9 0.312 ± 0.02 61.3 ± 0.42 65.05 ± 0.14
F2 2.95 ± 0.5 0.234 ± 0.11 65.1 ± 0.58 72.11 ± 0.11
free drug released after 30 and 40 min. Therefore, all the F3 3.77 ± 2.4 0.136 ± 0.14 65.5 ± 0.61 78.26 ± 0.09
formulations were subjected to purification by dialysis F4 4.08 ± 2.1 0.245 ± 0.22 69.4 ± 0.54 81.24 ± 0.21
technique for a period of 30 min. F5 5.16 ± 1.1 0.218 ± 0.10 72.7 ± 0.23 83.31 ± 0.05
F6 6.43 ± 2.3 0.308 ± 0.15 74.8 ± 0.39 85.57 ± 0.16
F7 7.58 ± 1.4 0.276 ± 0.21 79.2 ± 0.70 89.08 ± 0.16
Evaluation of niosomes F8 8.73 ± 2.7 0.225 ± 0.03 81.9 ± 0.37 93.44 ± 0.13
F9 10.09 ± 1.2 0.121 ± 0.07 85.4 ± 0.56 97.09 ± 0.11
Determination of vesicle size and zeta potential F10 3.98 ± 2.2 0.309 ± 0.18 57.4 ± 0.33 56.77 ± 0.03
F11 7.12 ± 1.8 0.248 ± 0.19 62.9 ± 0.59 61.46 ± 0.22
Niosomes and reference liposomal formulations were eval-
F12 13.99 ± 1.6 0.201 ± 0.12 67.5 ± 0.47 64.12 ± 0.10
uated for particle size, polydispersity index and zeta potential.
It was observed that the niosomal vesicles of
formulations (F1–F9) varied in size range of 2.87 ± 1.9 mm
to 10.09 ± 1.2 mm in size where as reference liposomal vesicles of particles within the formulation, whereas if it is greater than
(F10–F12) ranged between 2.54 ± 2.2 mm and 7.12 ± 1.6 mm 0.3, it dictates the heterogeneous nature of the particle
(Table 2). It was observed in niosomes that as the concentra- distribution (Maurya et al., 2010). It was found to be minimum
tion of span 60 increased, vesicular size of the particles also for F9 0.121 ± 0.07 among niosomal vesicles and
increased. Further it has also been evaluated that with 0.201 ± 0.120 (F12) among reference liposomal formulations
enhanced concentration of cholesterol, vesicle size also showing the homogeneous nature of the formulations. F9 and
increased. Availability of higher amount of raw materials F12 made with highest levels of cholesterol and span displayed
probably led to the formation of larger-sized vesicles (Zhaowu least PDI probably due to dominant surfactant action of span
et al., 2009). Similarly, in case of liposomal formulations, 60 (Akhilesh et al., 2012). Zeta potential is another parameter
vesicle size was dependent primarily on the amount of responsible for the thermodynamic stability. Niosomal ves-
cholesterol, as the concentration of cholesterol increases, icles had higher zeta potential 85.4 ± 0.56 mV as compared
the size of vesicles increased from formulation F10 to F12. to reference liposomal formulations, i.e. 67.5 ± 0.47 mV.
The results are found to be consistent with the earlier reports The negative charge on the vesicles might be the charge due to
made by Kamboj et al. where the authors reported an increase the drug. As buflomedil hydrochloride carries negative charge
in niosomal vesicular size on increasing cholesterol concen- concentrated on the Cl (chloride) atom and methoxy group
tration (Kamboj et al., 2014). The polydispersity index (PDI) (–OCH3) (Eicher et al., 2009). Cholesterol is electrically
specifies the distribution of vesicles as it defines the meas- neutral (Masterjohn, 2005). Span 60 also carries neutral charge
urement of homogeneity of a vesicular formulation. A PDI being a non-ionic surfactant (Akhilesh et al., 2012). So, the net
value of less than 0.3 indicates the homogeneous distribution charge on the vesicles may due to the drug. Analysis of zeta
 6 S. Arkvanshi et al.
potential values indicates higher magnitude of negative charge
on niosomal vesicles.
Entrapment efficiency
Among all the niosomal formulations, percent entrapment
efficiency (Table 2) was found to be maximum in formulation
F9 (97.09% ± 0.11%), where as in case of liposomes,
formulation F12 showed maximum percent entrapment effi-
ciency of 64.12% ± 0.10%. Thus, it was observed that
niosomes possessed higher drug entrapment as compared to
liposomes. The presence of span 60 had a positive impact on
percent drug entrapment of the vesicular carrier. The
percentage entrapment efficiency was found to be increased
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by increasing concentration of cholesterol. At a ratio of 1:1 of
span 60:cholesterol, entrapment efficiency was found to be
maximum (Kamboj et al., 2014).
Validation of experimental design
The 32 factorial design was validated using Design Expert
Software version 8.0.5 (Stat-Ease). A general statistical model
can be developed with respect to the data obtained from the
formulations subjected to optimization. The model depicted
can be characterized using the polynomial equation repre-
senting the respective response data. This can be given as
follows:
% Entrapment efficiency
For personal use only.
¼ þ85:14  8:16x1  1:45x2  4:29x21 þ 0:72x22
þ 1:24x1 x21 þ 2:36x2 x21  1:18x1 x22  1:35x2 x22
whereas the final transformed equation, Equation (2),
obtained by removing all the insignificant values was
% Entrapment efficiency ¼ þ85:14  8:61x1
From the above polynomial equations, response surface
plot was generated which was then used to predict the
response of dependent variables at the intermediate levels of
independent variables. From the response surface plot
(Figure 2), it was observed that as the levels of both
cholesterol and span 60 increased, entrapment efficiency
was also increased. The percent entrapment efficiency of the
extra design check point formulation (Fx) was found to be
87.62 ± 7.58%. Comparison with predicted value of 85.14%
obtained from the transformed polynomial equation indicated
no significant difference (p40.05) between the two values
when checked using pooled t test thus validating the design.
Selection of optimized formulation
The optimized formulation of niosomal formulation was
identified as F9 on the basis of highest entrapment efficiency
of 97.09% ± 0.11%. Similarly, the liposomal formulation F12
(reference) was selected on the basis of maximum entrapment
efficiency of 64.12% ± 0.10%.
Evaluation of optimized niosomes
Vesicular characterization by scanning electron microscopy
Vesicular characterization of optimized niosomes (F9) and
liposomes (F12) loaded with buflomedil hydrochloride was
J Liposome Res, Early Online: 1–11
Figure 2. Response surface plot showing the effect of levels of
cholesterol and Span 60 on entrapment efficiency.
done by scanning electron microscopy (SEM). SEM images,
in both cases, depicted the presence of almost uniform,
spherical-shaped vesicles and absence of aggregates
(Figure 3). However, niosomal vesicles more uniform as
compared to liposomal vesicles.
In vitro release study
In vitro release from the niosomes compared to the liposome
formulation was determined. Percent cumulative drug release
ð3Þ in case of niosomes and liposomes was observed to be
97.21 ± 1.21 and 63.78 ± 1.34, respectively, after 8 h (data not
shown). It was found that the drug release from niosomal
vesicles was more as compared to liposomes. This was due to
higher percent of drug entrapped in them, which increases the
cumulative amount of drug release.
ð4Þ
Stability studies
Stability studies were carried out by storing the samples at
4  C and 25  C to investigate the long-term stability of the
vesicles at its storage condition. The stability data had shown
in Table 3. As indicated, there was no significant difference in
vesicular size, zeta potential and percent entrapment effi-
ciency of niosomes before and after storage at refrigerated
temperature. This dictated good stability of niosomal vesicles
upon storage because of the combinatorial stable effect
exerted by the presence of both cholesterol and span 60. As
addition of higher concentration of cholesterol provides
stability to the vesicles (Das & Palei, 2011) and aggregation
of vesicles can be prevented by the addition of an amphiphile
(span 60). Thereby, stabilizing the system by repulsive steric
or electrostatic effects (Akhilesh et al., 2012). However, at
higher temperature (room temperature 25  C), significant
difference in vesicular size, zeta potential and percent
entrapment efficiency of niosomes before and after storage
was observed. Vesicles may tend to aggregate and form
vesicles of larger size. Thereby, vesicular size of vesicles was
found to be increased. Aggregation of these vesicles may
cause drug leakage, decreasing their entrapment efficiency.
Thus, it was concluded that storage under refrigerated
conditions showed greater stability as compared to the room
temperature (Sakthivel et al., 2012).
 DOI: 10.3109/08982104.2014.974058 Buflomedil hydrochloride niosomal patch for transdermal delivery 7
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Figure 3. Scanning electron microscopic images of (a) niosome (F9) and (b) Liposome (F12).

Table 3. Stability study data of the optimized buflomedil hydrochloride loaded niosomes (F9) and reference liposomes (F12) at refrigerated (4  C) and
room temperature (25  C).

Parameters

Formulation code Temperature ( C) Time period (Months) Vesicle size (mm) Zeta potential (mV) % Entrapment efficiency
F9 4 (refrigerated temperature) 0 10.09 ± 1.2 85.4 ± 0.56 97.09 ± 0.11
6 23.11 ± 3.4 71.9 ± 0.26 88.15 ± 0.08
F12 0 7.12 ± 1.6 67.5 ± 0.47 64.12 ± 0.10
6 35.88 ± 4.2 56.2 ± 0.35 53.16 ± 0.17
F9 25 (room temperature) 0 10.09 ± 1.2 85.4 ± 0.56 97.09 ± 0.11
6 47.33 ± 2.8 56.9 ± 1.10 66.75 ± 0.19
For personal use only.

F12 0 7.12 ± 1.6 67.5 ± 0.47 64.12 ± 0.10

6 65.14 ± 1.9 42.4 ± 0.73 45.31 ± 0.23

Evaluation of niosomal patches loaded with
buflomedil hydrochloride
Differential scanning calorimetry
Differential scanning calorimetric (DSC) studies were con-
ducted for drug, niosomal patch and liposomal patch. DSC
thermograms of pure drug, span 60, cholesterol and HPMC
K4M were shown in Figure 4. DSC thermograms of niosomal
patch, liposomal and control patch were shown in Figure 5.
It was observed that buflomedil hydrochloride (BH) exhibited
a sharp endothermic peak at 196.89  C, showing melting of
pure drug. The melting point of buflomedil hydrochloride was
reported to be 195–198  C (Clissold et al., 1987). Span 60 and
cholesterol showed a sharp endothermic peak at 56.25 and
148  C, respectively. These two peaks showing the melting
point of both the excipients, i.e. 54–57  C (Peltonen, 2001)
and 148–150  C (Ohvo-Rekila et al., 2002). Pure HPMC K4M
exhibited the slight broad peak at temperature range of
50–66.5  C. In case of both liposomal and niosomal patches, a
sharp endothermic peak of pure drug (BH) was disappeared.
A broad endothermic peak was observed in between 50 and
87.5  C. This may be attributed due to the dispersion of drug
in the polymeric matrix (Bhosale et al., 2011). No other sharp
endothermic peak was observed, indicating the fact that no
interaction took place between drug, span 60, cholesterol and
polymer (HPMC K4M). Thus, from this study, it was
concluded that for the preparation of liposomal and niosomal
patches, polymer and all other excipients (span 60,
cholesterol) were of optimum suitability because of their
compatibility with the drug (Verma & Chandak, 2009).
Scanning electron microscopy of patches
Scanning Electron microscopic images of niosomal and
liposomal patches highlighted the loading of vesicles within
the dermatological patch system. However, the distribution
of niosomes in patches was more uniform as compared to
liposomal vesicles (Figure 6).
Pharmacotechnical properties of patches
The pharmacotechnical properties of patches were given in
Table 4. Thickness was found to be 0.30 ± 0.48 mm in case of
niosomal patches, whereas liposomes possessed the thickness
of about 0.093 ± 0.31 mm. Thinner the patches, more flexible
it is (Mamatha et al., 2010). Niosomal patches
(4.83 ± 1.79 kg/cm2) showed more tensile strength as com-
pared to liposomal patches (4.30 ± 1.69 kg/cm2). It was
evaluated from the results that niosomes are more strong
and flexible as compared to liposomal patches (Rama &
Shantha, 2012). Percent moisture content was found to be less
for niosomal patches, i.e. 6.0 ± 2.10%. Low-moisture content
helps the formulation to remain stable and make them free
from being a brittle and dry film. Percent flatness was found
to be similar in both niosomal as well as liposomal patches
(100% for both). Flatness study revealed that the formulation
possess same length before and after the test. Thus, from this
 8 S. Arkvanshi et al. J Liposome Res, Early Online: 1–11
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For personal use only.

Figure 4. DSC thermogram of (a) pure drug; (b) cholesterol; (c) Span 60 and (d) HPMC K4M.

Figure 5. DSC thermogram of (a) control patch; (b) niosomal patch and (c) liposomal patch.

test, it was concluded that there was no constriction in patches

and had smooth and flat surface. It was expected that the
smoothness would be retained even after the application onto
the skin. Folding endurance was found to be 209 ± 1.8 which
showed that folding endurance was greater than liposomal
patches. This parameter specifies the fact that integrity of the
patches was maintained over the skin (Mamatha et al., 2010).
Percent drug content was found to be maximum in case
of niosomal patches (97.09 ± 8.04) due to maximum
drug entrapment possessed by the niosomes due to max-
imum concentration of cholesterol and span 60. As span
60 due to low-HLB value provided higher drug loading
(Dharashivkar et al., 2014).
In vitro release studies
In vitro permeability studies were conducted for liposomal
and niosomal patch using dialysis membrane in phosphate
buffer pH 7.4 up to 8 h (Liu et al., 2011). The in vitro release
parameters had shown in Table 5. Cumulative release profiles
of niosomal patches, liposomal and control patches were
compared (Figure 7). Regardless of whether transdermal
absorption has a systemic action or a local effect, a drug
should pass through the stratum corneum. However, due to
barrier effect of the stratum corneum, many drugs cannot play
a full role (Liu et al., 2011). Flux, release rate and release
coefficient were found to be higher in case of niosomal
 DOI: 10.3109/08982104.2014.974058
Journal of Liposome Research Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 11/18/14
For personal use only.
Figure 6. Scanning electron microscopic images of (a) control patch; (b) niosomal patch and (c) liposomal patch.
Table 4. Pharmacotechnical properties of niosomal loaded patches and reference liposomal patches.
Formulation code Niosomal patch (NP)
Parameters
Thickness (mm) 0.30 ± 0.48
Weight variation (mg/cm2) 0.483 ± 0.57
Flatness (%) 99.98 ± 0.10
Folding endurance 209 ± 1.80
Tensile strength (kg/cm2) 4.83 ± 1.79
Moisture content (%) 6.66 ± 2.10
Drug content (%) 97.09 ± 1.11
patches as compared to liposomal patches and control
patches. Surfactant present in niosomes act as a penetration
enhancer which contribute in the permeation enhancement of
buflomedil hydrochloride from niosomes. Further, niosomes
undergo fusion at the interface of stratum corneum and high-
drug concentration in bilayers leads to improved thermo-
dynamic activity of buflomedil hydrochloride in the stratum
corneum. These results are in accordance with the report
evaluated by Fang et al., in which it was described that
niosomal formulations was superior to liposomal and control
formulations in enhancing the release across the skin (Fang
et al., 2011). The percent cumulative amount of drug released
was found to be 95.12 ± 1.12 in case of niosomal patches,
which greatly enhances the effect of buflomedil hydrochlor-
ide. On comparing the release profiles with that of control
patch profile (reference) using one way ANOVA, significant
difference (p50.05) in permeability was observed. Similarity
(f2) and dissimilarity factor (f1) were also calculated. The f2
Buflomedil hydrochloride niosomal patch for transdermal delivery 9
Liposomal patch (LP) Control patch (CP)
0.091 ± 0.31 0.085 ± 0.24
0.325 ± 0.47 0.603 ± 0.63
97.12 ± 1.11 94.68 ± 0.55
196 ± 1.43 199 ± 1.51
4.30 ± 1.69 3.6 ± 1.55
7.76 ± 2.44 9.0 ± 2.26
75.12 ± 1.53 71.33 ± 1.11
value of both liposomal patch and niosomal patch was less
than 50 (Table 5) which implies dissimilarity among release
profiles from control patch. Also, with reference to liposomal
patch (LP), niosomal patch release profile showed higher
dissimilarity (f2 ¼ 39) between two profiles. The curve was
fitted by zero order, first order, Higuchi, Korsmeyer–Peppas
and Hixon–Crowell release kinetic model. The order for the
release was chosen to investigate the possible mechanism of
drug release and release rate as indicated by higher
determination coefficient (r2) (Sakthivel et al., 2012). The
coefficient of determination (r2) in case of zero and first order
was found to be 0.9906 and 0.9019, respectively, in case of
niosomal patches (Table 6). As r2 value was more in zero
order, specifying zero order controlled release pattern.
Further, in order to determine whether diffusion is involved
in the drug release, the data were subjected to Higuchi model.
The lines obtained were comparatively linear with r2 value of
0.9994 indicating that the diffusion may be the mechanism
 10 S. Arkvanshi et al. J Liposome Res, Early Online: 1–11

Table 5. In vitro release study of buflomedil hydrochloride loaded niosomal patch and liposomal patch across the dialysis membrane.

Formulation % Cumulative drug Flux Release rate Release coefficient

code released (8 h) (mg/cm2/min) (mg/cm2/min0.5) (cm/min  102) ER f2
NP 95.12 ± 1.19 16.67 ± 1.53 57.80 ± 1.15 0.531 ± 1.50 2.62 30
LP 82.21 ± 1.24 8.83 ± 1.28 45.62 ± 1.70 0.265 ± 1.440 2.07 49
Control patch* 70.10 ± 1.33 1.04 ± 1.31 22.05 ± 1.01 0.033 ± 1.24 1.00 100

ER Enhancement ratio.
*Reference formulation for calculation of f2

Thus, niosomal system signifies to be a better carrier for

effective and safe transdermal drug delivery.
Journal of Liposome Research Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 11/18/14

Acknowledgements
The authors are thankful to IFTM University, Moradabad, for
providing all the facilities required for conducting the
research work.

Declaration of interest
Authors declared no conflict of interest.

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