Biomedicine & Pharmacotherapy 93 (2017) 255–266

Available online at

ScienceDirect
www.sciencedirect.com

Formulation and optimization of lacidipine loaded niosomal gel for

transdermal delivery: In-vitro characterization and in-vivo activity
Mohd Qumbara , Ameeduzzafarb , Syed Sarim Imamc , Javed Alia , Javed Ahmadd ,
Asgar Alia,*
a
Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
b
Departments of Pharmaceutics, College of Pharmacy, Al-Jouf University, Al-Jouf, Saudi Arabia
c
Departments of Pharmaceutics, Glocal School of Pharmacy, Glocal University, Saharanpur, UP, India
d
Departments of Pharmaceutics, College of Pharmacy, Najran University, Najran, Saudi Arabia

A R T I C L E I N F O A B S T R A C T

Article history:
Received 15 March 2017 The aim of the present research work is to formulate lacidipine (LAC) loaded niosomes formulation for
Received in revised form 29 May 2017 the management of hypertension by thin film hydration technique. The developed formulations were
Accepted 13 June 2017 statistically optimized by four factors, three levels Box–Behnken design and were evaluated for vesicle
size, entrapment efficiency, and flux. The optimized LAC niosomes was further evaluated for permeation
Keywords: depth by confocal laser scanning microscopy (CLSM) and converted to gel formulation. Further, the
Lacidipine optimized LAC niosomes gel was evaluated for ex-vivo permeation study, skin irritation study, stability
Niosomes study and pharmacodynamics study. The optimized LAC niosomes formulation showed vesicle size,
Transdermal gel
entrapment efficiency and flux value of 676.98  10.92 nm, 82.77  4.34% and 38.43  2.43 mg/cm2/h
Confocal laser scanning microscopy
respectively, with spherical morphology. The comparative CLSM study showed that optimized LAC
Antihypertensive activity
niosomes formulation has shown maximum permeation (70.75 mm) as compare to LAC liposomes
formulation (58.26 mm). The optimized LAC niosomes gel showed skin permeation enhancement of 2.15
times as compare to control gel. Furthermore, in vivo antihypertensive activity showed significantly
higher (p < 0.001) reduction in blood pressure compared to oral suspension. Indeed, it was found that
niosomal vesicles represented to be an efficient nano vesicular carrier for transdermal delivery of
lacidipine.
© 2017 Published by Elsevier Masson SAS.

1. Introduction efficacy compared to other dihydropyridine calcium antagonists

Cardiovascular disease is leading cause of mortality and
responsible for one-third of all deaths globally. The majority of
cardiovascular disorders are caused not by single risk factor, but
rather a mixture of several factors like high levels of blood lipids,
obesity, physical inactivity, smoking, glucose intolerance/diabetes
and age. High blood pressure (BP) certainly represents a
amendable risk factor [10]. Lacidipine (LAC) is a dihydropyridine
calcium-channel blocker developed for oral administration and
widely used in therapy since the early 1990s. It is used in the
treatment of hypertension and atherosclerosis and also possesses
an antioxidant effect [6,35]. In addition to calcium-channel
mediated vasodilation, it exhibits significantly higher antioxidant
* Corresponding author.
E-mail addresses: zzafarpharmacian@gmail.com (Ameeduzzafar),
sarimimam@gmail.com (S.S. Imam), alipharm786@gmail.com (A. Ali).
[35]. It has a high degree of lipophilicity which deposits on the lipid
moiety and continuously releases into the binding site during the
clean up phase, so it has long duration of action [51]. It undergoes
extensive first-pass hepatic metabolism which result to a low oral
bioavailability (about 10%) [29].
Several conventional and novel approaches have already
explored for the formulation of lacidipine, including the using
the nanoformulation [37], self nanoemulsifying system [32]
lipotomes [33] and proniosomes [46] have reported limited
fruition. All these formulations have enhances the solubility and
bioavailability of poorly water soluble drug lacidipine but also
reported for lower loading capacity and drug expulsion during
storage. But among these different approaches, the use of
niosomes as formulation act as a solubilizing matrix and offer
several advantages, modify the particle composition or surface to
adjust the drug release rate, acting as permeation enhancer,
greater stability, or the affinity for the target site and longer shelf-
life [24]. However, the is no specific literature report available on

http://dx.doi.org/10.1016/j.biopha.2017.06.043
0753-3322/© 2017 Published by Elsevier Masson SAS.
256 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266
the lacidipine niosomal gel formulation using statistical design
approach for enhancing its antihypertensive activity on rat model.
Niosomes are vesicular nanocarriers and have gained much
attention as novel drug delivery systems in the last three decades
due to their unique characteristics for transdermal application.
Niosomes are self-assembling non-ionic vesicle systems also called
as also called as non-ionic liposomes formed from nonionic
surfactants in an aqueous environment. It has high potential to act
as carriers for poorly soluble drugs [7,41,25]. It is structurally
similar to liposomes [43], but due to the better stability, they are
ideal alternative to liposomes in drug delivery systems [18,21].
They are biodegradable and biocompatible, relatively nontoxic and
capable of loading both hydrophilic and lipophilic drugs [9,36].
Niosomes have lamellar structures composed of amphiphilic
molecules, known as surfactants, contain both hydrophobic (tails)
and hydrophilic (heads) groups and leading self-assembling
properties, aggregating into a variety of shapes like micelles or
into a planar lamellar bilayer [42]. Hydrophilic drugs are entrapped
inside of vesicular aqueous core or adsorbed on bilayer surfaces
while lipophilic substances are encapsulated by partitioning into
the lipophilic domain of the bilayers [48].
There are various nano drug delivery systems have been
reported as a promising approach to overcome the various
drawbacks of conventional delivery system. These novel delivery
system have reported in permeability enhancement of the drug
across the stratum corneum of skin or cornea of eye and also
enhances the bioavailability through different routes. The previous
experimental studies carried out on diverse nano sized delivery
systems for various diseases like arthritis [19,26], hypertension
[3,29], schizophrenia [44,48], hepatoprotective activity [22,45],
antifungal disease [47], Cancer [17,2] have vouched the utility of
these delivery systems through different routes. In fact, niosomes
act as therapeutic reservoirs for drug delivery in a controlled
manner to improve bioavailability and enhanced therapeutic
efficacy over extended period of time [34].
The current research work explored the feasibility of niosome
as a carrier for the transdermal application of poorly soluble drug
Lacidipine. The developed formulations were optimized using
four-factor three-level Box–Behnken statistical design and evalu-
ated for the vesicle size, shape, entrapment efficiency, in-vitro drug
release, transdermal flux, confocal laser scanning microscopy,
histopathological examination and in-vivo antihypertensive effi-
cacy.
2. Materials and methods
2.1. Materials
Lacidipine was received as gift sample from Unichem Pharma,
Ahmadabad, India. Cholesterol, Span 60, Carbopol 934 and soya
phosphatidylcholine 70 (SPL 70) had been procured from
Spectrochem, Mumbai, India and S.D. Fine Chemicals, Mumbai,
India respectively. All the other chemicals used for formulation
were of analytical grade and procured from S.D. Fine Chemical,
Mumbai, India. Albino Wistar rats (6–8 weeks/200–250 g) were
approved and provided by Institutional Animal Ethics Committee,
and CPSCEA (Committee for the purpose of control and supervision
of experiments on animals) (1021/IAEC/2014), Govt of India,
Central Animal House of Hamdard University. Animal experiments
were carried out according to the Guidelines for the Care and Use of
Laboratory Animals that was approved by the IAEC of Jamia
Hamdard. All the Albino Wistar rats were housed under controlled
environmental conditions (temperature, 25  2  C; humidity,
55  15% RH, 12 h light/dark cycle) and were nourished with pellet
diet (Lipton, Kolkata, India).
2.2. Methods
2.2.1. Formulation and optimization of niosomes
LAC niosomes were formulated and optimized by the thin-film
hydration technique [48]. Precisely, cholesterol, Span 60, SPL 70
and LAC were taken in a clean and dry round bottom flask and the
lipid mixture was dissolved in chloroform: methanol mixture
(1:1 v/v). The organic solvent was removed by rotary evaporator
above the lipid transition temperature. The traces of organic
solvents were removed under vacuum for the overnight period.
The deposited thin lipid film was hydrated with phosphate buffer
saline (PBS) with pH 7.4 by rotation at 150 rpm at room
temperature. The resulted vesicles were hydrated for 2 h at room
temperature to obtain large multilamellar vesicles (LMLV). To
prepare smaller vesicles, these vesicles were sonicated as
summarized in Table 1 at 4  C at 40% output frequency (at
40 W) (Titanium probe, Ultrasonicator, Model-UP100H, Hielscher
Ultrasonics GmbH, Berlin, Germany). Similarly Rhodamine Red
(RR) (0.03% w/v) loaded niosomes was prepared for confocal laser
microscopy study.
2.2.2. Experimental design
The developed formulations were optimized using 4-factor 3-
level Box-Behnken statistical design. The rationale behind this
Box Behnken design based on the salient principles of Design of
experiments and quality by design (QbD) approach. The design was
particularly selected because it needs less runs than a central
composite design in cases of three or four independent variables. It
provides understanding of the plausible interaction(s) among the
different levels of variables and helps in selecting “the best”
formulation with minimal expenditure of time, effort and
developmental cost vis-a‘-vis the traditional one factor at a time
(OFAT) approach [5]. The QbD methodology involves defining the
critical process parameters (CPPs) using screening and risk
assessment, optimization data analysis and optimum search
through response surface methodology (RSM) to embark upon
the design space and postulation of control strategy for continuous
improvement [38,4]. This property prevents a potential loss of data
in those cases. The design matrix generated the nonlinear
quadratic equation for the response as shown below;
Y = b0 + b1A + b2B + b3C + b12AB + b13AC + b23BC + b11A2 + b22B2 +
b33C2 . . . . . . . . . .
Where Y is the response related with each factor level combina-
tion; b0 is constant; b1, b2, b3 are linear coefficients, b12, b13, b23 are
interaction coefficients while b11, b22, b33 are quadratic coefficients
generated from the observed experimental values of response from
experimental runs, while A, B and C are the coded intensity of
independent variables. The terms A2, B2 and C2 (i = 1, 2 or 3)
represent the interaction and quadratic terms, respectively [27].
The concentration range of independent variables along with their
Table 1
Various independent and dependent variables used in LAC niosomes formulations
by Box-Behnken design.
Factor Level used, actual (coded)
Independent variable Low ( 1) Medium (0) High (+1)
A = span 60 (mM) 1 2 3
B = CHO (mM) 0.5 1.0 1.5
C = hydration time (min) 45 52.5 60
D = sonication time (min) 5 10 15
Dependent variables Goal
Y1 = Vesicle size (nm) Minimize
Y2 = Entrapment efficiency (%) Maximize
Y3 = flux (mg/cm2/h) Maximize
 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266 257

low, medium and high levels were showed in Table 1. The selected
independent variables for the experimental design were span 60
concentration (A), cholesterol concentration (B), hydration time
(C) and sonication time (D) and their effect were observed on
vesicle size (Y1), entrapment efficiency (Y2) and flux (Y3) with five
centre point were shown in Table 2. The resulting experimental
values of the responses were compared with predicted value and
linear regression plot between actual and predicted value of the
responses was plotted. The model was evaluated in terms of
statistically significant coefficient and R2 values and finally one
optimum formulation was selected from point prediction method
and converted to gel for further study.
3. Characterization of niosomes formulation
3.1. Niosomes size and size distribution
The vesicles size and size distribution were determined by
dynamic light scattering (DLS) method, using a computerized
inspection system (Zetasizer, HAS 3000; Malvern Instruments,
Malvern, United Kingdom). For vesicle size measurement, the
vesicular suspension was further diluted with the phosphate buffer
saline to avoid multiscattering events and the measurements were
conducted in triplicate [12].
process was repeated thrice to ensure that free drug was
completely removed [48]. The% EE of niosomes formulations
was calculated by the following equation.
%EE = (Total drug Drug in supernatant)/(Total drug)
3.3. Ex-vivo skin permeation studies
The permeation of LAC from niosomes and control was
measured through excised rat skin on diffusion cells. The study
was performed using rat abdominal skin on a diffusion cell with an
effective diffusional area of 0.785 cm2 having 10 mL of receiver
capacity. The filled in receiver chamber. The receiver fluid
ethanolic: phosphate buffered saline (30:70% v/v, pH 7.4) was
stirred with a magnetic stirrer at a speed of 200 rpm, and the
temperature was maintained 32  C  2  C in the whole study.
Ethanol was added in receiver sample to maintain sink conditions
due to the extreme poor solubility in water [1]. 2 mL of the
niosomes formulation (equivalent to 4 mg) was placed into donor
compartment and covered with aluminum foil to provide occlusive
conditions. Samples were withdrawn at regular intervals up to 48 h
and analyzed for drug content by HPLC method [29]. Each
experiment was replicated three times and concentration of LAC
permeated through the skin was calculated.

3.2. Entrapment efficiency 3.4. Niosomes morphology

The entrapment efficiency (% EE) of LAC niosomes formulations
were determined by the centrifugal method. The formulations
were centrifuged at 7000 rpm for 20 min and supernatant was
taken and diluted with PBS (pH 7.4). The drug concentration in the
resulting solution was assayed by UV method at 240 nm. This
Morphology of niosomes vesicle was visualized by using a
Morgagni 268D (Fei Electron Optics, Netherland) transmission
electron microscope (TEM), with an accelerating voltage of 100 kV.
The sample for TEM observation was prepared by placing few
drops of optimized niosomes dispersion that was previously

Table 2
Observed Box–Behnken Design LAC niosomes formulation runs with their actual and predicted experimental value of Y1 (particle size), Y2 (encapsulation efficiency), and Y3
(flux).

Runs Independent variables Dependent variables

A B C D (Y1) Mean  SD (Y2) Mean  SD (Y3) Mean  SD

Actual value Predicted value Actual value Predicted value Actual value Predicted value
LNF1 2.00 0.50 52.50 5.00 754.23  10.23 750.24 71.92  2.36 72.05 27.87  1.71 25.40
LNF 2 2.00 1.00 52.50 10.00 674.65  11.45 677.24 57.46  5.49 75.31 28.91  1.28 35.20
LNF 3 2.00 1.00 52.50 10.00 678.64  9.26 677.24 78.91  4.58 75.31 37.29  2.06 35.20
LNF 4 1.00 1.00 45.00 10.00 715.98  12.73 720.35 33.39  5.01 36.46 18.06  1.17 16.41
LNF 5 2.00 1.00 60.00 15.00 586.67  15.92 585.35 40.19  3.56 37.64 34.19  2.16 32.19
LNF 6 3.00 0.50 52.50 10.00 605.87  11.36 611.29 94.75  3.59 93.15 30.91  1.98 30.28
LNF 7 3.00 1.00 52.50 15.00 583.65  10.39 584.98 58.67  2.84 60.68 32.76  2.36 34.19
LNF 8 2.00 1.00 45.00 15.00 643.82  12.31 641.44 48.42  2.51 45.68 28.49  1.85 27.88
LNF 9 2.00 1.00 52.50 10.00 672.97  19.97 677.24 76.85  4.26 75.31 34.37  1.69 35.20
LNF10 2.00 0.50 45.00 10.00 613.98  15.84 608.59 66.28  3.49 67.05 14.56  2.54 15.87
LNF11 2.00 1.50 52.50 5.00 788.42  21.49 783.08 30.93  1.98 31.80 20.47  2.67 18.78
LNF12 3.00 1.00 52.50 5.00 697.92  13.63 703.15 58.98  2.27 60.66 21.73  1.78 24.13
LNF13 2.00 1.00 45.00 5.00 737.76  19.38 740.18 39.42  2.52 37.82 16.32  1.59 18.55
LNF14 1.00 1.50 52.50 10.00 760.38  16.29 756.07 36.68  3.65 34.12 17.84  1.79 18.70
LNF15 3.00 1.50 52.50 10.00 624.34  13.21 621.05 44.01  4.08 42.56 21.98  1.73 20.31
LNF16 2.00 0.50 60.00 10.00 648.45  11.62 643.31 66.85  4.13 69.31 35.65  2.53 34.20
LNF17 2.00 1.00 60.00 5.00 743.67  18.78 747.16 51.63  2.72 50.21 27.33  2.16 28.17
LNF18 2.00 1.00 52.50 10.00 682.98  14.23 677.24 80.54  3.24 75.31 36.98  2.35 35.20
LNF19 2.00 1.50 52.50 15.00 665.98  13.82 669.34 31.7  1.78 33.37 25.56  1.89 27.68
LNF20 2.00 1.50 45.00 10.00 712.57  16.79 717.24 30.93  2.11 30.82 21.26  1.74 22.83
LNF21 3.00 1.00 60.00 10.00 605.39  14.49 600.39 62.62  3.56 61.35 29.26  2.05 30.56
LNF22 1.00 1.00 52.50 5.00 812.45  20.27 810.65 39.99  2.89 40.33 21.65  1.87 20.33
LNF23 2.00 1.00 52.50 10.00 676.98  10.92 677.24 82.77  4.34 75.31 38.43  2.69 35.20
LNF24 1.00 1.00 52.50 15.00 673.98  9.25 668.28 34.92  2.59 35.59 25.91  2.52 23.62
LNF25 3.00 1.00 45.00 10.00 589.65  8.38 585.97 57.93  4.13 58.55 25.41  2.21 22.57
LNF26 1.00 1.00 60.00 10.00 653.76  10.43 656.81 36.82  2.38 38.01 19.85  1.95 22.35
LNF27 1.00 0.50 52.50 10.00 662.67  9.21 667.07 58.87  3.85 56.16 15.63  2.00 17.53
LNF28 2.00 0.50 52.50 15.00 598.71  10.47 603.42 64.82  4.27 65.76 28.52  2.45 29.86
LNF29 2.00 1.50 60.00 10.00 628.49  15.27 633.41 31.32  1.83 32.90 19.62  2.05 18.43

A = Cholesterol (mM); B = Span 60 (mM); C = Hydration time (min); D = Sonication time (min); Y1 = Particle size (nm); Y2 = Encapsulation efficiency (%); Y3 = Flux (mg/cm2/h).
258 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266

Table 3 skin of the rat. The experiment was run in diffusion cell as
Summary of regression analysis for responses Y1 (particle Size in nm), Y2
described under ex-vivo skin permeation study for 8 h. Both the
(encapsulation efficiency in%), and Y3 (flux in mg/cm2/h) for fitting to different
models. skin sample was removed, washed with distilled water and tested
for probe penetration [8,48]. The excised nude rat skin was placed
Model R2 Adjusted R2 Predicted R2 SD %CV
on the microscopic slide with the SC side facing the cover glass. The
Response (Y1) CLSM (Olympus Fluo View; FV1000) was carried out with an argon
Linear 0.8151 0.7843 0.7111 28.73 –
laser beam with excitation at 488 nm and emission at 590 nm. The
2FI 0.8899 0.8288 0.6385 25.60 –
Quadratic 0.9956 0.9911 0.9768 5.82 0.87
skin sample was sliced into sections of 5–10 mm thickness through
the z–axis by CLSM with Fluo view software.
Response (Y2)
Linear 0.5854 0.5163 0.4897 12.78 – 3.6. Formulation of optimized LAC niosomes gel
2FI 0.6203 0.4093 0.3732 14.13 –
Quadratic 0.9484 0.8968 0.8878 5.90 10.92
The optimized LAC niosomes formulation (LNF23) was con-
Response (Y3) verted into gel to get epidermal retention for prolonged period of
Linear 0.3551 0.2476 0.1578 6.10 – time. The selection of optimized LAC niosomes formulation
2FI 0.4883 0.2041 0.0667 6.28 –
(LNF23) was done on the basis of formulation showed maximum
Quadratic 0.9046 0.8093 0.6270 3.07 11.78
transdermal flux. Briefly, Carbopol-R 940 as gelling agent (1% w/w)
was added into water and kept overnight for complete humecta-
diluted 50-fold with double-distilled water onto a 400-mesh tion of polymer chains. The optimized LAC niosomes formulation
carbon film coated copper grid. Before the carbon film dried on the (LNF23) equivalent to 2 mg of LAC (transdermal dose of LAC for
grid, it was negatively stained with 1% phosphotungstic acid for 48 h) was added to hydrate carbopol solution with continuous
10 s. The sample was dried in air before TEM observation [15]. stirring. Other ingredients like 15% w/v polyethylene glycol-400
(PEG-400) and triethanolamine (TEA) (0.5%w/v) were added to get
3.5. Confocal laser scanning microscopy (CLSM) study homogeneous dispersion of gel (optimized LAC niosomes gel).

Confocal laser scanning microscopy (CLSM) used to scan the
fluorescence signal of developed formulations at different skin
depths. The optimized LAC niosomes formulation and control
formulation (liposome) prepared with rhodamine red (RR-0.03%)
and were applied homogeneously and non-occlusive to the dorsal
3.7. Characterization of optimized LAC niosomes gel
3.7.1. Viscosity
The viscosity of optimized LAC niosomes gel was determined at
25  2  C using Brook-field viscometer with spindle number C-50-

Fig. 1. Three-dimensional (A-B) and contour response surface plot (C-D) image showing influence of independent variables (A = Cholesterol; B = Span 60; C = Hydration time;
D = Sonication time) on particle size for LAC loaded niosomes.
 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266
1 (Brookfield Engineering Laboratories Inc., Middleboro, MA, USA),
at 50 rpm and the determinations were carried out in triplicate.
3.7.2. pH
The optimized LAC niosomes gel was diluted in double distilled
water and the pH was recorded using a pH meter (Mettler Toledo
MP 220, Greifensee, Switzerland) by bringing it in contact with the
optimized niosomes gel and allowed it to stand for 1 min to
equilibrate.
3.7.3. Drug content
For assay of LAC in niosomes gel, LAC was extracted from 10 g of
optimized niosomes gel with 100 mL of methanol; it was suitably
diluted and assayed by UV method at 240 nm. The analysis was
performed triplicate for each batch and the content of LAC in each
sample was determined in terms of percentage of drug content.
3.7.4. Ex-vivo skin permeation study
The skin permeation studies of optimized LAC niosomal gel was
performed on diffusion cell with an effective diffusional area of
0.785 cm2and 10 mL of receiver chamber capacity using rat
abdominal skin. After complete preparation and stabilization of
the skin 2gm of niosomal gel formulation was placed into donor
compartment and covered with aluminum foil to provide occlusive
conditions. After specific time interval samples were withdrawn
from receptor compartment at regular time intervals (1, 2, 4, 6, 12,
24, and 48 h), and analyzed for drug content by HPLC [29]. Similar
Fig. 2. Three-dimensional (A-B) and contour response surface plot (C-D) image showing influence of independent variables (A = Cholesterol; B = Span 60; C = Hydration time;
D = Sonication time) on encapsulation efficiency for LAC loaded niosomes.
259
experiments were performed with conventional liposome. To
determine the extent of skin permeation enhancement, enhance-
ment ratio (ER) was calculated as follows:
ER = steady state flux of formulation/steady state flux of control
3.7.5. Texture analysis of niosomal gel formulation
Gel characteristics such as stickiness and firmness of LAC
niosomes gel were analyzed by using spreadability ring that was
fitted on the texture analyzer (Stable Micro Systems Ltd.,
Godalming, Surrey GU7 1YL, UK). It consists of two probes: an
upper probe and a lower probe. Niosomes gel was allowed to
equilibrate for 30 min at room temperature before starting the test.
The test sample was kept in the lower cone of the equipment and
pressed to eliminate entrapped air and the excess was dragged off
to make a flat test area. Before testing, the upper cone probe was
calibrated alongside the lower probe in such a way that the starting
point can be kept at the same height. During testing, the
equipment was set to compression mode with a test speed of
3 mm/s. The upper cone probe was allowed to reach and penetrate
the sample up to a depth of 2 mm above the sample holder surface.
Thus, the probe moved a distance of 23 mm from its starting point.
3.7.6. In-vitro drug release and release kinetics
The release study was performed for the optimized niosomes
gel formulation (LNF23) by the paddle method, using ethanol:
phosphate buffer (30:70, pH 6.8) to simulate human skin pH. The
260 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266
calculated amount of niosomes gel formulation was placed in a
cellulose membrane (MW = 12000 Da) and was placed at the
bottom of the USP dissolution tester (Veego, VDA-8DR, Mumbai,
India). The vessel contained 500 mL buffer solution and the speed
were adjusted to50 rpm [50]. About 5 mL were withdrawn from
the release medium at different time intervals for 24 h. and
replaced by the equivalent volume of the medium. The amount of
drug released from the formulation was determined by HPLC. The
mean cumulative amount of drug released was plotted against
time and data obtained from the release studies were kinetically
analyzed for the order of drug release from the formulation. The
release evaluated to check the goodness of fit for zero-order model,
first-order model, Higuchi's matrix model [13] and Korsmeyer–
peppas model [39]. The correlation coefficient (R2) for each model
was calculated. Model giving R2 nearer to 1 was selected as the best
fit model for the drug release.
3.7.7. Skin irritation and histopathological studies
Skin irritation study was performed using Wistar rat to check
for skin irritation potential [16]. After application of optimized LAC
niosomes gel formulation and aqueous formalin solution (positive
control) [49,44], the application sites were examined for dermal
reactions. The primary irritation score was calculated following
study completion and compounds producing scores of 2 or less
were considered no skin irritation. The primary irritancy index
(PII) was calculated for each treated group by adding the edema
and erythema scores of each group. The score was accordingly
Fig. 3. Three-dimensional (A-B) and contour response surface plot (C-D) image showing influence of independent variables (A = Cholesterol; B = Span 60; C = Hydration time;
D = Sonication time) on flux for LAC loaded niosomes.
classified on the basis of scoring. [PII <2 (non-irritant); PII = 2–5
(irritant); PII = 5–8 (highly irritant)]. Further skins were tested for
histopathological study by sacrificing Albino Wistar rat. The
treated area and untreated area were collected was stored in 10%
formalin solution in phosphate buffer solution. The samples were
dehydrated using ethanol, embedded in paraffin and stained with
hematoxylin and eosin. The skin samples were then observed
under light microscope (Motic, Japan) and compared with the
control sample and evaluated for any skin irritation effect.
3.7.8. In-vivo antihypertensive study
The animals were trained for their stay in the restrainer because
slight movement and aggression by the animal would have led to
variation in BP reading. For this reason, a rat was inserted in the
restrainer headlong until the whole body was easily accommodat-
ed inside. The restrainer was locked from the open side and leaving
the tail outside the apparatus. The exercise was repeated several
times for 7–10 days before the actual experiment until the animals
learned to stay in the restrainer without aggression and was
familiar with the experimental conditions. The animals (Wistar
rats) were divided into 4 groups (Group A to D) of 4 rats each.
Group A was taken as control and remaining three groups (Groups
B to D) hypertension was induced by subcutaneous injection of
methylprednisolone acetate (MPA) (20 mg/kg/wk) for 2 weeks as
per reported method [23]). The BP of hypertensive rats from Group
B to D was measured before the drug administration on the day of
the actual experiment. Group B served as toxic control and received
 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266 261

Table 4
In vitro release kinetics data to different mathematical models for LAC loaded optimized niosomes and optimized niosomes gel formulations.

Optimized niosomes (LAC23) Optimized niosomal gel (LAC23 Gel)

2
Model Equation R Model Equation R2
Zero-order mo m = kt 0.8908 Zero-order mo m = kt 0.9391
First-order ln m = kt 0.9606 First-order ln m = kt 0.9773
Higuchi matrix mo m = kt1/2 0.9744 Higuchi matrix mo m = kt1/2 0.9962
Korsmeyer- peppas log (mo m) = log k + n logt 0.9774 Korsmeyer- peppas log (mo m) = log k + n logt 0.9826
Best fit model Korsmeyer- peppas Best fit model Higuchi model

no more treatment, Group C received an oral suspension of LAC
(1 mg/kg) with the help of feeding needle and group D received
optimized LAC niosomes gel formulation, respectively [11,40]. The
formulation was applied on the previously shaven abdominal area
of rat skin. The rat was then placed in the restrainer and the BP
from the tail was recorded at predetermined time intervals up to
48 h (Table 3).
3.7.9. Stability study of LAC niosomes gel formulation
The optimized LAC niosomes gel formulation was subjected to
stability studies to evaluate any physical or chemical changes in
storage. Gel formulation was kept at 25  2  C/60  5% RH and
40  2  C/75  5% RH for 1 month in borosil glass container ICH
Q1A (R2), [14]. The samples were analyzed for various parameters
at 0, and 1 month after storage like Clarity, pH, drug content and
spreadability.
4. Results and discussion
4.1. Formulation and optimization of LAC niosomes
A 4-factor, 3-level Box–Behnken statistical design was used to
preparation niosomes (Table 2) using Design Expert1 (Version
8.0.0.6, Stat-Ease Inc., Minneapolis, MN). All individual and
interactive effects of independent variables were investigated
and all the responses of these runs fitted to first order, second order
and quadratic models and found that best fit model was quadratic
(p < 0.0001). The Summary of results of regression analysis for
responses Y1, Y2 and Y3 for fitting to quadratic model and the
polynomial equation of each response and each model were shown
in Table 3. Three-dimensional plots showed the interaction effects
of the independent variables on the responses as well as their
usefulness in studying the effects of two factors on one response at
a time (Figs. 1–3).

Fig. 4. Linear correlation plots between actual and predicted values and the corresponding residual plots for all responses.
262 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266
4.1.1. Fitting of data to the model
The results of regression analysis of different responses are
given in Table 4. Larger values of the standard error for coefficients
shows that the quadratic nature of the relationship. From Table 2, it
is evident that the one independent variables viz. the concentra-
tion of the Span 60 have positive effects on the response Y2
(entrapment efficiency) and response Y3 (flux), whereas the
response Y1 (vesicle size) has an inverse relationship with Span
60. The concentration of cholesterol has the positive effect on
response Y1 (vesicle size) whereas the concentration of cholesterol
has inverse effect on response Y2 (entrapment efficiency) and on
response Y3 (flux). Hydration time and sonication time have an
inverse effect on response Y1 (vesicle size) whereas both have a
positive effect on response Y3 (flux).
4.1.2. Effect of independent variables on size
Small vesicular size is most important criteria for the effective
transdermal drug delivery of niosomes. The size of the vesicles was
found to vary between 583.65 to 812.45 nm (Table 2). Initially,
average vesicle size increased with increase in the concentration of
Span 60 from 1 to 2 mM. However, an additional increase in the
concentration of Span 60 from 2 to 3 mM leads to decrease in the
average vesicle size. This is due to the development of a micellar
structure instead of the vesicles, which are comparatively smaller
in size. This relationship is presented by the following equation.
Y1 (Particle Size) = 677.244 47.7A + 24.6892B 12.2775C
65.1367 D 19.8 AB + 19.49 AC + 6.05 AD 29.6375 BC + 8.27
BD 15.765 CD 11.5666 A2 1.81033 B2 24.7978 C2 + 26.0859
D2
Fig. 5. Optimized LAC niosomes (a) Particle size graph (b) Transmission electron micrograph (80,000) (scale bar indicates in nm).
The Model F-value of 224.63 implies the model is significant.
There is only a 0.01% chance that a “Model F-Value” this large could
occur due to noise. Values of “Prob> F” less than 0.05 indicated that
the model terms are significant. In this case, A, B, C, D, AB, AC, BC,
BD, CD, A2, C2, D2 are significant model terms. Values greater than
0.1 indicates the models are not significant. The “Lack of Fit F-
value” of 2.77 implies the “Lack of Fit” is not significant which is
relative to the pure error. There is only 16.91% chance that this large
“Lack of Fit F-value” could occur due to noise. There existed a direct
relationship between the Span 60 concentration and cholesterol on
the vesicle size, entrapment efficiency of the vesicles and
transdermal flux of vesicles loaded with LAC. This model can be
used to navigate the design space and result of this calculated
model for vesicle size represented by 3-dimentional plots and
contour plot as shown in Fig. 1 (A-D). The plot showed the effect of
two formulation factors on particle size at one time.
4.1.3. Effect of independent variables on EE
Entrapment efficiency (%EE) is the percentage fraction of the
total drug (LAC) entrapped into the vesicles. The maximum and
minimum entrapment efficiency obtained were 94.75% for LNF6
and 30.93% for LNF20 respectively (Table 2). It is observed from the
experimental design that entrapment efficiency has a direct
positive relationship with the concentration of Span 60 as revealed
by the following equation.
Y2 (Entrapment Efficiency) = 75.306 + 11.3575A 18.16
B + 1.08833C 1.17917 D 7.1375 AB + 0.315 AC + 1.19 AD 0.045
BC + 1.9675 BD 5.11 CD 10.1176 A2 8.6888 B2 16.5963
C2 15.8726 D2
 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266
The Model F-value of 18.38 implies the model is significant.
There is only a 0.01% chance that a “Model F-Value” this large could
occur due to noise. Values of “Prob> F” less than 0.05 indicated that
the model terms are significant. In this case, A, B, AB, A2, B2, C2, D2
are significant model terms. Values greater than 0.1 indicated that
the model terms are not significant. The “Lack of Fit F-value” of 0.07
implies the “Lack of Fit” is not significant which is relative to the
pure error. There is 99.97% chance that this large “Lack of Fit F-
value” could occur due to noise. This model can be used to navigate
the design space and result of this calculated model for entrapment
efficiency represented by 3-dimentional plots and contour plot as
shown in Fig. 2 (A-D). As the concentration of Span 60 increases, %
EE also increases. It was found that upon increasing hydration time
the% EE significantly increased from 36.82% (LNF26, with least
concentration cholesterol and Span 60) to 71.92% LNF1, medium
ratio cholesterol: Span 60 (2:0.5). According to [28] the entrap-
ment of drug occurred in both, bilayer and aqueous compartment
of the vesicles. When the lipid compartment and aqueous phase
becomes saturated with the drug, the vesicles provided limited
entrapment capacity. The entrapment of drug occurs in both the
bilayers and the aqueous compartment of the vesicles [20]. When
the lipid compartment and aqueous phase became saturated with
the drug, the vesicles provided limited entrapment capacity [31].
Hence, niosome could entrap LAC only to an optimum extent, after
which any further increase in hydration time would lead to leakage
of LAC from vesicles.
4.1.4. Effect of independent variables on flux
There is no significant effect of hydration time on transdermal
flux. The maximum and minimum transdermal flux were found to
be 38.43 (LNF 23) and 14.56 (LNF10) as shown in Table 2. It was
observed from the equation below that the transdermal flux of LNF
has a direct relationship with the concentration of cholesterol
content as revealed by the following equation.
Y3 (Flux) = 35.196 + 3.5925 A 2.2008 B + 3.4833C + 3.3383 D
2.785 AB + 0.515 AC + 1.692 AD 5.682 BC + 1.11 BD 1.327
CD 6.675 A2 6.8155 B2 5.5492 C2 2.9492 D2
The Model F-value of 9.49 implies that the model is significant.
There is only a 0.01% chance that this large “Model F-Value” could
occur due to noise. Values of “Prob> F” less than 0.05 indicated that
the model terms are significant. In this case, A, B, C, D, BC, A2, B2, C2,
Fig. 6. CLS micrographs of rat skin cross section images after application with rhodamine red loaded (0.3%) (a) optimized LAC niosomes gel formulation (b) LAC liposomal gel
formulation (control) (Scale bar indicates in mm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
263
D2 are significant model terms. The values greater than 0.1
indicated that the model terms are not significant. The “Lack of Fit
F-value” of 0.51 implies that this small “Lack of Fit F-value” is not
significant which is relative to the pure error. There is 82.42%
chance that this large “Lack of Fit F-value” could occur due to noise.
Effects of Independent variables on flux are presented by three-
dimensional graph and contour plot Fig. 3 (A-D).
4.2. Optimization
The optimized LAC loaded niosomal formulation was selected
based on the criteria of attaining the maximum value of
transdermal flux and entrapment efficiency whereas minimizing
the vesicle size by applying point prediction method of the Design
Expert1 software [30]. Upon ‘trading of’ various response variables
and comprehensive evaluation of feasibility search and exhaustive
grid search, the formulation composition with Span 60 (2 mM),
cholesterol (1 mM), hydration time (52.5 min) and sonication time
(10 min) was found to fulfil requisites of an optimum formulation
i.e. LNF 23. The optimized formulation has the vesicle size of
676.98  10.92 nm with entrapment efficiency 82.77 4.34% and
transdermal flux across rat skin of 38.43 2.69 mg/cm2/h,
respectively. Fig. 4 showed the quantitatively linear relationship
between resultant experimental values of the responses with that
of the predicted value of all dependents variables. Optimized LAC
niosomes formulation (LNF 23) was converted into gel formulation
and applied transdermally for in vivo antihypertensive studies.
4.3. Characterization and evaluation of niosomes
4.3.1. Vesicles size and size distribution
The least mean vesicles size was observed for LAC loaded
niosomes formulations LNF7 (583.65 nm) while maximum vesicles
size was obtained as 812.45 nm for LNF22 shown in Table 2. The
size of optimized formulation was found 683.8 nm and PDI was
found 0.329 as shown in Fig. 5a.
4.3.2. Vesicles shape
The transmission electron micrograph (TEM) of the optimized
niosomes formulation (LNF23) is shown in Fig. 5b. TEM micro-
graph revealed the formation of well identified spherical and
264 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266

Fig. 7. Haematoxylin and eosin stained cross section photomicrograph of rat skin (a) untreated (control), (b) formalin treated (c) optimized LAC niosomes gel treated.

Table 5
Comparative pharmacodynamic assessment on mean BP of various formulation of LAC in MPA induced hypertensive rat.

Group Treatment Systolic BP (mm Hg) % reduction in BP

0h 1h 2h 4h 6h 12 h 24 h 48 h
A N saline 115.4  5.4 116.3  9.2 115.2  7.1 114.6  4.6 118.2  6.3 115.7  5.1 117.3  4.9 118.2  5.6
B HC control 182.2  7.9 185.8  8.1 178.2  6.1 186.5  6.9 180.1  7.1 181.8  7.3 189.5  8.1 181.5  5.9
C OLS suspension 180  7.5 142.3  8.1 122.5  6.7 136.2  5.4 145.5  6.9 154.1  7.3 159.3  5.4 161.9  6.8 10.79
D OLNniosomal gel 181.2  7.5 161.6  5.9 143.5  8.1 134.6  5.1 126.2  4.9 122.3  4.1 118.7  3.9 141.5  6.4 22.04

NS:- normal saline; HC: Hypertensive control; OLS: Oral LAC Suspension; OLN: optimized LAC niosomal.

discrete vesicles with sharp boundaries after hydration of
niosomes. It shows the outline and core of the well identified
spherical vesicles, and displaying the retention of sealed vesicular
structure. The surface morphology of niosomes formulation
presents increased vesicle wall rigidity with increasing cholesterol
content.
4.3.3. Confocal laser scanning microscopy (CLSM) study
The comparative CLSM study between rhodamine loaded LAC
niosomes formulation and liposome formulation (control) was
performed to check the penetration depth. The result revealed that
rhodamine red loaded LAC niosomes formulation was uniformly
distributed to a greater extent throughout the subcutaneous, viable
epidermis, and dermis with high fluorescence intensity [50]. The
formulation exhibited significantly higher depth (70.75 mm) of
penetration compared to control formulation (38.25 mm) after
transdermal application as depicted in Fig. 6 (A-B). The promi-
nently efficient delivery of LAC by niosomal carriers suggested
their enhanced penetration and consequent fusion with the
membrane lipids in the depths of the skin, corroborating the
results of many researchers [8].
4.3.4. Characterization of optimized LAC niosomal gel
The drug content, viscosity, pH and spreadability of optimized
niosomal gel were shown 98.60%, 38.00 PaS, 6.7 and satisfactory
spreadability, respectively.
4.3.5. Texture of niosomal gel formulation
The various parameter of texture analysis like of were like
consistency, stickiness, firmness and work of adhesion were
evaluated of optimized LAC niosomal gel. The consistency,
stickiness, firmness and adhesion of optimized LAC-niosomal gel
was found to be 0.029  0.003, 0.032  0.002, 0.048  0.003 and
0.018  0.003 respectively.
4.3.6. Ex vivo permeation study
The ex-vivo permeation profile of niosomes yielded significantly
higher (p < 0.0001) flux value i.e. 38.43 mg/cm2/h (optimized LNF
23 gel) with enhancement ratio of 33.92 through rat skin. The
reason for this better performance of niosomes formulations is the
deformability and the ability to retain vesicle integrity, while the
aggregates undergo a dramatic change in shape in comparison
with liposomes and all these characteristics allow the niosomes to
pass the skin pores which are much smaller than the diameter of
niosomes [40]). Effects of independent variables on flux are
presented by three-dimensional graph and contour plot as shown
in (Fig. 3).
4.3.7. In-vitro drug release and release kinetics
The obtained in vitro release data for both optimized LAC
niosomes and LAC niosomes gel data was fitted to various release
kinetic models. The correlation coefficients (R2) values for different
models for both formulations are presented in Table 4. The model
showing highest value of R2 was considered as best model for
release mechanism. It was found that the best fit model for
optimized LAC niosomes (LAC23) was Korsmeyer-peppas model
and for LAC niosomes gel was Higuchi matrix model, respectively.
The highest R2 obtained for optimized LAC niosomes gel
formulation (LAC23) and optimized LAC niosomes gel formulation
were 0.9744 and 0.9962 respectively.
4.3.8. Histopathological study
Sections of untreated rat skin (control) revealed well defined
epidermal and dermal layers. The keratin layer was also well
formed and was presenting just adjacent to the top most layer of
the epidermis. Dermis was devoid of any inflammatory cells as
clearly seen in Fig. 7(A-C). There were no significant changes
observed in rat skin specimens treated with formulation in
comparison with the untreated SC suggest absence of any skin
 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266
irritation. The formalin treated skin showed marked damage to the
skin. The image shows marked damage to skin and extraction of
lipid has been seen in the formalin treated skin.
4.3.9. In vivo studies
The pharmacodynamic data of different treatments were
compared for statistical significance by Dunnet test, which showed
significant difference (P < 0.001) in BP values of normal control
(Group A) (Table 5). The oral administration of LAC suspension
significantly (p < 0.05) controlled the hypertension within 2 h,
with the maximum antihypertensive effect (122 mm Hg), beyond
2 h mean systolic blood pressure remained above the normal value
and ultimately reached to 161 mm Hg at 48 h. Oral suspension of
LAC acted rapidly and showed utmost changes in rat BP, but its
effect dipped rapidly and BP increased to above normal value. The
administration of LAC oral suspension resulted in a gradual
decrease of BP, with the maximum effect of 122.5 mm Hg, observed
at 6 h on treatment the experimental hypertensive rats (p > 0.05).
The result revealed that the systolic BP of hypertensive rats was
controlled up to 48 h; however at, 48 h time point BP, was
comparable to that of the normotensive rats (p > 0.05). The
decrease in systolic blood pressure by LAC niosomes gel was
gradual, not sharp as with oral suspension, because of controlled
release of LNF. The systems were effective and could control the BP
up to 48 h. The above results are in conformity with ex vivo release
study data. The above result suggested that the developed LAC
niosomes gel formulation hold promise for the management of
hypertension that needs to be validated by in vivo study and
clinical trial.
4.3.10. Stability studies
The influence of elevated temperature and humidity conditions
on the physical stability of LAC niosomes gel formulation was
performed in adherence to the ICH guidelines. After period of 0,
and 1 month clarity, pH, homogeneity and drug content were
determined in the formulation. In both storage conditions the
formulation remained physically stable. No significantly (p > 0.001)
changes were observed. The shelf life of the formulation was found
to be approx 1.8 h with degradation rate constant
5. Conclusion
The present study conclusively demonstrates the use of Box-
Behnken design in formulation and optimization of LAC niosomes
gel formulations to avoid its systemic toxicity. This study indicated
that niosomes can be optimized to achieve desired properties
using span 60 concentrations, cholesterol concentrations, sonica-
tion time, and hydration time. The optimized niosomes formula-
tion demonstrated spherical morphology, enhanced entrapment
efficiency, and reasonable polydispersity index and particle size.
Confocal laser scanning microscopy further confirmed the poten-
tial of the developed formulation by showing the high fluorescent
intensity in comparison to conventional liposomes. The optimized
LAC niosomal gel formulation was found to decrease the BP
significantly in experimental hypertensive rats, which was
maintained for 48 h. The developed niosomes system was found
to be a good carrier for LAC by transdermal delivery.
Conflict of interest
Authors have no conflict of interest
Acknowledgments
The authors are thankful to Unichem Pharma Pharmaceuticals
Ltd., India for providing gift sample of carteolol. The authors are
265
also grateful to AICTE, New Delhi, India, for providing senior
research fellowship.
References
[1] A. Ahad, M. Aqil, K. Kohli, Y. Sultana, M. Mujeeb, A. Ali, Formulation and
optimization of nanotransfersomes using experimental design technique for
accentuated transdermal delivery of valsartan, Nanomedicine 8 (2012) 237–
249.
[2] A. Khan, S.S. Imam, M. Aqil, A. Ahad, Y. Sultana, A. Ali, K. Khan, Brain targeting
of temozolomide via the intranasal route using lipid-based nanoparticles:
brain pharmacokinetic and scintigraphic analyses, Mol. Pharm. 13 (2016)
3773–3782.
[3] A. Mishra, S.S. Imam, M. Aqil, A. Ahad, A. Ali, Carvedilol Nano lipid carrier:
formulation, characterization and in-vivo evaluation, Drug Deliv. 23 (4) (2016)
1486–1494.
[4] B. Singh, R. Kapil, M. Nandi, N. Ahuja, Developing oral drug delivery systems
using formulation by design: vital precepts, retrospect and prospects, Expert
Opin. Drug Deliv. 8 (2011) 1341–1360.
[5] B. Singh, R. Kumar, N. Ahuja, Optimizing drug delivery systems using
systematic “design of experiments. ” Part I: Fundamental aspects, Crit. Rev.
Ther. Drug Carrier. Syst. 22 (2005) 27–105.
[6] C.R. Lee, H.M. Bryson, Lacidipine A review of its pharmacodynamic and
pharmacokinetic properties and therapeutic potential in the treatment of
hypertension, Drugs 48 (1994) 274–296.
[7] C.T. Lo, A. Jahn, L.E. Locascio, W.N. Vreeland, Controlled self-assembly of
monodisperse niosomes by microfluidic hydrodynamic focusing, Langmuir 26
(2010) 8559–8566.
[8] E. Touitou, N. Dayan, L. Bergelson, B. Godin, M. Eliaz, Ethosomes-novel
vesicular carriers for enhanced delivery: characterization and skin penetration
properties, J. Control. Rel. 65 (3) (2000) 403–418.
[9] E.R. Bendas, H. Abdullah, M.H.M. El-Komy, M.A.A. Kassem,
Hydroxychloroquine niosomes: a new trend in topical management of oral
lichen planus, Int. J. Pharm. 458 (2013) 287–295.
[10] G. Mancia, G. Grassi, S.E. Kjeldsen, Nadcisnienietetnicze podrecznik
European Society of Hypertension, VIA, MEDICA, Gdansk, 2009.
[11] G.E. Paget, J.M. Barnes, in: D.R. Laurence, A.L. Bacharach (Eds.), Evaluation of
Drug Activities: Pharmacometrices, vol.1, Academic press, New York and
London, 1964.
[12] G.M.M. El-Maghraby, A.C. Williams, B.W. Barry, Skin hydration and possible
shunt route penetration in controlled oestradiol delivery from
ultradeformable and standard liposomes, J. Pharm. Pharmacol. 53 (2001)
1311–1322.
[13] T. Higuchi, Mechanism of sustained- action medication. Theoretical analysis of
rate of release of solid drugs dispersed in solid matrices, J. Pharm. Sci. 52
(1963) 1145–1149.
[14] ICH Topic Q 1 A (R2), Stability Testing of New Drug Substances and Products
CPMP/ICH/2736/99, (2003) (August).
[15] J. Guo, Q. Ping, G. Sun, C. Jiao, Lecithin vesicular carriers for transdermal
delivery of cyclosposrine, Int. J. Pharm. 194 (2000) 201–207.
[16] J.H. Draize, G. Woodard, H.D. Calvery, Methods for the study of irritation and
toxicity of substances applied topically to the skin and mucous membranes, J.
Pharmacol. Exp. Ther. 82 (1944) 377–390.
[17] K. Wang, Q. Huang, F. Qiu, M. Sui, Nonviral delivery systems for the application
in p53 cancer gene therapy, Curr. Med. Chem. 22 (35) (2015) 41184136.
[18] K.M. Kazi, A.S. Mandal, N. Biswas, A. Guha, S. Chatterjee, M. Behera, K. Kuotsu,
Niosome: a future of targeted drug delivery systems, J. Adv. Pharm. Technol.
Res. 1 (2010) 374–380.
[19] L. Abidin, M. Mujeeb, S.S. Imam, M. Aqil, D. Khurana, Enhanced transdermal
delivery of Luteolin via non-ionic surfactant based vesicle: quality evaluation
and antiarthritic assessment, Drug Deliv. 23 (3) (2016) 1069–1074.
[20] L.B. Lopes, M.V. Scarpa, G.V.J. Silva, D.C. Rodrigues, C.V. Santilli, A.G. Oliveira,
Studies on the encapsulation of Diclofenac in small unilamellar liposomes of
soya phosphatidylcholine, Colloids. Surf. B: Biointerfaces 39 (2004) 151–158.
[21] L.D. Marzio, C. Marianecci, M. Petrone, F. Rinaldi, M. Carafa, Novel pH-sensitive
non-ionic surfactant vesicles: comparison between Tween 21 and Tween 20,
Colloids Surf. B 82 (2011) 18–24.
[22] M. Akhtar, S.S. Imam, M. Afroz, M. Mujeeb, A.K. Najmi, M. Aqil, Neuroprotective
study of Nigella sativa loaded oral provesicular lipid formulation: in-vitro and
ex-vivo study, Drug Deliv. 21 (6) (2014) 487–494.
[23] M. Aqil, A. Ali, Y. Sultana, K. Dubey, A.K. Najmi, K.K. Pillai, In vivo
characterization of monolithic matrix type transdermal drug delivery
systems of pinacidil monohydrate: a technical note, AAPS PharmSciTech 7 (1)
(2006) E6.
[24] M. Bragagni, A. Scozzafava, A. Mastrolorenzo, C.T. Supuran, Paola Mura
Development and ex vivo evaluation of 5-aminolevulinic acid-loaded
niosomal formulations for topical photodynamic therapy, Int. J. Pharm. 494
(2015) 258–263.
[25] M. Bragagni, N. Mennini, S. Furlanetto, S. Orlandini, C. Ghelardini, P. Mura,
Development and characterization of functionalized niosomes for brain
targeting of dynorphin-B, Eur. J. Pharm. Biopharm. 87 (2014) 73–79.
[26] M. Jamal, S.S. Imam, M. Aqil, M. Amir, S.R. Mir, M. Mujeeb, Transdermal
potential and anti-arthritic efficacy of ursolic acid from niosomal gel systems,
Int. Immunopharmacol. 29 (2015) 361–369.
266 M. Qumbar et al. / Biomedicine & Pharmacotherapy 93 (2017) 255–266
[27] M. Khajeh, Application of Box-Behnken design in the optimization of a
magnetic nanoparticle procedure for zinc determination in analytical samples
by inductively coupled plasma optical emission spectrometry, J. Hazard Mater.
172 (1) (2009) 385–389.
[28] M. Mokhtar, O.A. Sammour, M.A. Hammad, N.A. Megrab, Effect of
someformulation parameters on flurbiprofen encapsulation and release
rates of niosomes prepared from proniosomes, Int. J. Pharm. 361 (2008) 104–
111.
[29] M. Qumbar, Ameeduzzafar, J. Ali, S.S. Imam, Mohd Fazil, A. Ali, DOE-Based
stability indicating RP-HPLC method for determination of lacidipine in
niosomal gel in rat: pharmacokinetic determination, Pharm. Anal. Acta 5
(2014) 8.
[30] A. Ahad, M. Aqil, K. Kohli, Y. Sultana, M. Mujeeb, Enhanced transdermal
delivery of an anti-hypertensive agent via nanoethosomes: Statistical
optimization, characterization and pharmacokinetic assessment, Int. J. of
Pharm. 443 (2013) 26–38.
[31] M.Y. Ning, Y.Z. Guo, H.Z. Pan, H.M. Yu, Z.W. Gu, Preparation and evaluation of
proliposomes containing clotrimazole, Chem. Pharm. Bull. (Tokyo) 53 (2005)
620–624.
[32] N. Subramanian, S.P. Sharavanan, P. Chandrasekar, A. Balakumar, S.P. Moulik,
Lacidipine self nanoemulsifying drug delivery system for the enhancement of
oral bioavailability, Arch. Pharm. Res. 39 (4) (2016) 48191.
[33] N.A. El Kasabgy, I. Elsayed, A.H. Elshafeey, Design of lipotomes as a novel dual
functioning nanocarrier for bioavailability enhancement of lacidipine: in-vitro
and in-vivo characterization, Int. J. Pharm. 472 (1–2) (2014) 369–379.
[34] P.K. Lakshmi, S. Bhaskaran, Phase II study of topical niosomal urea gel an
adjuvant in the treatment of psoriasis, Int. J. Pharm. Sci. Rev. Res. 7 (2011) 1–7.
[35] P.L. McCormack, A.J. Wagstaff, Lacidipine A review of its use in the
management of hypertension, Drugs 63 (2003) 2327–2356.
[36] Q. Li, Z. Li, W. Zeng, S. Ge, H. Lu, C. Wu, L. Ge, D. Liang, Y. Xu, Proniosome derived
niosomes for tacrolimus topical ocular delivery: in vitro cornea permeation,
ocular irritation, and in vivo anti-allograft rejection, Eur. J. Pharm. Sci. 62
(2014) 115–123.
[37] C. Shirodkar, G.H. Misra, P. Chethan, Z. Shetty, S. Attari, S. Mutalik, Subacute
toxicity profile of lacidipine nanoformulation in wistar rats, Sci. World J. (2015)
(12 pages).
[38] R.A. Lionberger, S.L. Lee, L. Lee, et al., Quality by design: concepts for ANDAs,
AAPS J. 10 (2008) 268–276.
[39] R.W. Korsmeyer, R. Gurny, E. Doelker, P. Buri, N.A. Peppas, Mechanisms of
solute release from porous hydrophilic polymers, Int. J. Pharm. 15 (1983) 25–
35.
[40] S. Jain, B.H. Chaudhari, N.K. Swarnakar, Preparation and characterization of
niosomal gel for iontophoresis mediated transdermal delivery of
isosorbidedinitrate, Drug Deliv. Transl. Res. 1 (2011) 309–321.
[41] S. Kumar, D. Bharjava, A. Thakkar, S. Arora, Drug carrier systems for solubility
enhancement of BCS class II drugs: a critical review, Crit. Rev. Ther. Drug
Carrier Syst. 30 (2013) 217–256.
[42] S. Moghassemi, A. Hadjizadeh, Nano-niosomes as nanoscale drug delivery
systems:an illustrated review, J. Control. Release 185 (2014) 22–36.
[43] S. Moghassemi, E. Parnian, A. Hakamivala, M. Darzianiazizi, M.M. Vardanjani,
S. Kashanian, B. Larijani, K. Omidfar, Uptake and transport of insulin across
intestinal membrane model using trimethyl chitosan coated insulin niosomes,
Mater. Sci. Eng. C 46 (2015) 333–340.
[44] S.S. Imam, M. Aqil, A. Ahad, M. Akhtar, Y. Sultana, A. Ali, Formulation by design
based risperidone nano soft lipid vesicle as a new strategy for enhanced
transdermal drug delivery: in-vitro characterization, and in-vivo appraisal,
Mater. Sci. Eng. C 75 (2017) 1198–1205.
[45] S. Sayeed, S.S. Imam, A.K. Najmi, M. Aqil, M. Akhtar, Nonionic surfactant based
thymoquinone loaded nanoproniosomal formulation: in vitro
physicochemical evaluation and in vivo hepatoprotective efficacy, Drug Dev.
Ind. Pharm. (2017), doi:http://dx.doi.org/10.1080/03639045.2017.1318903.
[46] S.M. Soliman, N.S. Abdelmalak, O.N. ElGazayerly, N. Abdelaziz, Novel nonionic
surfactant proniosomes for transdermal delivery of lacidipine: optimization
using 2(3) factorial design and in vivo evaluation in rabbits, Drug Deliv. 23 (5)
(2016) 160822.
[47] S.R.M. Moghddam, A. Ahad, M. Aqil, S.S. Imam, Y. Sultana, Formulation and
optimization of niosomes for topical diacerein delivery using 3-factor:3-level
Box-Behnken design for the management of psoriasis, Mater. Sci. Eng. C 69
(2016) 789–797.
[48] S.S. Imam, M. Aqil, M. Akhtar, Y. Sultana, A. Ali, Formulation by design-based
proniosome for accentuated transdermal delivery of risperidone: in vitro
characterization and in vivo pharmacokinetic study, Drug Deliv. 22 (8) (2015)
1059–1070.
[49] U. Ubaidulla, M.V.S. Reddy, K. Ruckmani, F.J. Ahmad, R.K. Khar, Transdermal
therapeutic system of carvedilol: effect of hydrophilic and hydrophobic matrix
on in-vitro and in-vivo characteristics, AAPS Pharm. Sci. Tech. 8 (2009) 1065–
1070.
[50] B. Junyaprasert, P. Singhsa, A. Jintapattanakit, Influence of chemical
penetration enhancers on skin permeability of ellagic acid-loaded
niosomes, Asian J. Pharm. Sci. 8 (2013) 110–117.
[51] Y.L. Geng, J. Zhao Guo, B.P. Ma, Development of a supercritical fluid
chromatography-tandem mass spectrometry method for the determination
of lacidipine in beagle dog plasma and its application to a bioavailability study,
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 15 (945–946) (2014) 21–26.