Artificial Cells, Nanomedicine, and Biotechnology

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Invasomes of isradipine for enhanced transdermal

delivery against hypertension: formulation,
characterization, and in vivo pharmacodynamic
study

Gauhar R. Qadri, Abdul Ahad, Mohd. Aqil, Syed S. Imam & Asgar Ali

To cite this article: Gauhar R. Qadri, Abdul Ahad, Mohd. Aqil, Syed S. Imam & Asgar Ali (2017)
Invasomes of isradipine for enhanced transdermal delivery against hypertension: formulation,
characterization, and in�vivo pharmacodynamic study, Artificial Cells, Nanomedicine, and
Biotechnology, 45:1, 139-145, DOI: 10.3109/21691401.2016.1138486

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2017
VOL. 45, NO. 1, 139–145
http://dx.doi.org/10.3109/21691401.2016.1138486

Invasomes of isradipine for enhanced transdermal delivery against hypertension:

formulation, characterization, and in vivo pharmacodynamic study
Gauhar R. Qadria, Abdul Ahadb, Mohd. Aqila, Syed S. Imamc and Asgar Alia
a
Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi, India; bDepartment of Pharmaceutics,
College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; cGlocal School of Pharmacy, Glocal University, Saharanpur, Uttar Pradesh, India

ABSTRACT ARTICLE HISTORY

Context Isradipine is an effective calcium channel blocker used in the management of Received 19 October 2015
hypertension. It undergoes extensive first pass metabolism and has low oral bioavailability. Revised 31 December 2015
Hence we attempted to develop isradipine-loaded invasomes. Objective The purpose of this work Accepted 2 January 2016
was to prepare and characterize invasomes carrier for isradipine, and to evaluate the optimized Published online 29 January
formulation obtained for pharmacodynamic study. Materials and methods Isradipine-loaded 2016
invasomes were prepared by conventional thin layer evaporation technique using PhospholiponÕ KEYWORDS
90G, b-citronellene (terpene) and ethanol. Prepared formulations were characterized in terms of Hypertension; invasomes;
size, size distribution, morphology, entrapment efficiency, and antihypertensive activity. Results isradipine; transdermal
and discussion It was observed that prepared isradipine-loaded invasomes delivers ameliorated
flux, reasonable entrapment efficiency, and more effectiveness for transdermal delivery. The
optimized formulation presented the particle size of 194 ± 18 nm, entrapment efficiency (88.46%),
and attained mean transdermal flux of 22.80 ± 2.10 mg/cm2/h through rat skin. Confocal laser
scanning microscopy revealed an enhanced permeation of Rhodamine-Red-loaded isradipine
invasomes to the deeper layers of the rat skin. During antihypertensive study, the treatment group
showed a substantial and constant decrease in blood pressure, for up to 24 h. The isradipine
invasomes formulation was found to be effective, with a 20% reduction in blood pressure by virtue
of better permeation through Wistar rat skin. Conclusion It was concluded that the developed
isradipine invasomes accentuate the transdermal flux and the results obtained encouraged the use
of the isradipine-loaded invasomes as the formulation for the potential management of
hypertension.

Introduction approaches used, chemical penetration enhancers have been

Transdermal drug delivery systems are designed so that the
drug can be delivered at a predetermined and controlled rate
(Ahad et al. 2015b, 2016), which is particularly conducive to
treating chronic disorders like hypertension (Ahad et al. 2013a).
Correspondingly, the adverse effects and inconvenience con-
comitant with oral route are circumvented. In addition, a good
number of antihypertensive drugs undergo extensive first-pass
metabolism which too can be avoided by transdermal therapy.
Hence, antihypertensive agents for therapeutic and prophylac-
tic usages have been subjected to transdermal investigation.
Essential attempts have been made to develop transdermal
systems, to circumvent the drawbacks of conventional drug
delivery of antihypertensive drugs (Ahad et al. 2013a, 2015a,
2016, Gungor and Ozsoy 2012, Selvam et al. 2010). The biggest
challenges in the development of transdermal systems are to
mitigate the barrier property of skin without causing harmful
effects, principally local irritation. The obstacle function of the
skin is chiefly due to the stratum corneum, which is responsible
for the poor penetration of drugs into the skin. To overcome
this barrier, several proposals have been assessed. Among the
intensively investigated over the years (Ahad et al. 2009, 2010).
In recent years, vesicular systems have been intensively
studied as drug carrier systems for the dermal and transdermal
administration of drugs. Traditional liposomal formulations,
compared to conventional dosage forms, have shown in vitro
an enhanced cutaneous drug accumulation allowing a reduc-
tion of the dose applied onto the skin (Touitou et al. 1994). In
the last two decades, new classes of lipid vesicles were
introduced by different researchers. Cevc and Blume (1992)
introduced the first generation of the highly deformable, elastic
liposomes, referred as TransfersomesÕ . They consist of
phospholipids and a surfactant molecule, the so called ‘‘edge
activator’’, which destabilizes lipid bilayer and increases its
deformability. Subsequently, Touitou et al. (2000) developed
ethosomes, new soft vesicular carriers mainly consisting of
phospholipids, ethanol, and water. More recently, researchers
investigated the novel vesicular systems called as invasomes
(Dragicevic-Curic et al. 2008b, 2009, Verma et al. 2003a). Briefly,
invasomes contain not only phospholipids but also ethanol and
terpenes, which make the vesicles deformable, and also serve

CONTACT Dr. Mohd. Aqil aqilmalik@yahoo.com Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), M. B. Road,
New Delhi 110062, India; Dr. Abdul Ahad abdulahad20@yahoo.com, aahad@ksu.edu.sa

ß 2016 Taylor & Francis

140 G. R. QADRI ET AL.
as penetration enhancers. This system has shown to improve
skin penetration of hydrophilic and lipophilic drugs (Shah et al.
2015). Ethanol is a good penetration enhancer while terpenes
have also shown potential to increase the penetration of many
drugs by disrupting the tight lipid packing of the stratum
corneum (Aqil et al. 2007, Sapra et al. 2008).
The aim of the present study was to develop and charac-
terize isradipine-loaded invasomal drug carrier systems.
Isradipine has been previously identified as a promising
candidate for transdermal drug delivery (Al-Suwayeh 2003,
Tirunagari et al. 2010). Isradipine (Figure 1) has low oral
bioavailability of about 15–24% (Tirunagari et al. 2010). It has
low molecular weight (371.4 Da) and melting point (168–
170  C) with a log partition coefficient (4.28) and terminal
elimination half-life is often stated to be about 8 h although a
value of less than 4 h has also been reported (Ahad et al.
2013a). All the above characteristics make isradipine a good
candidate for transdermal delivery.
Materials and methods
Materials
Isradipine was procured from Novartis, Basel, Switzerland.
PhospholiponÕ 90G was received as a gratis sample from
Lipoid AG, Sennweidstrasse, CH-6312 Steinhausen, Switzerland.
b-Citronellene was purchased from Sigma-Aldrich Chemicals
Private Ltd., New Delhi, India. Ethanol was purchased from
Changshu Yangquan Chemical, Suzhou, China. High-
Performance Liquid Chromatography (HPLC) grade acetonitrile
was purchased from BDH, Poole, England. CarbopolÕ 934,
triethanolamine, and propylene glycol were purchased from S.
D. Fine Chemical, Mumbai, India, and Loba Chemie Pvt. Ltd.,
Mumbai, India, respectively. Double-distilled water was used for
all experiments.
Animals
Albino Wistar rats (180–200 g) were supplied by Central Animal
House of Hamdard University and inhabited under standard
laboratory conditions in 12 h light/dark cycle at 25 ± 2  C.
Animals were nourished with pellet diet (Lipton, India) and
water ad libitum. The animals were received after the study was
duly approved by the University Animal Ethics Committee and
Figure 1. The chemical structure of isradipine.
Committee for the Purpose of Control and Supervision on
Experiments on Animals (CPSCEA), Government of India. Hairs
on the skin of animal were removed with electrical clipper;
subcutaneous tissues were surgically removed, and dermis side
was wiped with isopropyl alcohol to remove residual adhering
fat. The skin was washed with phosphate buffer saline,
wrapped in aluminum foil and stored in a deep freezer
at 20  C until further use (used within two weeks of prepar-
ation). On the day of experiment, skin was brought to room
temperature and skin samples were mounted over the diffusion
cells in such a way that stratum corneum side faced the donor
compartment whereas the dermis faced the receiver
compartment.
Preparation of invasomes
Isradipine invasomes formulations were prepared by conven-
tional thin layer evaporation technique (Shah et al. 2015).
Briefly, PhospholiponÕ 90G, isradipine and terpene were taken
in a clean, dry, round bottom flask, and dissolved in chloroform,
methanol, 2:1 (v/v). The organic solvent was removed by rotary
evaporation (Rotary Evaporator, Model-HS-2005V-N, Hahnshin
Scientific Co., Bucheon-si, Korea). Final traces of solvent were
removed under vacuum overnight. The deposited lipid film was
hydrated with ethanolic–phosphate buffer saline (pH 7.4)
mixture by rotation at 60 rpm for 1 h at room temperature.
The resulting vesicles were swollen for 2 h at room temperature
to get large multilamellar vesicles. To prepare smaller particles,
large particles were probe sonicated at 4  C at 40% output
frequency (at 40 W) (Titanium probe, Ultrasonicator, Model-
UP100H, Hielscher Ultrasonics GmbH, Berlin, Germany). The
compositions of various formulations and characterization
parameters such as particle size, entrapment efficiency, and
transdermal flux are presented in Table 1.
Particle size and shape determination
The mean invasomes particles size and polydispersity index
were analyzed by dynamic light scattering technique using
Malvern Zetasizer (Zetasizer, HAS 3000; Malvern Instruments,
Malvern, UK). The sample was placed in quartz cuvette, diluted
with distilled water and size measurements were carried out at
25 ± 1  C and at a scattering angle of 90 . All observations were
recorded in triplicate for each formulation. The particle shape
was visualized by using a Philips, CM10 (Philips Research,
Hamburg, Germany) transmission electron microscope.
Samples were dried on carbon-coated grid and negatively
stained with aqueous solution of phosphotungstic acid. After
drying the specimen was viewed under the microscope at 10–
100 k-fold enlargements at an accelerating voltage of 100 kV
(Ahad et al. 2012, 2013b).
Determination of entrapment efficiency
Entrapment efficiency of isradipine-loaded invasomes particles
was determined by sephadex minicolumn centrifugation
method (Chourasia et al. 2011, El Maghraby et al. 2001). In
brief, sephadex G-50 (10% w/v) was placed in a beaker and

allowed to swell overnight. To prepare the minicolumn,
Whatman filter pad was inserted in the 1 ml syringe and the
swollen gel was poured into it. To avoid air entrapment this
assembly was centrifuged at 1000 rpm for 5 min and the
excessive amount of water was removed. Invasomal suspension
200 ml separately was then poured on to the prepared column
and centrifuged at 5000 rpm for 5 min. The same procedure
was repeated by adding 100 ml of water to obtain all the
vesicles containing entrapped drug. The eluted invasomal
vesicles were lysed by using Triton X-100 (0.5% v/v) and filtered
through a 0.45 mm nylon disk filter. The concentration of
isradipine was analyzed by UV–Visible spectrophotometry at
330 nm. The determination of entrapment efficiency was done
in triplicate, all maintained at 25  C using the following
equation.
% Entrapment efficiency ¼ CP =Ci  100
where CP: concentration of isradipine analyzed after lysing with
Triton X-100 as described in the above methods; Ci: the initial
concentration of isradipine loaded into the formulation.
In vitro skin permeation study
The in vitro skin permeation of invasomes system was studied
using locally fabricated Franz’s diffusion cell having an effective
permeation area and receiver cell volume of 2 cm2 and 15 ml,
respectively. The receptor cell contained 15 ml of phosphate
buffer saline pH 7.4 and ethanol (ratio 70:30), which was
constantly stirred with a magnetic stirrer at 100 rpm.
Experiments were carried out for 24 h at 32  C ± 1  C. Samples
were withdrawn through the receiver cell sampling port at 0.5,
1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 h and analyzed for drug content
by UV spectrophotometer at 330 nm. The receptor cell after
each withdrawal was replenished with an equal volume of fresh
vehicle (Ahad et al. 2014a, 2014b).
Confocal laser scanning microscopy
In order to determine the extent of penetration confocal laser
scanning microscopy was performed on the intact skin. Rats
were sacrificed and the abdominal skin was removed.
Invasomal formulation loaded with Rhodamine B dye was
applied to the excised abdominal skin for 18 h. The skin was
washed with distilled water and placed on the slide with
stratum corneum facing upward and observed under confocal
microscope (Olympus Fluoview TM-FV1000, Olympus, Melville,
NY) with an argon laser beam with excitation at 488 nm and
emission at 590 nm. The skin sample was sliced in sections of
6–10 mm thickness through the z-axis by laser confocal
microscope.
Table 1. Formulation details and characterization of isradipine-loaded invasomes.
Phospholipid Ethanol b-citronellene
Formulations 90G (% w/v) (% w/v) (% w/v)
F1 1 10 0.1
F2 1 10 0.5
F3 2 10 0.1
F4 2 10 0.5
F5 3 10 0.1
F6 3 10 0.5
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 141
Pharmacodynamic study
The rats (180–200 g) were randomly divided into two groups.
Hypertension was induced in both the groups (Group A and B)
by allowing them to drink 1% NaCl in drinking water and by
injecting deoxycorticosterone acetate (DOCA, 5 mg/kg in corn
oil) subcutaneously at every fourth day for two weeks (Detroja
et al. 2011, Majima et al. 1991). After two weeks, Group B was
subjected to isradipine-loaded invasomal transgel formulation.
Formulation was applied to the previously shaven abdominal
area of rat skin. The rat was then placed in the restrainer and
the systolic blood pressure was recorded using noninvasive
blood pressure system (NIBP 200A; Biopac System, Inc., Goleta,
CA) based on cuff tail technique at 0, 1, 2, 4, 8, 12, 24 h time
intervals.
Results and discussion
Preparation of invasomes
As a new type of vesicle structured drug carrier, invasomes
showed the characteristics of good flexibility, high entrapment
efficiency, satisfactory permeability, and reliable stability, which
enabled the delivery of drugs into deeper skin layers and/or the
systemic circulation. In the present work, invasomes containing
phospholipid (1, 2, and 3% w/v), 0.1 and 0.5% w/v of the
terpene (b-citronellene) and 10% w/v ethanol were prepared by
thin film hydration technique in order to accentuate the
transdermal penetration of isradipine. Invasomal system was
found to be easy to prepare and composed mainly of
phospholipids, ethanol, and terpene compounds commonly
found in pharmaceutical preparations.
Particle size and shape
Particle size analysis of the optimized formulation revealed a
mean particle size of 194 ± 18 nm (Figure 2) which is primarily
suitable for transdermal delivery. The observed polydispersity
index of the optimized formulation was found to be
0.272 ± 0.062 which also supported the presence of homogen-
eity and narrow distribution of particle size. Furthermore,
Figure 2A obtained by transmission electron micrograph
revealed the presence of spherical shape and smooth and
even surface of the isradipine-loaded invasomes formulation.
The amount of terpene used in formulations has an effect on
the particle size as can be seen in formulations F1 and F3
bearing 0.1% b-citronellene have smaller particle size as
compared to formulations F2 and F4, having 0.5% terpene
(Verma et al. 2003b). While this was not the case with
formulations F4 and F5; which might be due to the highest
phospholipid content in these formulations. Moreover the
Particle size Poly dispersity Entrapment Transdermal
nm (n ¼ 3) index efficiency (%) flux (mg/cm2/h)
200 ± 12 0.322 ± 0.002 84.14 ± 6.76 19.67 ± 0.11
220 ± 24 0.210 ± 0.008 87.60 ± 7.56 18.79 ± 1.23
194 ± 18 0.272 ± 0.062 88.46 ± 9.26 22.80 ± 2.10
228 ± 23 0.333 ± 0.018 87.56 ± 7.21 19.16 ± 2.16
207 ± 15 0.327 ± 0.026 86.46 ± 4.48 18.42 ± 0.15
202.6 ± 20 0.291 ± 0.0024 80.2 ± 9.12 14.78 ± 0.17
142 G. R. QADRI ET AL.
Figure 2. (A) Transmission electron micrograph of isradipine-loaded invasomes (F3). (B) Size distribution of optimized (F3) invasomal formulation loaded with
isradipine.
particles size depicted by duo formulations is not significantly
different.
Entrapment efficiency
Entrapment efficiency is a direct commentary on the ability of
drug to integrate with lipoidal content to form particles of
suitable integrity. The entrapment efficiency for all formulations
(F1–F6) isradipine-loaded invasomes systems was more than
80% (Table 1). The entrapment efficiency was least found with
80.2% for the formulation F6 and the formulation F3 with a
moderate level of phospholipid content (2.0%), and least
concentration of terpene (0.1%) showed maximum entrapment
(88.46%) as compared to rest of the formulations. In addition to
high entrapment efficiency; F3 also elicited a least particle size
of 194 nm with a low polydispersity index of 0.272, indicating
homogeneous populations of particles.
In vitro skin permeation studies
The in vitro skin permeation profile shows that invasomes
formulation (F3) presents maximum transdermal flux value, i.e.
22.80 ± 2.10 mg/cm2/h through rat skin. The reason for this
better performance of invasomes is that it contains not only
phospholipids but also ethanol and terpenes. Terpenes are
considered as potent penetration enhancers, and they have
been shown to enhance the percutaneous absorption of
hydrophilic and lipophilic drugs (Ahad et al. 2011a, 2011b,
Aqil et al. 2007). The presence of ethanol and terpenes makes
the particles deformable and may also serve as penetration
enhancers. The permeation enhancing effect of invasomes has
been explained by fluidizing the stratum corneum lipid bilayer
structure, thus disturbing the organization of stratum corneum
lipids. These phenomena, i.e. an increased deformability of
particles and a disorganized stratum corneum bilayer structure,
are thought to facilitate the penetration of invasomes. Thus,
invasomes may follow the transepidermal osmotic gradient,
into and through the stratum corneum of the skin (Dragicevic-
Curic et al. 2008a).
The effect of variables such as phospholipid, ethanol and
terpene on transdermal flux is shown in Table 1. However, to
the contrary of our expectation, the transdermal flux value of
isradipine delivered by invasomes with 0.5% terpene was
smaller, but not significantly, compared to the flux delivered by
invasomes with 0.1% terpene. The optimized formulation was
selected based on the criteria of attaining the maximum value
of transdermal flux and entrapment efficiency; minimizing the
particle size. The formulation F3 was found to fulfill requisites
of an optimized formulation.
Confocal laser scanning microscopy
To visualize the permeation modes of the invasomes through
the stratum corneum, invasomes were further investigated by
using confocal laser scanning microscopy. Figure 3 shows the
penetration of Rhodamine B-loaded invasomes at different
depth levels through the skin. As observed by the confocal
laser scanning microscopy, the Rhodamine B-loaded invasomes
formulation (F3) traversed the skin thickness to an extent of
approximately 171.18 mm with high fluorescence intensity
(Figure 3). This prominently efficient delivery of isradipine-
loaded invasomes particles proposes their increased penetra-
tion and subsequent fusion with the lipid membrane in the
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 143

Figure 3. Confocal laser scanning micrographs after application of Rhodamine B-loaded invasomes system optical cross sections perpendicular to the rat skin surface.

Figure 4. Showing blood pressure lowering effect of applied isradipine-loaded invasomal transgel in DOCA induced hypertensive rats.

depths of the skin confirming the hypothesis of researchers
(Ahad et al. 2012, 2013b, Godin and Touitou 2004, Jain et al.
2008, Touitou et al. 2000). Overall, invasomes could enhance
the delivery of isradipine in terms of depth and quantity.
Pharmacodynamic study
The optimized invasomes formulation (F3) was selected for
further pharmacodynamic study. For ease of application and
longer contact time; the F3 formulation was converted into gel.
Concisely, carbopol 934 (2% w/v) was soaked in minimum
amount of water for an hour for complete humectation of
polymer chains (Das et al. 2013). The formulation F3 was added
slowly to the swollen polymer under stirring (Ahad et al. 2014a).
Additional constituents like polyethylene glycol 400 as plasti-
cizer and triethanolamine (0.1% (w/v)) were added to get
uniform gel formulation and named as isradipine-loaded
invasomal transgel. The prepared gel formulation presented
good homogeneity with absence of lumps. The pH value of 6.4
was observed for developed gel formulation. The drug content
144 G. R. QADRI ET AL.
was found to be 99.80%. During pharmacodynamics study,
administration of DOCA raised the blood pressure to 144.18–
155.24 mm Hg in normotensive rats. However, the application
of the isradipine-loaded invasomal transgel resulted in a steady
reduction of blood pressure. It was noticed that the isradipine-
loaded invasomal transgel showed a 17.43% decrease in blood
pressure at a 4 h time point. Figure 4 discloses that after
application of isradipine-loaded invasomal transgel, the blood
pressure of DOCA induced hypertensive rats was controlled for
up to 24 h. It was observed that at 8–24 h time point the
applied invasomal transgel was found to maintain the blood
pressure in propinquity of the normal blood pressure value and
the effect was continued till 24 h. It was observed that the
isradipine invasomal transgel was found productive in retro-
verting the rat blood pressure to normal values in DOCA
induced hypertensive rats. The above results suggest that the
prepared isradipine invasomal transgel admits promise for the
management of hypertension.
Conclusion
An invasomes system capable of delivering isradipine
through rat epidermis was successfully developed. The
flexible invasomes stimulated an increase of the percutan-
eous permeation of isradipine, thus improving the antihy-
pertensive activity in DOCA induced hypertensive rats. In
conclusion, the invasomes delivery systems may be a
promising carrier for transdermal delivery of isradipine for
the management of hypertension.
Disclosure statement
All authors have approved the final manuscript and the authors
declare that they have no conflicts of interest to disclose.
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