International Journal of Pharmaceutics 487 (2015) 135141

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Propranolol hydrochloride-loaded liposomal gel for transdermal

delivery: Characterization and in vivo evaluation
Yuanyuan Guan, Tiantian Zuo, Minglu Chang, Fang Zhang, Ting Wei, Wei Shao,
Guimei Lin *
School of Pharmaceutical Science, Shandong University, Jinan 250012, China

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 24 January 2015
Accepted 10 April 2015
Available online 13 April 2015

This study was to develop propranolol hydrochloride (PRO)-loaded liposomal gel as a topical drug
delivery system. Scanning electron microscope (SEM) revealed that the liposomes were spherical and
scattered in the surface of the gel. Pseudoplastic ows of PRO liposomal gel were showed after stored at
three different temperatures. Besides, PRO liposomal gel showed non-irritating to the skin of rabbit. Skin
deposition studies in vivo demonstrated that PRO liposomal gel can apparently increase drug content in
skin compared with PRO gel. Histopathology examination showed that PRO liposomal gel could obviously
weaken the barrier function of stratum corneum (SC) by comparison with PRO gel. What is more, the
plasma pharmacokinetic showed the maximum concentration in plasma was 0.41 mg/mL and 1.17 mg/mL
after topical and oral administration respectively. However, tissue distribution study showed PRO
liposomal gel obviously changed drug distribution in tissues, signicantly increased drug concentration
in skin with about 74 folds compared with PRO gel. In conclusion, liposomes-based gel could be a
promising vehicle as a transdermal delivery system of PRO.
2015 Elsevier B.V. All rights reserved.

Keywords:
Propranolol hydrochloride
Liposomal gel
Transdermal delivery
Pharmacokinetic
Tissue distribution

1. Introduction
Propranolol hydrochloride (PRO) is one of b-blockers which can
antagonize the b-adrenergic pathway, blocking receptors such as
in heart, pancreas, liver, and in peripheral blood vessels and
bronchi (Snchez-Carpintero et al., 2011). Since Laut-Labrze
et al. (2008) rst described its occasional antiproliferative effect on
severe infantile hemangioma (IH), PRO has gradually become the
rst-line therapy for IH. IH is one of the most common tumors in
infants, which can lead to deformities when they are located in the
facial areas of the lip, nasal tip or the ear (Matuszczak et al., 2013).
Therefore, it is necessary to treat IH timely. However, PRO given
orally shows signicant rst pass metabolism, some side effects on
heart and poor patient compliance (Lawley et al., 2009). As a result,
we need to seek for another administration route to solve these
problems.
Transdermal drug delivery offers potential to overcome
disadvantages caused by oral administration. Unfortunately, the
stratum corneum (SC) which is recognized as the primary barrier

* Corresponding author at: School of Pharmaceutical Science, Shandong

University, 44 Wenhuaxi Rd. Jinan, Shandong 250012, China.
Tel.: +86 53188382007; fax: +86 53188382548.
E-mail address: guimeilin@sdu.edu.cn (G. Lin).
http://dx.doi.org/10.1016/j.ijpharm.2015.04.023
0378-5173/ 2015 Elsevier B.V. All rights reserved.

for transdermal drug delivery makes it difcult for most drugs to

enter into skin by this route. In recent years liposomes have been
intensively studied as drug carrier systems for topical delivery.
Several in vivo and in vitro transport studies reported that
liposomes can improve skin permeability (Verma et al., 2003;
Dragicevic-Curic et al., 2008; Vyas et al., 2013) and enhanced the
skin deposition with reduction in systemic absorption (Patel et al.,
2000), which suggested that liposomes were useful for topical
drug delivery. As liposomes and biomembranes of skin have lipid
bilayer membrane structure in common, liposomes have a good
biocompatibility with skin. The major disadvantage of using
liposomes topically lies in the liquid nature of the preparation. To
achieve the viscosity desirable for topical application, liposomes
should be incorporated into a suitable vehicle. It has been well
established that liposomes are fairly compatible with carbopol
(Elnaggar et al., 2014), which has a good bioadhesive properties
and the ability to prolong the retention of the formulation on the
mucosal surface.
The purpose of this study was to develop a topical delivery
carrier for the use of PRO. As a result, scanning electron microscope
(SEM), rheological study, skin irritation test, in vivo skin deposition
studies, histopathology examination, plasma pharmacokinetic
properties and biodistribution characteristics were carried out
to evaluated PRO liposomal gel.

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Fig. 1. The SEM photomicrographs of liposomal gel. A (55); B (20,000).

2. Materials and methods

2.1. Materials
PRO was purchased from Wuhan Ding Lixin Chemical Co. Ltd.,
China (99.90%). Phosphatidyl ethanolamine (PE) was obtained
from Jianglaibio, Shanghai, China. Cholesterol (Chol) was supplied
by Aobox Biotechnology, Beijing, China. Carbopol 934 was obtained
from Jingxifang Technology Co. Ltd., Beijing, China. Methanol used
was of chromatographic grade and others were of analytical grade.

2.2.3. Rheological measurements

Rheological tests of liposomal gel were performed by a
rotational viscosimeter (RheoStress 6000, Thermo Scientic,
Germany). The ow properties of the developed gel were
conducted at 25  1  C (room temperature) by using a cone/
plate-measuring device (cone with D = 35 mm, 1 ). The measurements were carried out by increasing the shear rate from 0.1 s 1 to
100 s 1. In order to explore the effect of storage conditions on the
gel properties, rheological measurements were performed after
production and 1 week of storage at three different temperatures
(4  C, 25  C and 40  C).

2.2. Methods
2.2.1. Preparation of PRO liposomes and liposomal gel
PRO liposomes and PRO liposomal gel were prepared as detailed
in our previous work (Guan et al., 2014).
2.2.2. SEM of PRO liposomal gel
PRO liposomal gel was prefreezed in the ultra-low-temperatue
refrigerator of 80  C (DW-86L, Haier, China) for 24 h before they
were freeze-dried by the use of freeze drying equipment for 24 h.
The nal products were xed on a platform and sprayed with gold
and observed by the JSM-6700F scanning electron microscope.

2.2.4. Primary skin irritation test

To evaluate and compare skin irritation of PRO liposomal gel
and blank liposomal gel, the study was carried out on rabbits. New
Zealand rabbits weighing 2.53.0 kg were fed with food and water
for 1 week to adapt to the environment before the study. The hairs
of the dorsal portion were removed with the help of shaver at 24 h
prior to application of the formulations. The rabbits were divided
into three groups of 3 rabbits each as follows:
Group 1: no application (control).
Group 2: blank liposomal gel.
Group 3: PRO liposomal gel.
PRO liposomal gel (1.0 g) and blank liposomal gel (1.0 g) were
applied on the hairless skin of the rabbits by uniform spreading
within the area of 2 cm  2 cm, respectively. The preparations were
removed after 24 h and each of the area was observed for any
changes such as erythema or edema. To study the cumulative effect
of repeated applications, the respective preparations were applied
once daily for next 7 days on the same area of hairless skin. Draize
scale was applied to evaluate the skin irritation (Draize et al., 1944).
The irritation scores between 0 and 4 were used to grade stimulus
intensity which range from no response to a severe response.

Table 1
The irritation scores of gels to the rabbit skin after administration of 7 days.
Formulation

Fig. 2. Shear stressshear rate curves of PRO liposomal gel after production and
1 week of storage at three different temperatures.

Control (Group 1)
Blank liposomal gel (Group 2)
PRO liposomal gel (Group 3)
Stimulus intensity

Irritation index
24 h

Day 7

0
0
0
Nonirritant

0
0
0
Nonirritant

Y. Guan et al. / International Journal of Pharmaceutics 487 (2015) 135141

137

Fig. 3. The irritation photos after administration of preparations to skin.

A control (24 h); B control (Day 7).
C PRO liposomal gel (24 h); D PRO liposomal gel (Day 7).
E blank liposomal gel (24 h); F blank liposomal gel (Day 7).

2.2.5. In vivo skin deposition studies

2.2.5.1. Animals. Studies were performed on Kunming strain mice
(25  2) g, which were provided by the Experimental Animal
Center of Shandong University (Jinan, China). All animals were
acclimatized for 1 week before use. Prior to formal experiments,
mice were kept under food fasting but free access to water
overnight. All studies were performed in accordance with the
ethics and regulations of animal experiments of pharmaceutical
sciences, Shandong University, China.
2.2.5.2. Skin sample processing. The Kunming strain mice were
randomly divided into two treatment groups of 4 mice each. PRO
liposomal gel and PRO gel were administered transdermally at a
dose of 30 mg/kg, respectively. Then animals were sacriced by

cervical dislocation at the established post-administration time

point (0.5, 1, 2, 4, 6, 12, 24 h). Skin samples were collected from the
back of the mice and then washed in normal saline and dried using
lter paper. All the samples were stored at 20  C until further
analysis.

2.2.6. Histopathology examination by light microscopy

PRO liposomal gel was given to Kunming strain mice (25  2) g
for 24 h. Afterwards, the skins were immediately collected and
xed in 10% formaldehyde solution for 24 h. Then samples were
dehydrated using ethanol, embedded in parafn wax and followed
by stained with hematoxylin and eosin (H&E). At last, skins were
cut by a cryostat into 34 mm and evaluated under light
microscopy.

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Fig. 4. Mean concentrationtime curves of PRO after topical administration of PRO

liposomal gel and PRO gel to mice at 30.0 mg/kg (mean  SD; n = 4).

2.2.7. Pharmacokinetic and tissue distribution study

The principle of treating with Kunming strain mice was the
same as described above except that PRO liposomal gel and PRO
suspension were administered transdermally and orally at a dose
of 75 mg/kg, respectively. Besides, blood samples were collected
from the ocular vein into heparin-containing eppendorf tubes at
0.5, 1, 2, 4, 6, 8 h and then immediately centrifuged at 4000 rpm for
20 min to obtain supernate. The dorsal skins beneath the drug
application site and the organs (heart, liver, spleen, lung and
kidney) were separated, washed in normal saline and dried with
lter paper. Plasma and tissue samples were stored at 20  C until
further analysis. The concentrations of PRO at different time points
were expressed as mean values and standard deviations, and the
mean concentrationtime curves were plotted. Pharmacokinetic
parameters were calculated using the Drug and Statistics software
(DAS, version 2.0).
2.2.8. High performance liquid chromatography (HPLC) analysis
The quantication of PRO was performed on an Agilent HPLC
system (Agilent, USA). The system was equipped with a variable
wavelength UV detector with being set at 289 nm in this study and
a reversed-phase C18 column (5 mm, 4.6 mm  250 mm, Welch
Materials, Inc.). The mobile phase was consisted of methanol and
0.01 mol/L dipotassium phosphate solution (50:50, v/v) and was
used at a ow rate of 1.0 mL/min for chromatographic separation
and analysis. The column temperature was set at 25  C and samples
were injected into the column at a constant volume of 20 mL.
3. Results and discussion
3.1. SEM of liposomal gel
SEM was used to observed liposomes surface morphology.
Fig. 1A showed that liposomes were scattered in the surface of the
gel. It can clearly show that liposomes were spherical and about
260 nm in size when observed under a greater magnication
(Fig. 1B). In other words, liposomes had no obvious change in
morphology and sizes after they were incorporated into the gel
matrix compared with our previous date monitored by transmission electron microscope (TEM, Hitachi, Japan) and DelsaTM Nano C
Particle Analyzer (Beckman Counter Ltd., USA) respectively.

Fig. 5. Photomicrographs of the skin structure of using different preparations

(400). A skin of no preparations; B skin treated with PRO liposomal gel; C skin
treated with PRO gel.

3.2. Rheological measurements

The rheological properties play an important role for topical gel
formulations in delivering the molecules onto or across the skin
because they can greatly affect spreadibility, adhesiveness, drug
release from semisolid formulations, and subsequent penetration

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139

Table 3
The targeting disposition of PRO after topical administration of PRO liposomal gel
and oral administration of PRO suspension (n = 4).
AUC (mg h/g)

Tissue

PRO liposomal gel

Heart
Liver
Spleen
Lung
Kidney
r = (AUC

51.31
20.84
87.38
141.80
52.11







0.26
1.35
7.01
6.15
6.37

r
PRO suspension
68.99
78.06
92.19
242.08
92.29







7.98
9.82
8.73
22.52
18.86

0.74
0.27
0.95
0.59
0.56

topica/AUCoral).

3.3. Skin irritation test

One of the major disadvantages associated with transdermal
drug delivery system is skin irritation of preparations. To study
security of products, rabbits are often used for the prediction of
skin irritation effects in humans because of their high sensitivity to
irritations (Ishii et al., 2013). The results of the skin irritation
studies were listed in Table 1. It indicated that PRO liposomal gel
and blank liposomal gel resulted in no skin irritation compared
with control group after 24 h of application and 7 days of repeated
applications. Besides, the irritation photos of rabbits (Fig. 3) clearly

Fig. 6. Mean plasma (A) and skin (B) concentrationtime curves of PRO after topical
and oral administration to mice at 75.0 mg/kg (mean  SD; n = 4).

into the skin (Elnaggar et al., 2014). Fig. 2 clearly showed that the
slope of rheogram (namely the viscosity of the gel) was decreased
with the increase of shear rate, which indicated that the developed
gel showed pseudoplastic ow. In fact, with the shear rate
increasing the structure of the gel began to disrupt and the
particles started to align until it started owing. On the other hand,
the viscosity of the developed gel was high (255.2 Pas at shear rate
of 0.1 s 1, date was not shown) to adhere to the skin, which was
suitable for the transdermal administration. Besides, no obvious
change of the viscosity was observed post 7 days of storage at three
different temperatures compared with that of gel on the 0 day seen
from Fig. 2. All this indicated that preparations were suitable for
the transdermal administration and stable when stored at 40  C.

Table 2
The pharmacokinetic parameters of PRO in mice after topical and oral application.
Cmax mg/mL(or mg/g) Tmax (h) AUC0t mg h/mL
Plasma
Skin

Topical
Oral
Topical
Oral

0.41  0.04
1.17  0.13
265.10  23.34
13.37  0.93

6.5
1.25
4.0
0.5

1.72  0.17
2.67  0.23
1618.50  109.07
21.84  1.31

Fig. 7. The concentration level of PRO in tissues of mice over 0.58 h after topical (A)
and oral (B) administration PRO.

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Y. Guan et al. / International Journal of Pharmaceutics 487 (2015) 135141

showed no oedema and erythema after administration of 24 h

and7 days. Thus, PRO liposomal gel demonstrated evident
advantage of non-irritating to skin, which indicated their potential
in improving patient acceptance and topical delivery.
3.4. In vivo skin deposition studies
The cumulative PRO concentration in skin was showed in Fig. 4.
PRO liposomal gel increased rapidly from 0 to 4 h compared with
PRO gel group. The max drug concentration was 123.34 mg/g for
PRO liposomal gel, obviously remarkable than that of PRO gel
(75.65 mg/g). As Fig. 4 showed that PRO liposomal gel was more
able to increase drug content in skin, as a result it can provide
effective drug concentration for a long time in comparison with
PRO gel. The differences can be attributed that the presence of the
liposomes. Yokomizo and Sagitani (1996) found that phospholipids
had an effect on the permeability barrier of SC. In fact when
liposomes act on the skin they can successively fuse with the SC by
mixing with the intercellular polar lipids and loosening the
bilayered structure of the intercellular mortar of the SC (Sinico and
Fadda, 2009).
3.5. Histopathology examination by light microscopy
Skin is the outermost and largest organ in the body, which from
outside to inside is consisted of epidermis, dermis and subcutis
(Lai-Cheong and McGrath, 2009). As the outmost layer of
epidermis to protect skin from invasion, SC is composed of about
10 layers of attened corneocytes. They have a thick insoluble cell
envelope, which is consisted of loricrin and involucrin as well as
intercellular lipids (Alexander et al., 2012). As a result, SC becomes
the major challenge for the penetration of drugs across the skin. As
was shown in Fig. 5A, the skin of control group displayed a clear
delineation among epidermis, dermis and subcutis. The SC cells
were arranged neatly with no inammation being observed. After
the administration of PRO liposomal gel, the intercellular gaps of
SC were increased and the SC structure was loose (Fig. 5B), while
PRO gel slightly changed the tight junction of the SC (Fig. 5C).
Besides, there being no obvious change in dermis and subcutis of
all samples demonstrated that the preparation did not cause
irritation and was safe to patients. In a conclusion, PRO liposomal
gel can signicantly weaken the barrier function of SC and promote
drug permeation, which explain the phenomenon that PRO
liposomal gel had a higher drug deposition in skin than PRO gel.
The change was attributed to that liposomes produced an
enhancing effect because their lipid components may penetrate
deep into the SC or militate with skin lipids to loosen their
structure (El Maghraby et al., 2008).
3.6. Plasma and tissue concentrationtime curves
As is shown in Fig. 6A, plasma concentrationtime curves of
PRO liposomal gel and PRO suspension were signicantly different.
The maximum concentration in plasma appeared at a 6.5 h
(Cmax = 0.41 mg/mL) and 1.25 h (Cmax = 1.17 mg/mL) after topical
and oral administration respectively (Table 2), which indicated
that the extension of time for PRO absorbed in the systemic
circulation. The maximum concentration in plasma post oral
administration was 2.85 folds of that topical administration, while
AUC08h of PRO after oral administration was 1.55 folds compared
with topical administration, both of which indicated that lower
lever of PRO was absorbed in the systemic circulation after topical
administration compared with oral treatment. Besides, after 4 h
drug concentration post topical administration was higher than
that of oral administration. The reason can be attributed to the
drug repository effect of skin.

As was shown that maximum drug concentration in skin

(265.00 mg/g) after local application was signicantly higher than
oral application (13.37 mg/g, Table 2). The AUC08h value post local
application was 74-fold higher than oral application, which
demonstrated that the topical application was more effective than
oral application at the aspect of increasing drug concentration in
skin. Besides, PRO can maintain an effective drug concentration for
a long time after topical application (Fig. 6B). As a result PRO
liposomal gel can prolong time of acting on body and improve
patient compliance.
The targeting disposition of PRO after topical administration of
PRO liposomal gel and oral administration of PRO suspension were
shown in Table 3. The re value of heart, liver, spleen, lung, kidney all
less than 1, which indicated a lower bioavailability after topical
administration compared with oral treatment. What's more, lower
drug disposition in skin would reduce the side effects on the organs
especially heart mainly reecting in such as bradycardia, hypotension, and hypoglycemia. In order to more visually show the
biodistribution characteristics of PRO after administration, concentration distribution histograms over the time course of 0.58 h were
plotted. Fig. 7 showed that PRO liposomal gel obviously changed drug
distribution in tissues, relatively increased drug concentration in
skin, and decreased drug concentration in other tissues.
4. Conclusion
The present work demonstrated that PRO liposomal gel as a
transdermal preparation was prepared successfully. It could
obviously increase drug content in skin to reduce dosing frequency
compared with PRO suspension. Besides, it decreased drug content
in the systemic circulation. As a result, side effects of PRO on organs
especially the heart can be reduced. Thus, the liposomal gel could
be a promising vehicle for topical delivery of PRO.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China(Grant No.: 21203112) and the Natural Science
Foundation of Shandong Province (Grant No.: ZR2012BQ002).

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