Review

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Skin permeation behavior of

elastic liposomes: role of
formulation ingredients
1.

Introduction

2.

Basic ingredients

3.

Optional ingredients

4.

Conclusion

5.

Expert opinion

Jun Chen, Wen-Li Lu, Wei Gu, Shan-Shan Lu, Zhi-Peng Chen &
Bao-Chang Cai

Nanjing University of Chinese Medicine, School of Pharmacy, Nanjing, PR China

Introduction: With the incorporation of edge activators into the lipid bilayer
structure, elasticity properties are given to liposomes. Regardless of the
debate over the precise permeation mechanism of elastic liposomes, these
vesicles have been proven to enhance drug permeation into or through skin
in most cases.
Areas covered: This article provides an overview of the formulation ingredients of elastic liposomes and their relationship with skin permeation behavior. The ingredients are divided into two categories of basic and optional
ingredients. The effect of stability on permeation behavior of the vesicles
is highlighted.
Expert opinion: More attention should be paid to the stability of elastic liposomes. The different stability properties of the elastic liposomes following
administration can induce different skin permeation behaviors of the vesicles.
It is necessary to select the optimum composition of the elastic liposomes in
order to control the stability and permeation behavior of the vesicles into or
through the skin. Moreover, for the development of elastic liposomes, particular attention should also be paid to the drug leakage from the vesicles during long-term storage. The application of optional ingredients to improve
the stability and/or elasticity of the elastic liposomes is becoming a new trend.
Keywords: dermal delivery, elastic liposomes, formulation ingredients, skin penetration
behavior, stability, transdermal delivery
Expert Opin. Drug Deliv. (2013) 10(6):845-856

1.

Introduction

Skin, the largest organ of human body, can offer many advantages as a route for
drug administration. For topical drug delivery, high concentrations of drugs can
be localized at the site of action, reducing the systemic drug levels and, therefore,
also reducing the systemic side effects. For transdermal drug delivery of systemically
acting drugs, the advantages include avoidance of hepatic first-pass metabolism,
decrease of fluctuations in drug plasma levels and good patient compliance.
However, it is difficult for most drugs to partition into and diffuses through
the skin.
The stratum corneum (SC), the skins outermost layer and interface with the outside world, is now well recognized as the effective barrier of intact skin that prevents
unwanted materials from entering the body. The structure of the SC is often
depicted in the so-called brick and mortar structure [1,2], where the corneocytes
of hydrated keratin (bricks) are embedded in the intercellular matrix enriched in
non-polar lipids (mortar) and organized as lamellar lipid layers. In SC, 3 -- 10
neighboring corneocyte columns are found to form a cluster. Two quantitatively
different hydrophilic pathways are found in SC: an inter-cluster route with low penetration resistance and an inter-corneocyte pathway that resists penetration better
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J. Chen et al.

Article highlights.
.

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.
.

The stability following administration of the elastic

liposomes can be used to explain their different
permeation behaviors.
Due to the usage of unsaturated PC and EA, the
bilayers of the liposomes are highly permeable and the
drug molecules are easily leaked from the vesicles.
Therefore, the long-term stability of elastic liposomes is
found to be the main drawback of the vesicles,
especially at room temperature.
Flexible modification of Tm might be a potential method
to obtain high elasticity and stability of the vesicles at
the same time.
Further research needs to investigate the final site elastic
liposomes can arrive at with an intact structure.
The application of optional ingredients has become a
new trend to improve stability and/or elasticity.

This box summarizes key points contained in the article.

and is more abundant ( 80% of the pathway area). The latter

route is strongly tortuous, as it goes between all the corneocytes in a cluster [3]. Underneath the SC resides the viable epidermis in which the main cell type is the keratinocyte. The
dermis, located under the viable epidermis, is 5 -- 20-times
thicker than the epidermis and contains blood vessels, lymph
vessels, nerves and an abundant level of collagen fibers.
Beneath the dermis lays the subcutaneous fat tissue, an
assembly of adipocytes linked by collagen fibers.
Past decades have witnessed significantly increased interest in the exploration of new techniques to enhance drug
permeation into or through the skin. One of the efficient
strategies is the use of vesicles. Topical delivery of drugs
by liposomes has evoked considerable interest [4]. In
1980, Mezei and Gulasekharam first reported a four- to
fivefold greater permeation of the steroid triamcinolone
acetonide from a liposomal lotion as compared with an
ointment containing an equal drug concentration [5].
Now it becomes evident that conventional liposomes are
of little or no value as carriers for transdermal drug delivery, for they generally accumulate in the upper layers of
the SC with minimal permeation to deeper tissues or the
systemic circulation [6].
In order to facilitate the passage of the drugs across the SC,
a new type of liposome, elastic liposomes, was first introduced
by Cevc in 1992 [7]. Unlike the conventional liposomes, edge
activators (EA) incorporate into the lipid bilayer increasing
the resulting vesicle elasticity. Due to the elasticity, they are
claimed to be able to squeeze through channels one-tenth
the diameter of the vesicles [8], allowing them to spontaneously penetrate across the SC by the intercellular route.
Under different names (Transfersomes owned by IDEA
AG [Munich, Germany], flexible liposomes, deformable
liposomes, ultradeformable liposomes, ultraflexible liposomes, etc.), numerous research groups have been working
with elastic liposomes.
846

Application of dispersed elastic liposomes in an aqueous

suspension on the skin leads to the following sequence of
events [9-12]. First, the water in suspension starts immediately
to evaporate, increasing the concentration of all nonvolatile
components on the skin. When saturation is reached, the
hydration gradient along the skin barrier becomes available
to the elastic liposomes. The water concentration increases
from around 10 to 30% at an air-exposed SC surface to
around 75% in the viable epidermis. Based on this transepidermal hydration gradient, the liposomes high elasticity and
hydro-affinity allow and prompt the drug-loaded vesicles to
pull across the skin barrier. The process continues until the
vesicle has reached the water-rich viable epidermis. Then the
pull can be replaced by a push on the liposome which
already crossed the SC by diffusion-based re-distribution.
The push is exerted by the elastic liposomes still sensing
the hydration gradient in the SC; intercellular fluid motion
in living skin may also be influential. It should be noted
that elastic liposomes do not penetrate the SC without a transcutaneous hydration gradient. Consequently, elastic liposomes should not be applied under occlusion because this
would decrease the hydration gradient [12].
Several studies supported that elastic liposomes may act
as carrier systems. Application of oestradiol entrapped in
the elastic liposomes resulted in a 14 -- 17-fold increase in
flux compared with the control, while pretreatment of skin
membranes with blank elastic liposomes only slightly
(1.6 -- 2.4-fold) enhanced oestradiol flux [13]. Cevc et al. suggested that elastic vesicles penetrate the skin intact, that is,
without permanent disintegration [14]. Further research needs
to be carried out to find the final site that elastic liposomes
can arrive at with an intact structure.
The penetrant properties of the elastic liposome itself can
be considered as another mechanism accounting for the
improved skin permeation of drugs. Some specific components of elastic liposomes may change the skin barrier effect.
The transepidermal water loss (TEWL) value, which is considered a good indicator of the functional status of the SC,
is a measurement of the diffusion rate of water molecules
across the skin. The elastic liposome formulation showed a
2.1-fold increase in TEWL value 24 h after the liposome
application. However, after 48 h the value reverted to
1.2-fold, showing the reversible protection property of the
SC barrier [15].
Currently, the skin permeation enhancing ability of elastic
liposomes is generally considered to be due to the synergistic
effect of elastic liposomes acting as drug carriers, as well as,
permeation enhancers. However, based on the results of the
most recent literature, the former mechanism may be more
important than the latter.
Typically, elastic liposomes are composed of phospholipids
such as phosphatidylcholine (PC) and a surfactant, and consist of at least one inner aqueous compartment surrounded
by a lipid bilayer. The surfactant acts as EA that destabilize
the lipid bilayer and increase the elasticity of the vesicle.

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Skin permeation behavior of elastic liposomes

Moreover, other materials such as a charged lipid, PEG-lipid,

ethanol, cyclodextrin and gel ingredients are also reported to
be included in the formulation. The ingredients of elastic liposomes must be designed and optimized on a case-by-case
basis.
It is evident that drug permeation behavior is chiefly
affected by the liposomal composition. Liposomal composition influences their physicochemical properties and, therefore, their efficacy as drug delivery systems [16]. Depending
on their composition, elastic liposomes may penetrate
through the SC with intact structure, or fuse and mix with
skin lipids to lose their structure. Therefore, different permeation behaviors of the elastic liposomes are mostly due to the
difference in formulation ingredients.
It should be noted that there is a difference between
in vitro and in vivo skin permeation behavior. In fact,
in vitro skin permeation tests are widely used to evaluate
the permeation behavior of elastic liposomes because of their
convenience [17]. However, the results of recent experiments
revealed significant differences between in vitro and in vivo
skin permeation behaviors of elastic liposomes [12]. More
comparison studies of in vitro and in vivo skin permeation
behavior should be carried out when researching elastic
liposomes.
Recently, it has been proposed that many different
amphipats can be combined into highly elastic bilayer
vesicles [18]. However, in the present review, only elastic
bilayer vesicles containing phospholipids or 1,2-dioleoyl-3trimethylammonium-propane (DOTAP) are discussed.
2.

Basic ingredients

Phosphatidylcholine
The membrane of liposomes is mainly composed of PC,
amphipathic molecules in which a glycerol bridge links a
pair of hydrophobic acyl hydrocarbon chains with a hydrophilic polar headgroup of phosphocholine. The PC acyl chain
length and saturation degree have an effect on the phase transition temperature (Tm) of liposomal membrane. Above Tm,
lipid chains partially fluidize, owing to their thermally driven
conformational isomerization. It is also almost always true
that the fluid-chains vesicles with a rather elastic bilayer promote drug transport across skin barrier better than the more
rigid liposomes [19]. Therefore, the most common PC used
to prepare elastic liposomes is unsaturated PC (i.e., soybean
phosphatidylcholine [SPC] or egg phosphatidylcholine
[EPC]) with a much lower Tm (< 0 C).
Elastic liposomes of sumatriptan succinate were prepared
by 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC)
and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
respectively. It was found that DLPC vesicles proved to be
more elastic and provided a higher sumatriptan transdermal
flux than vesicles formulated with DOPC [20]. The observed
difference might be due to different bilayer thickness of
DLPC and DOPC, which makes DLPC bilayers more elastic
2.1

than DOPC vesicles. It seems the length of acyl chain of PC

(DLPC: C12; DOPC: C18) has a significant effect on the
elasticity and skin permeation behavior of the fluid-state
elastic liposomes.
It is possible that purity of PC affects the permeation
behavior of elastic liposomes. Sodium cholate and PC with
different purities were used for the preparation of elastic liposomes, namely 95, 78.6 and 50% PC [21]. The best permeation behavior was obtained with 95% PC. Similar results
were also found in conventional liposomes without EA.
Hofaland et al. investigated three types of liposomes using
various purities of PC [22]. The formulation using 85% PC
was found to have the greatest effect on the ultrastructure of
the SC compared with the other two formulations (10 and
28% PC). It was proposed that PC could penetrate into the
SC, mix with the SC lipids and induce a penetration-enhancing effect by changing the ultrastructure of intercellular
lamellae.
PC composition can exert a significant effect on the stability of liposomes [23]. For elastic liposomes, the usage of unsaturated PC with lower Tm (< 0 C) leads to higher fluidity and
lower stability of vesicles during storage. In addition, freezedrying is a technique used to dehydrate conventional liposomes to increase stability during storage. However, it was
found that elastic liposomes suffered irreversible aggregation
when rehydrated after freeze-drying, even in a high sugar ratio
to lipid mass of 4:1 [24]. Therefore, the long-term storage stability problem of elastic liposomes, especially the significant
drug leakage from the vesicles, should be given particular
attention.
As the Tm of liposomes can be flexibly modified by changing the ratio of different PCs in the liposomal membrane [25,26], the effect of Tm on permeation behavior and
stability of the vesicles should be intensively investigated.
For example, the Tm values can be set as 30 C. During storage (25 C), the rigid elastic liposomes are stable because the
Tm is higher than storage temperature. Following administration to skin, the liposomal membrane turns into a liquid
state due to the skin temperature (32 C). In this way, both
satisfactory stability and elasticity can be obtained
simultaneously.
Edge activator
EA is usually a kind of surfactant, which destabilizes the lipid
bilayer of the elastic liposomes and increases elasticity of the
bilayer simultaneously. Among EAs, sodium cholate, sodium
deoxycholate, Span 80, Tween 80 and Tween 20 were commonly used. El Maghraby et al. [27] showed that incorporation
of surfactants into liposomes significantly reduced the main
Tm, which induced fluidization of the lipid bilayer. Furthermore, El Maghraby et al. suggested that some chemical penetration enhancers such as oleic acid can also be used as EA to
replace the commonly used surfactant [28].
The elasticity properties of elastic liposomes depend on the
EA. Only at the optimal balance between the amount of EA
2.2

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J. Chen et al.

Table 1. Basic parameters of the most commonly used EAs.

EA

Molecular weight

Span 80
Tween 80
Tween 20
Sodium cholate
Sodium deoxycholate
Oleic acid

428.59
1309.7
1227.5
430.55
414.55
282.46

Formula

HLB

CMC (mM)

C24H44O6
C64H124O26
C58H114O26
C24H39O5Na
C24H40O4Na
C18H34O2

4.3
15
16.7
16.7
16
1

0.016 -- 0.019
0.012
0.06
9 -- 14
2 -- 6
*

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*Oleic acid is a water-insoluble oil at room temperature without CMC, but in its ionized, surfactant-like form, it has CMC around 1 mM.
CMC: Critical micelle concentration; HLB: Hydrophilic/lipophilic balance.

and the amount of bilayer forming PC, are the vesicles elastic.
If the EA level in the vesicles is too low, the vesicles are rigid
and if the concentration of EA is too high, most vesicles turn
into micelles [29,30]. In comparison to elastic liposomes, the
mixed micelles are reported to be less elastic in nature and
also have less skin permeation ability because they are much
less sensitive to the water activity gradient in skin [10]. The
existence of mixed micelles also leads to lower drug
entrapment due to their higher rigidity and smaller size [31,32].
EA plays an important role in determining the skin
permeation behavior of elastic liposomes. An overview of
the difference among EAs is helpful for the selection of an
ideal EA for the optimal formulation.
Comparison of different EAs
For the optimization of elastic liposomes containing diclofenac
sodium (DS), different EAs were employed, resulting in elastic
vesicles with diverse membrane characteristics [33]. Regardless
of the type of EA, liposomes prepared using 85:15% (w/w
PC:EA) ratio showed the highest elasticity. Among EAs,
Tween 80 was found to be superior to bile salts (sodium cholate and sodium deoxycholate) and Spans (Span 80 and Span
85). The elastic liposomes with Tween 80 showed the highest
transdermal flux values. This could be attributed to the highly
flexible and non-bulky hydrocarbon chains of Tween 80.
The EA with the highest elasticity showed the highest drug
leakage. It was found that Tween 80 showed the highest elasticity and Span 85 showed the least elasticity for the elastic
liposomes containing DS. However, after storage in glass vials
at 4 C for up to 3 months, liposomes containing Tween
80 showed the lowest percentage of drug retained after
90 days (48.01%) while Span 85 showed the highest percentage drug retained (70.53%) [33]. Therefore, the investigation
into optimal EA type and concentration should also cover
the influence of EA on the stability of the vesicles.
EA also affects entrapment efficiency (EE) of the vesicles.
Elastic liposomes of griseofulvin, a lipophilic drug, were prepared by a thin-film hydration method and the formulation
was optimized for types and concentrations of EA [34].
When the ratio of PC to EA is 85:15 (w/w), the liposomes
showed the highest EE. Comparing different EAs at the PC:
EA ratio of 85:15, Span 85 had the highest EE of (63.44
0.45%), followed by Span 80 (59.36 0.32%), Tween 80
2.2.1

848

(55.52 0.35%) and then sodium deoxycholate (49.16

0.56%). The results can be explained on the basis of lipophilicity of the EAs. Span 85 and Span 80 being lipophilic
in nature possess higher affinity for lipids, which resulted in
higher drug entrapment compared with the hydrophilic
EAs. The optimized elastic liposomes made with Span
85 illustrated remarkably higher drug permeation and skin
retention when compared with conventional liposomes.
The hydrophilic/lipophilic balance (HLB) value of EA
should be taken into account when different EAs are compared. For the preparation of elastic liposomes containing
melatonin, three different EAs (Span 80, sodium cholate
and sodium dodecylsulphate) were investigated. The formulation bearing Span 80 at a lipid:surfactant ratio of 85:15 (w/w)
was proved to be the best considering all the parameters studied. The transdermal fluxes of melatonin across human
cadaver skin were determined to be 59.5 4.12, 51.2
2.21, 49.4 3.12 and 10.9 1.65 g/cm2/h for elastic liposomes containing Span 80, sodium cholate, sodium dodecylsulphate and conventional liposomes without EA,
respectively. The best performance of Span 80 among these
three EAs could be attributed to its lower HLB value and
the best interaction with phospholipid bilayer [35].
The HLB values, which give a measure of the physicochemical properties of surfactants in terms of their affinity for, or
solubility in, water or lipids, are listed in Table 1 for the
most commonly used EAs.
It was also found that the use of EAs with lower HLB
resulted in smaller sized vesicles [33]. The relationship
observed between vesicle size and surfactant HLB has been
attributed to the decrease in surface energy obtained with
increasing hydrophobicity, thus, resulting in smaller vesicles.
For transdermal gene delivery, different EAs were also compared. Lee et al. reported the effects of EAs (sodium cholate,
sodium deoxycholate and Tween 80) on the formation and
permeation behavior of elastic liposomes containing gene therapy drug [36]. Following topical application onto mice, DNA
complexed with vesicles containing either sodium cholate or
sodium deoxycholate showed substantial transdermal absorption. In contrast, DNA complexed with Tween 80-based
vesicles did not show in vivo transdermal absorption.
Cytotoxicity following incubation of human keratinocyte
with elastic liposomes was investigated [37]. The toxicity was

Expert Opin. Drug Deliv. (2013) 10(6)

Skin permeation behavior of elastic liposomes

Table 2. Formulation ingredients and skin permeation behavior of charged elastic liposomes.
Liposomal composition (w/w)

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DOTAP/Tween 20 = 86.9/13.1 (cationic)

EPC/Tween 20 = 86.9/13.1 (neutral)
EPC/DCP/Tween 20 = 82.9/4/13.1 (anionic)
DOTAP/Tween 20/Tocopherol = 5/0.9/0.05 (cationic)
EPC/Tween 20/Tocopherol = 5/0.9/0.05 (neutral)
EPC/DOPA/Tween 20/Tocopherol = 4.2/0.8/0.9/0.05 (anionic)
SPC/SA/Tween 20/mTHC = 81.35/3.25/15.60/1.5 (cationic)
SPC/Tween 20/mTHC = 84.50/15.60/1.5 (neutral)
SPC/DCP/Tween 20/mTHC = 78.26/6.34/15.60/1.5 (anionic)

Aqueous
composition

Permeation behavior
sequence

Refs.

LMWH in PBS (pH 7.4)

Steady-state flux through hairless

mice skin: cationic > neutral > anionic

[58]

5-ALA in PBS (pH 5.0)

Transdermal flux through hairless

mice skin: cationic > anionic > neutral

[59]

PBS (pH 7.4)

Amounts of the drug into deeper skin:

cationic > neutral > anionic

[60]

5-ALA: 5-Aminolev-ulinic acid; DCP: Dicetyl phosphate; DOPA: 1,2-Dioleoyl-sn-glycero-3-phosphate; DOTAP: 1,2-Dioleoyl-3-trimethylammonium-propane;
EPC: Egg phosphatidylcholine; LWMH: Low molecular weight heparin; PBS: Phosphate buffer saline; SA: Stearylamine; SPC: Soybean phosphatidylcholine;
mTHC: Temoporfin.

only observed in the case of Tween 80-containing formulation. Vesicles containing sodium deoxycholate and sodium
cholate did not affect cell viability. More work should be
done on investigating and comparing the toxicity of EAs.
Span and Tween
Span, which is lipophilic in nature, has a high affinity for lipid
bilayers. Different Span surfactants have been compared with
EA. Elastic liposome formulations of propranolol hydrochloride were prepared using different Span types (Span 80, Span
60 and Span 40) and different concentration levels (SPC:
EA = 95:5, 90:10, 85:15, 80:20 and 75:25, w/w) [32]. Transdermal flux first increased with increasing EA concentration
(SPC:EA = 95:5 to 85:15) and then decreased (SPC:
EA = 85:15 to 75:25), a common phenomenon seen with
all three surfactants. The transdermal fluxes of the drug across
human cadaver skin were determined to be 16.19 1.5,
12.34 1.5, 11.12 1.2 and 3.24 0.6 g/cm2/h for elastic
liposomes containing Span 80, Span 60, Span 40 and conventional liposomes without EA, respectively. Finally, the optimum ratio (PC:Span 80 = 85:15, w/w) was verified by other
reports using Span 80 as the EA [15,35,38,39].
Hepatitis B surface antigen (HBsAg)-loaded elastic liposomes were utilized as a carrier for enhanced immunity
against the antigen. Elastic liposomes with different Span
80 concentrations (SPC:Span 80 = 90:10, 88:12, 86:14,
84:16, 82:18, 80:20, w/w) were prepared by conventional
rotary evaporation method and characterized for various
parameters [40]. The formulation composed of SPC and
Span 80 at a ratio of 86:14 showed the maximum elasticity
and cumulative antigen permeation.
Tween 80, a non-ionic surfactant with a large head group
(containing about 20 polyoxyethylene units), is soluble in
water. After encapsulated into the liposomes, it is considered
to be distributed between the lipid bilayer and inner aqueous
phase.
Elastic liposomes composed of SPC and Tween
80 (84.5:15.5, w/w) were used for dermal delivery of ketotifen
(KT). It was found that the cumulative KT permeated
2.2.2

through skin or deposited into skin after 24 h, with 1.6and 2.2-fold increase over KT solution control, respectively.
The cumulative KT permeated through skin after 24 h from
elastic liposomes was also found to be significantly higher
(1.7-fold) than that from conventional liposomes without
Tween 80 [41].
Tween 20, sorbitan monolaurate, has higher HLB values
(Table 1) compared with Tween 80. Interestingly, for cationic
elastic liposomes, Tween 20 is mostly applied as an EA
(Table 2).
Tween 20 can also be included in neutral elastic liposomes.
The enhanced skin permeation and bioavailability of lowmolecular-weight-heparin (LMWH) can be achieved by elastic liposomes compared with ethosomes [42]. LMWH loaded
elastic liposomes (EPC:Tween 20 = 86:14, w/w) were formulated for transdermal delivery, and their physicochemical and
pharmacokinetic parameters were compared with LMWH
loaded ethosomes. Elastic liposomes had similar particle sizes
to ethosomes, but their elasticity was higher than that of ethosomes (76.7 vs. 46.8%). In vitro elastic liposomes demonstrated a 2.6-fold higher permeability coefficient than
ethosomes. In vivo, after the topical application of LMWH
elastic liposomes to hairless mice, the [anti-Xa] max was
1.11 IU/mL, while that of ethosomes was only 0.32 IU/mL.
Moreover, the area under the curve (AUC0--24 h) value of
elastic liposomes was 2.5-fold higher than ethosomes.
Sodium deoxycholate and sodium cholate
Sodium deoxycholate is a water-soluble ionic surfactant. The
valsartan-loaded elastic liposomes containing sodium deoxycholate as the EA was investigated [43]. The optimal formulation was composed of SPC (85 mg), sodium deoxycholate
(15 mg) and valsartan (60 mg). Alvia et al. reported the
same results of sodium deoxycholate for 5-FU loaded elastic
liposomes [44]. Different ratios (% w/w) of SPC and sodium
deoxycholate were taken for the preparation of 5-fluorouracil
(5-FU) elastic liposomes. It was observed that the optimal
liposomal composition (SPC:sodium deoxycholate = 85:15,
w/w) produced vesicles of 153.2 10.3 nm in diameter
2.2.3

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J. Chen et al.

with a maximum EE of 82.4 4.8% and the highest elasticity

index of 130.4 4.2.
Elastic vesicles containing sodium cholate as the EA have
been reported to penetrate intact skin in vivo and to transfer
therapeutic amounts of drugs with similar efficiency to
subcutaneous administration [45,46].
For bleomycin-loaded elastic liposomes, the optimal
ratio of sodium cholate to PC was reported to be
16:84 (w/w) to obtain the highest elasticity and dermal
permeation behavior [47].
The effect of the sodium cholate concentration on the stability of elastic liposomes was investigated [48]. It was demonstrated that a higher percentage (4 -- 6% in final formulation)
of sodium cholate resulted in a larger mean diameter of the
particles or more instability of the formulations. For these
formulations, the concentration of sodium cholate was
much higher than its CMC, which might be the reason for
instability of the formulations.
Drug or fluorescent probe
Elastic liposomes have been used successfully as carriers for a
range of drugs, including small molecules, peptides, proteins
and genes. The vesicles can load high concentrations of hydrophobic or hydrophilic drugs in a monomeric state, which do
not perturb the matrix elasticity [49].
Interaction between the drug and the EA can affect the EE
of the elastic liposomes. The EE of colchicine was reported to
be 66.3 2.2% for elastic liposomes containing Span 80,
33.4 2.8% for conventional liposomes and 31.6 1.4% for
a niosome formulation, respectively. Span 80 was shown to
form hydrogen bonds with the amido group of colchicine,
which might lead to better retention in elastic liposomes [50].
On the contrary, the addition of sodium deoxycholate significantly decreased the EE from 97.8 5.4 to 74.6 2.5% for liposomes containing betamethasone in their lipid bilayer. This
can be explained by a competition between betamethasone
and sodium deoxycholate in the lipid bilayer [51].
Besides drugs, a lot of fluorescent probes have been encapsulated into elastic liposomes to investigate the mechanism of
action of the vesicles. Calcein (log P = -5.02) is a highly
hydrophilic dye, which can be encapsulated in the aqueous
compartment of liposomes. Based on the results of recent
investigations [51-53], calcein, which can be easily leaked from
the vesicles, might not be suitable for the research of elastic
liposomes.
Bahia et al. used calcein to probe the physicochemical and
pharmacokinetic characteristics of elastic liposomes [52]. In
vitro skin permeation and in vivo percutaneous absorption
studies were performed using hairless mice. Both studies indicated that elastic liposomes reduced the transdermal flux of
calcein when compared to solution. It was also found that
elastic liposomal membranes made from natural PC and
sodium cholate were highly permeable to calcein.
Gillet et al. investigated the skin penetration behavior of
two parts of elastic liposomes: the aqueous cavity and lipid
2.3

850

bilayer. Firstly, the aqueous cavity was made to be fluorescent by the incorporation of 16 mM calcein solution in
order to avoid the self-quenching phenomenon that
occurs at higher concentrations. Secondly, the lipid bilayer
became fluorescent through the incorporation of 1.33%
1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]
dodecanoyl}-snglycero-3-phosphocholine (NBD-PC). After
administration to the pig ear skin in vitro, confocal microscopic observations revealed that calcein in the inner cavity
of liposomes did not penetrate the epidermis, while NBDPC did penetrate the epidermis [51]. The results indicated
that most calcein leaked from elastic liposomes before
penetrating the deepest layers of the skin.
The stability of calcein-loaded elastic liposomes composed
of SPC and sodium deoxycholate was investigated. After
1 month of storage at 4 C, the increase in the percentage of
calcein released compared with day 0 was 9.43% (5.38)
and 23.75% (4.36) for conventional and elastic liposomes,
respectively [53]. It seemed that calcein was easily leaked
from the elastic vesicles. Therefore, the use of elastic liposomes cannot help calcein to permeate through or into the
skin. In addition, it should be noted that the skin contains
divalent ions, which might quench many fluorescent probes.
On the contrary, if the drug is very stable in elastic liposomes, after passing through SC, the drug is still retained in
the vesicles. As the vesicles are too big to be absorbed into
the blood circulation directly, the drug, such as paromomycin
sulfate [48], will be found to be mainly accumulated in the skin
region and permeate less through the skin compared with free
drug. In this case, elastic liposomes might act as dermal
carriers rather than transdermal carriers.
In summary, the stability of the drug or fluorescent probe
in the vesicles is a key factor affecting the permeation behavior of the elastic liposomes. With the significant leakage of
drug from the elastic liposomes, the amounts of the drug
that can be delivered into or across the skin barrier will be
decreased and the efficacy of the drug will also be decreased
correspondingly.
Aqueous phase ingredients
The addition of water-soluble solutes into liposome dispersions may alter the interaction between the phospholipid molecules at the bilayer surface. Thereby, osmotic gradients
change the bilayer properties of liposomes such as elasticity,
permeability and partition coefficients, perhaps by altering
the area per lipid at the membrane surface [54].
The pH value of the formulation plays a role in optimizing
the elastic vesicle delivery system. It was found that there was a
strong correlation between the drug transport and the amount
of drug incorporation, both of which were clearly influenced
by the pH of the vesicle system. For the preparation of
pergolide-loaded elastic liposomes, three pH values (5.0,
6.0 and 7.0) of aqueous phase were compared.
It was found that formulations at a pH of 5.0 showed a significant higher steady-state flux as compared with those at pH
2.4

Expert Opin. Drug Deliv. (2013) 10(6)

Skin permeation behavior of elastic liposomes

6.0 and pH 7.0 (p < 0.01). There was more than a fourfold
difference between the highest flux at pH 5.0 and the lowest
flux at pH 7.0 [55]. Therefore, the apparent pK of bilayer components and their relationship to the pH should be noted for
the preparation of elastic liposomes.
3.

PEG-lipid
Poly-(ethylene glycol) (PEG) has been widely used as a steric
stabilizer of liposomes. A PEGylated elastic liposomal formulation containing zidovudine was obtained by incubating the
vesicular dispersion with activated monomethoxy PEG2000
(MPEG) [56]. An amide formed between activated MPEG
and the terminal-NH2 group of phosphatidylethanolamine
(PE) located in the liposomal membrane. Zeta potential for
plain and PEGylated elastic liposomes was found to be
-2.8 0.4 and -18.2 0.8 mV, respectively. This significant
change in zeta potential value verified the PEGylation of elastic liposomes. Significantly, an increase in elasticity of the vesicle membrane was observed after PEGylation (55.5 5.0 and
68.9 5.8, respectively, for plain and PEGylated elastic liposome formulation). Correspondingly, in vitro flux of zidovudine across rat skin from drug solution, plain and
PEGylated elastic liposome formulation was found to be
5.7 0.3, 98.8 5.8 and 119.5 5.2 g/h/cm2, respectively.
N-(carbonyl-methoxypoly(ethylene
glycol)-2000)-1,2distearoyl-sn-glycero-3-phosphoethanolamine
(PEG2000DSPE) was added into the elastic liposomal composition to
prepare PEGylated elastic liposomes for the dermal delivery
of calcipotriol, a drug for the treatment of psoriasis [57].
It was found that inclusion of 0.5, 1 and 5 mol%
PEG2000-DSPE in the membrane enhanced the colloidal stability of the elastic liposomes. The results also showed an
increased negative zeta potential with increasing concentrations
of PEG2000-DSPE. The skin permeation behavior of the
PEGylated elastic liposomes was further compared. The highest
amount of calcipotriol delivered into the skin was obtained
with liposomes containing 1 mol% PEG2000-DSPE, where
PEG was expected to adopt a mushroom-like configuration.
It seems that PEG coating of elastic liposomes can increase
skin permeation and there could be several explanations for
this. Firstly, the PEG coating of elastic liposomes could
increase the stretching property of the composite membrane
with respect both to area dilation and bending of the liposomal membrane. This might help to increase the elasticity
of vesicle membrane [56]. Secondly, PEG could increase the
permeation into the skin by binding to water molecules,
which leads to increased hydration of the SC and results in
enhanced permeation of the SC barrier [57].
Furthermore, the application of PEGylated elastic liposomes for dermal administration provides the option for
active targeting strategies to specific cell types in the skin
through conjugation of receptor-specific ligands to the distal
ends of the PEG chains.
3.1

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Optional ingredients

Charged lipid
In recent years, there has been increased interest in developing
charged liposomes as carriers for transdermal or dermal drug
delivery [54]. Though the zeta potential of liposomes can be
affected by many other factors, the addition of charged materials,
especially charged lipids, is regarded as the key factor.
The formulation ingredients and skin permeation behavior
of elastic liposomes containing differently charged lipids are
summarized in Table 2 [58-60].
It is evident that positively-charged elastic liposomes with
sufficiently high surface charge densities provide greater skin
permeation parameters. The positive charged lipids, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and stearylamine (SA), were usually added to the liposomal composition
to obtain cationic elastic liposomes. Cationic elastic liposomes
were also reported with the composition of SPC, DOTAP and
Span 80 (9:3:1, molar ratio) [61]. Positively-charged vesicle
components are likely to interact with charges in the skin
(which is, on the average, anionic). This can lead to changes
to the skin barrier and improve the skin permeation behavior
of positively-charged liposomes.
Elastic liposomes containing DOTAP have been shown to
be a promising tool for transcutaneous gene therapy [62]. Elastic liposomes containing different DOTAP/sodium cholate
ratios (6:1, 8:1 and 10:1, w/w) were prepared and their
corresponding complexes with siRNA were characterized.
Transfection experiments indicated that the efficacy of gene
silencing clearly differed among different complexes. Elastic liposomes containing DOTAP/sodium cholate in a 6:1 (w:w)
ratio showed the best results.
In addition to improved permeation behavior, the presence
of charges on the surface of liposomes favors the electrostatic
repulsion among these vesicles, creating a stable formulation.
After storage of 90 days, the EE of 5-aminolevulinic acid
(5-ALA) in cationic elastic liposomes decreased by 15% at
4 C. On the other hand, the EE of 5-ALA in neutral and
anionic elastic liposomes decreased markedly by > 30% at
4 C. Thereby, cationic elastic liposomes were physically
more stable than other vesicles [59].
Reasonable application of charged lipids can lead to
improved skin permeability behavior and stability of elastic
liposomes. However, it should also be noted that the addition
of charged lipids might affect the EE of vesicles. For example,
the addition of SA was found to decrease markedly the EE of
DS elastic liposomes [33]. Moreover, after the addition of SA,
the Tm values of the liposomes were significantly reduced,
which indicated that SA perturbed the packing characteristics
of lipid bilayers and then fluidized them [33].
3.2

Ethanol
Ethanol is known to act as an efficient skin-penetration
enhancer. It can interact with the polar head group region
of the lipid molecules, resulting in the reduction of the melting point of the SC lipids, thereby increasing their fluidity
and cell membrane permeability. In 2000, ethanol was
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Expert Opin. Drug Deliv. (2013) 10(6)

851

J. Chen et al.

Though the ethanol has been proved to be an effective

ingredient of elastic liposomes, it should be noted that ethanol
might be detrimental to the stability of the vesicles, especially
at higher concentrations.
Cyclodextrin
Liposomes are able to encapsulate hydrophilic drugs in their
aqueous compartment, while hydrophobic drugs are encapsulated in their lipid bilayer. However, the accommodation
of hydrophobic drugs in the bilayer can be problematic as
the encapsulated drugs may interfere with bilayer formation
and affect stability. In 1994, entrapping water-soluble
drug--cyclodextrin inclusion complexes in the aqueous
compartment of liposomes was proposed in order to avoid
such drawbacks [67]. In 2008, a new delivery system for
cutaneous administration combining the advantages of drug-cyclodextrin inclusion complexes and those of elastic liposomes (Figure 1) was first reported by Jain et al. and named
drug-in-cyclodextrin-in-elastic liposomes (DCEL) [68].
To prepare DCEL, cyclodextrins should have a higher
affinity for drug molecules than for membrane lipids, allowing the entrapment of highly concentrated drug--cyclodextrin
inclusion complexes with or without minimal interaction with
lipid components of the liposomal membrane.
The encapsulation of cyclodextrins was found to have little
effect on the elasticity of liposomes. The elasticity values were
found to be 8.2 0.6 and 8.0 0.4 for the betamethasone
DCEL and elastic liposomes, respectively [51]. The use of
betamethasone-hydroxypropylated-g-cyclodextrin complexes
was shown to enhance the drug retention in liposomes.
Similar results were also obtained by Singh et al. [69].
A decrease in release rate was observed with elastic liposomal
formulations prepared with a colchicine-b-cyclodextrin complex (65.9% at 24 h) compared with that of normal elastic liposomes (79.2% at 24 h). The release experiments clearly
indicated the sustained release of colchicine from DCEL.
Compared with drug solution, the amount of drug deposited
in skin was found to be 12.4-, 8.4- and 3.4-fold higher after
24 h administration of colchicine DCEL, colchicine elastic
liposomes and colchicine-b-cyclodextrin complexes, respectively. In addition, the lipid perturbation effect was significantly less in skin treated with colchicine elastic liposomal
formulation compared with that treated with DCEL.
A skin retention study was carried out with the objective to
determine the depot effect of DCEL containing isotretinoin [70]. It was found that the amount of drug deposited
was 21-fold higher in the case of DCEL than drug solution.
The observed significantly higher skin deposition of hydroxypropyl-b-cyclodextrin-loaded formulation was due to the
increase in solubility, the higher EE and the sustained release
of the drug. In summary, DCEL seems to be a satisfactory
carrier for dermal application due to its good reservoir effect.

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3.4

Cyclodextrin

Phsophatidylcholine
Edge activator

Drug molecule

Figure 1. The schematic illustration of elastic liposomes

encapsulating a drug--cyclodextrin inclusion complex.

included in liposomes to form ethosomes which were composed of PC and a high proportion of ethanol (generally
> 30%) [63].
After addition of 10% ethanol in the aqueous phase, the
properties of 18b-glycyrrhetic acid-loaded elastic liposomes
composed of SPC, sodium deoxycholate and a-tocopherol (3.5:0.62:0.1, w/w) were investigated [64]. The mean particle size of elastic vesicles with 10% ethanol (v/v) was 87.3
1.5 nm, which was smaller compared with elastic vesicles without ethanol. Elasticity values of elastic liposomes with or without ethanol were determined to be 212.8 29.7 and 25.9
2.7, respectively, but the EE was not affected by the addition
of 10% ethanol. Therefore, to increase elasticity of the vesicles,
ethanol can be added as a formulation ingredient. The
concentration of ethanol is usually set at 7% (v/v) [15,34,41,65].
Ethanol is doomed to disappear from the skin surface soon
after application owing to evaporation and diffusion into the
skin interior, so the effect of ethanol on the elasticity of
liposomes might only be maintained in for a very short time
after administration.
Song et al. regarded elastic liposomes with higher concentration ethanol (30%) as a novel carrier which they named
transethosomes (TELs). TELs are composed of PC, ethanol,
water and EA. The composition and permeation behavior of
TELs containing 0.3% (w/w) voriconazole were compared
between different concentrations (7 or 30%) of ethanol.
TELs of 30% ethanol showed higher elasticity, permeability
and deposition, which is due to the synergic effect of ethanol
and EA. In addition, the TELs appeared as irregular spherical
shapes, which indicated that the lipid bilayer was disturbed by
the presence of both EA and ethanol [66].
852

Gel ingredients
In order to make them suitable for administration, the elastic
liposomes are sometimes dispersed in gel. Gel is needed to
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Skin permeation behavior of elastic liposomes

apply large amounts of lipids on the skin surface, as in the case

of deep local delivery. If chosen well, the gel neither affects
skin surface hydration nor captures lipid vesicles and, thus,
does not affect vesicle-mediated drug delivery.
Griseofulvin-loaded elastic liposomes were incorporated in
Carbopol 980 NF (0.5% by weight) pre-gelled with triethanolamine by gentle levigation [34]. In the in vitro permeation
studies, the elastic liposome suspension showed 59.54
1.04% drug permeation in 24 h. After incorporated of gel,
the elastic liposomes showed slightly lower drug permeation
of 51.47 1.16%. The lower permeation behavior may be
attributed to the slow diffusion of the drug through the
gel network.
Following the preparation of insulin-loaded elastic liposomes, 2% v/v dimethyl sulfoxide (DMSO) was added to
the suspension as a chemical permeation enhancer. The suspensions were transferred to 5% w/v methylcellulose gel and
showed a good permeation result with in vitro permeation
flux of 13.50 0.22 g/cm2/h through porcine ear skin. An
in vivo study of elastic liposome gel demonstrated a prolonged
hypoglycemic effect in alloxan-induced diabetic rats over 24 h
after transdermal administration [71].
Furthermore, it has been proved that gel ingredients do
not affect the improved permeation behavior of elastic liposomes compared with conventional liposomes. For 5-FU,
elastic liposomes (SPC:sodium deoxycholate = 85:15, w/w),
conventional liposomes (SPC:CH = 7:3, mol/mol) and
niosomes (Span 80:CH = 7:3, mol/mol) were prepared. Carbopol 941 gel (0.5% w/v) was prepared in water and gently
stirred for the gel to swell. A volume of 5 mL of triethanolamine or water were alternatively added with continuous stirring to form a transparent gel; then the three vesicles were
transferred to the carbopol gel. Rheological analysis indicated
that there was no significant difference in viscosity of the different vesicles when incorporated into a gel base. The amount
of 5-FU permeated across rat abdominal skin in 24 h in vitro
was found to be 68.25 5.36, 49.85 4.65 and 41.14
3.69% for elastic liposomes, conventional liposomes and niosomes in gel, respectively; whereas only 7.0 0.9 and 11.77
1.21% of 5-FU was permeated from 5-FU gel and 5-FU
solution, respectively [44]. As studies only focus on the skin
permeation behavior, more attention should be paid to
rheological properties of elastic liposome gels.

4.

Conclusion

Various ingredients, basic and optional, have been used to

prepare elastic liposomes, leading to different skin permeation
behaviors of the vesicles. For the choice of ingredients, their
effects on drug loading, zeta potential, elasticity and stability
of the vesicles should be taken into account to obtain satisfactory skin permeation behavior. More attention should be paid
to drug leakage from the vesicles, whether following administration or during long-term storage, for the development of

elastic liposomes. In addition, more optional ingredients are

applied for the preparation of elastic liposomes.
5.

Expert opinion

Despite the long history of intensive research, conflicting

results continue to be published concerning the effectiveness
of elastic liposomes. However, the stability during administration of the vesicles can be used to explain their different permeation behaviors. Following administration, if most drug
molecules (e.g., calcein) are immediately leaked from the
vesicles on the skin surface, reduced permeation behavior
will be obtained with the elastic liposomes. However, if the
vesicles can retain the drug long enough, on, in and also below
the skin barrier, the vesicles will be found to be mainly accumulated in the viable epidermis and dermis regions as drug
reservoirs for sustained release. In this case, elastic liposomes
will be suitable for topical application. As these blood vessels
are non-fenestrated and possess tight junctions between endothelial cells, it is believed that the intact vesicles are not
allowed to enter into the bloodstream. Therefore, after elastic
liposomes cross the SC, if most drug molecules can release
from the vesicles and diffuse into blood circulation, elastic liposomes will be found to be suitable for transdermal drug
delivery. It should be emphasized that the explanation based
on vesicle stability does not exclude the mechanism of elastic
liposomes as penetration enhancers.
Elastic liposomes are mainly composed of unsaturated PC
and EA. The unsaturated PC has lower Tm and the lipid
bilayers are in liquid-crystal state, so the liposomal membrane is highly permeable to encapsulated drug molecules
during storage. Furthermore, it is well known that EA destabilizes lipid bilayers of the liposomes and increases permeability of the bilayers. Therefore, more attention should be
paid to the stability of the vesicles, especially the storage stability. The long-term stability of elastic liposomes is found
to be the main drawback of the vesicles, especially under
room temperature; therefore, the vesicles are stored at 4 C
in most cases.
If the Tm of elastic liposomes can be flexibly modified by
changing the ratio of different PCs in the liposomal bilayer,
the Tm values can be set between storage and skin temperature,
for example 30 C. During storage under room temperature
(25 C), the rigid elastic liposomes are stable. After administration to skin, the liposomal membrane turns into a liquid state
due to the skin temperature (32 C) being higher than its Tm
and the higher elasticity of liposomes is obtained. Thus, satisfactory elasticity and stability can be obtained at the same time.
Following elastic liposome administration, structural
changes in the SC have been identified and intact elastic
liposomes visualized within the SC lipid lamellar regions,
but no intact vesicles have been identified in the deeper viable
tissues. Further research needs to be carried out to find the
final site that elastic liposomes can arrive at with their structure intact. That is to say, the relationship between stability

Expert Opin. Drug Deliv. (2013) 10(6)

853

J. Chen et al.

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of the vesicles and the permeation depth into skin should

be emphasized.
The composition of elastic liposome has a great effect on its
skin permeation behavior. It is necessary to modify the composition of elastic liposomes in order to enhance the penetration properties of the vesicles into or through the skin. The
best carrier composition has to be determined experimentally
for each drug separately to acquire appropriate vesicles with
the highest elasticity and stability.
The application of optional ingredients has become a
new trend to improve stability or elasticity or both. The
Bibliography

application of ethanol can lead to higher elasticity, while the

application of charged lipids and cyclodextrin can lead to
higher stability. The use of PEG--lipid can lead to both higher
stability and elasticity. In the future, new ingredients are
expected to be proposed to significantly enhance skin permeation behavior.

Declaration of interest
The authors state no conflict of interest and have received no
payment in preparation of this manuscript.

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Expert Opin. Drug Deliv. (2013) 10(6)

Jun Chen1, Wen-Li Lu2, Wei Gu1,

Shan-Shan Lu1, Zhi-Peng Chen1 &
Bao-Chang Cai3

Author for correspondence

1
Nanjing University of Chinese Medicine,
School of Pharmacy, Nanjing, PR China
2
University of Tennessee Health Science Center,
Department of Pharmaceutical Sciences,
Memphis, TN, USA
3
Professor,
Nanjing University of Chinese Medicine,
School of Pharmacy, Nanjing, PR China
Tel: +86 25 86798281;
Fax: +86 25 86798281;
E-mail: [email protected]