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Mechanism of transdermal permeation promotion

of lipophilic drugs by ethosomes
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
26 April 2017
Number of times this article has been viewed

Li Yang 1,* Abstract: Ethosomes can promote the penetration of lipophilic drugs into the skin, but the
Lifang Wu 1,* underlying mechanism is still unknown. The purpose of this study was to investigate the mecha-
Dongze Wu 2 nism of transdermal permeation promotion of lipophilic drugs by ethosomes. The formulation of
Deshun Shi 3 ethosomes was optimized using the Box–Behnken experimental design, in which Rhodamine B
Tai Wang 4 and 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-
phosphocholine were used to simulate a model lipophilic drug and act as a fluorescent tracer
Xiaoliang Zhu 4
of ethosomal phospholipids, respectively. Liposomes with the same phospholipid concentra-
1
Department of Dermatology,
tion and a hydroethanolic solution with the same ethanol concentration were also prepared as
Nanfang Hospital, Southern Medical
University, Guangzhou, 2Department controls. The percutaneous progression of the above fluorescent preparations was observed by
of Medicine and Therapeutics, The confocal laser scanning microscopy, and the fluorescence intensity of the images was analyzed.
Prince of Wales Hospital, The Chinese
University of Hong Kong, Hong Kong,
The optimized ethosome formulation consisted of 2.45% yolk phospholipids, 30% ethanol,
3
Department of Dermatology, The and 67.55% distilled water. The percutaneous permeation of Rhodamine B in the optimized
First People’s Hospital of Foshan, ethosomes was superior to that in hydroethanolic solution (P,0.05) and liposomes (P,0.05).
Foshan, 4Department of Dermatology,
Nanfang Hospital, Southern Medical The ethosomes could penetrate the skin via the percutaneous pathway of the hair follicle and
University, Guangzhou, People’s stratum corneum, while during the process of penetration, the vesicles were broken and the
Republic of China phospholipids were retained in the upper epidermis, with the test compounds penetrating gradu-
*These authors contributed equally ally. The superior percutaneous penetration of ethosomes was linked to the synergistic effects
to this work of their ingredients. The percutaneous pathways of ethosomes included open hair follicles
and stratum corneum pathways. In addition, the vesicles might break up during percutaneous
penetration in the superficial layer of the skin, allowing the test compounds to keep permeating
into the deeper layer alone, while the phospholipid was retained in the upper epidermis.
Keywords: ethosomes, mechanism, percutaneous, confocal laser scanning microscopy,
CLSM

Introduction
Many lipophilic drugs, including fusidic acid,1 bifonazole,2 valaciclovir,3 corticosteroid,4
and tacrolimus,5 are used in the treatment of various skin diseases. Formulating
these drugs as creams or emulsions could have advantages over other methods of
administration, including avoidance of first-pass metabolism, a lower frequency of
administration, and improved patient compliance. However, topical skin formulations
remain ineffective due to a low percutaneous penetration through the stratum corneum.
Correspondence: Xiaoliang Zhu
Department of Dermatology, Nanfang There are increasing efforts to overcome the powerful barrier of the stratum corneum.
Hospital, Southern Medical University, Such approaches include the use of iontophoresis,6 electroporation,7 microneedles,8
1838 North Guangzhou Avenue,
Guangzhou, Guangdong 510515, People’s
sonophoresis,9 surfactants,10 and liposomal-based nanoparticles;11 however, the defi-
Republic of China ciencies associated with these methods, such as complicated processing, skin injury,
Tel +86 20 6278 6258
Fax +86 20 6164 1049
patient compliance, poor stability, and high cost, have significantly limited their
Email [email protected] widespread applications in clinical practice.

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As a novel lipid vehicle, ethosomes have shown superb and humidity (50%±2%), with free access to standard
effectiveness in percutaneous drug delivery. In addition, they laboratory chow and tap water in an iron cage. The animal
have superior pharmaceutical properties, including stability at use and care protocols were reviewed and approved by the
room temperature, high entrapment efficiency, and improved Institutional Animal Care and Use Committee of Southern
compatibility with the stratum corneum, thus promoting the Medical University. All procedures of the animal experi-
effective penetration of lipophilic drugs into the skin.12–14 In ments adhered to the Principles of Laboratory Animal Care
our preliminary study, we have proven that ethosomes can guidelines of the Laboratory Animal Center, Southern Medi-
promote transdermal permeation of lipophilic drugs, such cal University, following the principles of the ­Declaration
as lidocaine, with a shorter onset time and a longer duration of Helsinki.
in vivo than lidocaine-based liposomes or lidocaine delivered
in a hydroethanolic solution.15 Subsequently, ethosomes have Experimental design to optimize the ethosome
been used in photodynamic therapy,16,17 cosmeceuticals,18 formulation
and a vaccine.19 Using the vesicle size as a dependent variable, we optimized
However, the mechanisms by which ethosomes promote the following independent variables: yolk phospholipid con-
skin penetration of drugs remain elusive.20,21 Therefore, tent (1%–5%, w/v), ethanol content (20%–30%, v/v), and
the following three key questions are critical: Which key ultrasonic treatment time (6–8 min), with each being set at
ingredient(s) in ethosomes is responsible for their excellent low (-1), medium (0), and high (+1) levels. We employed a
properties in promoting percutaneous penetration? Can etho- Box–Behnken experimental design,22 with three factors, three
somes penetrate into the skin with an intact vesicle structure? levels, and 17 runs in the optimization study, using Design-
What is the detailed mechanism by which ethosomes loaded Expert.V8.0.6 (Stat-Ease, Inc., Minneapolis, MN, USA).
with lipophilic drugs penetrate into the skin? Three-dimensional response surface plots were generated
In this study, we adopted Rhodamine B to simulate a for the main effects and interactive responses to obtain the
model lipophilic drug and 1-palmitoyl-2-{12-[(7-nitro-2- optimized vesicle size.
1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-
phosphocholine (NBD-PC) as a tracer of phospholipids to Preparations of ethosomes and other formulations
prepare fluorescent ethosomes. The aims of this study were To optimize the composition of the ethosomes, various for-
to evaluate the percutaneous efficiency of model drug-loaded mulations of ethosomes based on the above Box–Behnken
ethosomes as well as to determine the mechanisms related design were prepared by applying the sonication method
to lipophilic drug penetration into the skin. reported previously.23 Yolk phospholipid was dissolved along
with Rhodamine B (0.02%, w/v) in ethanol in a sealed glass
Materials and methods bottle with magnetic stirring at 700 rpm, and distilled water
Chemicals and reagents was added slowly at a constant rate of 200 μL⋅min-1 at room
The compound NBD-PC was purchased from Avanti Polar temperature. After the addition of water, stirring was contin-
Lipids (Alabaster, AL, USA). Rhodamine B, hexyl ester, per- ued for an additional 5 min. The mixed solution was placed
chlorate was obtained from AAT Bioquest (Sunnyvale, CA, in an ultrasonic apparatus in an ice bath for predetermined
USA). High-purity yolk phospholipids (98%) were provided by time intervals (150 W, 5 s and 3 s apart). The ethosomes were
Nippon Fine Chemical Co., Ltd. (Tokyo, Japan). Chloroform filtered through a 0.22 μm microporous membrane filter,
and 100% ethanol were purchased from GuangHua Company protected from light, and then stored at 4°C until use. Blank
(Guangzhou, People’s Republic of China). All other reagents control ethosomes were prepared using the same procedure,
and solvents used in this study were of analytical grade and but without Rhodamine B.
were obtained from local companies. Liposomes were prepared using the thin layer evapora-
tion technique,24 in which the contents of yolk phospholipids
Animals and Rhodamine B were identical to the optimized ethosome
Specific pathogen-free male Sprague Dawley rats, weigh- formulation. Briefly, a chloroform solution of the required
ing 220–250 g and aged 7–8 weeks, were obtained from the lipid mixture was first dried under vacuum overnight, and
Laboratory Animal Center, Southern Medical University, the resultant lipid film was hydrated with distilled water
Guangzhou, People’s Republic of China. The animals were to prepare the lipid suspension. The suspension was then
housed in a room under a controlled temperature (25°C±2°C) sonicated for the same time as the optimized ethosomal

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Dovepress Mechanism of drug permeation by ethosomes

preparation, protected from light, and stored at 4°C until use. 556 nm. The blank group ethosomes were used as a control.
For preparation of the Rhodamine B hydroethanolic solution, All procedures were carried out in the dark to prevent the
the contents of ethanol and Rhodamine B were identical to influence of ambient light. The fluorescence intensity of each
those in the optimized ethosomal preparation. image was measured by Image-Pro Plus software (Image-Pro
Plus 6.0; Media Cybernetics, Washington DC, USA).
Separation of nonentrapped Rhodamine B from
ethosomes Penetration mechanism study in rats
Rhodamine B-loaded, NBD-PC-labeled ethosomes Sprague Dawley rats were randomly divided into three groups
(phospholipid:NBD-PC =100 mole:1 mole) were prepared (n=5 per group) for different treatment durations: 1, 4, and 8 h.
using the aforementioned methods.25 Nonentrapped Rhod- Rhodamine B-loaded, NBD-labeled ethosomes were adminis-
amine B was separated from the NBD-PC-labeled ethosomes tered as above, and the skin tissue samples were collected at
by centrifugation (Thermo Scientific Sorvall Legend Mach; 1, 4, and 8 h after drug application, and processed as above for
Thermo Fisher Scientific, Waltham, MA, USA). In brief, observation by CLSM. In this study, the two probes used could
the ethosomes were added to an ultrafiltration tube (EMD be visualized with different excitations. A helium–neon laser
Millipore, Billerica, MA, USA), with a cut-off molecular (excitation wavelength: 558 nm) was applied to visualize the
weight of 3,000 Da, and centrifuged at 11,185 rpm and 4°C vesicle and Rhodamine B distribution in the treated skin samples
for 60 min. The filtrate was removed, and the retentate tube (red), and an argon laser (excitation wavelength: 478 nm) was
was then inverted into a new collection tube to collect the applied to visualize NBD-PC in the ethosomes (green).
entrapped ethosomes, which was centrifuged at 2,990 rpm
and 4°C for 5 min. The entrapped Rhodamine B-loaded, Statistical analysis
NBD-PC-labeled ethosomes were used immediately in the All quantitative data are expressed as the mean ± standard
subsequent skin penetration experiments. deviation of three measurements. Statistical analyses were
accomplished by analysis of variance or the Student’s t-test,
Characterization of the ethosomes using SPSS software (SPSS Statistics 20.0; IBM Corporation,
The vesicle size and polydispersity index of Rhodamine Armonk, NY, USA). A value of P,0.05 was considered
B-loaded ethosomes and liposomes were determined using statistically significant.
a dynamic light scattering method, with a Zetasizer Nano
ZS90 (Malvern Instruments Ltd., Malvern, UK) without Results
dilution or concentration. Ethosomal optimization and
characteristics of the vesicles
In vivo skin penetration study in rats To determine the optimal formulation for the ethosomes, a
The time-dependent permeation behavior of Rhodamine total of 17 runs of the Box–Behnken experimental design
B from the ethosomal system in rats (n=6 per group) was were performed. The results of the responses (vesicle size) are
measured by confocal laser scanning microscopy (CLSM)26 shown in Table 1, and the three-dimensional plots are shown
and compared with two control systems: liposomes and a in Figure 1. All three independent variables, including yolk
hydroethanolic solution. A mini-funnel with a diameter of phospholipid content, ethanol content, and ultrasonic time,
3 cm, which served as a drug pool, was stuck with glue to the had interactive effects on the vesicle size of the ethosomes.
rat abdominal skin that had been shaved carefully. Different The optimal design was determined to be 2.45% (w/v) yolk
formulations (200 μL) were added into the funnels. Three phospholipids, 30% (v/v) ethanol, and an ultrasonic time of
rats from each group were selected randomly, and the treated 8 min. The predicted value of the vesicle size was 132.4 nm,
skin samples were removed at 1, 4, and 8 h after test com- and the measured mean value was 125.7 nm.
pound application. The experiments for the other three rats The vesicle size and polydispersity index of the
in each group lasted for 8 h, and then the test compound was Rhodamine B-loaded ethosome vesicles were determined
removed. The application areas were cleaned, and the treated by dynamic light scattering. The mean vesicle size of the
skin samples were collected at 1, 4, and 8 h. The skin tissue ethosomes was 125.7±3.2 nm (n=3), while the mean poly-
samples were embedded in embedding medium, sectioned at dispersity index was 0.199±0.014 (n=3). The mean size of
8 μm thicknesses immediately, with a cryo-ultramicrotome, the liposomes was 185.9±1.8 nm (n=3), while the mean
and observed with CLSM, with an excitation wavelength of polydispersity index was 0.289±0.012 (n=3).

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Table 1 Box–Behnken study design and experimental results in Figure 3. Enhanced delivery of Rhodamine B in terms
Run Yolk phospholipid Ethanol Ultrasonic Particle of depth (viable epidermis, dermis, hair follicle, and even
(w/v) (v/v) time (min) size (nm) adipose tissue) was observed when it was loaded in etho-
1 0 1 -1 182.8 somes. In contrast, the penetration of Rhodamine B from
2 -1 1 0 176.5
conventional liposomes was limited to the upper layer of the
3 0 0 0 187.2
4 0 0 0 188.7 skin (stratum corneum), with slight or negligible penetration
5 -1 0 -1 178.9 observed in the viable epidermis and dermis (Figure 3C).
6 1 1 0 197.2 Furthermore, the presence of fluorescence in the skin append-
7 1 0 1 213.4
ages pathway indicated that Rhodamine B-labeled ethosomes
8 0 -1 -1 198.1
9 0 0 0 183.7 penetrated the skin via this pathway. A 30% hydroethanolic
10 0 0 0 189 solution of Rhodamine B (Figure 3B) showed much weaker
11 0 1 1 123.5 fluorescence in comparison with the ethosomal formulation
12 0 -1 1 195.2
(Figure 3A), but it displayed a stronger fluorescence than
13 -1 -1 0 188.2
14 1 -1 0 225.4 liposomes (Figure 3C). The control ethosome samples did
15 -1 0 1 175.3 not show any fluorescence (data not shown).
16 1 0 -1 236.3
17 0 0 0 183.4 Mechanistic observation of skin
penetration
Skin penetration properties CLSM with double fluorescence-labeled ethosomes illus-
The quantitative data from the experiments are shown in trated the permeation process of ethosomes loaded with
Table 2 and Figure 2. Fluorescent photomicrographs of lipophilic compounds into the deep layers of the skin
the skin treated with Rhodamine B-labeled ethosomes, (Figure 4). The presence of red and green fluorescence in the
liposomes, and 30% hydroethanolic solution are shown skin tissues indicated that both Rhodamine B and NBD-PC








 
 
 
 
 

 
 
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Figure 1 Three-dimensional response surface plots showing the effects of the independent variables (phospholipid, ethanol, and ultrasonic time) on the vesicle size of
ethosomes.

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Dovepress Mechanism of drug permeation by ethosomes

Table 2 The mean fluorescence intensity (AU, mean ± standard deviation, n=3) of tissue slices of Rhodamine B-loaded ethosomes,
liposomes, and hydroethanolic solution at 1, 4, and 8 h after test compound administration and 1, 4, and 8 h after removal of the test
compounds from the skin
Formulation Time (h)
Time after transdermal test compound delivery Time after test compound removal from the skin
1h 4h 8h 1h 4h 8h
Ethosomes 57.479±7.331 68.08±3.158 87.997±3.985 55.524±1.586 31.065±1.626 23.777±2.052
Liposomes 20.207±0.564 25.435±0.573 28.551±1.276 25.258±0.125 15.982±0.523 9.24±0.900
Hydroethanolic 22.808±0.725 28.399±1.028 35.412±0.696 30.005±0.485 20.051±0.499 14.413±0.543
solution

could penetrate through the skin. If their intensities appeared in the viable epidermis, indicating that the distribution dif-
differently or only one fluorescent color existed in the deep ference between the two fluorescent compounds emerged.
layers, it would indicate that the vesicles were broken and At 8 h, a broader distribution of red fluorescence without
that the entrapped drug was released into the tissues alone. green fluorescence was seen in the dermis or deep layer of
At 1 h after test compound administration, the red and green the skin (adipose tissue). In addition, the distribution of
fluorescence exhibited a slight, continuous strip distribution, Rhodamine B and NBD-PC appeared obviously different.
which was consistent in the stratum corneum and viable Moreover, the distribution pattern of ethosomes was dif-
epidermis. In hair follicles of the superficial layer, the red ferent from that of liposomes because the liposomes were
fluorescence was distributed in the perifollicular epithelial confined in the upper layers of the stratum corneum and
tissues and connective tissues, while the green fluorescence even prevented the test compounds from passing through
was distributed in the hair root and hair shaft; separation of the deeper layer.
fluorescent colors was observed in the hair follicle, but not in
the stratum corneum. Four hours later, the fluorescence from Discussion
both Rhodamine B and NBD-PC became brighter: bright Many factors influence the delivery of drugs into the skin
red fluorescence became visible in the deep layer of the from topically applied vesicles; the vesicle size and encap-
dermis and hair follicles and green fluorescence remained sulation efficiency are the key parameters.27 Smaller vesicles

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Figure 2 Mean fluorescence intensity of Rhodamine B-loaded ethosomes, hydroethanolic solution, and liposomes at 1, 4, and 8 h after application (A) as well as 1, 4, and 8 h
after the test compound was removed from the skin (B). *P0.05 for the comparison with hydroethanolic solution, +P0.05 for the comparison with liposomes.

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3361
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Yang et al Dovepress

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Figure 3 Confocal laser scanning microscopy images of rat skin samples from the skin penetration study of Rhodamine B-loaded ethosomes (A), hydroethanolic solution
(B), and liposomes (C) at 1, 4, and 8 h after application (A1, A4, A8, B1, B4, B8, C1, C4, and C8) as well as 1, 4, and 8 h after the test compound was removed from the
skin (A-1, A-4, A-8, B-1, B-4, B-8, C-1, C-4, and C-8).

are able to deliver their encapsulated drugs into the deeper the vesicles with flexible characteristics that allow them to
layers of the skin,28 and like other studies, the vesicle size penetrate easily into deeper layers of the skin;32,33 the second
was chosen as an index for efficiency and feasibility in our one is that phospholipids between the stratum corneum and
experiments. Our data (Figure 1) show that the vesicle size ethosomal vesicle fuse easily, thus changing the transition
of the ethosomes decreased as the ethanol concentration temperature, leading to enhanced drug penetration.34 In our
increased, but it increased as the phospholipid concentration research, we interestingly found that even though the lipid
increased, indicating that the factors, in descending order, that content in the liposomes and the ethanol content in the
could influence the vesicle size were the yolk phospholipid hydroethanolic solution were equal with those in the etho-
content, ethanol content, and ultrasonic treatment time. somes, after percutaneous penetration, a greater fluorescence
Our previous study15 and subsequent other studies have intensity in the skin of the ethosomal system was observed,
proven that ethosomes can improve percutaneous permeation compared with that in the hydroethanolic solution and lipo-
of drugs.29–31 In terms of possible mechanisms for ethosomes somes (P,0.05; Figure 2B). These results further confirmed
to promote percutaneous permeation of drugs, two viewpoints that alcohol or phospholipid content was not the only driving
have been presented: the first one is that ethanol is a known force for promoting effective drug penetration into the skin;
penetration enhancer, which can disturb the multilamellar therefore, we speculate that there is a synergistic mechanism
and ordered lipid domain, lower the structure density, and among lipids, ethanol, and the skin, by which skin penetration
enhance the fluidity of the stratum corneum, thus providing of drugs can be obviously promoted. Also, ethosomes can

3362 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2017:12

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Dovepress Mechanism of drug permeation by ethosomes

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Figure 4 Confocal laser scanning microscopy images of rat samples from the skin penetration study of Rhodamine B-loaded, NBD-PC-labeled ethosomes at 1, 4, and 8 h
after application (A1, A4, A8, B1, B4, B8, C1, C4, and C8).
Notes: (A) Red fluorescence of Rhodamine B; (B) green fluorescence of NBD-PC; (C) overlay of A and B.
Abbreviation: NBD-PC, 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphocholine.

penetrate into the skin via skin appendages; the hair follicle superiority over liposomes and hydroethanolic solution, due
and stratum corneum have a remarkably longer retention to the synergistic effect of their ingredients with the skin
time compared with the other two preparations. Nevertheless, structures. Of note, during the percutaneous process, the ves-
we did not find the existence of green fluorescence-labeled icles might break up in the superficial layer of skin, allowing
phospholipids in the dermis. This difference reminded us that drugs to permeate into the deeper layer alone, thus allowing
ethosomes experience sphere separation and drug release in the phospholipid to be retained in the upper epidermis.
the superficial epidermis.
Compared with liposomes and the hydroethanolic solu- Acknowledgments
tion, ethosomes have a particularly longer duration in skin We are grateful to all of our colleagues working in the
appendages and the dermis, suggesting that ethosomes are Department of Dermatology, Nanfang Hospital, Southern
especially suitable to be the carrier of some drugs through the Medical University for their help. We would also like to
skin, such as the drugs for hair follicle and sebaceous gland acknowledge the useful comments on this paper that were
diseases, photodynamic therapy, and anesthetics. received from reviewers. This work was supported by the
Natural Science Foundation of Guangdong Province, China
Conclusion (Grant number 2014A030313320), the Medical Scientific
Based on our results from this study, we conclude that etho- Research Foundation of Guangdong Province, China (Grant
somes as a percutaneous drug carrier showed transdermal number A2016544), Science and Technology Development

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Program of Southern Medical University (C1033041), and 15. Zhu X, Li F, Peng X, Zeng K. Formulation and evaluation of lido-
caine base ethosomes for transdermal delivery. Anesth Analg. 2013;
the Science and Technology Planning Project of Guangzhou, 117(2):352–357.
China (Grant number 201510010120). 16. Rkein AM, Ozog DM. Photodynamic Therapy. Dermatol Clin. 2014;
32(3):415–425.
Disclosure 17. Abrahamse H, Hamblin MR. New photosensitizers for photodynamic
therapy. Biochem J. 2016;473(4):347–364.
The authors report no conflicts of interest related to this 18. Ganesan P, Choi DK. Current application of phytocompound-based
work. nanocosmeceuticals for beauty and skin therapy. Int J Nanomedicine.
2016;11:1987–2007.
19. Jain S, Tiwary AK, Sapra B, Jain NK. Formulation and evaluation
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