Colloids and Surfaces B: Biointerfaces 172 (2018) 323–329

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Colloids and Surfaces B: Biointerfaces

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Hyaluronic acid-containing ethosomes as a potential carrier for transdermal T

drug delivery
⁎ ⁎
Jiesi Xiea,b, Yujie Jia,b, Wei Xuea,b, Dong Maa,b, , Yunfeng Huc,
a
Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China
b
Guangdong Provincial Engineering and Technological Research Center for Drug Carrier Development, Jinan University, Guangzhou 510632, China
c
Department of Dermatology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou 510630, Guangdong, China

A R T I C LE I N FO A B S T R A C T

Keywords: A hyaluronic acid-containing ethosomes (HA-ES) as the transdermal drug delivery system was prepared in this
Transdermal drug delivery work, and rhodamine B (RB) was used as a model drug to be encapsulated. The obtained HA-ES-RB was then
Hyaluronic acid characterized by the surface morphology, entrapment efficiency, drug loading and the stability. Results showed
Ethosomes that the prepared HA-ES-RB was spherical and showed good dispersion as well as the stability, with a particle
size of below 100 nm. The skin permeation experiments were carried out in vitro with the Franz diffusion cells
and the rat dorsal skins were used. It was found that the penetration effect of HA-ES-RB was much better than
that of ES-RB. The fluorescence microscopy image showed that HA-ES-RB penetrated into the deepest dermis.
The excellent transdermic drug delivery effect of HA-ES-RB maybe attributed from its smaller size, hydration of
hyaluronic acid as well as greater potential targeting to skin and skin appendages of liposomal carriers.
Moreover, the HA-ES delivery system showed non-cytotoxicity to normal cells, indicating a good biocompat-
ibility. This work provded a hyaluronic acid-containing ethosomes which can offer a quick, high efficient, safe
and self-administered transdermal drug delivery system.

1. Introduction chemical enhancers, iontophoresis, electroporation, sonophoretic and

Transdermal drug delivery system (TDDS) is a route of adminis-
tration that drugs are applied on the skin surface and continuously
delivered into the body through the skin, followed realizing the topical
superficial or systemic therapy effects. Compared with the traditional
injection and oral administration methods, TDD showed some ad-
vantages as follows: reducing the pain caused by injection, releasing the
medication at a constant rate, avoiding the liver first-pass effect and
preventing drug degradation in the gastrointestinal route et al. [1,2].
Stratum corneum is the outermost layer of the epidermis, which is di-
rectly contact with the external environment and is the most important
protective structure of the skin. Stratum corneum cells have a property
similar to the semipermeable membrane, which resulted into that the
hydrophilic macromolecules are hard to be delivered across the skin
and the transdermal rate cannot meet the treatment requirements.
Therefore, some methods are attempted to increase the efficiency of
transdermal administration: change the structure of the stratum cor-
neum, deliver the medication by external force or modifies/wraps the
medication. In the past several decades, various technologies have been
utilized to improve transdermal or topical drug delivery, including the
magnetophoresis [3–5].
Ethosomes (ES) have been reported as the carriers for TDDS to in-
crease the amount of drug permeation through skin [6,7]. ES showed
the higher capacity of liquidity and deformability as well as the high
encapsulation efficiency. In addition, ES can significantly promote the
drug to penetrate the skin, increase the drug accumulation in the skin
[8]. The ethanol contained in ES can change the tight arrangement of
stratum corneum lipid molecules and enhance the lipid fluidity.
Moreover, the high concentration of ethanol also enhances the flex-
ibility and fluidity of the ES membrane [9]. However, its poor stability
and low entrapment efficiency limit its application in TDD.
Recently, it has been reported that hyaluronic acid (HA) was used as
an effective transdermal delivery carrier [10,11]. HA is a natural
polysaccharide that has been widely used in biomedicine with excellent
biocompatibility, biodegradability and hydrophilicity [12,13]. It is a
significant component of extracellular matrix and is present in the skin
at high concentrations. As the TDDS, HA has the high adhesion to some
biological macromolecular drugs which could not only delay the drug
release but also improve the transdermal absorption ability and tar-
geting [14]. HA can form the hydration film on the surface of the skin to

⁎
Corresponding authors.
E-mail addresses: tmadong@jnu.edu.cn (D. Ma), 103257712@qq.com (Y. Hu).

https://doi.org/10.1016/j.colsurfb.2018.08.061
Received 26 May 2018; Received in revised form 23 August 2018; Accepted 27 August 2018
Available online 29 August 2018
0927-7765/ © 2018 Elsevier B.V. All rights reserved.
J. Xie et al.
increase the moisture content of stratum corneum, thereby changing
the permeability of the skin stratum corneum. In addition, HA showed
the amphiphilic property due to it contained the hydrophobic patch
domain of CH group. Some reports have shown that the structural hy-
drophobic patch domain of HA could make a complex with phospho-
lipids [15]. Therefore, HA can complex with ES to increase the ability of
drugs to penetrate the skin.
In this study, we prepared a HA-ES by using HA to make a complex
with ES to improve the transdermal delivery effect of the drugs. To
enhance the transdermal efficiency of HA-ES, three different mass ratios
of HA to ES were designed. At the same time, rhodamine B (RB) was
used as the model drug for the carrier to facilitate the observation of the
carrier in various representations. Transmission electron microscopy
(TEM) and confocal laser scanning microscopy (CLSM) were used to
characterize the structure and morphology of HA-ES and to determine
whether the drug was encapsulated. The encapsulation efficiency and
drug loading rate were determined by UV–vis spectrophotometer. In
vitro transdermal delivery assay was used to evaluate the permeation of
HA-ES. Finally, the biocompatibility and cytotoxicity of ES were de-
termined by CCK-8 assays.
2. Materials and methods
2.1. Materials
Lecithin (from soybean, > 98%), cholesterol (AR, > 95%) and
Rhodamine B (RB) were purchased from Aladdin (Shanghai, China).
Doxorubicin hydrochloride was obtained from Guangzhou Wandong
Biological Technology Co., Ltd (Guangdong, China). Sodium forms of
HA (Mw = 150 kDa) was bought from Macklin Biochemical Technology
Co., Ltd (Shanghai, China). Dulbecco’s Modified Eagle’s Medium
(DMEM), fetal bovine serum (FBS) and Trypsine-EDTA (0.25%) were
purchased from Gibco (BRL, MD, USA). Cell counting kit-8 (CCK-8) was
purchased from Beyotime Institute of Biotechnology (Shanghai, China).
Ethanol and isopropanol were of analytical reagent grade.
2.2. Preparation of ES-RB, HA-ES-RB and FITC-HA
ES were prepared by the ethanol injection method. Lecithin (2%, w/
v), cholesterol (0.5%, w/v) and RB were mixed into 3 mL of ethanol
solvent and then heated by water bath to make it completely dissolved.
After that, the mixture was added dropwise into 7 mL of phosphate
buffer solution (PBS), and the mixture was stirred for 30 min at 50 °C
and 300 rmp. Finally, the suspension was homogenized and downsized
by ultrasonic dispersion, and then ES-RB was obtained.
HA-ES-RB was prepared by mixing HA and ES with different mass
ratio (ES:HA = 10:1, 5:1 and 2.5:1) in PBS.
FITC-HA was obtained according to the reported method [16].
Subsequently, FITC-HA-ES and FITC-HA-ES-RB were also prepared ac-
cording to the above method.
2.3. Characterization of ES-RB and HA-ES-RB
The particle sizes and zeta potentials of the ES-RB, HA-ES-RB
(ES:HA = 10:1, 5:1 and 2.5:1) and free RB in alcohol were determined
by dynamic laser scattering using a Malvern Zetasizer Nano ZS
(Malvern Instruments, UK), the detection temperature was 25 ℃ and
each sample test was repeated for three times. The physical stability of
ES-RB and HA-ES-RB (ES:HA = 5:1) were investigated by monitoring
the particle sizes during the storage at various time points (1, 2, 3, 4, 5,
6 and 7 days) after the preparation. The ES-RB and HA-ES-RB
(ES:HA = 5:1) from three batches were stored at 4 °C for 7 days. The
surface morphology and particle distributions of the samples were
analyzed by the transmission electron microscopy (TEM). A drop of
samples dispersion was applied to a carbon film-covered copper grid,
and the samples were negatively stained with one drop of 3% aqueous
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Colloids and Surfaces B: Biointerfaces 172 (2018) 323–329
solution of sodium phosphotungstate before the observation.
2.4. Confocal laser scanning microscopy and fluorescence microscope
observation
FITC-HA-ES and FITC-HA-ES-RB were prepared with FITC-labeled
HA. The freshly prepared samples were added to a laser confocal cul-
ture dish and imaged with the FV10i Fluoview confocal microscopy. In
the confocal images, FITC-labeled HA displayed green fluorescence
with excitation/emission wavelengths at 539/570–620 nm. Those
stained with RB presented red fluorescence with excitation/emission
wavelengths at 470/490–540 nm.
2.5. Drug loading
The drug loading amount (DL) and entrapment efficiency (EE) for
HA-ES-RB were studied. The DL refers to the unit weight or unit volume
of the drug loaded by the microspheres [17]. The EE was the percentage
fraction of the total amount of RB found in the studied formulations at
the end of the preparation procedure. To assess the DL and EE, fresh
samples were centrifuged at 8000 rpm for 30 min at 20 °C and then the
supernatant was collected. The supernatant solution was appropriately
diluted and detected the absorbance by UV–vis spectrophotometer at
375 nm. The concentration of the unloaded RB was then calculated
from the standard curve (y = 0.0069 x + 0.0029, R² = 0.999) con-
structed with freshly prepared RB solution. The DL and EE were cal-
culated by the following formula:
Intial amount of drug – drug in the supernatant
EE (%) =
Initial amount of drug
Initial amount of drug – drug in the supernatant
DL (%) =
Weight of the liposomes
2.6. In vitro skin permeation assay
2.6.1. Skin preparation
The hairs of dead rat dorsal surface were removed by a razor and the
full thickness of skin was surgically removed. The dermis side was
wiped with isopropanol to remove the residual adhering subcutaneous
fat. Subsequently, the obtained skin was repeatedly washed with PBS
(pH = 7.4) and wrapped in aluminum foil and stored at - 20 ℃ until
further use.
2.6.2. Cumulative drug delivery
In vitro permeation experiments of HAeESeRB and ESeRB were
carried out through the rat dorsal skin using the Franz diffusion cells
with a permeation area of 1.767 cm2 (Fig. 1). The receptor compart-
ments were filled with PBS (pH = 7.4), which were stirred continuously
at 200 rpm and maintained at the temperature of 37 ℃ by a circulating
water bath. The appropriate size of the skin specimen was sandwiched
between the donor and receptor of the Franz diffusion cell system, with
the stratum corneum facing the formulations [18]. HAeESeRB with
different ES/HA ratio (10:1, 5:1 and 2.5:1) were carried out. ESeRB,
aqueous free RB and free RB in 30% alcohol were used as control
groups. For all formulations, 2 mL of those was added in the donor
compartment above the skin, which was then covered with plastic wrap
to prevent the evaporation. At appropriate interval, 1 mL of the re-
ceptor fluid was withdrawn via the sampling port at and then replaced
by equal volume of fresh buffer. The receptor solutions were analyzed
for drug content by a UV–vis spectrophotometer.
The cumulative drug permeation (Qt) through the skin was calcu-
lated as follows:
t−1
Qt = Vr Ct + ∑ VsCi
i=0
J. Xie et al.
Fig. 1. Scheme of in vitro skin permeation assay using the Franz diffusion cell. (A) Franz diffusion cell.(B)Rat dorsal skin. (C) The subcutaneous tissue of the rat dorsal
skin after the skin surface in contact with formulations directly after 4 h. (D) The skin surface in contact with formulations directly after 4 h.
Where Ct is the drug concentration of the receiver solution at each test
time, Ci is the drug concentration of the ith sample, Vr is the volume of
the transdermal cup, and Vs is the sample volume.
Data were expressed as the cumulative drug permeation per unit of
skin surface area, Qt/S. The steady-state fluxe (Jss, μg/ (cm2 h)) was
calculated as below:
ΔQt
Jss =
Δt × S
Where Δ Qt is the cumulative drug permeation within certain period (Δ
t).
Apparent permeability coefficients (Kp, ×10−3 cm/h) was calcu-
lated according to the equation:
Jss
Kp =
Cd
Where Cd is the active ingredient concentration in the donor com-
partment. It was assumed that under the sink conditions, drug con-
centration in the receiver compartment was negligible compared to that
in the donor compartment.
2.6.3. Penetration depth and distribution of HA-ES-RB in the skin
According to the method described above, 4 ml of the prepared HA-
ES-RB was administered to skin tissues for 4 h using the Franz diffusion
cell. After that, the skin was removed from the Franz diffusion cell and
the surface of skin was washed three times with distilled water to en-
sure that no samples were left on the skin surface. Skin tissues were
dried and sectioned into 1 cm2 pieces, embedded by an optimal cutting
temperature compound (O.T.C compound) and processed in a cryostat.
The frozen skin tissue was sectioned vertically at a thickness of ap-
proximately 10 μm by using a cryostat microtome at -20 ℃. Skin tissue
slices were placed on the glass slides and observed right with a positive
fluorescent microscope equipped with an AxioCam MRc camera. Red
fluorescence (Rhodamine B) was observed with a long-pass 560 nm
emission filter with excitation at 546 nm [19].
2.7. Cytotoxicity assays
Experimental samples included different concentrations of HA-ES,
HA-ES-ADM and ES-ADM (HA-ES, HA-ES-ADM and ES-ADM were re-
spectively added to the serum-free medium at different volume ratios
and mixed well). Cell viability after treatment with experimental
samples was determined using CCK-8 assay. 3T3 cells were collected
and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) media
containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and
100 μg/ml streptomycin within a humidified incubator containing 5%
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Colloids and Surfaces B: Biointerfaces 172 (2018) 323–329
CO2 at 37 °C. 3T3 cells were seeded in 96-well plates at a density of
5 × 103 cells per well in complete DMEM and kept overnight for at-
tachment. The next day the culture medium was replaced with ex-
perimental samples for 24 h incubation. After that, the cells were wa-
shed with PBS to remove the sample residues and CCK-8 was added to
measure the cell viability. The cell viability (%) was defined relative to
untreated control cells.
A−B
Cell viability (%) = ⎛ ⎞ × 100
⎝C−B⎠
Where A, B and C are the absorbance of the tested sample, background
and a fresh control group.
2.8. Statistical analysis
All data were given as the mean ± the standard deviation.
GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was
used to perform statistical analysis (one-way analyses of variance,
ANOVA). The significance level was 0.05, and the data were indicated
with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001.
3. Results and discussion
3.1. Physicochemical characterization
3.1.1. Particle size, zeta potential and stability of the HA-ES-RB
Amphipathic ethosome was prepared by ethanol injection method,
and then thoroughly mixing HA to form the HA-ES-RB. To characterize
its physical properties, particle size and zeta potential were tested
firstly. As shown in Fig. 2A, the particle size of both HA-ES-RB and ES-
RB were significantly smaller than that of free Alcohol-RB, which in-
dicated that ethosomes showed small size with the advantage of pe-
netrating the skin. For zeta potential, it was found that the zeta po-
tential of ES and HA-ES were significantly different, which may due to
the addition of HA that increased the negative charge of ethosome.
Different mass ratios of ES to HA also gave the differences in the par-
ticle size and zeta potential. Results showed that the particle size of HA-
ES-RB increased with the increase of HA content, with the size in-
creased from 593.8 nm to 916.2 nm. This may be attributed to that
more RB wrapped in HA-ES due to the hydrophilic of HA. In addition,
the zeta potential of HA-ES-RB was higher than 10 mV, which was
beneficial to the stability of the carrier. Stability was essential for HA-
ES-RB as a transdermal carrier. The stability of ES-RB and HA-ES-RB
(ES:HA = 5:1) were monitored during a 7 days storage at 4 °C. The
particle size of ES-RB increased obviously with time, while HA-ES-RB
(ES:HA = 5:1) remained basically unchanged (Fig. 2B). This result
J. Xie et al.
Fig. 2. (A) Particle sizes and zeta potentials of alcohol-RB, ES-RB and different mass ratios of HA-ES-RB. The results are expressed as mean ± SD (n = 3). (B) Particle
size of ES-RB and HA-ES-RB (ES:HA = 5:1) during the storage. The results are expressed as mean ± SD (n = 3).
suggested that HA-ES-RB (ES:HA = 5:1) had desirable stability. Com-
pared with free Alcohol-RB and ES-RB, HA-ES-RB was physically stable
with relatively narrow size distributions and negative charge [20].
3.1.2. Drug loading and encapsulation efficiency
As shown in Table 1, different mass ratios of ES to HA resulted in the
difference in the encapsulation efficiency and drug loading rate of HA-
ES-RB. It was found that with the increase of HA content in HA-ES-RB,
the encapsulation efficiency significantly increased from 1.71% to
2.81% and the drug loading rate increased from 11.14% to 22.50%. The
increase of EE% and DL% might be due to the hydrophilic domain of-
fered by HA, which could load more hydrophilic drugs. Data analysis
showed that the particle size of HA-ES-RB increased with the increase of
HA input, which was closely related to the increased of drug loading
and entrapment efficiency.
3.1.3. Morphology
In order to observe the morphology of HA-ES-RB, TEM was per-
formed and the images were shown in Fig. 3. It can be seen that both
ES-RB and HA-ES-RB (ES:HA = 10:1, 5:1 and 2.5:1) formed the sphe-
rical morphology with good dispersion, displaying the sealed vesicular
structure. According to the 200 nm bar in the images, HA-ES-RB was
found larger than ES-RB. This may due to the addition of HA, which was
involved in the assembly of ethosome and increased its particle size.
Moreover, the particle size of HA-ES-RB increased with the increased
HA content, even if the sizes were still relatively small and not much
variation. The image analysis showed that the size of the ES-RB was
about 20 nm, and the sizes of HA-ES-RB with different mass ratio of HA
to ES were below 100 nm. This was not consistent with the average
particle size measured by the dynamic laser scattering. The reason may
be that the size of the nanoparticles measured from TEM was in the dry
state of the copper network, and the Malvern Zetasizer Nano ZS was
performed in the solution. In addition, the increase of HA content in
Table 1
Encapsulation efficiency (EE%) and drug load in rate (DL%) of ES-RB and HA-
ES-RB with different mass ratios.
ES-RB HA-ES-RB HA-ES-RB HA-ES-RB
(ES:HA = 10:1) (ES:HA = 5:1) (ES:HA = 2.5:1)
Encapsulation 2.70 1.71 2.45 2.81
efficiency
(EE%)
Drug loading 16.21 11.14 17.15 22.50
rate
(DL%)
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Colloids and Surfaces B: Biointerfaces 172 (2018) 323–329
HA-ES-RB resulted in the enhancement of hydration effect and the in-
crease of particle size. Therefore, whether in dry state or in aqueous
solution, the particle size of HA-ES-RB increases with the increase of the
HA content, and its hydration effect thereof also increased.
3.1.4. Confocal laser scanning microscope and fluorescence microscope
For CLSM observation, HA-ES-RB was labeled by FITC through
using the FITC-labeled HA. The CLSM results showed that the overlap of
green fluorescence (FITC-HA) and red fluorescence (RB) gave the
yellow spheres, meaning that RB was successfully entrapped within HA-
ES to form the HA-ES-RB (Fig. 4A) [21]. As illustrated by the figure,
FITC-HA-ES showed the small green fluorescent points. After adding
RB, FITC-HA-ES-RB showed a larger green fluorescent sphere. The
larger particle size probably arisen from adding RB, which was wrapped
in HA-ES. Moreover, as shown in Fig. 4B, green spheres can be observed
under fluorescence microscope, indicating that HA indeed participated
in the formation of ethosomes and form the basic structure of HA-ES-
RB.
3.2. In vitro skin penetration
3.2.1. Cumulative drug delivery
Transdermal drug permeation of HAeESeRB across rat dorsal skin
was quantitatively studied using the Franz diffusion cell. Free alcohol-
RB solution and aqueous RB solution were used as the control groups
and PBS as the receptor isotonic solution. As shown in Fig. 5A, over 8 h
exposure, the cumulative drug permeation (Qt, μg) through the skin
increased with time for all formulations. Particularly, after 8 h of pe-
netration, only small amount of RB penetrated through the skin in the
control groups. For the groups of ESeRB and HAeESeRB, both of them
showed good transdermal drug permeation effect. It was found that
both HAeESeRB and ESeRB passed through the skin at the initial
0.5 h, and the cumulative drug permeation of both HAeESeRB and
ESeRB were significantly higher than that of the control groups from
0.5 to 8 h, indicating that HAeESeRB and ESeRB showed a favorable
percutaneous permeability. This may be attributed that the ethosomes
as exogenous lipid bilayers changed the structure of the keratinocytes
after entry into the skin and disrupted the arrangement of the hydro-
phobic tails in the lipid bilayer. Drugs in the ES were diffused into the
cell gap after capillary action [22]. It also can be seen that HAeESeRB
penetration more effectively than ESeRB, which indicated the ad-
vantage of HA in penetration. HA is an ideal moisturizing factor that
can form a hydrated film on the skin surface and increase the moisture
of the stratum corneum. This allowed the keratinocytes swell and then
reduced the denseness of the structure after absorbing a certain amount
J. Xie et al. Colloids and Surfaces B: Biointerfaces 172 (2018) 323–329

Fig. 3. TEM images of ES-RB and HA-ES-RB with different mass ratios.

of moisture, thereby changed the penetration of the stratum corneum of and other characteristics. Obviously, the effective penetration effi-
the skin and promoted the penetration of the drug into the skin. The ciency of the drug increased with the smaller particle size of the carrier
skin permeability of the transdermal drug delivery is mainly affected by and the higher loading of the drug. In the HAeESeRB groups, the
particle size, structure, hydrophilicity, flexibility, drug loading capacity transdermal effect differed with the different mass ratio of ES to HA. For

Fig. 4. (A) CLSM images of FITC-HA-ES, ES-RB and FITC-HA-ES-RB. Red fluorescence corresponds to rhodamine B and green fluorescence to FITC-HA. (B) An
enlargement fluorescence microscope image of the FITC-HA-ES. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article).

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J. Xie et al. Colloids and Surfaces B: Biointerfaces 172 (2018) 323–329

Fig. 5. (A) The cumulative drugs permeation of different formulations through the entire skin of rat. (B) The images of skin tissues after treated with different
formulations for 4 h.

Table 2 RB sample, its penetration effect was poor and RB mainly stayed in the
In vitro percutaneous permeation parameters of different formulations stratum corneum boundary. For both ES-RB and HA-ES-RB samples, RB
(mean ± SD, n = 3). penetrated into the dermis layer and HA-ES-RB showed much wider and
Formulation Jss (μg/ (cm2 h)) Kp (× 10−3 cm/ h) deeper distribution. In addition, the fluorescence intensity of the image
was also quantified using the Image J software. The average fluores-
Alcohol-RB 23.07 ± 0.93 23.07 ± 0.93 cence intensities of HA-ES-RB and ES-RB were 17.3 and 10.1 respec-
ES-RB 129.76 ± 0.99 129.76 ± 0.99
tively. This result indicated that both HA-ES-RB and ES-RB were pe-
HA-ES-RB (ES:HA = 10:1) 156.34 ± 0.94 156.34 ± 0.94
HA-ES-RB (ES:HA = 5:1) 172.37 ± 0.90 172.37 ± 0.90 netrated into the skin through the stratum comeum after 4 h
HA-ES-RB (ES:HA = 2.5:1) 125.91 ± 0.95 125.91 ± 0.95 infiltration, and the penetration effect of HA-ES-RB was significantly
better than ES-RB, indicating the advantage of HA in penetration.
Therefore, it can be inferred that HA could improve the transdermal
HAeESeRB with a mss ratio of 5:1, its steady-state fluxes (Jss, effect of ethosomes. The efficient transdermal delivery of HA-ES was
172.03 ± 0.90 μg/(cm2 h)) was significantly higher than that of orther speculated to depend on the following factors: (1) HA had a moistur-
two mass ratios. HAeESeRB (ES:HA = 5:1) showed a favorable per- izing effect that increased the water content of the skin surface and
cutaneous permeability (Table 2). This may attribute to the increasing enhanced the hydration of the stratum corneum [23]; (2) ethosomes
HA content with higher hydration effect as well as a relative small altered the liposolubility of the stratum corneum and increased the
particle size than HAeESeRB with a ratio of 2.5:1. fluidity of lipid molecules [24].

3.2.2. The penetration distribution in rat skin 3.3. Cytotoxicity

Besides cumulative drug permeation, the penetration depth and
distribution of RB in rat skin were also the significant parameters for
evaluating the penetration effect of the HA-ES system. Fluorescence
microscope photos showed the penetration situation directly. RB, an
effective fluorescence label, was used as the model drug for HA-ES
system. The percutaneous penetration behaviors of HA-ES-RB could be
visualized by penetration depth and distribution situation of fluores-
cence. It was found that all formulations were well distributed in the
skin with high fluorescence intensity as shown in Fig. 5B. For alcohol-
To identify the cytotoxicity of carriers, 3T3 cells were used and all
formulations with different concentrations were incubated with 3T3
cells for 24 h. The cell viability (%) was defined relative to untreated
cells [25]. According to the results shown in Fig. 6, the survival rate of
cells in different concentrations of HAeES was more than 90%, in-
dicating that the carrier has good biocompatibility. To evaluate the real
effect of drug loaded in HAeES, adriamycin (ADM) was loaded to form
the HA-ES-AMD. It was found that after ADM was loaded, a significant

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J. Xie et al.
Fig. 6. In vitro cell viability determination by CCK-8 assay performed on 3T3
cells treated with HA-ES, HA-ES-ADM and ES-ADM in the dark for 24 h (n = 5).
decrease in the cell survival rate was detected. Comparing the data of
the experimental groups of HA-ES-ADM and ES-ADM, it can be seen
that the cell survival rate of the both groups remained close. HA-ES-
ADM and ES-ADM showed the increased cytotoxicity with the increase
of the concentration. This indicates that the carrier could deliver drugs
and the drugs could play effectively.
4. Conclusions
In this study, a HA-containing ethosomes was successfully prepared
and its transdermal administration effects were verified by loading the
model drug of RB. HA is intertwined by chains to form a three-di-
mensional network structure, which participated in the formation of the
ethosomes, thereby improving the stability of the ethosomes. In addi-
tion, the result suggested that HA is an amphiphilic polymer whose
hydrophilic structure allowed HA diffuse into the epidermis through
hydration with the stratum corneum, which interacted with lipids in the
stratum corneum and then destroyed the barrier function of the skin.
The introduction of HA enhanced the penetration efficiency of the drug
obviously. Moreover, the HA-ES delivery system showed non-cyto-
toxicity in this work, indicating a good biocompatibility. This work
suggested that HA-containing ethosomes have great potential for drug
penetrating into the subcutaneous tissue through the stratum corneum.
Acknowledgements
This study was financially supportedby the National Natural Science
Foundation of China (51573071) and the Chinese Fundamental
Research Funds for the Central Universities.
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