Journal Pre-proofs

Development of curcumin-loaded composite phospholipid ethosomes for en-

hanced skin permeability and vesicle stability

Yu Li, Fei Xu, Xiang Li, Si-Ying Chen, Lin-Yu Huang, Yao-Yao Bian, Jia
Wang, Ye-Ting Shu, Guo-Jun Yan, Jie Dong, Shao-Ping Yin, Wei Gu, Jun
Chen

PII: S0378-5173(20)30921-2
DOI: https://doi.org/10.1016/j.ijpharm.2020.119936
Reference: IJP 119936

To appear in: International Journal of Pharmaceutics

Received Date: 8 June 2020

Revised Date: 23 September 2020
Accepted Date: 27 September 2020

Please cite this article as: Y. Li, F. Xu, X. Li, S-Y. Chen, L-Y. Huang, Y-Y. Bian, J. Wang, Y-T. Shu, G-J. Yan,
J. Dong, S-P. Yin, W. Gu, J. Chen, Development of curcumin-loaded composite phospholipid ethosomes for
enhanced skin permeability and vesicle stability, International Journal of Pharmaceutics (2020), doi: https://
doi.org/10.1016/j.ijpharm.2020.119936

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier B.V.

Development of curcumin-loaded composite phospholipid ethosomes

for enhanced skin permeability and vesicle stability

Yu Li a,b#, Fei Xu a#, Xiang Li a, Si-Ying Chen a, Lin-Yu Huang a, Yao-Yao Biand, Jia

Wange, Ye-Ting Shu a,b, Guo-Jun Yan a,b, Jie Dong a,b, Shao-Ping Yin a,b, Wei Gu a,b*,

Jun Chen a,b,c*

a School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR

China
b Jiangsu Provincial Key Laboratory of Chinese Medicine Processing, Nanjing

University of Chinese Medicine, Nanjing 210023, PR China

c Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia

Medica, Nanjing 210023, PR China

d School of Nursing, Nanjing University of Chinese Medicine, Nanjing 210023, PR

China

e School of Traditional Chinese Medicine，Nanjing University of Chinese Medicine,

Nanjing 210023, PR China

# Yu Li and Fei Xu contributed equally to this work and should be considered co-first

authors.

*Corresponding author

Mail address: School of Pharmacy, Nanjing University of Chinese Medicine, 138

Xianlin Avenue, Nanjing 210023, PR China

Tel.: +86-25-85811611

Fax.: +86-25-85811632

E-mail address: guwei@njucm.edu.cn (W. Gu)

1
E-mail address: chenjun75@163.com (J. Chen)

2
3
 Abstract

Ethosomes are widely applied as the carriers for the transdermal delivery of

hydrophobic and hydrophilic drugs. Herein, curcumin-loaded ethosomes (CE) with

different phospholipid composition were formulated and thoroughly compared. A

significant interaction between the unsaturated phosphatidylcholine (PC) and saturated

hydrogenated phosphatidylcholine (HPC) was found by molecular simulation and

differential scanning calorimetry (DSC), which led to the reduction of PC peroxidation

with the presence of HPC. Subsequently, the composite phospholipid ethosomes

containing curcumin were prepared for the first time to evaluate their properties in

comparison with the conventional ethosomes composed of PC (CE-P) or HPC (CE-H).

CE with PC/HPC ratio of 1:1 (CE-P1H1) with the best vesicle stability and flexibility

significantly decreased the uptake by HaCaT cells compared to CE-H and free

curcumin, indicating reduced skin cell toxicity. Compared with free curcumin, CE-

P1H1 had the highest transdermal efficiency (p<0.001), followed by CE-P (p<0.05),

partly due to the fact that CE-P1H1 could disturb lipid domain of stratum corneum

(SC). Moreover, CE-P1H1 was found to promote curcumin for deep penetration of the

skin via the hair follicles route. Our study has shown that using composite phospholipid

ethosomes as lipid vesicular carriers could enhance transdermal penetration of drugs

and increase in the vesicle stability.

Key word: ethosomes, phosphatidylcholine, hydrogenated phosphatidylcholine,

transdermal, curcumin

4
1. Introduction

Transdermal delivery, an alternative route for systemic delivery of drugs, offers

many advantages as a route for drug administration, including avoidance of first-pass

metabolism, decrease of fluctuations in drug plasma levels, good patient compliance

and the option of immediate termination of the therapy, etc. However, due to the

excellent barrier function of skin, it is difficult for most drugs to partition into and

diffuse through the skin. The skin acts as an effective barrier to protect the human body

against chemicals, microorganisms and other threats. It is well known that the physical

barrier of the skin is mainly located in the stratum corneum (SC), the outermost layer

of the skin (Proksch et al., 2008). The SC is a composite of proteins and lipids in which

protein-rich corneocytes are surrounded by lipid bilayers. The barrier function of the

SC results from the ordered structure and low permeability of these lipid bilayers

(Karande et al., 2006).

Several strategies have been developed to improve the percutaneous absorption,

including incorporation of the drug within flexible liposomal vesicles such as

ethosomes (Ashtikar et al., 2016). Ethosomes can be defined as “the vesicular carriers

consisting of hydro-alcoholic or hydro/alcoholic/glycolic phospholipid with relatively

high alcohol concentration of (20-40%)” (Das et al., 2018). The addition of small-chain

biocompatible alcohols can increase the fluidity of the phospholipid membrane of

liposomes. Ethosomes with flexible membrane is conducive to the deep penetration of

the skin to increase percutaneous absorption. This unique composition makes

ethosomes suitable for the application of transdermal drug delivery system.

Additionally, ethosomes can weaken the skin barrier, thereby enhancing the penetration

of the drug through the skin. Following administration to skin, it was found that

ethosomes could disturb lipid domain of SC, dissolve and extract the intercellular lipid,

5
as well as change the transition temperature of SC (Niu et al., 2019).

It is well-known that phospholipids are essential for the preparation of liposomes and

ethosomes. Most ethosomes have been prepared with phosphatidylcholine (PC), an

unsaturated phospholipid with a phase transition temperature of approximately -20 °C.

However, some ethosomes have been formulated with hydrogenated

phosphatidylcholine (HPC), a saturated phospholipid with a phase transition

temperature of 52 °C (Zhang et al., 2019; Zhao et al., 2013). However, compared to

PC, HPC offered both stronger rigidity and lower flexibility of the phospholipid

membrane, enhancing vesicle stability while theoretically inhibiting the penetration

efficiency of the prepared ethosomes. In our previous studies, it was proved that the

combination of PC and HPC could improve the characteristics of drug-loaded

liposomes, including their drug loading capacity, vesicle stability, anti-tumor activity

and oral bioavailability (Chen et al., 2006; Chen et al., 2010; Chen et al., 2016; Zeng et

al., 2016). The liposomes composed of both PC and HPC were called composite

phospholipid liposomes (Zeng et al., 2016). However, composite phospholipid

ethosomes have not been explored to date. In addition, the mechanism of the interaction

between PC and HPC is still unclear.

Curcumin is a natural polyphenolic compound extracted from the turmeric plant.

As a multifunctional compound, curcumin possesses a variety of pharmacological

activities, including anti-inflammatory, antitumor, antioxidant and anti-virus activities.

Moreover, curcumin can also promote wound healing and protect against pulmonary

toxicity, hepatocellular injury and cataract formation (Mehanny et al., 2016).

Unfortunately, the low aqueous solubility and rapid intestinal and hepatic metabolism

of curcumin severely limits its application via the oral route (Slika & Patra, 2019).

Hence, the transdermal route is a potential alternative for the delivery of curcumin into

6
the systemic circulation. Various vesicles, including ethosomes, have been used to

improve the skin permeation behavior of curcumin (Pathan et al., 2018; Zhang et al.,

2019; Zhao et al., 2013). In the present study, the interaction between PC and HPC was

first investigated by molecular simulation and verified by DSC analysis and anti-

oxidation studies. Then, the novel composite phospholipid ethosomes with different

PC/HPC ratios were prepared for curcumin loading. Using curcumin ethosomes

composed of only PC or HPC as controls, experiments were designed to evaluate their

particle size, zeta potential, encapsulation efficiency, vesicle stability, cellular uptake

and transdermal efficiency. Finally, the penetration enhancement mechanism of the

optimized composite phospholipid ethosomes was further investigated.

2. Materials and methods

2.1. Materials

Phospholipids PC-98T, e.g. phosphatidylcholine (PC, purity>98%), hydrogenated

soybean phosphatidylcholine (HPC, purity>98%) and cholesterol were purchased from

Shanghai A.V.T. Pharmaceutical Co., Ltd (Shanghai, China). Curcumin (purity >98%)

was purchased from Nanjing Spring & Autumn Biological Engineering Company

(Nanjing, China). Acetonitrile and methanol of HPLC-grade were supplied by Tedia

(Fairfield, OH, USA). Milli-Q water was used throughout the experiment. All other

reagents used were commercially available and of analytical grade.

2.2. Animals

Male Sprague-Dawley rats (180~220 g) were obtained from Nanjing Qinglongshan

Laboratory Animal Co. Ltd. (Nanjing, China) with license number SCXK (Jiangsu)

2017-0001. The rats were housed under a 12 h light/12 h dark cycle at 22 ± 2 °C with

a relative humidity of 55 ± 5% and were given free access to food and water. The animal

7
experiments were performed in accordance with the Principles of Laboratory Animal

Care and Use in Research (Ministry of Health, Beijing, China). The protocols of the

animal experiments were approved by the Animals Ethics Committee of Nanjing

University of Chinese Medicine (No. ACU1711102).

2.3. Interaction between PC and HPC

2.3.1. Molecular simulation

The PC and HPC molecules were first sketched using Chemdraw Ultra (Cambridge,

MA), and then, the molecular simulations were performed on Discovery Studio 2.5 (DS

2.5, Accelrys, San Diego, CA), which was used to generate the three-dimensional

structures of these compounds. The ‘CDOCKER’ module of the DS 2.5 software

package was used for semiflexible docking applying a CHARMm (Chemistry at

HARvard Macromolecular Mechanics) force field and for molecular modeling. The

process parameters of the simulation were set as ‘‘Standard Dynamics Cascade’’,

including minimization, minimization 2, heating, equilibration and production. The ten

conformations obtained during docking were used for energy minimization calculations

to determine the most likely conformation, after which the PC-HPC complex could be

formed. The negative interaction energy values suggested that the interaction of the PC

and HPC molecules was stable.

2.3.2 Effect of HPC on the peroxidation of PC

The calculated amounts of phospholipids (PC, HPC or the mixture of PC and HPC

at different weight ratios) were dissolved in chloroform to obtain a PC concentration of

20 mg/mL. The solvent was volatilized under nitrogen flow to yield a film of even

thickness. After storage at 37 °C for 120 h, the films were dissolved in anhydrous

ethanol, and the degree of lipid peroxidation was estimated based on the

malondialdehyde (MDA) concentration according to the modified 2-thiobarbituric acid

8
method (Yang et al., 2018).

2.3.3. Differential scanning calorimetry (DSC) analysis

Using a Diamond DSC apparatus (Perkin Elmer; Waltham, Massachusetts, USA),

DSC was conducted to determine the phase transition temperature (Tm) values of the

PC, HPC and PC-HPC complexes (1:1) in the film. To investigate the interaction

between PC and HPC in the presence of cholesterol, cholesterol was added to the PC,

HPC or PC-HPC mixture (1:1) at a weight ratio of 1:7 before the preparation of the

film.

In brief, 10 mg of the complex powder was placed into aluminum pans, which were

subsequently sealed and analyzed. The samples were scanned from 25 to 200 °C at

5 °C/min heating rate under nitrogen flow.

2.4. HPLC analysis of curcumin

The HPLC system (Shimadzu Corporation, Kyoto, Japan) for analysis was equipped

with two LC-20AT pumps, a SPD-20A UV-VIS detector and a SIL-20A autosampler.

The mobile phase consisted of acetonitrile and 0.4% acetic acid (65:35, v/v). Separation

was carried out at 30 ± 0.1 °C using a reverse-phase C18 column (Kromasil, 5 μm, 4.6

mm × 250 mm, Hanbang Corp., Huaian, China). The detection wavelength was 426 nm

and a flow rate of 1.0 mL/min was employed. A sample volume of 10 μL was injected.

2.5. Preparation and characterization of curcumin-loaded ethosomes

2.5.1. Preparation of ethosomes

Curcumin-loaded ethosomes were prepared by the ethanol injection method. The

compositions of the ethosomes are shown in Table 1. Briefly, curcumin, PC, HPC and

cholesterol were dissolved in 3 mL anhydrous ethanol. Then the ethanol solution was

added slowly into 7 mL phosphate buffered saline (PBS) at pH 6.6 with constant

rotation at 700 rpm for 20 min. It was then ultrasonicated (240 W, 1 min), using a JY92-

9
IIDN probe ultrasonicator (Xinzhi Biotechnology Co., Ltd, Ningbo, China) equipped

with a tapered microtip. Then the curcumin-loaded ethosomes were filtered through a

0.45 μm micropore filter to remove the titanium fragments.

Table 1

Components of curcumin-loaded ethosomes.

Components
Ethosomes
Curcumin PC HPC Cholesterol

CE-P 7 mg 210 mg - 30 mg

CE-P1H3 7 mg 52.5 mg 157.5 mg 30 mg

CE-P1H1 7 mg 105 mg 105 mg 30 mg

CE-P3H1 7 mg 157.5 mg 52.5 mg 30 mg

CE-H 7 mg - 210 mg 30 mg

CE: curcumin-loaded ethosomes.

2.5.2. Particle size and zeta potential

The particle size and particle distribution index (PDI) values of the prepared

ethosomes were measured by dynamic light scattering using the Zetasizer Nano

instrument (Nanozs 90, Marlvern Panalyitcal, UK) and the zeta potential was measured

simultaneously.

2.5.3. Entrapment efficiency

The entrapment efficiency (EE) of the tested curcumin ethosomes was measured by

the ultrafiltration method. After dissolving the ethosomes in methanol after vortexing,

the total drug content of the samples was determined by HPLC analysis. Another

aliquot of the sample was filtered through an ultrafiltration tube (molecular weight cut-

10
off at 100 kDa) at 3000 rpm for 15 min, and the free curcumin content in filtrate was

also determined by HPLC analysis. The EE was calculated according to the following

equation:

EE (%) = (W1-W2)/W1×100%

where W1 is the total amount of curcumin in the tested ethosomes, and W2 is the

amount of free curcumin in the filtrate.

2.5.4. Deformation index

The deformability of vesicles was determined by the extrusion method (Campani et

al., 2016). Briefly, curcumin ethosomes were extruded through a polycarbonate

membrane of 50 nm at a pressure of 1 MPa by a thermobarrel extruder. The

deformability of the ethosomes was expressed as the deformation index (DI) through

the following equation:

DI=J(d0/p)(1/|d1-d0|)

where J is the fraction of the suspension recovered after extrusion, d0 and d1 are the

mean diameters of the ethosomes before the passage through the extruder and after the

extrusion, respectively, and p is the pore size of the extruder membrane.

2.5.5. Vesicle stability

The prepared curcumin-loaded ethosomes were stored at 4 °C. To evaluate the

vesicle stability, the particle size, PDI and zeta potential values were measured after

being stored for 0, 14 and 28 d. In addition, the MDA content of the ethosomes were

also measured.

2.6. Cellular uptake

HaCaT (epidermal keratinocytes) cell lines were obtained from KeyGen Biotech Co.

(Nanjing, China). The cells were incubated in minimum essential medium (MEM

Eagles with Earle’s Balanced Salts) supplemented with 10% heat-inactivated fetal

11
bovine serum and 100 U/mL penicillin/streptomycin in a humidified incubator at 37 °C

and 5% CO2.

The cells were seeded at a density of 106 cells/well in a 6-well plate and incubated

with free or ethosomal curcumin (final concentration, 8 μg/mL) for 8 h at 37 °C. After

incubation, the cells were washed twice using cold PBS and 0.25% trypsin. The cells

were collected by centrifugation, washed with cold PBS, and resuspended in 0.5 mL of

PBS. The fluorescence intensity of the cells was measured by flow cytometry (BD

Accuri C6, Becton Dickinson Company, San Jose, CA, USA) (Zhang et al., 2019).

2.7. In vitro drug release study

The in vitro release behavior of different curcumin ethosomes were estimated

determined using dialysis membranes placed in Franz diffusion cells. The regenerated

cellulose membrane (molecular weight cut-off at 35 kDa) was pre-activated in double

distilled water and clamped between the donor and the receptor chamber with an

effective diffusion area of 3.14 cm2. Ethosomes (0.5 mL) were added into the donor

chamber and normal saline containing 0.5% Tween 80 was selected as release medium

in receptor chamber maintained at 37 ± 0.5 °C and stirred with a magnetic bar at 500

rpm. Samples (0.3 mL) were collected at different time points from the receptor

chamber and then replaced with an equal volume of fresh medium. The concentration

of curcumin in release medium was determined by HPLC

2.8. In vitro skin permeation study

After sacrificing the rats with excess diethyl ether inhalation, the abdominal skin

fragment used for the experiment was excised from rats and the adhering fat and other

tissues were removed. The prepared full thickness skin was subsequently washed with

physiological saline solution three times and stored at -20 °C with the restriction of

usage within two weeks.

12
 The skin was clamped between the donor and the receptor chamber of a Franz

diffusion cell with an effective permeation area of 3.14 cm2 and a receiver cell volume

of 8 mL normal saline containing 0.5% Tween 80 was used as the receptor solution.

The Franz diffusion cell was incubated at 37 ± 0.2 °C using a water bath with a magnetic

stirrer at 500 rpm. Ethosomes with different phospholipid composition (containing 50

μg curcumin) were respectively added onto the surface of skin in the donor chamber

with equivalent free curcumin in 30% ethanol-PBS served as control. The transdermal

receiving solution (0.3 mL) were withdrawn from the receptor chamber at

predetermined time intervals (1, 2, 4, 6, 8, 10, 12 and 24 h) and then replaced with an

equal volume of fresh medium. The curcumin concentrations in receptor fluid samples

were diluted by ethanol and then determined by LS55 fluorescence spectrometer

(Perkin Elmer, Waltham, Massachusetts, USA) with excitation and emission

wavelengths at 430 and 530 nm, respectively.

After sampling at 24 h, the skin surface was wiped with normal saline to remove the

residual ethosome, cut into pieces, and treated with 1 mL methanol under sonication.

The extract was centrifuged at 12000 rpm for 10 min and the supernatant was analyzed.

The cumulative amount of drug permeated through a unit area of skin was plotted

against time. The Steady state flux (ng/cm2/h) values were calculated from the slope of

the linear portion of the plot. To compare the permeation enhancement capacities of

different curcumin-loaded ethosomes, the enhancement ratio (ER) was determined as

follows: ER = (flux of curcumin ethosome)/(flux of free curcumin).

2.9. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-

FTIR) studies

Using 30% ethanol-PBS as control, different curcumin ethosome formulations (CE-

P, CE-P1H1 and CE-H) were applied onto the excised rat skin mounted on Franz Cells

13
as described in 2.7. After 12 h of treatment, the skin samples were dismounted and

frozen at -20 °C. ATR-FTIR studies were performed in a manner similar to the

previously reported method (Campani et al., 2016). On the day of the experiment, skin

samples were thawed and the infrared spectra of skin samples were obtained using FTIR

spectroscopy (FTIR-230 spectrometer, JASCO Co., Tokyo, Japan) with an ATR unit

(ATR-500/M, JASCO Co., Tokyo, Japan). The spectrum recorded represents an

average of 64 scans obtained with a resolution of 2 cm−1 at room temperature. The

spectra were collected in the wave number range of 4000 ~ 650 cm−1. The internal

reflectance element (IRE) used in this study was a zinc selenide trapezoid having 45°

entrance and exit faces. Skin was carefully mounted onto the IRE.

2.10. Fluorescence microscopy imaging

Different ethosome samples (CE-P, CE-P1H1 and CE-H) were applied onto the

excised rat skin mounted on Franz Cells as described in 2.7. After 12 h of treatment,

the skin samples were washed thoroughly and frozen at -80 °C. The treated skin samples

were cut vertically at the length of 6 μm thickness using a cyro-microtome (CM1850,

Lecia Microsystems GmbH, Wetzlar, Germany). The frozen sections were evaluated

using fluorescence microscopy (Axio Vert A1, Carl Zeiss, Jena, Germany) to visualize

the distribution of curcumin. All the procedures were performed in the dark to avoid

the impact of ambient light.

3. Results and discussion

3.1. Interaction between PC and HPC

Molecular simulation was employed to explore the possible interaction between PC

and HPC. For control, the interaction between HPC and HPC as well as PC and PC was

also performed. The minimum energy complexes are shown in Figure 1 to 3. The

14
docking energy values were calculated to be -69.63, -51.04 and -36.58 kcal/mol. The

lower the interaction energy, the more stable the complexes. Therefore, the interaction

force between PC and HPC was obviously stronger compared to that between HPC and

HPC as well as PC and PC.

PC is commonly used as the main phospholipid material in the formulation of

ethosomes. PC extracted from egg yolk has two major unsaturated fatty acids, oleic

acid (18:1) and linoleic acid (18:2). Due to its high degree of unsaturation, peroxidation

of PC can be easily induced. Consequently, MDA is produced by degradation of

polyunsaturated fatty acids (Cui and Decker, 2016). Serving as a common index for

determining the degree of the peroxidation reaction, the production of MDA are shown

in Fig.4. After incubation at 37 °C for 120 h, the most MDA was found in the presence

of pure PC, suggesting the highest peroxidation degree. However, with the addition of

saturated HPC in the mixture, the peroxidation of PC was significantly inhibited when

the HPC composition ratio was higher than 25%. It was demonstrated that the saturated

HPC protected unsaturated PC from peroxidation due to their interaction. This finding

indicates the importance of PC/HPC ratio in the preparation of composite phospholipid

ethosomes.

With the presence of cholesterol, the thermal behavior of PC, PC-HPC (1:1) and HPC

in the state of film was characterized by DSC analysis. As shown in Fig.5, no

endothermic peak was observed for unsaturated PC. In contrast, for saturated HPC,

several endothermic events were found from 53.57 °C to 151.07 °C. However, for the

complex of PC-HPC at 1 to 1 ratio, only one endothermic peak was found with the

single phase transition temperature of 60.49 °C. The disappearance of the endothermic

peaks of HPC could probably be explained by the potent interaction between PC and

HPC.

15
 It is well-known that phospholipid composition has a significant effect on the

characteristics of liposomes. To improve the poor drug payload of the conventional

paclitaxel liposomes, Kan et al. developed a formulation combining both unsaturated

PC and saturated HPC in liposomes, which have significant differences between their

phase transition temperatures (Kan et al., 2011). It was speculated that separate phases,

namely, a gel phase and a liquid-crystal phase, formed in the liposomal bilayer

membrane at room or body temperature. Thus, a combination of two phospholipids,

including PC and HPC, was expected to produce liposomes with the many segregated

microdomains coexisting in the membrane (Kan et al., 2011). However, in the present

study, PC and HPC were found to easily interact with each other, as revealed by

molecular simulation and DSC analysis. The interaction between PC and HPC resulted

in the inhibition of PC peroxidation, which lead to the increased storage stability of the

composite phospholipid ethosomes. Consequently, it is highly possible that PC and

HPC could combine into one phase instead of coexisting in separated phases in the

liposomal membrane.

3.2. Characterization of ethosomes

The particle sizes of curcumin-loaded ethosomes are shown in Table 2. Five

ethosome formulations, including three composite phospholipid ethosomes (CE-P3H1,

CE-P1H1 and CE-P1H3) and two conventional ethosomes (CE-P and CE-H), were

prepared with the variation in the phospholipid composition. The size of curcumin-

loaded ethosomes increased as the HPC ratio increased from 50% to 100%, possibly

due to the fact that the increase of HPC ratio made the vesicular structure less flexible.

However, the increase in size was not significant when HPC ratio was lower than 50%.

Except for CE-H, the PDI data of the other four ethosome formulations was lower than

0.15, indicating a narrow size distribution. The zeta potential of all the curcumin-loaded

16
ethosomes were recorded negative. This may be attributed to the fact that ethanol

possesses the negative charge which contributes to the negative zeta potential of the

system (Pathan et al., 2018). The negatively zeta potential would favorably enhance the

colloidal stability of the formulation. As shown in Table 2, regardless of the

phospholipid composition, the curcumin-loaded ethosomes were produced with EE

values in the range of 93.76%~97.26%. This can be explained by the fact that curcumin

is a lipophilic molecule, so it was easily accommodated in the phospholipid bilayer of

the composite ethosomes.

The deformation index values of CE-P, CE-P1H1 and CE-H were measured and

compared. The deformability of these nanocarriers was found to be in the following

order: CE-P1H3≈CE-H<CE-P<CE-P3H1<CE-P1H1. Due to the high ethanol content

and the moderate phase transition temperature, CE-P1H1 exhibited characteristics of

phospholipid bilayer in fluid state and high membrane elasticity.

Table 2

Characterization of curcumin-loaded ethosomes with different PC to HPC ratios (n=3).

Polydispersity Zeta potential Encapsulated Deformation

Ethosomes Particle size (nm)
index (PDI) (mV) efficiency (%) index

CE-P 133.43±0.25 0.07±0.02 -10.48±1.33 94.00±2.96 0.35±0.03

CE-P3H1 139.27±1.01*** 0.04±0.01 -8.47±1.02 93.76±1.25 0.47±0.07

17
CE-P1H1 132.37±1.53 0.08±0.04 -13.40±0.56* 94.38±2.00 0.66±0.07**

CE-P1H3 171.70±1.15*** 0.11±0.01* -12.20±0.79 96.23±0.84 0.20±0.04**

CE-H 256.70±11.48*** 0.50±0.07*** -8.80±0.23 97.26±1.33 0.26±0.06

CE: curcumin-loaded ethosomes

Significant differences compared to CE-P are indicated by asterisks: *p<0.05, **p<0.01,

***p<0.001.

The results of stability are shown in Fig.6. Significant alterations in particle size

(Fig.6A) and PDI (Fig.6B) were detected in CE-P1H3 and CE-H. In contrast, little

change was observed in the other three formulations, including CE-P, CE-P3H1 and

CE-P1H1. It was obvious that the addition of HPC affected the particle size and size

distribution of curcumin-loaded ethosomes when the HPC ratio was higher than 50%.

With the presence of 30% ethanol, the viscosity of CE-H and CE-P1H3 might be

significantly increased resulting in the remarkable increase in vesicle size and size

distribution during storage.

Ethosomes, which was proposed by Touitou et al., have been proven to enhance drug

permeation through the skin due to the presence of high concentration of ethanol.

Instead of damaging the structure of the bilayer, the inclusion of ethanol enhances the

fluidity and flexibility of the ethosomes composed of PC (Touitou et al., 2000).

However, the addition of ethanol at high concentration (>20%) might change the

solubility and hydrophobic interaction of HPC which possesses higher hydrophobicity

than PC. The ethanol molecules can displace some of the water molecules associated

to the interfacial region bilayer and penetrate to some extent into the bilayer membrane

of HPC ethosomes. As a result, the HPC hydrophobic tails are partially tilted to form

the inner part of the membrane to the solvent phase, leading to the interdigitated phase

18
(Castangia et al., 2015; Manca et al., 2014). Therefore, when the HPC ratio was higher

than 75%, ethanol could induce the formation of interdigitated vesicle structure,

resulting in the significant increase in the particle size and size distribution during

storage.

Zeta potential of all the ethosomes has shown slight changes on storage for 28 d

(Fig.6C). As a commonly used index for determining the degree of the peroxidation

reaction, the production of MDA are shown in Fig.6D. The addition of HPC can inhibit

the peroxidation of PC in ethosome formulations. In summary, based on the stability

results, CE-P1H1 was demonstrated to possess the best vesicle stability among all the

tested formulations, including physical stability (particle size and size distribution) and

chemical stability (anti-peroxidation effect).

It should be noted that the obtained curcumin-loaded composite ethosome, e.g. CE-

P1H1, possessed higher EE values (>93%) and better vesicle stability compared with

some of the existing curcumin nanocarriers. Curcumin-loaded ethosome vesicle

without surface modification presented a lower EE value (about 80%) and the vesicle

size increased with the extension of time in 15 days (Zhang et al., 2019). Other lipid

based formulations of curcumin such as liposome (Manca et al., 2015) and lipid-

hybridized cellulose nanofiber film (Kang et al., 2018) were also showed lower EE of

approximately 60%.

3.3. Cellular uptake

Using flow cytometry analysis, cell-associated fluorescence was assessed to evaluate

intracellular delivery of curcumin-loaded ethosomes to the HaCaT cells. As seen in

Fig.7, the fluorescence intensity of the blank cells was much lower than that of the cells

incubated with curcumin-loaded ethosomes, indicating negligible background

fluorescence. The fluorescent intensity of the free curcumin and CE-H treated HaCaT

19
cells was much higher than that of the composite phospholipid ethosomes (CE-P3H1,

CE-P1H1 and CE-P1H3) and CE-P treated cells within the test period. This suggested

that the composite phospholipid ethosomes mainly pass through the intercellular space

rather than intracellular compartments during the active SC transdermal process (Niu

et al., 2019).

Based on the results of ethosome characterization and cellular uptake, the optimized

composite phospholipid ethosomes containing curcumin were determined to be CE-

P1H1 which was composed of PC and HPC at the weight ratio of 1 to 1. Moreover,

since curcumin has been confirmed to inhibit the proliferation of HaCaT cells and

induce apoptosis, the topical skin toxicity of CE-P1H1 was expected to be reduced

(Zhang et al., 2019).

3.4. In vitro drug release study

The release behavior of curcumin from different ethosomes was examined over 24 h

(Fig. 8). The accumulative release ratio of curcumin from CE-P was remarkably higher

than that of CE-P1H1 and CE-H in 24h (p < 0.001), and a sustained release of curcumin

is desirable for in vivo administration. It was well known that the phase transition

temperature of PC is lower than 0 °C. As shown in Fig.5, no obvious phase transition

was found from 20 to 200 °C. However, the mixture of PC and HPC (1:1) as well as

HPC possessed higher phase transition temperature. The reduced release rate of CE-

P1H1 and CE-H was probably due to the higher rigidity of lipid bilayer membrane

compared with CE-P.

3.5. In vitro skin permeation study

In vitro skin permeation for the different curcumin-loaded ethosomes was studied in

comparison with free curcumin through full thickness rat skin. The permeation profiles

were generated by plotting amount of drug permeated through unit area of skin against

20
time. As shown in Fig.9 and Table 3, there was significant difference among different

curcumin-loaded ethosomes. Compared with free curcumin, the composite

phospholipid ethosomes containing curcumin (CE-P1H1) had the highest transdermal

efficiency (p<0.001), followed with CE-P (p<0.05). However, for the ethosomes

containing curcumin (CE-H) composed of saturated HPC, the transdermal delivery

efficiency was slightly decreased compared with free curcumin. Larger vesicle size and

less deformability might reduce the penetration efficiency for CE-H compared to CE-

P1H1 and CE-P.

Table 3

Percutaneous permeation parameters of different curcumin-loaded ethosomes through

excised rat skin (n=5).

Flux
Formulations Enhancement ratio (ER)
(ng/cm2/h)

Free curcumin 8.88±0.93 1.00

CE-P 10.18±0.83* 1.15
CE-P1H1 19.54±4.14*** 2.20
CE-H 8.30±2.67 0.93
*p<0.05, **p<0.01,***p<0.001 vs. the free curcumin group.

Since curcumin is hardly dissolved in normal saline, surfactant was added in the

receptor fluid to ensure sink condition during skin permeation studies. With the addition

of 0.5% Tween 80 in normal saline, the solubility values of curcumin increased from

0.12 μg/mL to 72.23 μg/mL. The addition of both 0.5% Tween 80 and 20% ethanol

could further increase the solubility of curcumin (Chen et al., 2012). Ethanol is

considered as an effective permeation enhancer. It can increase the SC hydration, the

21
alteration of proteinaceous corneocyte components, the disturbance or extraction of

intercellular lipid arrangement and so on (Zhao et al., 2013). Thus the ethanol was not

added in receptor fluid since it can significantly facilitate the penetration of curcumin

and its formulations.

3.6. ATR-FTIR studies

ATR-FTIR has already been proved to be a promising tool for non-destructive

characterizing SC at the molecular level. To elucidate the effect of ethosomes on the

intercellular lipids in the SC, ATR-FTIR stretching peaks near 2850 cm-1 (C-H

symmetric stretching absorbance frequency peak) and 2920 cm-1 (C-H asymmetric

stretching absorbance frequency peak) were measured after the application of

ethosomes with different phospholipid compositions to skin (Table 4). It should be

noted that the intercellular spaces of the SC are the preferential pathway for lipophilic

curcumin to penetrate through the SC. The shift of a higher frequency occurs when the

CH2 groups along the alkyl chain of lipids change from trans to gauche conformation,

suggesting that the SC lipid is disturbed (Zhang et al., 2007). The magnitude of blue

shift in the peak position of the asymmetric and symmetric stretching vibration

absorbance is correlated with an increase number of gauche conformers in the lipid acyl

chain. After the skin was treated by ethosomes, obvious shifts were observed to CH2

symmetric and asymmetric stretching frequencies which were approximately 2.31 to

3.05 cm-1 higher than those observed with solvent alone (control). The shift in the peak

position of lipid compared with control group followed a decreasing trend as CE-P1H1

> CE-P> CE-H, indicating the increase in the fluidity of the SC lipids with the

composite phospholipid ethosomes which could facilitate the skin permeation of

curcumin.

22
 Table 4

Peak positions of SC lipids after treated with different ethosomes (n = 3).

Peak position of lipid (cm-1) The shift of peak position (Δcm-1) a

Asymmetric Symmetric
Formulations Asymmetric Symmetric Total of the
C-H C-H
C-H stretching C-H stretching increase
stretching stretching

30% ethanol-PBS 0 0 0
2922.08±0.79 2853.12±0.56
CE-P 2.89 0.16 3.05
2924.97±3.43 2853.28±0.56
CE-P1H1 3.61 1.09 4.70
2925.69±1.83* 2854.21±0.46*
CE-H 2.21 0.10 2.31
2924.29±4.25 2853.22±2.30
a Shift in peak position compared to the untreated skin treated (Blank).

* p<0.05 vs the 30% ethanol-PBS group.

3.6. Fluorescence microscopy imaging

Curcumin is a hydrophobic fluorescent dye that can be used as a probing compound.

For the effective transdermal delivery of curcumin, higher drug penetration into the

deeper layers of the skin following ethosome administration is essential. Fluorescence

microscopy images of rat skin after 12 h of treatment with CE-P, CE-P1H1 and CE-H

are shown in Fig. 10. The CE-P showed fluorescence was confined only to the

superficial SC layer. In the case of CE-P1H1, the significant increase in the

fluorescence intensity in the deeper layers of skin indicates the effective delivery of the

composite phospholipid ethosomes to these deeper layers of the skin. In contrast to CE-

P and CE-P1H1, very weak fluorescence was observed in the SC after treatment with

CE-H. The skin irritation study was also performed. As shown in Fig.S1, compared

with control, all of the three curcumin-loaded ethosomes demonstrated negligible

changes in rat skin specimens after administration for 8 h, suggesting no stimulation to

skin.
23
 The morphological changes of skin specimen after treated with CE-P, CE-P1H1, CE-

H and saline are shown in Fig.S1. Compared with control,

The percutaneous pathways of ethosomes included open hair follicles and SC

pathways (Yang et al., 2017). Following dermal application of CE-P, the vesicles might

break up during percutaneous penetration in the superficial layer of the skin, resulting

in the retention of curcumin mainly in the epidermis although it could permeate slowly

into the deeper layer. However, after the administration of CE-P1H1, the significant

distribution of curcumin into hair follicles, especially hair root, was observed.

Therefore, the hair follicles route might be one of the main permeation pathways for

the transdermal delivery of composite phospholipid ethosomes.

4. Conclusions

The objective of this research was to develop the novel composite phospholipid

ethosomes formulation that can possess both the improved percutaneous permeability

and vesicle stability compared to the conventional ethosomes composed of only PC or

HPC. The results of molecular simulation and DSC analysis revealed the significant

interaction between PC and HPC molecules, which might account for the reduced

peroxidation of PC with the presence of HPC. Then the composite phospholipid

ethosomes containing curcumin with different phospholipid compositions (PC to HPC

ratio) were prepared and the optimal ratio was determined to be 1to 1 (PC to HPC ratio)

based on the evaluation of vesicle properties. The results of the skin permeation studies

revealed that the composite phospholipid ethosomes (CE-P1H1) can deliver more

curcumin across the skin when compared with the conventional ethosomes composed

of PC or HPC (CE-P or CE-H). In addition, CE-P1H1 could disturb lipid domain of SC

and allow curcumin to penetrate easily into deeper layers of the skin. Interestingly, the

24
hair follicles route was found to be one of the main permeation pathways for the

transdermal delivery of composite phospholipid ethosomes. Therefore, these findings

suggest that the composite phospholipid ethosomes can be successfully exploited as a

potent vesicle for transdermal drug delivery.

Acknowledgements

This research was funded by the Project of the Priority Academic Program

Development of Jiangsu Higher Education Institutions (2019YSHL077) and the Open

Project Program of Jiangsu Key Laboratory for Pharmacology and Safety Evaluation

of Chinese Materia Medica (No. JKLPSE201808).

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1

References:

Ashtikar, M., Nagarsekar, K., Fahr, A., 2016. Transdermal delivery form liposomal

formulations-evolution of the technology over the last three decades. J. Control.

Release. 242, 126-140.

Campani, V., Biondi, M., Mayol, L., Cilurzo, F., Franzé, S., Pitaro, M., De Rosa, G.,

2016. Nanocarriers to enhance the accumulation of vitamin K1 into the skin. Pharm.

Res. 33, 893-908.

Castangia, I., Menca, M.L., Matricardi, P., Catalán-Latorre, A., Nácher, A., Diez-Sales,

O., Fernàndez-Busquets, X., Fadda, A.M., Manconi, M., 2015. Effects of ethanol and

diclofenac on the organization of hydrogenated phosphatidylcholine bilayer vesicles

25
 and their ability as skin carriers. J. Mater. Sci: Mater. Med. 26, 137.

Chen, J., Ping, Q.N., Guo, J.X., Chu, X.Z., Song, M.M., 2006. Effect of phospholipid

composition on characterization of liposomes containing 9-nitrocamptothecin. Drug

Dev. Ind. Pharm. 32, 719-726.

Chen, J., Lin, A.H., Chen, Z.P., Wang, W., Zhang, T., Cai, H., Cai, B.C., 2010.

Ammonium sulfate gradient loading of brucine into liposomes: effect of

phospholipid composition on entrapment effiency and physicochemical properties in

vitro. Drug Dev. Ind. Pharm. 36, 245-253.

Chen, J., Lin, A.H., Peng, P., Wang, Y., Gu, W., Liu, Y.M., 2016. Lipid composition

has significant effect on targeted drug delivery properties of NGR-modified

liposomes. Drug Deliv. 23, 1426-1433.

Chen, Y., Wu, Q., Zhang, Z., Xuan, L., Liu, X., Zhou, L., 2012. Preparation of

curcumin-loaded liposomes and evaluation of their skin permeation and

pharmacodynamics. Molecules. 17, 5972-5987.

Cui, L., Decker, E.A., 2016. Phospholipids in foods: prooxidants or antioxidants? J.

Sci. Food Agric. 96, 18-31.

Das, S.K., Chakraborty, S., Roy, C., Rajabalaya, R., Mohaimin, A.W., Khanam, J.,

Nanda, A., David, S.R., 2018. Ethosomes as novel vesicular carrier: An overview of

the principle, preparation and its applications. Curr Drug Deliv. 15, 795-817.

Kan, P., Tsao, C.W., Wang, A.J., Su, W.C., Liang, H.F., 2011. A liposomal formulation

able to incorporate a high content of paclitaxel and exert promising anticancer effect.

J. Drug Deliv. 2011:629234.

Kang, N., Kim, M.H., Sohn, S., Kim, K.T., Park, J.H., Lee, S.Y., Lee, J.Y., Kim, D.D.,

2018. Curcumin-loaded lipid-hybridized cellulose nanofiber film ameliorates

imiquimod-induced psoriasis-like dermatitis in mice[J]. Biomaterials. 182, 245-258.

26
Karande, P., Jain, A., Mitragotri, S., 2006. Relationships between skin’s electrical

impedance and permeability in the presence of chemical enhancers. J. Control.

Release. 110, 307-313.

Manca, M.L., Castangia, I., Matricardi, P., Lampis, S., Fernàndez-Busquets, X., Fadda,

A.M., Manconi, M., 2014. Molecular arrangements and interconnected bilayer

formation induced by alcohol or polyalcohol in phospholipid vesicles. Colloids Surf.

B Biointerfaces. 117, 360-367.

Manca, M.L., Castangia, I., Zaru, M., Nácher, A., Valenti, D., Fernàndez-Busquets, X.,

Fadda, A.M., Mancon, M., 2015. Development of curcumin loaded sodium

hyaluronate immobilized vesicles (hyalurosomes) and their potential on skin

inflammation and wound restoring[J]. Biomaterials. 71:100-109.

Mehanny, M., Hathout, R.M., Geneidi, A., Mansour, S., 2016. Exploring the use of

nanocarrier systems to deliver the magical molecule: Curcumin and its derivatives.

J. Control. Release. 225, 1-30.

Niu, X.P., Zhang, D.P., Bian, Q, Feng, X.F., Li, H., Rao, Y.F., Shen, Y.M., Geng, F.N.,

Yuan, A.R., Ying, X.Y., Gao, J.Q., 2019. Mechanism investigation of ethosomes

transdermal permeation. Int. J. Pharm. Doi: https// doi.org/10.1016/j.ijpx.2019.

100027.

Pathan, I.B., Jwware, B.P., Shelke, S., Ambekar, W., 2018. Curcumin loaded

ethosomes for transdermal application: Formulation, optimization, in-vitro and in-

vivo study. J. Drug Deliv. Sci. Tec. 44, 49-57.

Proksch, E., Brandner, J.M., Jensen, J., 2008. The skin: An indispensable barrier. Exp.

Dermatol. 17, 1063-1072.

Slika, L., Patra, D., 2019. A short review on chemical properties, stability and nano-

technological advances for curcumin delivery. Expert Opin. Drug Deliv. Doi:
27
 10.1080/174252147.2020. 1702644.

Touitou, E., Dayan, N., Bergelson, L., Godin, B., Eliaz, M., 2000. Ethosomes-novel

vesicular carriers for enhanced delivery: characterization and skin penetration

properties. J. Control. Release. 65, 403-418.

Yang, L., Wu, L., Wu, D., Shi, D., Wang, T., Zhu, X., 2017. Mechanism of transdermal

permeation promotion of lipophilic drugs by ethosomes. Int. J. Nanomed. 12, 3357-

3364.

Yang, Y., Guo, Y., Tan, X., He, H., Zhang, Y., Yin, T., Xu, H., Tang, X., 2018.

Vincristine-loaded liposomes prepared by ion-paring techniques: Effect of lipid, pH

and antioxidant on chemical stability. Eur. J. Pharm. Sci. 111, 104-112.

Zeng, C., Jiang, W., Tan, M., Yang, X., He, C., Huang, W., Xing, J., 2016.

Optimization of the process variables of tilianin-loaded composite phospholipid

liposomes based on response surface-central composite design and pharmacokinetic

study. Eur. J. Pharm. Sci. 85, 123-131.

Zhang, C.F., Yang, Z.L., Luo, J.B., Zhu, Q.H., Zhao, H.N., 2007. Effects of cinnamene
enhancers on transdermal delivery of ligustrazine hydrochloride. Eur. J. Pharm.
Biopharm. 67, 413-419.
Zhang, Y., Xia, Q., Li, Y., He, Z., Li, Z., Guo, T., Wu, Z., Feng, N., 2019. CD44 assists

the topical anti-psoriatic efficacy of curcumin-loaded hyaluronan-modified

ethosomes: A new strategy for clustering drug in inflammatory skin. Thernostics. 9,

48-64.

Zhao, Y.Z., Lu, C.T., Zhang, Y., Xiao, J., Zhao, Y.P., Tian, J.L., Xu, Y.Y., Feng, Z.G.,

Xu, C.Y., 2013. Selection of high efficient transdermal lipid vesicle for curcumin

skin delivery. 454, 302-309.

28
Figure caption:

Fig. 1. The binding mode between PC and HPC. Red means negatively charged, and

blue means positively charged.

Fig. 2. The binding mode between HPC and HPC. Red means negatively charged, and

blue means positively charged.

Fig. 3. The binding mode between PC and PC. Red means negatively charged, and

blue means positively charged.

Fig. 4. The malondialdehyde (MDA) values of PC with the presence of different ratios

of HPC (n=3). Significant differences compared to PC are indicated by asterisks:

*p<0.05, **p<0.01, ***p<0.001.

Fig. 5. DSC thermograms of PC, PC-HPC (1:1) and HPC in the state of film with the

presence of cholesterol.

Fig. 6. Stability (A, particle size; B, PDI; C, zeta potential; D, MDA) of curcumin-

loaded ethosomes with different PC to HPC ratios (n=3). A, B, C: Significant

differences compared to the corresponding group at 0 d are indicated by asterisks:

*p<0.05, **p<0.01, ***p<0.001. D: Significant differences compared to the CE-P at

the same time are indicated by asterisks: *p<0.05, **p<0.01, ***p<0.001.

Fig. 7. The flow cytometric measurement of curcumin uptake from composite

phospholipid ethosomes (CE-P1H1) and conventional ethosomes (CE-P and CE-H) by

HaCaT cells at 8 h.

29
Fig. 8. The release profiles of curcumin from different ethosomes (n = 3).

Fig. 9. In vitro skin permeation profiles of different curcumin-loaded ethosomes (n=5).

Fig. 10. Fluorescence microscopy images of samples (cross-sections) treated with

different curcumin-loaded ethosomes for 12 h.

30
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

31
32
33
34
35
 Credit Author Statement

The detailed contributions of authors to this paper is accurately described in the

following text with permission of all the authors.

Yu Li: Conceptualization, Methodology, Software, Writing - Original Draft.

Fei Xu: Conceptualization, Methodology, Data curation.

Xiang Li: Methodology, Validation.

Si-Ying Chen: Methodology, Data Curation.

Lin-Yu Huang: Resources, Investigation.

Yao-Yao Bian: Software, Investigation.

Jia Wang: Methodology, Formal analysis.

Ye-Ting Shu: Software, Data Curation.

Guo-Jun Yan: Formal analysis.

Jie Dong: Supervision, Data Curation.

Shao-Ping Yin: Software, Data Curation, Writing-Reviewing and Editing.

Wei Gu: Conceptualization, Data Curation, Supervision, Funding acquisition,

Validation.

Jun Chen: Conceptualization, Data Curation, Writing-Reviewing and Editing,

Project administration, Funding acquisition.

36
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

37