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ISSN: 0265-2048 (print), 1464-5246 (electronic)

J Microencapsul, Early Online: 1–9

! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.973072

ORIGINAL PAPER

Development and characterisation of a novel chitosan-coated

antioxidant liposome containing both coenzyme Q10
and alpha-lipoic acid
Guo Dong Zhao1,2, Rui Sun1,2, Shi Lei Ni1,2, and Qiang Xia1,2,3
1
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, PR China, 2Suzhou Key
Journal of Microencapsulation Downloaded from informahealthcare.com by Dokuz Eylul Univ. on 11/06/14

Laboratory of Biomedical Materials and Technology, Suzhou, PR China, and 3Suzhou Nanohealth Biotech. Corp. Limited, Suzhou, PR China

Abstract Keywords
This article describes the physicochemical properties of chitosan-coated liposomes containing Alpha-lipoic acid, antioxidant liposome,
skin-protecting agents, coenzyme Q10 and alpha-lipoic acid (CCAL). CCAL had a spherical shell- chitosan, coenzyme Q10, skin permeation,
core structure and liposomes inverted the surface charge from negative to positive after stability
coating with chitosan. Compared with the uncoated liposome, CCAL had higher zeta potential,
larger droplet size and long-term stability. Fourier transform infrared spectroscopy (FTIR) study History
showed that the driving force for chitosan coating the liposomes was enhanced via hydrogen
bonding and ionic bond force between the chitosan and the alpha-lipoic acid. While the Received 22 January 2014
encapsulation efficiency (EE) of alpha-lipoic acid also increased by interacting with the chitosan Revised 22 July 2014
shell. In vitro antioxidant activity study showed an excellent hydroxyl radical scavenging activity Accepted 8 September 2014
For personal use only.

of CCAL. In vitro release study displayed a sustained drug release, and in vitro penetration Published online 20 October 2014
studies promoted the accumulation of drugs in rabbit skin.

Introduction rapidly taken up by cells and reduced to dihydrolipoic acid

2 (DHLA) intracellularly (Weerakody et al., 2008). ALA and
Skin is the largest organ with an area of approximately 2 m in
DHLA are effectively preventing damages caused by ROS (Bilska
the human body (Gokce et al., 2012), and its main function is
and Wlodek, 2005; Golbidi et al., 2011). Meanwhile, DHLA is
to protect body from pathogens and damage between the internal
able to reduce CoQ10 to ubiquinol, and DHLA also reduces the
and the external environment (Kohen, 1999). However, aging
oxidised form of CoQ10, which can increase the antioxidant
of skin will weaken the protection. Unlike other organs, skin is in
capacity in biomembranes (Smith et al., 2004). In the study of
direct contact with the environment and, therefore, the main rea-
Jiankang Liu, co-administration of ALA with other mitochondrial
son of skin aging is environmental damage, among which
nutrients, such as acetyl-L-carnitine and CoQ10, appears more
the primary factor is UV radiation from sunlight (Fisher et al.,
effective in reducing oxidative mitochondrial dysfunction (Liu,
2002). After the skin absorbs UV light, ultraviolet A (UVA)
2008). Wagner et al. (2012) demonstrated that when supply
initiates the formation of reactive oxygen species (ROS), which
CoQ10 and ALA was combined of with skeletal muscle cells,
can do harm to lipids, proteins and nucleic acid (Matta et al.,
energy homeostasis, stress response and antioxidant defense
2001).
mechanisms might improve (Wagner et al., 2012). Therefore,
Coenzyme Q10 (CoQ10) is an important component in a
complementary combination of CoQ10 along with ALA is a more
variety of cellular processes (Hsu et al., 2003), and it shows great
effective strategy for protecting human skin from the damage by
antioxidant properties by clearing ROS and protecting cells from
UV light. However, CoQ10 and ALA both exhibit poor water
oxidative stress (Yue et al., 2010). As UVA induced skin ageing,
solubility and low stability under UV radiation (Ruktanonchai
CoQ10 is a potential preventive medication against UV radiation
et al., 2009; Zhang and Wang, 2009), and their bioavailability is
(Zhou et al., 2010), in particular, CoQ10 was determined to be
poor due to the barrier function of stratum corneum. Therefore, a
effective against UVA-mediated oxidative stress in human
proper carrier should be carried out to facilitate delivery of drugs
keratinocytes (Rona et al., 2004). Alpha-lipoic acid (ALA) is an
and improve their stability.
important and powerful biological antioxidant that can directly
Several years ago, liposome has been applied as a drug
scavenge free radicals and protect cells from oxidative damage
delivery system to enhance the absorption of drugs into skin
(Scott et al., 1994). In cells containing mitochondria, free ALA is
(Scott et al., 1994; El Maghraby et al., 2008). Furthermore, the
unique ability of liposome to entrap drugs both in an aqueous and
Address for correspondence: Xia Qiang, State Key Laboratory of
in a lipid phase makes such delivery system attractive for both
Bioelectronics, School of Biological Science & Medical Engineering, hydrophilic and hydrophobic drugs (Suntres, 2011). However, due
Southeast University, Nanjing 210096, China. Tel: +86 512 62867117. to the fluid nature of the liposome membrane, common liposome
Fax: +86 512 62867117. E-mail: xiaq@seu.edu.cn has relatively poor physical stability. Chitosan (CH) is a cationic
 2 G. D. Zhao et al.
polymer polysaccharide extensively used in many applications
because of its biocompatibility, biodegradable, fungicidal and
fungistatic properties (Dutta et al., 2004; Rinaudo, 2006).
Numerous studies have shown that CH is able to be a potential
enhancer for carrying drugs across the skin barrier by opening the
tight junctions (Thanou et al., 2001; Brandner et al., 2010;
Kirschner et al., 2011). It was reported that the CH would interact
with the negative charge on the surface of skin to improve drug
diffusion to deeper skin region and enhance physicochemical
stabilities of liposome by forming a polymer layer on the surface
(Li et al., 2011; Park et al., 2014).
In this study, liposome containing both CoQ10 and ALA
coated with CH for enhancing skin delivery was developed. The
characterisation and the physicochemical stabilities of CCAL
were studied. Afterwards, FTIR study, in vitro drug release, skin-
permeation efficiency and skin deposition were evaluated in an
attempt to understand the behaviour of CCAL as a transdermal
Journal of Microencapsulation Downloaded from informahealthcare.com by Dokuz Eylul Univ. on 11/06/14
delivery system. This formulation was developed in order to
improve the application of CoQ10 and ALA in cosmetics.
Material and methods
Materials
Soy phosphatidylcholine (PC) was purchased from Shanghai
Aikang Fine Chemical Co., Ltd. (Shanghai, China). Chitosan
(deacetylation degree 95.0% and molecular weight 50 000), KBr
(Spectrography degree), methanol, ethanol, cholesterol, Tween 80
and glacial acetic acid were purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). CoQ10 was purchased from
Haotian Bioengineering Technology Co., Ltd. (Xi’an, China).
For personal use only.
ALA was purchased from Changshu Fushilai Medicine &
Chemical Co., Ltd. (Changshu, China). All other reagents were
of analytical grade and used without further purification.
Preparation of liposome
Liposome containing both CoQ10 and ALA (CAL) was prepared
by using the ethanol injection method modified from a previous
study (Dua et al., 2012). Accurately weighed quantities of the PC,
cholesterol, Tween 80, CoQ10 and ALA in a mass ratio of
1.5:1.44:0.24:0.96:0.48, i.e. the lipid mixture, was dissolved in
absolute ethanol and incubated at 55  C for 20 min, then lipid
solution of ethanol was rapidly injected to phosphate buffer
(pH ¼ 7.4), followed by stirring at 800 rpm with the temperature
maintained at 55  C for 15 min.
Preparation of CCAL
CH solution was prepared by using appropriate amount of
CH dissolved in a glacial acetic acid solution (0.5%, v/v). For
getting optimal ratios, 5 mL of liposome suspension was added to
10 mL of CH solution (concentrations ranging between 0.1% and
1%, w/v) under controlled stirring rate at room temperature
(25  C), followed by incubation at room temperature for 2 h. The
PC and the CH in a mass ratio range from 15:2 to 15:20. The
dropping and stirring rates were 1 mL/min and 400 rpm,
respectively.
HPLC assay
The loading of CoQ10 remaining in CCAL, CAL and CoQ10
ethanol solution was obtained by High Performance Liquid
Chromatography (HPLC; Perkin-Elmer, LC-200U, Shelton, CT).
The HPLC conditions were as follows: a metering pump, an auto-
injector, a model UV detector and a C18 analytical chromatog-
raphy column (Agilent, Santa Clara, CA; 4.6 mm  250 mm). For
CoQ10 analysis, the mobile phase consisted of 10% methanol and
J Microencapsul, Early Online: 1–9
90% ethanol. The sample injection volume was 20 mL. The mobile
phase was at a flow rate of 1.5 mL/min. The column was
maintained at 35  C in the column oven. The content of CoQ10
was detected by the absorption intensity of wavelength at 275 nm.
A calibration curve (peak area versus drug concentration) was
constructed by running standard CoQ10 solutions in a methanol/
ethanol (1:9, v/v) for each series of chromatograph samples in the
range of 0–0.4 mg/mL and a good linearity was obtained
(r ¼ 0.9991).
A similar process was used to analyze ALA. However, the
mobile phase was replaced with 25% phosphate buffer (pH ¼ 8.0)
and 75% methanol. The mobile phase was at a flow rate of 1 mL/
min with the column maintained at 40  C. The UV wavelength for
detecting ALA was set at 330 nm. The calibration curve had a
good linearity (r ¼ 0.9997) with chromatograph samples in the
range of 0–2 mg/mL.
Determination of vesicle size and zeta potential
Analysis of the vesicle size, polydispersity index value (PDI) and
zeta potential was performed by dynamic light scattering (DLS,
Malvern Zetasizer ZS90, Malvern Instruments, Malvern, UK).
Before vesicle size and PDI measurements, in order to avoid
multiscattering phenomena, the vesicular suspensions were suit-
ably diluted with deionised water. While for zeta potential
analysis, the vesicular suspensions were suitably diluted with
phosphate buffer (pH ¼ 7.4). The dynamic light scattering data
were collected by averaging of three measurements at an angle of
90 in 1 cm diameter cells at 25  C.
pH determination
The pH of sample was determined by a pH meter with the E-201-
C composite electrode (Leici Instrument, PHSJ-3 F, Shanghai,
China) at room temperature.
FTIR spectroscopy
FTIR spectra of lyophilised samples of CAL and CCAL deposited
in KBr disks were recorded on a FT/IR 460 plus spectrometer
(Thermo Nicolet, Nicolet 5700, Thermo Electron Scientific
Instruments Corp., Madison, WI). The scanning was done in the
range 390–4000 cm1 with a speed of 2 mm/s at a resolution of
4 cm1 at room temperature. The peaks were analyzed based on
peak frequencies and domain.
Transmission electron microscopy (TEM) analysis
The micro-morphology of CCAL was observed by TEM (Tecnai
G2 F20 S-Twin, FEI, Hillsboro, OR). The CCAL suspension was
suitably diluted with deionised water and then dropped on small
mesh grids. The excess liquid was tapped with a filter paper. After
drying, the specimen was analyzed with TEM at 200 kV.
EE measurement
The free and encapsulated drugs were separated by ultrafiltration
centrifugal filter tubes with a molecular weight cut-off of 30 kDa
(Millipore, Billerica, MA) (Xiang et al., 2011; Wang et al., 2012).
The free drugs and the sample suspensions were ruptured using a
sufficient volume of ethanol, and the concentration of drugs was
determined using HPLC. Then the EE of the CCAL was
calculated by the following equation:
Total amount of grugs  Free amount of drugs
EE% ¼  100
Total amount of drugs
ð1Þ
 DOI: 10.3109/02652048.2014.973072
Stability studies
Photo-stability study
Photo-stability of the samples was determined upon exposure to
sunlight with a high-level solar UV radiation for 30 d. During this
period, the mean daily temperature was between 28.8  C and
37.4  C. Samples were characterised for their retention ratio at a
definite time by the following equation:
Wt
Retention Rate % ¼ 100
W0
where W0 is the initial drug content and Wt is the drug content
after a definite period.
Storage stability study
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The selected CCAL and CAL were stored at room temperature
(25  C) and 4  C for 60 d, vesicles were withdrawn at a definite
time and characterised for their mean size. The storage stability
was measured by the following equation:
R  R0
K% ¼  100
R0
where K is the vesicle size change rate; R0 is the initial mean size
and R is the mean size after a definite period.
In vitro antioxidant activity study
For personal use only.
The in vitro antioxidant activity of CCAL was studied following
the method described previously (Jin et al., 1996; Amarowicz,
2004). First, CCAL and CAL were diluted for different times (20,
30, and 40) with phosphate buffer (pH ¼ 7.4). The reaction
mixture contained 2 mL phosphate buffer (pH ¼ 7.4), 1 mL water,
1 mL 1,10-phenanthroline solution (0.75 mM), 1 mL FeSO4
(0.75 mM) and 1 mL H2O2 (0.01 %). And then the mixture was
incubated at 37  C for 60 min as the blank solution. A similar
process was carried out to prepare the control solution and the
sample solution. However, in the control solution, 1 mL of water
was added instead of H2O2, and in the sample solution, 1 mL of
sample was added instead of water as compared with the blank
solution. The absorbance value of the blank solution (Ablank), the
control solution (Acontrol) and the sample solution (Asample) was
recorded at 536 nm. The capability of scavenging hydroxyl radical
was computed by the following equation:
Asample  Ablank
Hydrogen Peroxide scavenging activity %¼
Acontrol  Ablank
In vitro release of drugs from CCAL and CAL
In vitro release study of CoQ10 and ALA from CCAL and CAL
was determined by the method modified from a strip technique
(Hsu et al., 2003). About 2 mL of vesicular suspensions were
placed in a dialysis bag (MW: 8000–14 400, BIOSHARP,
Bayonne, NJ). The dialysis bag was immersed in 200 mL of the
release medium (Tween 80:ethanol:phosphate buffer
(pH ¼ 5.8) ¼ 5:20:75, v/v/v), The release medium was maintained
at 37  C in a shaking water-bath (200 rpm). At predetermined time
point, 3 mL of the release medium was withdrawn and immedi-
ately another 3 mL of fresh medium was added. ALA ethanol
solution (ALA-ES) and CoQ10 ethanol solution (CoQ10-ES)
were used as a control group for this study. The concentrations of
CoQ10 and ALA were determined using HPLC.
Chitosan-coated liposomes contain skin-protecting agents 3
In vitro skin permeation study
The in vitro skin permeation study of CCAL was carried out by
the method modified from previous studies (Li et al., 2009; Park
et al., 2014) in order to evaluate the permeation effect of drugs
from CCAL, compared with CAL, CoQ10-ES and ALA-ES. Skin
was obtained from the abdominal skin of a healthy New Zealand
White rabbit. Hair was removed with the help of a shear and
electric shaver. The abdominal skin was washed thoroughly with
the normal saline and subcutaneous fat and bristles were carefully
ð2Þ removed from the skin, then stored at 80  C and used within
1 week. Vertical Franz diffusion cells (RYJ-6B, Huanghai
Medicine & Drug Testing Instruments, China) with a diffusion
area of 2.8 cm2 and a receptor compartment with a volume of
6.5 mL were used for the in vitro skin permeation study. The
rabbit skin was sandwiched between the two halves of the Franz
cell, with a stratum corneum facing the donor compartment. The
receptor compartment consisted of receptor phase (Tween
80:ethanol:phosphate buffer (pH ¼ 5.8) ¼ 5:20:75, v/v/v) and
the apparatus was maintained at 37  C by circulating water
through it. By applying 200 mL of CCAL or CAL suspension on
rabbit’s skin, 100 mL receptor solution was collected at pre-
determined time point and replaced with the same volume of
ð3Þ phosphate-buffered solution. The concentrations of CoQ10 and
ALA were determined using HPLC.
Skin deposition study
At the end of the permeation study (24 h), the rabbit skins were
removed from receptor cells and washed with the phosphate
buffer solution (pH ¼ 5.8) and dried with a filter paper. The skins
were cut into small pieces and homogenised with 5 mL ethanol
and sonicated for 30 min using a uultrasonic sonicator (KQ-
250DE, Kun Shan, Jiangsu, China). Then it was left at 25  C for
2 h. Next, the solution was centrifuged at 10 000rpm for 30 min
and the supernatant was analyzed.
Statistical analysis
All reported data were expressed as means ± standard deviation
(SD, n ¼ 3). Data were subjected to statistical analysis by analysis
of variance (ANOVA) which was used to substantiate statistical
differences between groups, and Student’s t-test was used for
comparison between two samples at the significant level of
p50.05.
Results and discussion
Production of CAL and CCAL
100
Previous studies revealed that the pH of skin ranges from 5 to 6
ð4Þ (Akhtar et al., 2008), and CH is insoluble at physiological pH
(Zhang and Wang, 2009). Results of pH analysis indicate that the
pH of CAL is 5.2 ± 0.1, which is attributed to the hydrogen ion
produced by ALA. Hence, CH could diffuse in the CAL
suspension smoothly. Results of pH study also showed that the
pH of CCAL is 5.4 ± 0.1, which is within the limits of normal skin
pH range.
The vesicle size, PDI, Zeta potential of CAL and CCAL were
measured by DLS shown in Table 1. For the optimisation of CH
ratio, the CAL was coated by varying concentrations of CH (0.1–
1%, w/v). Compared with CAL (mean size 183.4 ± 2.1 nm and
PDI 0.28 ± 0.03.), CCAL had significantly larger sizes and PDI
(Table 1) where the CH concentration was increased from 0.1 to
0.3 (w/v %). When increasing the CH content to 0.5 (w/v %), the
vesicle size was increased to 279.9 ± 2.3 nm with PDI dropping to
0.28 ± 0.04. However, when the CH concentration was increased
up to 0.7 (w/v %) and 1 (w/v %), the PDI and the size of CCAL
 4 G. D. Zhao et al. J Microencapsul, Early Online: 1–9

Table 1. Physicochemical properties of CAL and CCAL. Each data point represents the mean ± SD (n ¼ 3).

Encapsulation efficiency (%)

Formulations CH (w/v%) Vesicles size (nm) PDI Zeta potential (mV) CoQ10 ALA
CAL – 183.4 ± 2.1 0.28 ± 0.03  6.9 ± 0.36 95.40 ± 2.40 86.42 ± 2.30
CCAL-1 0.1% 235.6 ± 2.3 0.34 ± 0.02 16.7 ± 1.07 95.01 ± 0.94 87.17 ± 1.76
CCAL-2 0.2% 243.9 ± 2.6 0.34 ± 0.05 17.2 ± 0.90 97.19 ± 1.21 87.37 ± 1.09
CCAL-3 0.3% 256.2 ± 3.1 0.37 ± 0.03 23.6 ± 0.36 96.41 ± 1.69 87.56 ± 1.70
CCAL-4 0.5% 279.9 ± 2.3 0.28 ± 0.04 29.4 ± 1.21 97.81 ± 1.19 88.61 ± 2.04
CCAL-5 0.7% 302.6 ± 2.2 0.38 ± 0.03 29.3 ± 1.68 96.70 ± 1.86 89.23 ± 1.03
CCAL-6 1.0% 308.0 ± 6.9 0.36 ± 0.06 30.2 ± 0.85 97.56 ± 1.71 91.28 ± 1.50
Journal of Microencapsulation Downloaded from informahealthcare.com by Dokuz Eylul Univ. on 11/06/14
For personal use only.

Figure 1. Mean size and PDI of CCAL-3, CCAL-4, and CCAL-5 in a

period of 30 d under 4  C. Each data point represents the mean ± SD Figure 2. FTIR spectra of ALA, CH, CAL and CCAL.
(n ¼ 3).

concentrations, the surface charge of liposome was increasing

both showed upward trends again (Table 1). In the coating
process, a low concentration of CH was used to coat the liposome
resulting in the coating shell formed to be loose, the uncoated
region of vesicles interacted with CH chains from nearby CAL
through electrostatic force. In this case, the particle distribution in
the CCAL dispersion was not uniform and led to relatively high
PDI. When CH concentration was at 0.5 (w/v %), the CH on the
surface of CAL might have reached a saturation state, hence, the
particle distribution in the CCAL dispersion was uniform and
showed a lower PDI value. Higher CH concentrations form a
thicker layer (Park et al., 2014), hence, the particle size of CCAL
showed significant increase with the increase of CH. When the
concentration was above 0.5 (w/v %), the vesicle size and PDI
increased further again, this might due to redundant CH in the
aqueous phase, whereas there was no notable increase in PDI
between CCAL-5 and CCAL-6. In the literature, when PDI
greater than 0.3 indicates high heterogeneity (Bayat et al., 2008),
the concentration of 0.5 (w/v %) CH might be the proper
concentration for CCAL in theory.
In order to further evaluate the effect of CH concentration on
vesicle size and PDI, CCAL-3, CCAL-4 and CCAL-5 were stored
at 4  C for 30 d. As shown in Figure 1, the vesicle size and PDI of
CCAL-4 had no significant change after 30 d, and the vesicle size
of CCAL-3 remained relatively stable with a high PDI value.
However, the vesicle size of CCAL-5 increased to 334.0 ± 9.7 nm
and PDI reached 0.40 ± 0.03 after 30 d. Those results also
indicated that CCAL-4 might be the optimal formulation.
Table 1 shows that the CAL had a slightly negative
charge (6.9 ± 0.36 mV). After being coated with CH at various
positively as the CH concentration was increased from 0.1 to 0.3
(w/v %) (Table 1), then it came to a relatively constant value
(Table 1). The data also indicated that CH on the liposome surface
might have reached saturation when the concentration was above
0.5 (w/v %). Zeta potential is an important value to investigate
certain physical properties of transdermal delivery vehicles. It was
reported that nanoparticles with a zeta potential above (±) 30 mV
were stable in the suspension (Sonavane et al., 2008), thereby the
stability of nanoparticles can be improved by preventing aggre-
gation and fusion of the particles by high surface charge. The
CCAL-4 had a lower PDI value, more stable vesicle size and
higher zeta potential, therefore, the concentration of 0.5 (w/v %)
was believed to be the optimal CH concentration and CCAL-4
was chosen for further study.
FTIR spectroscopy
FTIR spectra of CCAL and control group are shown in Figure 2.
The absorbance at 3430 cm1 (–OH and –NH stretching)
indicated that there is a considerable change in the peak
intensities between the control group and CCAL. This change
may be attributed to hydrogen bonding between hydroxyl/amino
groups of CH and carboxylic acid groups of ALA. A small
shoulder peak at 1641 cm1 appeared in the spectrum of the
CCAL. This may be due to overlapping of the carboxylic acid
group in ALA with the amide group in CH. These modifications
to the peak intensities were in good coincidence with recent
studies, which shows hat there are interactions between CH and
ALA (Weerakody et al., 2008; Bai and Hu, 2014).
 DOI: 10.3109/02652048.2014.973072 Chitosan-coated liposomes contain skin-protecting agents 5
Journal of Microencapsulation Downloaded from informahealthcare.com by Dokuz Eylul Univ. on 11/06/14

Figure 3. TEM images of CAL (a) and CCAL (b).

Previous studies have revealed that CH could interact with Table 2. The entrapment efficiency of CoQ10 and ALA from CAL and
liposomes with an electrostatic attraction (Li et al., 2011; Park CCAL-4 after stored at different temperatures for 60 d. Each data point
et al., 2014), while by adding ALA to liposomes, the driving force represents the mean ± SD (n ¼ 3).
for CH coating with the liposomes was enhanced. This mechan-
ism may rely on the following factors: ALA as a lipophilic drug is EE at 4  C (%) EE at 25  C (%)
usually dissolved in the lipid phase of liposomes, hence, CH could Formulations 30 d 60 d 30 d 60 d
For personal use only.

interact with the liposome via the hydrogen bonding and ionic
interactions between CH and ALA. CAL
CoQ10 93.42 ± 2.78 80.74 ± 1.42 80.48 ± 4.23 72.36 ± 2.23
ALA 83.27 ± 3.32 71.30 ± 3.81 60.08 ± 3.98 45.82 ± 3.19
TEM CCAL-4
CoQ10 92.69 ± 1.40 90.79 ± 0.49 90.28 ± 1.66 80.74 ± 1.62
Figure 3 shows TEM images of CAL (Figure 3a) and CCAL-4 ALA 84.33 ± 2.34 76.54 ± 2.14 77.89 ± 3.43 60.81 ± 2.76
(Figure 3b). In all cases, spherical-shaped particles were found.
As to the CCAL-4, the presence of polymer layers surrounding
the CAL led to the appearance of larger vesicles.
was effective to inhibit the decomposition and the fusion of
EE measurement CCAL-4 and CAL.
The EE of CAL and CCAL containing both CoQ10 and ALA was
calculated by HPLC. CoQ10 and ALA have poor water solubility Stability studies
(Ruktanonchai et al., 2009), so most drugs should be incorporated
Photo-stability of CCAL
within the phospholipid bilayer and kept at higher EE. As shown
in Table 1, the EE of CoQ10 from CAL was 95.40 ± 2.40% and The drug retention ratios of CCAL-4, CCL (CH-coated CoQ10
the EE of CoQ10 from other CCAL formulations was around liposome) and CAL were studied for evaluating the photo-
97%. The EE of ALA from CAL was 86.42 ± 2.30%, while an stability. ALA-ES and CoQ10-ES were investigated as the
increase in the CH concentration gave rise to an associated control group. Figure 4(a) presents the retention ratios of CCAL-
increase in the EE of ALA from CCAL (Table 1), which might 4, CAL, CCL and control group after 30 d. In Figure 4(a), the
due to the interaction between CH and free ALA in water. CoQ10 from CoQ10-ES was degraded rapidly at 4.57 ± 3.18%
Meanwhile, ALA is a weak acid molecule with amphiphilic after 30 d. While the retention ratio of CoQ10 from CAL and
properties (Femiano and Scully, 2002) which can be encapsulated CCAL-4 had great improvement, especially the CCAL-4 had a
either in the aqueous phase or in the lipid bilayer of liposome. In superior retention ratio at 44.37 ± 2.82%. In contrast, the ALA in
the literature, the EE of hydrophilic drug is usually lower than that ALA-ES was decomposed completely within only 5 d upon
of the hydrophobic drug (Kita and Dittrich, 2011), as a result, the exposure to solar UV radiation (not shown). However, because
EE of ALA was lower than the EE of CoQ10. of the protection of liposome and CH coating, the ALA from
Table 2 shows the EE of CoQ10 and ALA from CAL CAL and CCAL-4 could still be detected after 30 d.
and CCAL-4 after stored at 4  C and 25  C for 60 d, respectively. Furthermore, the ALA from CCAL-4 could remain as high as
After 60 d, the EE of CCAL-4 was only slightly decreased. 26.86 ± 1.98%. These results indicated that CH could protect
Particularly, ALA from CCAL-4 displayed superior EE compared drugs against damage from outside by forming a polymer film
with that of CAL at 25  C, which was in excellent agreement with around vesicles. Figure 4(a) also indicates that the retention ratio
the FTIR study. It was also observed that formulations have higher of CoQ10 from CCAL-4 is higher than that from CCL because
EE when stored at 4  C after 60 d. This is due to hydrolysis and the reducibility of ALA is stronger than CoQ10 (Smith et al.,
oxidation of the lipids at 25  C which consequently induce 2004), in that case, ALA can protect CoQ10 from the damage of
drug leakage and decrease in the EE. Thereby, low temperature UV and pyrolysis.
 6 G. D. Zhao et al.
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For personal use only.
Figure 4. Physicochemical stability of CCAL and CAL stored under
different conditions. (a) Drug retention rate (%) of CoQ10 and ALA from
CCAL-4, CAL, CCL, CoQ10-ES and ALA-ES stored under sunlight for
30 d. (b) Change of the vesicle size of CAL and CCAL-4 after stored at
4  C and 25  C for 60 d, respectively. Each data point represents the
mean ± SD (n ¼ 3).
Figure 5. Hydroxyl radical scavenging activity by different concentra-
tions of CAL or CCAL-4 aqueous dispersion. Each data point represents
the mean ± SD (n ¼ 3).
Storage stability of CCAL
Figure 4(b) presents the mean size of CAL and CCAL-4 after
being stored at 4  C and 25  C for 60 d, respectively. At
refrigerated condition (4  C), both CAL and CCAL-4 remained
stable throughout the entire analysis period with a low K value
J Microencapsul, Early Online: 1–9
Figure 6. In vitro release profiles of drugs from CCAL, CAL, CoQ10-ES
or ALA-ES. (a) The in vitro release profiles of ALA from different
samples. (b) The in vitro release profiles of CoQ10 from different
samples. Each data point represents the mean ± SD (n ¼ 3).
(5.95% and 3.75%, respectively). Those results suggested that low
temperature could significantly improve vesicles stability by
decreasing the fluidity of the membrane and inhibiting the fusion
of CAL and CCAL-4. It was observed in upward trends in mean
sizes of all formulations at 25  C, this was perhaps due to the
fusion of vesicles. However, the K value of CAL (65.75%) was
almost 3-fold in comparison with the K value of CCAL-4
(22.91%) at 25  C. These results suggest that the CH-coated
liposome was more stable than the uncoated liposome, which was
perhaps due to the fact that CCAL exhibited higher zeta potential.
In addition, CH could form a polymer former around the surface
of liposome to prevent oxidation of lipid to improve the stability
of liposome.
In vitro antioxidant activity study
Hydroxyl radical as the most reactive among oxygen radicals can
induce a severe damage to the biomolecules, therefore, hydroxyl
radical scavenging activity is an essential factor to evaluate the
antioxidant activity of CCAL. As shown in Figure 5, both CAL
and CCAL-4 exhibited the hydroxyl radical scavenging activity,
and the addition of CH did not affect hydroxyl radical scavenging
activity of liposome, even a boost effect was observed, though not
to a significant level. For example, the scavenging activity for the
CAL suspension which was diluted 20 times was 59.28 ± 4.28%,
while the scavenging activity for the CCAL-4 suspension which
 DOI: 10.3109/02652048.2014.973072
Journal of Microencapsulation Downloaded from informahealthcare.com by Dokuz Eylul Univ. on 11/06/14
For personal use only.
Figure 7. In vitro skin permeation profiles and the drug deposition rate of different samples. (a) The skin permeation profile of ALA. (b) The skin
permeation profile of CoQ10. (c) The drug deposition rate of different samples in rabbit skin after 24 h. Each data point represents the mean ± SD
(n ¼ 3).
was diluted 20 times was 64.02 ± 3.86%. Which is because amino
or acetamino (CH3CONH–) groups in CH have the capability
against hydroxyl radicals (Yang et al., 2010).
In vitro release of drugs from CCAL and CAL
Because of the poor water-solubility of CoQ10 and ALA, a
phosphate buffer (pH ¼ 5.8) of 20% ethanol (v/v) and 5% Tween
80 (v/v) was used as the medium in the present study. The
temperature was controlled at 37  C and the medium was in a
weak acid condition corresponding to the skin surface condition.
As shown in Figure 6(a), the release of ALA from ALA-ES was
much faster than ALA from CCAL-4 and CAL, and the ALA
had been delivered completely after 8 h. Whereas CAL and
CCAL-4 released only about 41.03% and 15.17% of ALA
contents after 48 h. The strong retardation of ALA from CCAL
release is based on ionic interactions between ALA and CH
(Nishiura et al., 2013). Figure 6(b) presents the release profile of
CoQ10 from CCAL-4, CAL and CoQ10-ES. As expected, the
result showed a rapidly release profile of CoQ10 from CoQ10-
ES. The relatively rapidly release of CoQ10 was observed over
the first 12 h from CAL and CCAL-4. However, the release of
CoQ10 from CAL and CCAL-4 was apparently slowed down
after 12 h, especially for CoQ10 from CCAL-4. The fluidity of
lipid bilayer is a core factor affecting the drug release rate (Hsu
et al., 2003), while CH forms a layer on the surface probably
restricting the bilayer fluidity and the drug release rate. The
Chitosan-coated liposomes contain skin-protecting agents 7
studies of Cheng-Hsuan Hsu et al reported that only 16% CoQ10
was released from nanoparticles after 7 d (Hsu et al., 2003),
which was in good coincidence with the results in this study.
Furthermore, the release profiles in this study also agreed well
with the previous studies that drug release profiles from
liposomes characteristically present an initial fast drug release
followed by slower rates of drug release (Henriksen et al., 1995;
Pavelic et al., 2001).
In vitro skin permeation study and skin deposition study
As shown in Figure 7(a) and (b), all samples can transport the
drug through the skin. While CCAL-4 showed apparent advantage
as a transdermal drug delivery carrier. The highest ALA
accumulation in receptor phase of CCAL-4 was about 2.2-fold
and 8.2-fold in comparison with ALA from CAL and ALA-ES.
While for CoQ10 from CCAL accumulation, the receptor phase
was 1.6-fold and 9.5-fold compared with CoQ10 from CAL and
CoQ10-ES. Meanwhile, we could detect drugs from CCAL-4
receptor phase since second time point (2 h). However, the
corresponding component could only be detected after 4 h from
the CAL receptor phase. Ethanol has been used to enhance drugs
through human skin in vitro, but higher levels of the ethanol
brought down permeation (Megrab et al., 1995), it was concluded
that at higher ethanol levels, dehydration of the biological
membrane reduced the permeation across the skin (Barry, 2002;
Williams and Barry, 2012).
 8 G. D. Zhao et al.
Figure 7(c) shows the residual amounts of ALA in skin from
CCAL-4 that was about 2.0-fold and 2.4-fold compared with that
of CAL and ALA-ES, while the residual amounts of CoQ10 in
skin from CCAL-4 were 1.6-fold and 2.4-fold compared with that
of CAL and CoQ10-ES. These results implied that CH-coating
liposome can improve accumulation of formulations in skin.
There are several mechanisms which can be used to explain
that CCAL enhances transdermal drug delivery. First of all, the
CAL contains Tween 80 and ethanol, hence CAL combines the
advantages of deformable liposome and ethosome: both of them
tend to fluidise membranes and make them very elastic, so that
the resulting vesicle enters more easily into the intercellular lipid
pathway of the stratum corneum layer and delivers the drug into
the deeper layers and through the skin (Elsayed et al., 2006;
Gonzalez-Rodriguez and Rabasco, 2011). The second mechanism
may correlate to hydrated outer environment provided by CCAL,
which might increase hydration of the stratum corneum that could
Journal of Microencapsulation Downloaded from informahealthcare.com by Dokuz Eylul Univ. on 11/06/14
facilitate permeation of drug into the epidermis. CH also could act
as a permeation enhancer that would open the tight junctions of
the cell which increases intercellular space to promote delivery of
drugs (Brandner et al., 2010; Tan et al., 2011). Furthermore, the
surface charge of liposome changes from negative to positive,
while skin surface acts as a negative membrane that could interact
with the CCAL by the electrostatic attraction, and the bioadhesive
of CH may promote CCAL contact with the skin and enhance
drug uptake (Felt et al., 1998; Guo et al., 2003).
Conclusions
CoQ10 and ALA both play an important role in cellular process,
For personal use only.
and combining CoQ10 along with ALA could enhance antioxi-
dant defense mechanisms, but their applications were limited
because of poor water solubility and instability. CH is an excellent
bioadhesive material-coated vesicle because its positive charge
can change the surface charge of liposome and overcome the skin
barrier to improve the delivery of drugs. In this work, CH-coating
liposome containing both CoQ10 and ALA as a potential
transdermal drug delivery system was developed. It was found
that CH could form a polymer shell around the vesicle and
significantly improve the chemical and physical stabilities of
CCAL. The FTIR study showed that the driving force for CH
coating the liposomes was enhanced via hydrogen bonding and
ionic bond force between CH and ALA. CCAL also promotes the
permeation and the accumulation of CoQ10 and ALA evidently in
rabbit skin compared with CAL and controls. Furthermore, CCAL
displays a preferable hydroxyl radical scavenging activity than
CAL. Therefore, we suggest that CCAL could be used as an ideal
transdermal delivery system for the antioxidant defense system to
resist skin aging.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the article. This work was
supported by International Cooperation Project (Grant no.
2008DFB50060) of China.
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