Colloids and Surfaces A: Physicochem. Eng.

Aspects 444 (2014) 54–62

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Preparation, characterization and evaluation of bufalin liposomes

coated with citrus pectin
Ying Li a , Hang Zhao a , Lin-Rui Duan a , Hua Li a , Qian Yang a , Hong-Hai Tu b ,
Wei Cao a,∗ , Si-Wang Wang a,∗∗
a
Department of Natural Medicine & Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, 169 West Changle Road, Xi’an
710032, China
b
Institute for Drug and Instrument Control, Xinjiang Military Area Command, Urumqi, Xinjiang, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• A pectin-coated liposomal formula-

tions was prepared by forming an
ion-complex.
• The pectin-coated liposome has an
excellent stability and mucoadhe-
siveness properties.
• The pectin-coated liposome would be
a promising drug carrier system for
colon cancer.

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to investigate the potential effect of pectin for liposomal drug delivery systems.
Received 16 June 2013 An orthogonal L9 (33 ) test was designed to optimize the preparation condition of cationic bufalin lipo-
Received in revised form somes coated with commercially available citrus pectin (CPL). The change in particle size, zeta potential,
25 November 2013
entrapment efficiency, stability, mucoadhesion and anticancer effect were evaluated. The results showed
Accepted 7 December 2013
Available online 31 December 2013
that CPL had an excellent stability and mucoadhesive properties, and the drug release in vitro was modest
prolonged and sustained. Furthermore, the inhibition effect of liposomes on SW480 colon cancer cells
was dramatic enhanced due to a block of cell cycle at G0/G1 phase, and CPL had a higher inhibition rate
Keywords:
Citrus pectin than bufalin liposomes (BFL) because of the anticancer effect of citrus pectin. It is concluded that CPL is
Liposomes a potentially promising drug carrier system treatment for colon cancer.
Bufalin © 2013 Elsevier B.V. All rights reserved.
Colon cancer

1. Introduction recurrence is high. Chemotherapy remains the primary cure or con-

Colon cancer is one of the most prevalent cancers throughout
the world, accounting for over 1 million new cases and about half
a million deaths every year [1]. Currently, the effective cure rate
of colon cancer has not been achieved with surgery, and the risk of
∗ Corresponding author. Tel.: +86 29 84773752; fax: +86 29 8322 4790.
∗∗ Corresponding author. Tel.: +86 29 84774748; fax: +86 29 8322 4790.
E-mail addresses: caowei@fmmu.edu.cn, 776472382@qq.com (W. Cao),
wangsiw@fmmu.edu.cn (S.-W. Wang).
trol strategy for patients in colon cancer phase III [2,3]. Therefore, it
is important to develop novel potent chemotherapeutic agents for
the treatment of colon cancer [4].
One of such chemotherapeutic agent is bufalin, which is puri-
fied from Chinese traditional medicine Chan Su [5]. Some recent
researches have revealed that bufalin exhibits significant activity
against a wide variety of malignancies, including gastric cancer,
prostate cancer, ovarian cancer and colon cancer [6]. However, it is
still somewhat controversial that the health benefits of oral admin-
istration of bufalin are often overshadowed by its drawbacks such
as high toxicity [7], poor water solubility [8], short half-life, narrow

0927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfa.2013.12.006
 Y. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 54–62
therapeutic window and the little difference between the effec-
tive dose and the toxic dose. Therefore, other substitutive agents
with the same positive effect and fewer side-effects are needed to
explore alternative treatment strategies.
In the past few decades, nano-scale therapeutic systems have
become novel therapeutic modalities for combating cancers [9].
Liposomal drugs are agents which are first approved and widely
used in the pharmaceutical field since it has many advantages
including the affinity to biological membrane and the ability
to improve the unfavorable physicochemical properties of drugs
[10–12]. However, the disadvantages of liposomal drugs such as
the aggregation during storage, fusion of the membranes, loss of
encapsulated material, hydrolysis and oxidation of the lipids show a
balance number of risks and benefits [13–15]. In recent years, these
potential drawbacks have stimulated the research of alternatives
to the classical liposomal drugs. More recently, some studies have
been reported on stabilize liposomes by using substrates, including
silicon particles [16]; gold nanocages [17] and polyelectrolyte-
based capsules [18,19]. Moreover, several researches indicated
that modifying by attaching polymers on the bilayer surface could
improve the stability of liposome as well as avoid the uptake of
liposomes by macrophages of the reticuloendothelial system. Many
polymers have been employed in the surface coating of liposome,
including pectin, amylopectin, dextran and pullulan [20]. As the
results, the toxicity of drugs might be diminished whereas the
mucoadhesive properties increased [21]. From the aforementioned
advantages, pectin can be a good polymer candidate for bonding
onto liposomes.
Citrus pectin (CP), as a promising polymer, is non-toxic,
biodegradable and biocompatible material, in addition to the
capability to form gels. It is a natural occurring polysaccharide,
including mainly of d-galacturonic acid units with varying degrees
of methylesterified carboxyl groups [22]. CP has a great potential
to be a colonic drug delivery carrier due to its prolonged reten-
tion in gastro-intestinal tract and nearly complete degradation by
the bacteria living in colon [23]. More importantly, the role of CP
as an inducer of malignant cell apoptosis cannot be ignored. The
study of Bergman et al. showed that CP exhibited a dose-dependent
anti-proliferation effect, which was compatible with the results of
Olano-Martin et al. [24–26].
In this study, we aim to design the citrus pectin-liposome nano-
complexes for improving the stability and mucoadhesiveness of
liposome, meanwhile enhance the anti-colon cancer effectiveness.
2. Materials and methods
2.1. Materials
l-␣-Phosphatidylcholine (Egg PC), cholesterol, citrus pectin,
mucin (extracted from porcine stomach, type II) and MTT (3-[4,5-
dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) from
porcine stomach (type II) were purchased from Sigma–Aldrich
(St. Louis, MO, USA). The cationic lipid dipalmitoyl trimethylam-
moniumpropane (DPTAP) and rhodamine-phosphoethanolamine
(Rb-PE) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL,
USA). Bufalin was purchased from ChenGuang Technology Devel-
opment Co. Ltd. (BaoJi, China).
2.2. Optimization of BFL preparation conditions by orthogonal
design method
2.2.1. Preparation of BFLs
BFLs were prepared according to the ethanol injection method
and their lipid compositions were as follows: EPC, cholesterol
and bufalin = 20:4:4.8 (mass ratio, total 14.4 mg total lipid/mL
55
ethanol), and 5.0 mol% cationic lipid DPTAP was added to prepare
cationic liposomes. Briefly, the lipids with the above composi-
tions were dissolved in 2 mL of ethanol, and rapidly injected
into 20 mL of phosphate buffer saline (PBS, pH 7.4), magnetic
stirred at 60 ◦ C for 1 h. The characteristic opalescence of colloidal
dispersions was occurred as soon as ethanolic solution was in con-
tact with the aqueous phase. Liposomes formed spontaneously
after further volatilizing the residual ethanol. The liposomes
suspension was immediately extrusion with a Lipex extruder
(LipoFast-Pneumatic, Avestin, Canada) five times using polycarbo-
nate membranes (0.2 ␮m pore size, Whatman, UK) to achieve the
desired size.
2.2.2. Single-factor test of liposomes preparation
Liposomes preparation was dependent on numerous factors,
and effects of four single factors (ratio of bufalin to lipid, ratio of
cholesterol to EPC, hydration media and volume of media) on the
encapsulation efficiency rate of liposomes were observed.
2.2.3. Orthogonal test of preparation of liposomes
Based on our previous research and the single-factor experi-
ments, the three factors, ratio of drug to lipid (w/w) (A), ratio of
cholesterol to EPC (w/w) (B), and the volume of media (C) were
mainly effective factors on encapsulation efficiency rate of lipo-
somes. Therefore, an orthogonal L9 (33 ) test design was used for
optimizing the preparation condition of liposomes and the encap-
sulation efficiency was chosen as determination index. Three levels
per factor were used and nine reacting conditions were designed
according to orthogonal test as L9 (33 ). These three factors at three
levels were as follows: the ratio of drug to lipid (1:5, 1:10 and 1:15,
w/w), the ratio of cholesterol to EPC (1:3, 1:4 and 1:5, w/w), the
volume of media (10, 20 and 30 mL).
In addition, several verification experiments were carried out
according to the optimal condition, and the encapsulation effi-
ciency was test in order to investigate whether the experimental
results were consistent with regression model.
2.3. Preparation of CPLs
2.3.1. Purification of CP
Purity of the commercially available CP was needed before use.
A stock solution of the CP was freshly prepared by dissolved in PBS
to a concentration of 2.0% (w/v), and then centrifuged for 30 min at
4000 rpm. The CP solution was further diluted with PBS to achieve
the final concentration of 0.4% (w/w) for each experiment. The solu-
tion was filtered through a 2 ␮m polycarbonate membrane filter
and stored in 4 ◦ C.
2.3.2. Coating of the liposomes with pectin
For the preparation of CPL, an equal volume of the cationic lipo-
somal suspension was added dropwise into the pectin solution and
mixed under continuous magnetic stirring for 0.5 h.
2.4. Size, charge, morphology and entrapment efficiency of
liposomes
The average particle size and charge of the liposomes were ana-
lyzed using a zeta potential analyzer (Malvern Zetasizer Nano ZS90,
Malvern, UK). The sample was diluted with deionized water prior
to the determination of surface properties. Mean size and poly-
dispersity index were obtained from 70 times measurements. The
morphologies of liposomes were observed by transmission electron
microscope (TEM) (Hitachi H-7650, Tokyo, Japan). For TEM studies,
carbon coated samples were placed over a copper grid and samples
were negatively stained with 1% phosphotungstic acidshape.
56 Y. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 54–62
The formulation was centrifuged at 14,000 rpm for 20 min to
remove the unloaded drug. The entrapment efficiency of bufalin in
the liposomes was expressed as the ratio of experimentally mea-
sured amount of drug before and after centrifugation. Bufalin was
quantified by an Xtimate C18 column (250 mm × 4.6 mm, 5 ␮m)
and detected by a validated HPLC with photodiode array detector
method. Mobile phase was composed of (A) monopotassium phos-
phate (0.5%, phosphoric acid adjust pH to 3.2) and (B) acetonitrile
using a gradient elution and performed at 40 ◦ C with a flow rate of
1.0 mL/min.
Encapsulation efficiency (EE) was calculated according to the
formula:
total amount of drug − free drug
Encapsulation efficiency(%) =
total amount of drug
× 100.
2.5. Mucin adsorption studies
The mucoadhesive properties of BFL and CPL were measured
by evaluating the adsorption of mucin on the surface of liposomes
[27]. The solution of mucin (1 mg/mL) was prepared in PBS by
stirred at room temperature overnight. The mucin standard solu-
tion was diluted into different contents (62.5, 125, 250, 500 and
1000 ␮g/mL) and prepared for mucin calibration curve. Bradford
reagent was added to the samples, and the absorbances of solu-
tions were detected at 595 nm by microplate reader after incubated
at 37 ◦ C for 10 min. 2 mL of BFL and CPL were stirred with 2 mL
mucin solution (1 mg/mL) at 37 ◦ C for 0.5 h respectively. Then, the
suspension was centrifuged at 14,000 rpm for 30 min at 4 ◦ C, and
the amount of free mucin in supernatant was determined using
Bradford colorimetric method.
The adsorption was estimated by using the mathematical for-
mula:
total amount of mucin used − free mucin
Adsorption(%) = × 100.
total amount of mucin used
2.6. Stability study
Since liposomes may aggregate during storage, resulting in
fusion of membranes and loss of encapsulated materials, the suffi-
cient stabilization turned into a challenge obviously. In addition, the
hydrolysis and oxidation of lipids might occur frequently. There-
fore, the formulations store in the form of freeze-dried powder was
an optimum selection. The stability of liposomal freeze-dried pow-
der at 4 ◦ C, which added 10% of sucrose as a cryoprotective agent
was evaluated. The particle size and encapsulation efficiency of BFL
and CPL were determined at day 0, day 7, day 15, 1 month and 3
months to estimate the stability.
2.7. In vitro drug release
The sustained-release properties of bufalin, BFL and CPL were
tested. 2 mL of BFL and CPL solutions in dialysis tube (molecu-
lar weight cut-off, 8 kDa) were directly immersed into 18 mL of
phosphate-buffered saline (PBS, pH = 7.4) at 37 ◦ C and subjected to
horizontal shaking (120 rpm/min). The dislysate of BFL and CPL was
withdrawn from the medium and replenished with equal volume
of fresh PBS at indicated durations 0, 1, 2, 4, 8, 12 and 24 h. The
bufalin content was measured by HPLC.
2.7.1. Cell cytotoxicity study
Cytotoxicities of free bufalin, BFL and CPL on SW480 colon can-
cer cells were evaluated by the MTT method. Briefly, SW480 colon
cells were plated in 96-well tissue culture plates at 1 × 104 cells
per each well and incubated overnight prior to drug treatment.
The culture medium was replaced with 100 ␮L respective differ-
ent amounts of free bufalin, BFL and CPL, with final concentrations
of 6.25, 12.5, 25, 50 and 100, 200 ␮mol/L, the blank liposomes as
negative control and the culture medium as blank control. After
incubated for 24 h, 20 ␮L of MTT stock solution (5.0 mg/mL) was
added to each well and the plates were incubated for 4 h at 37 ◦ C.
Subsequently, the medium was removed and 150 ␮L of dimethyl
sulfoxide added to dissolve the blue formazan crystals converted
from MTT. Cell viability was assessed by measuring absorbance at
490 nm on a microplate reader (Bio Rad Model 680, USA) with a
reference wavelength of 690 nm. The cell viability was calculated
as the ratio of the number of surviving cells in the drug-treated
samples to that of the control.
2.7.2. Cellular uptake studies
The cellular uptake experiment was observed using con-
focal laser scanning microscopy (CLSM). 0.5 mol% fluorescent
rhodamine-labeled Rb-PE, instead of bufalin, was incorporated
in liposomes as a fluorescence probe, while hoechst 33342 as
a nucleus marker. For CLSM observations, SW480 colon cancer
cells (105 cells per dish) were seeded in glass bottom petri dishes
(35 mm × 10 mm, Corning Inc., New York) and cultured for 24 h.
Then the cells treated with medium containing rhodamine-labeled
BFL and CPL. After incubation for 0.5, 2, 24 h, the media was
removed, and the cells were washed thrice with D-Hank’s solu-
tion to remove the residual nanoparticles. Then, 0.5 mL of hoechst
33342 solutions (0.5 mg/L) was added, and incubated for 10 min
to stain the nuclei. After incubation, the cells were softly washed
twice to remove excessive hoechst 33342 solution, and then fixed
with ethanol for 20 min. The cells were visualized under a CLSM.
2.7.3. Cell flow cytometry
To evaluate the effects of bufalin, BFL and CPL on cell cycle
distribution, the DNA contents of the cells were assessed by flow
cytometry. Briefly, cells were harvested and suspended at a con-
centration of 2 × 105 cells/well in six-well plates overnight, treated
with of free bufalin, BFL and CPL, contained equivalent dose of
12.5 or 50 ␮mol/L, and incubation for 24 h. The cells were digested
with pancreatin, and washed by PBS for twice, suspended by cul-
ture medium to analyze the apoptosis and fixed with 70% ice-cold
ethanol for cell cycle.
2.8. Statistical analysis
Data expressed as the mean ± SD, were representative of at least
three parallel experiments. The results of orthogonal test and anal-
ysis of variance were performed by statistical software SPSS 13.0.
The other statistical analysis was performed on Graphpad prism
5.0. Statistically differences were determined using the Student’s
t-test, and a one-way ANOVA followed by a posteriori testing with
Dunnett correction. A value of P less than 0.05 was considered sta-
tistically significant.
3. Results and discussions
3.1. Effect of singe factor on the encapsulation efficiency
3.1.1. Effect of ratio of bufalin to lipid on the encapsulation
efficiency
The ratio of bufalin to lipid was changed to 1:5, 1:10, 1:15, 1:20
and 1:25, when other three factors (the ratio of cholesterol to EPC,
the hydration media and the volume of media) were fixed at 1:5,
 Y. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 54–62 57

PBS and 20 mL. The results showed that the encapsulation efficien- Table 1
Analysis of L9 (33 ) test results.
cies were 66.05%, 66.27%, 47.66%, 40.16% and 37.52%, respectively.
No. Factors Encapsulation efficiency (%)
3.1.2. Effect of the ratio of cholesterol to EPC on the encapsulation
A B C
efficiency
1 1:5 1:3 10 78.29
The ratio of cholesterol to EPC was changed to 1:2, 1:3, 1:4,
2 1:5 1:4 20 63.40
1:5 and 1:6, when other three factors (the ratio of bufalin to lipid, 3 1:5 1:5 30 74.79
the hydration media and the volume of media) were fixed at 1:10, 4 1:10 1:3 20 43.29
PBS and 20 mL. The results showed that the encapsulation efficien- 5 1:10 1:4 30 52.26
cies were 23.48%, 47.80%, 56.69%, 47.70% and 45.14% in mentioned 6 1:10 1:5 10 75.94
7 1:15 1:3 30 39.20
condition. 8 1:15 1:4 10 39.62
9 1:15 1:5 20 40.11
3.1.3. Effect of ratio of the hydration media on the encapsulation
K1 72.157 53.593 64.617
efficiency K2 57.163 51.757 48.930
The hydration media was adjusted to distilled water, PBS, nor- K3 39.643 63.613 55.417
mal saline, 6% mannitol solution, when other three factors (the ratio R 32.514 11.856 15.687
of bufalin to lipid, the ratio of cholesterol to EPC and the volume Abbreviations: A, ratio of drug to lipid (w/w); B, ratio of cholesterol to EPC (w/w); C,
of media) were fixed at 1:10, 1:5 and 20 mL. The results showed the volume of media (mL).
that the encapsulation efficiencies were 33.43%, 51.01%, 46.33% and
34.43% in mentioned condition.

3.1.4. Effect of ratio of the volume of media on the encapsulation
efficiency
The volume of media was changed to 10, 20, 30, 40 and 50 mL,
when other three factors (the ratio of bufalin to lipid, the ratio of
cholesterol to EPC and the hydration media) were fixed at 1:10, 1:5
and PBS. The results showed that the encapsulation efficiency were
62.74%, 51.01%, 47.47%, 44.88% and 40.51% in mentioned condition.
3.2. The optimization of the preparation of liposomes
The major advantages of liposomal drug delivery are the pro-
tection of active compounds from degradation, the modification
of drug release, the increase in circulation time and the possi-
bility to achieve partial site. As we know, the most commonly
used preparative methods employed to prepare liposomes were
thin film hydration, reverse phase evaporation and ethanol injec-
tion [28]. In this study, the ethanol injection method was selected,
since the main advantage is the possibility to acquire small lipo-
somes with narrow distribution by simply injecting an ethanolic
lipid solution in water, and some industrial preparations have been
obtained by this way [29]. The encapsulation efficiency is most
widely employed to evaluate and control its quality. Depending
on the experiment results of the single-factor, we choose three
factors each with three variation levels. The results of orthogonal
test and extreme difference analysis were performed by statistical
software SPSS 13.0, presented in Table 1. The influence to encap-
sulation efficiency listed in a decreasing order as follows: A > C > B
according to the R values. The maximum encapsulation efficiency
was obtained when ratio of drug to lipid, ratio of cholesterol to
EPC, and the volume of media were 1:5, 1:5, 10 mL respectively
(A1 B3 C1 ).
The analysis of variance table of orthogonal test was shown in
Table 2. The F value of ratio of drug to lipid, ratio of cholesterol to
EPC, and the volume of media were 21.543, 3.313, 5.054, and the F
value of ratio of drug to lipid was larger than F critical value, which
meant a significant effect on the encapsulation efficiency.
Considering the feasibility and repeatability of experiment, the
optimized preparation condition of liposomes was confirmed, and
we got the high encapsulation efficiencies that were 77.52%, 74.88%
and 76.83%.
3.3. Characterization of the particles
cost. It has been extensive used in mucoadhesive delivery sys-
tems to increase the retention time in the gastro-intestinal tract,
and thus enhance drug absorption after oral administration. The
CPL was prepared by forming an ion-complex between pectin and
cationic lipid in the liposomal formulations. The previous stud-
ies found that size of pectin-coated liposomes depending on the
type and the concentration of pectin, and the influence factors
including molecular weight, branching of the polymer, electrical
charge, and hydrophobicity of the polymers. The particle size dis-
tribution and zeta potential of liposomes, measured by dynamic
light scattering (DLS), were summarized in Fig. 1. The average par-
ticle size of BFL was 136.4 ± 58.2 nm with narrow size distribution
(PI = 0.145). After coated by pectin, the average particle size turned
to be 296.3 ± 77.4 nm (PI = 0.171) was relatively larger than that
of cationic liposomes which was probably due to the interaction
between polar hydroxyl groups of the sugar units outwards and
water molecules.
Coating of the liposomes with pectin resulted in a signifi-
cant shift from positive to negative zeta potentials. The cationic
liposomes showed positive charge, 29.5 ± 9.63 mV, and the oppo-
site charge was appeared −18.1 ± 5.33 mV, after coated by pectin.
Pectin, a negatively charged polysaccharide, could interact with the
positively charged liposomes though the electrostatic interaction
between protonated amine groups (NH3 + ) of DPTAP and carboxyl-
ate anion (COO− ) of pectin [30].
The morphology of liposomes was examined by TEM (Fig. 1).
The liposomes had an almost spherical shape and well dispersed.
The gray area in the core of liposomes indicated the entrapped drug,
and the light transparent on the surface indicated the pectin-coated
layer.
The entrapment efficiency of bufalin was carried out by mea-
sure the difference of bufalin amount in the liposomal suspension
before and after centrifugation. The entrapment efficiency of BFL
and CPL were 68.59 ± 9.46% and 72.31 ± 5.84%, respectively, a lit-
ter higher after coated by pectin. This may be due to the result
Table 2
Analysis of variance table of orthogonal test.
Factor Sum of square of deviation Deviation F F critical value Significance
*
A 1588.867 21.543 19.0
B 244.354 3.313 19.0
C 372.788 5.054 19.0

Error 73.75
Pectin is a polysaccharide well-known for its long and safe use
*
in drug delivery due to the lack of toxicity and low production A significant effect on the encapsulation efficiency.
58 Y. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 54–62

Fig. 1. Characterization of bufalin liposomes with or without pectin. TEM images of bufalin liposomes (BFL) (A) and coated with citrus pectin (CPL) (B), with a uniform
spherical shape. Particle size distribution of BFL (C) and CPL (D), measured by dynamic light scattering. Zeta potentials of BFL (E) and CPL (F), displayed narrow widths.

of interpenetration and entanglement of bufalin with the polymer delivery system [5]. The addition of cryoprotectants is necessary
chains. to maintain the initial formulation characteristics. In this study,
the suitability of several cryoprotectants (mannitol, sucrose and
lactose) has been investigated to preserve the structure of lipo-
3.4. Mucin adsorption studies
somes, and 10% of sucrose was selected eventually (data not
shown). The particle size and encapsulation efficiency of BFL and
Liposomes have been extensively investigated for parenteral
CPL in the form of freeze-dried powder were estimate after store
use and local application of drugs in oral cavity. However, one
at 4 ◦ C. After rehydrated by PBS, the stabilities of formulations
of the problems associated with local application of liposomes is
were measured (Table 3). The size and encapsulation efficiency
the short residence time. This can be improved by the develop-
were barely changed during storage, suggesting the potential long-
ment of a mucoadhesive pharmaceutical formulation [22]. Mucin,
term stability of liposomes stored as the freeze-dried powder, even
the major component of mucus, takes responsibility for the vis-
though the decrease of encapsulation efficiency to a certain extent,
coelastic properties of mucus, which is primarily used to evaluate
after freeze-dry was observed. In addition, the barely changed sizes
the mucoadhesive properties. It is well known that wetting and
confirmed the refractory pectin layer.
swelling of the polymer can allow an intimate contact with the
tissue. In addition, the negatively charged mucin was close to the
zeta potential of CPL, indicating that the surface of CPL had a similar
charge density as mucin, which confirmed the interaction between
mucin and the CPL [23]. The mucoadhesive property of liposomes
was determined by the adsorption of mucin on the surface of lipo-
somes. As illustrated in Fig. 2, the amount of mucin adsorbed on
the surface of pectin-coated liposomes was 4-fold higher than that
of uncoated liposomes, which implied a significantly increased
mucoadhesive property after coated pectin and prolonged time
retained in the cavity compared to uncoated liposomes.

3.5. Stability study

Generally, aggregation, fusion, phospholipid hydrolysis and

leakage of the encapsulated drugs may occur during a long period
of storage in an aqueous medium, and freeze-drying appears Fig. 2. Mucoadhesiveness of liposomes (mean ± SD, n = 3).*p < 0.05, compared to the
as one of the most suitable methods to stabilize liposomes uncoated liposome.
 Y. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 54–62
Table 3
Stability of liposomes (mean ± SD, n = 3).
Formulation Property Day 0
Size (nm) 108.23 ± 5.75
BFL
Entrapment efficiency (%) 43.39 ± 1.62
Size (nm) 271.43 ± 16.12
CPL
Entrapment efficiency (%) 41.48 ± 1.36
3.6. In vitro drug release
The controlled drug release is one advantage of drug delivery
systems which improves the drug bioavailability and reduces the
side effect of toxic drugs. The in vitro cumulative drug-release pro-
file was shown in Fig. 3. BFL was released much more slowly than
free drug indicating that bufalin was steadily incorporated into the
lipid bilayer due to its semblable structure of cholesterol. A steric
protection effect endowed by pectin on the surface of liposome
could minimize the drug premature leakage lead to even slowly
release of CPL. The release behavior of bufalin in the liposomal
formulations could be divided into two stages: a fast release and
a slow sustained release. In the first 12 h, the cumulative bufalin
release in free drug was about 90%, while the liposome was about
50%. The drug-release in BFL started at 2 h, while CPL started at
6 h. The 90% cumulative release in both liposomal formulations all
need 36 h, and then the difference of release property between BFL
and CPL was barely detectable. The fast release of drug was asso-
ciated with those drug molecules located close to the lipid bilayer
surface, which could easily access to the aqueous medium. Since
permeate through CP need a litter long-term diffusion, the drug-
release started a little later [31]. In addition, the sustained release
of the drug might mainly contribute to the degradation rate of the
liposomal structure. Collectively, these results implicate that CPL is
a mild and suitable delivery system.
3.6.1. Cell cytotoxicity study
In the cytotoxicity assay, SW480 colon cancer cells were chose as
model cell, which were incubated with the liposomal formulations
at a dose equivalent of the free bufalin, and the result was shown
in Fig. 4. The cytotoxicity showed bufalin had a strong inhibitory
effect on cell proliferation with a dose-dependent manner, and the
effect of liposomal formulations was higher than free bufalin. A
possible reason for these effects was efficient uptake of liposome
Fig. 3. In vitro release profiles of bufalin from BFL and CPL by dialysis against phos-
phate buffer solution (PBS), compared to the dialysis of freely soluble bufalin. The
results are a representative of 3 independent experiments.
59
Day 7 Day 15 1 month 3 month
113.47 ± 8.59 115.33 ± 8.45 110.35 ± 3.29 114.58 ± 9.63
41.08 ± 0.65 45.62 ± 0.89 39.86 ± 1.21 42.70 ± 0.94
293.53 ± 10.03 284.30 ± 6.37 286.61 ± 12.58 295.14 ± 9.86
43.49 ± 1.92 42.76 ± 1.05 45.51 ± 0.73 49.23 ± 1.49
by cells through endocytosis, resulting in a higher drug concentra-
tion accumulated in cells. Furthermore, the cytotoxicity of CPL was
even more than BFL due to the inhibition of colon carcinoma cells
proliferation by CP, suggesting CPL would be potential for colon
cancer therapy.
3.6.2. Cellular uptake studies
In order to visualize the amount of drug taken up by cells, the
distribution of two liposomal formulations were tested by confocal
laser scanning microscope (CLSM) (FV1000 Olympus, Tokyo, Japan)
(Fig. 5). The liposomal formulations were mainly internalized into
the cells via endocytosis or phagocytosis. After incubation of 0.5,
2 and 24 h, liposomal label was localized at the cell nucleus and
cytoplasm of the SW480 colon cancer cells. The red fluorescence
could be found around the cell nucleus and the fluorescence exhib-
ited was markedly increased as time went by. The Rb fluorescence
intensities of the cells incubated with BFL and CPL were not sig-
nificantly different, suggesting coated with pectin had not affect
cellular uptake much.
3.6.3. Cell flow cytometry analysis
To study how bufalin formulations affect the cell cycle pro-
gression of cancer cells, the cellular DNA content at each stage
was measured with PI using flow cytometer (Becton Dickinson,
San Jose, CA, USA) (Fig. 6). After SW480 colon cancer cells incu-
bated with 50 ␮mol/L concentrations of free bufalin, BFL and CPL
for 24 h, a reduction in the fraction of cells in G0/G1 phase and
concomitantly accumulation of cells in S phase were observed.
Since the liposomal formulations could increase the cellular uptake
and the drug concentration in cells through endocytosis, the
drug induced G0/G1 arrest might be more prominent. The CPL
Fig. 4. Cellular viability of SW480 colon cancer cells after 24 h culture with free
bufalin, BFL and CPL. *p < 0.05, data are presented as the mean ± standard deviation
(n = 6).
60 Y. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 54–62

Fig. 5. Confocal images of SW480 colon cancer cells after treat with rhodamine-BFL or rhodamine-CPL for 0.5, 2, 24 h.The red fluorescence could be found around the cell
nucleus and the fluorescence exhibited was markedly increased as time went by. (For interpretation of the references to color in figure legend, the reader is referred to the
web version of the article.)

exhibited a higher S phase percent (53.23%) compared with BFL
(41.19%) and free bufalin (35.43%), whereas the untreated control
cells was 24.91%. The percentage of cells in G0/G1 phase increased
from 31.01%, 39.44%, 44.04%, while the control is 57.53%, which
indicated that the growth inhibitory effect of bufalin was the result
of a block of cell cycle at G0/G1 phase on SW480 colon cancer
cells.
Apoptosis was another important mechanism involved in can-
cer therapy. The numbers of apoptotic cells were assessed using
the Annexin V-FITC Apoptosis Detection kit. When SW480 colon
cancer cells treated with different bufalin formulations, the CPL
showed a much higher increase in the early apoptotic cell per-
centage (34.1%) compared to BFL (17.3%), bufalin (15.5%) and
the control cells (8.7%). While the late apoptotic cell proportions
 Y. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 54–62
Fig. 6. Flow cytometry analysis of SW480 colon cancer cells after treat with free bufalin, BFL and CPL. The cellular DNA contents at each stage were measured with PI using
flow cytometer and apoptotic cells were assessed using the Annexin V-FITC Apoptosis Detection kit.
were also decreased to 1.7%, 1.3%, 0.7%, and 0.3% in CPL, BFL,
bufalin, control groups respectively, consistent with the result of
cytotoxicity study.
4. Conclusions
In the present study experiment, a natural polyelectrolyte with
negative charge to stabilize positively charged liposomes as drug
delivery systems for anticancer treatment in vitro was successfully
established. Bufalin is a kind of steroids purified from Chinese tra-
ditional medicine Chan Su, used for curing a wide variety of tumors,
and the related research of liposomal bufalin formulations has not
been reported. An orthogonal L9 (33 ) test was used to optimize the
preparation condition of cationic bufalin liposomes coated with cit-
rus pectin, and the adsorption of pectin could be demonstrated by
an increase in particle size and a shift of the zeta potential from pos-
itive to negative. Based on the data, CPL has an excellent stability,
mucoadhesiveness and improved anti-colon cancer effectiveness,
which would be a promising drug carrier system treatment for
colon cancer.
Acknowledgment
This work was supported by the grant from the National Natural
Science Foundation of China (81173513).
References
[1] J. Cerny, A. Hassan, C. Smith, B. Piperdi, Coronary vasospasm with myocardial
stunning in a patient with colon cancer receiving adjuvant chemotherapy with
FOLFOX regimen, Clin. Colorectal Cancer 8 (2009) 55–58.
[2] J. Lin, Y. Yu, S. Shigdar, D.Z. Fang, J.R. Du, M.Q. Wei, A. Danks, K. Liu, W.
Duan, Enhanced antitumor efficacy and reduced systemic toxicity of sulfatide-
containing nanoliposomal doxorubicin in a xenograft model of colorectal
cancer, PLoS ONE 7 (2012) e49277.
[3] A. Sala-Vila, P.C. Calder, Update on the relationship of fish intake with prostate,
breast, and colorectal cancers, Crit. Rev. Food Sci. Nutr. 51 (2011) 855–871.
[4] C.M. Xie, W.Y. Chan, S. Yu, J. Zhao, C.H. Cheng, Bufalin induces autophagy-
mediated cell death in human colon cancer cells through reactive oxygen
species generation and JNK activation, Free Radic. Biol. Med. 51 (2011)
1365–1375.
61
[5] F. Li, T. Wang, H.B. He, X. Tang, The properties of bufadienolides-loaded nano-
emulsion and submicro-emulsion during lyophilization, Int. J. Pharm. 349
(2008) 291–299.
[6] P. Yin, Y. Wang, Y. Qiu, L. Hou, X. Liu, J. Qin, Y. Duan, P. Liu, M. Qiu, Q.
Li, Bufalin-loaded mPEG-PLGA-PLL-cRGD nanoparticles: preparation, cellular
uptake, tissue distribution, and anticancer activity, Int. J. Nanomed. 7 (2012)
3961–3969.
[7] R.J. Bick, B.J. Poindexter, R.R. Sweney, A. Dasgupta, Effects of Chan Su, a tradi-
tional Chinese medicine, on the calcium transients of isolated cardiomyocytes:
cardiotoxicity due to more than Na, K-ATPase blocking, Life Sci. 72 (2002)
699–709.
[8] P.F. Liu, M.J. Zhu, Y.G. Li, H.Z. Wang, Y.R. Duan, The research on cytotoxicity
and cellular uptake of mPEG-PLGA-PLL nanoparticles, the International Con-
ference on Advanced Fibers and Polymer Materials, Shanghai, China, 2009, pp.
1190–1192.
[9] N.H. Segal, L.B. Saltz, Evolving treatment of advanced colon cancer, Annu. Rev.
Med. 60 (2009) 207–219.
[10] D.C. Li, X.K. Zhong, Z.P. Zeng, J.G. Jiang, L. Li, M.M. Zhao, X.Q. Yang, J. Chen, B.S.
Zhang, Q.Z. Zhao, M.Y. Xie, H. Xiong, Z.Y. Deng, X.M. Zhang, S.Y. Xu, Y.X. Gao,
Application of targeted drug delivery system in Chinese medicine, J. Control.
Release 138 (2009) 103–112.
[11] H.K. Han, H.J. Shin, D.H. Ha, Improved oral bioavailability of alendronate via the
mucoadhesive liposomal delivery system, Eur. J. Pharm. Sci. 46 (2012) 500–507.
[12] A.Z. Wang, R. Langer, O.C. Farokhzad, Nanoparticle delivery of cancer drugs,
Annu. Rev. Med. 63 (2012) 185–198.
[13] G. Smistad, S. Boyum, S.J. Alund, A.B. Samuelsen, M. Hiorth, The potential of
pectin as a stabilizer for liposomal drug delivery systems, Carbohydr. Polym.
90 (2012) 1337–1344.
[14] M.E. Davis, Z.G. Chen, D.M. Shin, Nanoparticle therapeutics: an emerging treat-
ment modality for cancer, Nat. Rev. Drug Discov. 7 (2008) 771–782.
[15] K. Park, Nanotechnology: What it can do for drug delivery, J. Control. Release
120 (2007) 1–3.
[16] L. Gao, Y. Cui, Q. He, Y. Yang, J. Fei, J. Li, Selective recognition of co-assembled
thrombin aptamer and docetaxel on mesoporous silica nanoparticles against
tumor cell proliferation, Chem. A Eur. J. 17 (2011) 13170–13174.
[17] L. Gao, J. Fei, J. Zhao, H. Li, Y. Cui, J. Li, Hypocrellin-loaded gold nanocages with
high two-photon efficiency for photothermal/photodynamic cancer therapy in
vitro, ACS Nano 6 (2012) 8030–8040.
[18] C. Du, J. Zhao, J. Fei, L. Gao, W. Cui, Y. Yang, J. Li, Alginate-based microcapsules
with a molecule recognition linker and photosensitizer for the combined cancer
treatment, Chem. An Asian J. 8 (2013) 736–742.
[19] Q. He, Y. Cui, J. Li, Molecular assembly and application of biomimetic microcap-
sules, Chem. Soc. Rev. 38 (2009) 2292–2303.
[20] S. Nguyen, S.J. Alund, M. Hiorth, A.L. Kjoniksen, G. Smistad, Studies on pectin
coating of liposomes for drug delivery, Colloids Surf. B Biointerfaces 88 (2011)
664–673.
[21] S.J. Alund, G. Smistad, H. Hiorth, A multivariate analysis investigating different
factors important for the interaction between liposomes and pectin, Colloids
Surf. A Physicochem. Eng. Aspects 420 (2013) 1–9.
62 Y. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 54–62
[22] T. Klemetsrud, H. Jonassen, M. Hiorth, A.L. Kjoniksen, G. Smistad, Studies on
pectin-coated liposomes and their interaction with mucin, Colloids Surf. B
Biointerfaces 103 (2013) 158–165.
[23] J. Jung, R.D. Arnold, L. Wicker, Pectin and charge modified pectin hydrogel beads
as a colon-targeted drug delivery carrier, Colloids Surf. B Biointerfaces 104
(2013) 116–121.
[24] E. Olano-Martin, G.H. Rimbach, G.R. Gibson, R.A. Rastall, Pectin and pectic-
oligosaccharides induce apoptosis in in vitro human colonic adenocarcinoma
cells, Anticancer Res. 23 (2003) 341–346.
[25] H. Ohkami, K. Tazawa, I. Yamashita, T. Shimizu, K. Murai, K. Kobashi, M.
Fujimaki, Effects of apple pectin on fecal bacterial enzymes in azoxymethane-
induced rat colon carcinogenesis, Jpn. J. Cancer Res. 86 (1995) 523–529.
[26] K. Tazawa, H. Okami, I. Yamashita, Y. Ohnishi, K. Kobashi, M. Fujimaki, Anti-
carcinogenic action of apple pectin on fecal enzyme activities and mucosal or
portal prostaglandin E2 levels in experimental rat colon carcinogenesis, J. Exp.
Clin. Cancer Res. 16 (1997) 33–38.
[27] F. Qiang, H.J. Shin, B.J. Lee, H.K. Han, Enhanced systemic exposure of fexofe-
nadine via the intranasal administration of chitosan-coated liposome, Int. J.
Pharm. 430 (2012) 161–166.
[28] S. Yang, J. Chen, D. Zhao, D. Han, X. Chen, Comparative study on preparative
methods of DC-Chol/DOPE liposomes and formulation optimization by deter-
mining encapsulation efficiency, Int. J. Pharm. 434 (2012) 155–160.
[29] J. Song, F. Shi, Z. Zhang, F. Zhu, J. Xue, X. Tan, L. Zhang, X. Jia, Formu-
lation and evaluation of celastrol-loaded liposomes, Molecules 16 (2011)
7880–7892.
[30] N. Thirawong, J. Thongborisute, H. Takeuchi, P. Sriamornsak, Improved intesti-
nal absorption of calcitonin by mucoadhesive delivery of novel pectin-liposome
nanocomplexes, J. Control. Release 125 (2008) 236–245.
[31] Q. Tian, X.H. Wang, W. Wang, C.N. Zhang, P. Wang, Z. Yuan, Self-assembly
and liver targeting of sulfated chitosan nanoparticles functionalized with gly-
cyrrhetinic acid, Nanomedicine 8 (2012) 870–879.