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Cholesterol succinyl chitosan anchored liposomes: preparation,

characterization, physical stability, and drug release behaviorYinsong Wang, MDa,, Shaoli Tu, MSb, Rongshan Li, MSa, XiaoYing Yang, PhDa,

Lingrong Liu, MDb, Qiqing Zhang, PhDb,c

aCollege of Pharmacy, Tianjin Medical University, Tianjin, People's Republic of ChinabInstitute of Biomedical Engineering, Chinese Academy of Medical Science, Peking Union Medical College, Tianjin, People's Republic of China

cResearch Center of Biomedical Engineering Medical School Xiamen University, Xiamen, People's Republic of China

Received 2 June 2009; accepted 16 September 2009

Abstract

The purpose of this study was to prepare cholesterol succinyl chitosan anchored liposomes (CALs) and to investigate theircharacterization, physical stability, and drug release behavior in vitro. Three cholesterol succinyl chitosan (CHCS) conjugates with different

substitution degrees (DS) of the cholesterol moiety were synthesized and used as the anchoring materials to coating on the liposome surface

by the incubation method. CALs were almost spherical and had a classic shell-core structure. Compared with plain liposomes and chitosan-

coated liposomes (CCLs), CALs had larger sizes, higher zeta potentials, and better physical stability after storage at 4 2C and 25 2C.

Epirubicin, as a model drug, was effectively loaded into CALs and exhibited the more sustained release in both phosphate buffer solution

(pH 7.4) and 1% (vol/vol) aqueous fetal bovine serum compared to plain liposomes and CCLs.

From the Clinical Editor: Cholesterol succinyl chitosan anchored liposomes (CAL) as delivery vehicles are characterized in this work,

including their physical stability and drug release behavior in vitro. Epirubicin as a model drug, was effectively loaded into CALs, and

exhibited sustained release behavior both in phosphate buffer solution (PBS, pH 7.4) and 1% (V/V) aqueous fetal bovine serum (FBS).

2010 Elsevier Inc. All rights reserved.

Key words: Cholesterol succinyl chitosan; Drug carrier; Epirubicin; Polysaccharide anchored liposome

Liposomes, the lipid bilayer vesicles, have gained attention as

drug carriers because they can reduce the toxicity and increase

the therapeutic efficacy of various drugs.1 However, the

applications of liposomes in drug delivery systems are currently

rather limited because of their relatively short blood circulation

time,2 and therefore various liposome formulations with

prolonged circulation time in blood have been studied.3-5

Recently, many investigations have shown that some hydro-

phobically modified polysaccharides such as palmitoylated

pullulan6 and amylopectin derivatives7,8 can anchor their

hydrophobic modified groups into the phospholipid bilayer of

liposomes by hydrophobic interaction and form a hydrophilic

shell on the liposome surface. This novel kind of liposome,

termed polysaccharide anchored liposome, has attracted in-

creasing interest for its following advantages in drug delivery

systems9,10: The hydrophilic polysaccharide shell can not only

increase the physical stability of liposomes but also provide

steric protection for liposomes to escape the adsorption of

opsonins and the phagocytosis of mononuclear macrophage,

thereby prolonging their circulation time in blood. Moreover,

there are many functional groups such as hydroxyl, amino, and

carboxyl groups in polysaccharide molecules, so that some

biologically active molecules (eg, ligand, monoclonal antibody

and biosensor) can be introduced to the polysaccharide anchored

liposomes by covalent bonds.

Chitosan, a homopolymer of (1,4)-linked 2-amino-2-deoxy-

-glucan, is produced by the deacetylation of chitin, which is the

second most abundant, renewable natural polysaccharide after

cellulose. Chitosan and its derivatives have been used in many

biomedical applications because of their excellent properties

such as biocompatibility, biodegradability, nontoxicity, and

bioadhesivity.11-13 In this study, chitosan was selected as the

polysaccharide material and was hydrophobically modified by

cholesterol to obtain the amphiphilic cholesterol succinyl

chitosan (CHCS) conjugates, which were used as the anchoring

materials to coat on the liposome surface by the incubation

method. The characterization and the physical stability in vitro of

chitosan anchored liposomes (CALs) were studied. Furthermore,

BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 471477

Original Articlewww.nanomedjournal.com

This work was supported by the National Natural Science Foundation of

China (grant no. 30900303).Corresponding author: College of Pharmacy, Tianjin Medical Univer-

sity, Tianjin 300070, People's Republic of China.

E-mail address: [email protected](Y.S. Wang).

1549-9634/$ see front matter 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.nano.2009.09.005

Please cite this article as: Y.S. Wang, S.L. Tu, R.S. Li, X.Y. Yang, L.R. Liu, Q.Q. Zhang, Cholesterol succinyl chitosan anchored liposomes: preparation,

characterization, physical stability, and drug release behavior. Nanomedicine: NBM2010;6:471-477, doi:10.1016/j.nano.2009.09.005
mailto:[email protected]://dx.doi.org/10.1016/j.nano.2009.09.005http://dx.doi.org/10.1016/j.nano.2009.09.005http://dx.doi.org/10.1016/j.nano.2009.09.005http://dx.doi.org/10.1016/j.nano.2009.09.005mailto:[email protected]


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epirubicin (EPB) was chosen as a model drug to assess the drug

loading and release behavior of CALs.

Methods

Materials

Biomedical grade chitosan (deacetylation degree was 92%,

viscosity average molecular weight was 8.0 104 Da) was

supplied by Yuhuan Ocean Biochemical Co., Ltd. (Zhejiang,

China). Three CHCS conjugates were synthesized by the method

that we previously reported.14 The substitution degrees (DS) of

the cholesterol moiety of CHCS, defined as the amount of

cholesterol moieties per 100 glucosamine units of chitosan, was

determined by the colloid titration method,15 and the DS values

of three CHCS conjugates were 2.80% (CHCS-1), 5.58%

(CHCS-2), and 8.00% (CHCS-3), respectively. EPB was

supplied by Hisun Pharmaceutical Co. (Zhejiang, China).

Phosphatidylcholine (Lipoid S100) was purchased from Lipoid

(Ludwigshafen, Germany). Cholesterol was purchased fromDingGuo Biotechnology Co., Ltd. (Beijing, China). Aqueous

fetal bovine serum (FBS) was purchased from the Institute of

Blood Disease of Peking Union Medicine College (Beijing,

China). Sephadex G-50 was purchased from Amersham

Biosciences (Uppsala, Sweden).

Preparation of CALs

The thin-film hydration method16 was developed to prepare

the plain liposomes. Phosphatidylcholine (50 mg) and choles-

terol (15 mg) were dissolved in chloroform. The mixture was

dried under reduced pressure using a Eyela rotary evaporator

(model N-1000; Eyela, Tokyo, Japan) at 40C to form a thinlipid film, and then trace solvent was removed by holding the

lipid film under high vacuum overnight. The lipid film was

hydrated by the addition of 5.0 mL 0.3 M citric acid buffer

solution (pH 4.0) and then vortexed and ultrasonicated for 9

minutes (with two intervals of 2 minutes after every 3 minutes)

using an ultrasonic processor (model UH-500A; Automatic

Science Instrument Co., Ltd, Tianjin, China) at 40 W to produce

multilamellar vesicles (MLVs). Then, MLVs were respectively

filtered through 0.45 m and 0.22 m porosity membrane filters

to obtain small-size liposomes.

CHCS-1, CHCS-2, and CHCS-3 anchored liposomes,

respectively named as CAL-1, CAL-2, and CAL-3, were

prepared by the incubation method. Briefly, different amounts

of CHCS (12.5, 33, 75, and 200 mg) dissolved in 2% aqueous

acetic acid solutions were respectively added to 5 mL of the

above plain liposomal suspensions, and the polysaccharide/lipid

weight ratios were 1/4, 2/3, 3/2, and 4/1. Then, the mixture

solutions were incubated at 20C with gentle shaking for 1 hour

and followed by storage at 4C for 24 hours until use.

Furthermore, CCLs as the control were also prepared by the

same method as above.

Characterization of CALs

The sizes and size distributions of CALs were measured by

dynamic laser light scattering (DLLS) with a Brookhaven digital

autocorrelator (model BI-90 Plus; Brookhaven Instruments,

Holtsville, New York) at a scattering angle of 90 degrees, a

wavelength of 633 nm, and a temperature of 25 0.1C. The zeta

potentials of CALs were determined using a Brookhaven

electrophoretic light-scattering spectrometer (model BI-Zeta-

plus; Brookhaven Instruments, Holtsville, New York).

Transmission electron microscopy (TEM) observations were

performed after negative staining of CALs with phosphomolyb-

dic acid. Briefly, the liposome samples were dropped onto

carbon-coated grids (100 mesh) and drawn off with a piece of

filter paper. Then, the grids were immersed for 1 minute in 2%

phosphomolybdic acid aqueous solution and washed twice with

200 mL distilled water. Finally, the grids were dried and imaged

using a Philips transmission electron microscope (model

EM400ST; Philips, Eindhoven, The Netherlands).

Study of physical stability of CALs

CALs were stored at 4 2C, 25 2C, and 37 2C for a

period of 5, 10, 15, 20, and 30 days. As above, the sizes and size

distributions of CALs were evaluated by DLLS using aBrookhaven digital autocorrelator (model BI-90 Plus; Brookha-

ven Instruments). The morphologic changes of CALs were

observed after 30 days of storage by a Philips transmission

electron microscope (model EM400ST; Philips).

Drug loading and release studies

EPB was first loaded into the plain liposomes by the modified

pH gradient method.17 Briefly, 5 mL of the plain liposome

suspension prepared above was applied to a Sephadex G-50

column (2 60 cm) and eluted with phosphate buffer solution

(PBS; pH 7.4) to obtain liposomes with a pH gradient between

their interior and exterior. Then, different volumes (62.5, 125,250, and 500 L) of 10 mg/mL EPB in PBS (pH 7.4) were added

to these pH gradient liposomes and incubated at 4C for 12, 24,

36, and 48 hours, respectively. The resulting liposome dispersion

passed through the Sephadex G-50 column with PBS (pH 7.4) as

the eluent to separate free EPB from the plain EPB-loaded

liposomes (PELs). The fluorescence intensity of free EPB was

measured by a Shimadzu fluorescence spectrophotometer

(model RF-4500; Shimadzu, Kyoto, Japan). The excitation

wavelength (ex) and the emission wavelength (em) were set at

470 and 585 nm, respectively. The entrapment efficiency of EPB

in liposomes was calculated according to the following equation:

EE = W0W1

W0100k

where EE is the entrapment efficiency, W0is the total amount of

EPB initially added, andW1is the amount of free EPB.

Then, PELs were incubated with CHCS conjugates to prepare

CHCS anchored EPB-loaded liposomes (CAELs) with the

polysaccharide/lipid weight ratio of 3/2 using the same method

for preparing CALs. At the same time, chitosan-coated EPB-

loaded liposomes (CCELs) as the control were also prepared as

the same method for preparing CCL.

In vitro release behaviors of EPB from CAELs were studied

by the dialysis method18 both in PBS (pH 7.4) and 1% (vol/vol)

aqueous FBS. Briefly, 2 mL liposome suspension was placed

472 Y. Wang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 471477


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into the dialysis tube (molecular weight cutoff 8 to 14 kDa;

Millipore, Bedford, Massachusetts) and dialyzed against the

above release media (10 mL) at 37 0.5C in an air-bath shaker

at 100 rpm. At scheduled time intervals, the whole of the release

media was collected and then the fresh release media were added.

The release amount of EPB was determined by fluorescence

spectrophotometry as described earlier. The accumulative release

percentage of EPB (RE %) was calculated according to the

following equation:

RE k = D0t

D0

100

where D0-tis the amount of drug released from liposome

suspension from the beginning to the scheduled time, and D0is the total amount of drug in liposome suspension.

Results

Preparation and characterization of CALs

In our study, the different polysaccharide/lipid weight ratios

were investigated to prepare CAL.Figure 1shows the effects of

polysaccharide/lipid weight ratio on the size (Figure 1,A) and the

zeta potential (Figure 1,B) of CAL, and the same trend occurred

among CALs with different DS of the cholesterol moiety.

Compared with the plain liposomes (size 148.2 3.0 nm and zeta

potential4.75 mV), CALs had significantly larger sizes and

positive zeta potentials, which indicated that CHCS conjugates

with different DS of the cholesterol moiety successfully coated on

the surface of the plain liposomes to form the polysaccharide

shells. The sizes and the zeta potentials of CALs evidently

increased with the polysaccharide/lipid weight ratio increasing

from 0 to 3/2 but slightly changed when the polysaccharide/lipid

weight ratio continued increasing, which indicated that CHCS

conjugates coating on the liposome surface reached a saturation

state. Therefore, 3/2 was believed to be the optimal polysaccha-

ride/lipid weight ratio to prepare CALs. Moreover, as shown in

Figure 1, CALs had evidently larger sizes and significantly higher

zeta potentials compared with those of CCL under the same

polysaccharide/lipid weight ratio. For example, the zeta poten-

tials of CAL-1, CAL-2, and CAL-3 were respectively +25.48

mV, +22.06 mV, and +21.09 mV at the polysaccharide/lipid

weight ratio of 3/2, whereas the zeta potential of CCL was only

+7.81 mV. Therefore, it could be deduced that CALs had betterstability than CCL due to their higher zeta potentials.

Figure 1, A also shows that the size of CAL increased when

the DS of the cholesterol moiety of CHCS increased from 2.8%

to 8.0%; for example, the size of CAL-3 was 262.5 6.5 nm

under the polysaccharide/lipid weight ratio of 2/3, evidently

larger than the size of CAL-1 (205.8 1.2 nm) and the size of

CAL-2 (232.5 9.1 nm), which suggested that CHCS with larger

DS of the cholesterol moiety could be easier to coat on the

surface of the plain liposomes.

Figure 2shows TEM images of the plain liposomes (Figure 2,

A1and A2), CAL-2 with the polysaccharide/lipid weight ratio of

3/2 (Figure 2, B1 and B2), and the physical mixture of plain

liposomes and CHCS-2 with the same polysaccharide/lipid

weight ratio of CAL-2 (Figure 2, C). Morphologically, plain

liposomes were nearly spherical (Figure 2, A1) and had an

obvious multilamellar (onion-like) structure characterized by

multiple membrane bilayers, each separated from the next by anaqueous layer (Figure 2, A2); CAL-2 also had a spherical shape

(Figure 2, B1) and a classic shell-core structure (Figure 2, B2)

with a shell thickness of about 30 nm. However, the plain

liposomes were wrapped with a thin polymer film formed by

CHCS-2 under the TEM observation (Figure 2, C) when they

were simply mixed with CHCS-2. Furthermore, the size of CAL-

2 determined by DLLS was about 245.4 8.1 nm, obviously

larger than the size determined by TEM images. We believed this

was because the polysaccharide shell of CAL-2 was highly

hydrophilic in nature and would be likely to swell in aqueous

media, thus the size determined by DLLS was a hydrodynamic

diameter and was larger than the size measured by TEM in the

dried state. Other CALs had similar morphologic characteristics

Figure 1. Effects of polysaccharide/lipid weight ratio (wt/wt) on the (A) size

and(B)zeta potential of liposomes. Each data point represents the mean of at

least three independent experiments (--, CCL; --, CAL-1; --, CAL-2;

--, CAL-3).

473Y. Wang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 471477


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to CAL-2. All the above results suggested that CHCS conjugates

could effectively be coated on the surface of liposomes by the

incubation method in this study.

The physical stability of CAL

CALs were stored at 4 2C, 25 2C, and 37 2C for a

period of 5, 10, 15, 20, and 30 days and then were determined by

DLLS and TEM to evaluate their physical stability. The plain

liposomes and CCL were also investigated as the controls.Figure3shows the effect of storage conditions on the size of liposomes.

An insignificant difference (PN.05) was found in the sizes of all

liposomal formulations stored at 4 2C (Figure 3, A) for 30

days. But a significant (Pb .05) increase in the size of the plain

liposomes was observed after 30-day storage both at 25 2C

(Figure 3, B) and 37 2C (Figure 3, C), which was due to the

fusion of the plain liposomes. No significant (PN .05) changes in

the sizes of CALs and CCL were observed when they were

stored at 25 2C up to 30 days (Figure 3, B). However, the

sizes of CALs and CCL all significantly (Pb.05) increased after

30-day storage at 37 2C. These results indicated that CALs

and CCL exhibited good stability on storage for at least 30 days

at 4 2C and 25 2C.

Figure 4shows the morphologies of CAL-2 and CCL after30-day storage at 25 2C. CAL-2 (Figure 4, A) retained its

spherical shape, and no evident aggregation phenomenon was

observed; the other two CALs had similar morphology to CAL-

2. CCLs (Figure 4, B) were irregularly spherical, and a

significant aggregation phenomenon took place. Therefore, it

could be concluded that CALs had better storage stability than

CCL, which was perhaps due to their higher zeta potentials

compared with that of CCL as discussed earlier.

Drug loading and release behaviors of CALs

EPB, as a weak base (pKa = 7.7), was first loaded into the

plain liposomes by the modified pH gradient method.17 This

method is often used to load weak bases such as doxorubicin and

Figure 2. TEM images of(A1, A2) plain liposomes, (B1, B2) CAL-2, and

(C)the physical mixture of liposomes with CHCS-2.

Figure 3. Particle sizes of plain liposomes, CCL, and CALs on time of storage

at(A) 4 2C,(B) 25 2C, and(C) 37 2C. All values are expressed as

mean SD (n = 3).



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vincristine,19-21 which coexist in aqueous solutions in neutral

and charged forms, into liposomes using pH gradient as a driving

force. In this study, the preparation conditions such as the weight

ratio of EPB to lipid and the incubation time were investigated to

obtain an optimal drug encapsulation. As shown in Figure 5,

EPB encapsulation efficiency increased with the weight ratio of

EPB to lipid increasing from 0.01 to 0.025, and then decreased

with the weight ratio further increasing, which suggested that the

ability of liposomes to load EPB reached saturation. Figure 5

also shows there was no significant difference (PN .05) of EPB

encapsulation efficiency among the incubation times (12 to 48

hours) when the weight ratio of EPB to lipid was less than 0.05,

but an obvious increase of EPB encapsulation efficiency

occurred with the incubation time increasing when the weight

ratio of EPB to lipid was larger than 0.05. However, when the

incubation time was longer than 36 hours, a significantly larger

size of liposomes was observed, which was perhaps due to the

fusion of liposomes. Therefore, considering the preparation

conditions prescribed above, 0.025 (EPB to lipid weight ratio)

and 24 hours (incubation time) were chosen to prepare PELs, and

EPB encapsulation efficiency was high, to 96.8%.

Then, PELs were incubated with CHCS conjugates to prepare

CAELs at the polysaccharide/lipid weight ratio of 3/2. CAELs

had similar morphology to CALs such as the spherical shape and

the classic shell-core structure under TEM observation, but a

slight decrease on EPB content was measured after PEL surface

coating with CHCS conjugates. We believed this was because

EPB passively leaked out of liposomes in the incubation period,

and EPB leakage rates were 2.65%, 1.98%, and 3.04% for

CAEL-1, CAEL-2, and CAEL-3, respectively.

EPB release behavior from CAEL was studied in vitro by the

dialysis method in PBS (pH 7.4) and 1% aqueous FBS,

respectively. EPB release from PELs and CCELs was also

investigated as the controls. The cumulative EPB release profilesare shown in Figure 6. InPBS (pH 7.4) (Figure 6,A), EPB release

from PEL showed a rapid pattern, about 52% EPB released in 24

hours; but only about 37%, 33%, and 41% EPB released

respectively from CAEL-1, CAEL-2, and CAEL-3, indicating

that CAEL significantly sustained the release of EPB. In 1% FBS

(Figure 6,B), EPB release rates from PELs and CAELs markedly

accelerated; nearly 100% EPB released from all liposome

formulations in 24 hours. We believe this was because the

bioactive substances such as proteins and enzymes in FBS

destroyed the liposome structure and polysaccharide coating

shell. However, CAELs also showed a significant sustained drug

release in the first 12 hours, about 73.6%, 67.7%, and 79.5% EPB

released respectively from CAEL-1, CAEL-2, and CAEL-3

Figure 4. TEM images of(A) CAL-2 and(B) CCL after 30-day storage at

25 2C.

Figure 5. Effect of preparation conditions on the encapsulation efficiency of

EPB. Each data point represents the mean of at least three independent

experiments (-

-, incubation time 12 hours; -

-, incubation time 24 hours;--, incubation time 36 hours; --, incubation time 48 hours).



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compared with 100% EPB from PELs in 1% FBS ( Figure 6,B).

The above results suggested that CAELs had better stability than

PELs in vitro due to CHCS shells on the surface of PELs.

Moreover, compared with CCEL, EPB release from CAELs more

or less slowed both in PBS (pH 7.4) and 1%FBS, which indicated

that CHCS coating shells were more stable than chitosan coating

shell on the liposome surface.

Discussion

Recently, many investigations into CCL have been performed

to enhance the stability of liposomes and increase their intestinal

uptake.22-24 We previously hoped to use CCL as the carrier of

anticancer drugs, but the chitosan layer formed by the

electrostatic attractive interaction between the positively charged

amino groups in chitosan molecules and the negatively charged

surface of liposomes was not effective enough to significantly

stabilize liposomes in vitro, so that the anchoring method using

hydrophobically modified chitosan derivatives (eg, CHCS and

palmitoyl chitosan conjugates) as anchoring materials was then

applied to increase the stability of liposomes. In our later research,

cholesterol modified groups showed better hydrophobic anchor-

ing properties for preparation of surface coating liposomes

compared with alkylated modified groups, which was consistent

with the previous report of Kang et al.10

Compared with CCL, CALs had larger sizes, higher zeta

potentials, better physical stability, and more sustained drug

release behavior under the same study conditions. We believe

this was due to the difference of coating mechanisms between

CHCS conjugates and chitosan on the liposome surface. It was

clear that chitosan coating on the liposome surface mainly

depended on the electrostatic attraction force. The amino

groups of chitosan molecules carrying positive charges

neutralized the negative surface charges of liposomes, so that

the zeta potential of liposomes changed from negative to

positive after chitosan coating, and its absolute value was

lower than that of some chitosan particles (eg, +28.44 mV

previously reported).25 However, in addition to the electro-

static attraction force, the process of CHCS coating alsoinvolved the cholesterol modified groups anchoring into the

phospholipid bilayer of liposome, thus a thicker and more

stable polysaccharide shell formed on the liposome surface.

Comparison of drug release profiles of CAELs both in PBS

(pH 7.4) and 1% FBS (Figure 6) showed that EPB release

behavior from CAEL was significantly influenced by the DS of

the cholesterol moiety of CHCS. EPB release rate from CAEL

decreased with the DS of the cholesterol moiety increasing from

2.8% (CHCS-1) to 5.6% (CHCS-2). This was because CHCS

with higher DS of the cholesterol moiety had more cholesterol

modified groups to anchor into the liposome phospholipid

bilayer, and thus a more stable polysaccharide coating shell was

obtained. However, with DS of the cholesterol moiety

increasing from 5.6% (CHCS-2) to 8.0% (CHCS-3), EPB

release rate evidently increased, such as EPB release from

CAEL-3 being even faster than that from CAEL-1 both in PBS

(pH 7.4) and in 1% FBS. We believed this was perhaps because

more cholesterol modified moieties anchoring into the liposome

phospholipid bilayer resulted in a higher liposome membrane

permeability. Therefore, CHCS-2 with DS of the cholesterol

moiety of 5.6% was considered an optimal hydrophobically

modified chitosan derivative to prepare polysaccharide anchored

liposomes in this study.

EPB was chosen as a model anticancer drug to assess the

potential of CAL as a novel carrier of anticancer drugs. As ananthracycline anticancer agent, EPB has a wide range of

antitumor activity and is used to treat various carcinomas.

However, EPB therapy may cause some serious side effectssuch

as allergic reactions, cardiotoxicity, and blood problems.26,27

Therefore, CAL was used as a carrier of EPB in an attempt to

sustain its release, prolong its circulation time, enhance its

therapeutic index, and decrease its toxic effects. Our current

results clearly showed that CHCS anchoring on the surface of

PELs could significantly improve their physical stability and

sustain the release of EPB in vitro, but investigations in vivo are

required to further confirm the advantages of CAL as the carrier

of EPB over the plain liposomes and CCL, and related

experiments are now in progress.

Figure 6. Dynamic release profiles of EPB from PEL (--), CCEL (--),

CAEL-1 (--), CAEL-2 (--), and CAEL-3 (--) in(A) PBS (pH 7.4) and

(B)1% FBS.



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Acknowledgment

The authors thank Drs. Xindu Yang, Wenzhi Yang, and

Hongli Chen for their helpful discussion and suggestions.

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