ORIGINAL ARTICLES
reduced pressure at room temperature to form a thin film. The film was kept
in vacuum over night to remove the residual chloroform followed by a redis-
persion of the film to the added TES buffer (pH ¼ 7.4), thus produced the
multilamaller liposomes. Then the liposome suspensions were treated with a
probe ultrasonic producer to form the single unilamelar liposomes.
3.4. Formation and optimization of LHRHa-liposomes complex
In order to investigate the LHRHa and liposome binding, it was necessary
to calculate the ratio of the LHRHa binding to the negatively charged lipid
material (molar to molar). Different amounts of LHRHa were added to the
optimized liposomes and incubated for 30 min in a 37 ꢀC thermostatic
water bath to complete the static adsorption. The amount of LHRHa addi-
tion was controlled according to the ratio of LHRHa to monocholesterolsu-
cinate (molar to molar). When positively charged LHRHa were adsorbed
to the surface of the negatively charged liposomes, the zeta potential of the
liposomes would increase until saturated. Besides, a minor change in parti-
cle size would also take place. Thus zeta potential and particle size change
were taken into consideration in this study for the optimization of LHRHa
addition in the preparation of LHRHa-liposomes complex.
Fig. 7: A549 cell line uptake of different fluorescein-liposomes in different
time course
interaction between the receptor and the ligand. The recep-
tor-identifying drug delivery system can let the therapeutic
drugs concentrate in the target organ, tissue or cells, and
efficiently decrease side effects. Both chemical coupling
and electrostatic adsorption were utilized for coupling the
ligand with drug delivery systems (Dharap et al. 2003).
Although the chemical coupling proved more stable, the
process for preparation was either complicated or the steric
structure of the complex might be changed. By direct che-
mical coupling, the attachment might be adversely af-
fected the recognition properties of ligand for the target
antigen (Kocbek et al. 2007). Static adsorption was com-
monly used in gene delivery systems. A successful exam-
ple was implemented by linking the transferrin (TF) with
liposomes. The absolute value of negative zeta potential of
the liposomes was decreased after adding LHRHa, which
showed that the LHRHa had been linked with the lipo-
somes successfully. Still, further studies need to be carried
out on the stability of the complex as well as its target-
ability in vivo.
3.5. LHRHa-fluorescein-liposomes preparation and cell uptake study
Fluorescein instead of docetaxel was encapsulated into liposomes accord-
ing to the optimized formulation method, and then LHRHa-fluorescein-
liposomes were prepared by interacting with LHRHa. The LHRHa-fluores-
cein-liposomes were added in to the cell plates and incubated with differ-
ent cells. After incubating for a certain time, the supernatant in the cell
plants were removed and the cells were rinsed with PBS buffer (pH ¼ 7.4)
for three times to stop the phagocytosis and the remnant unphagocytosed
liposomes were removed. Each well of the cell plate was viewed by an
Axiovert 40 CFL microscope to investigate the uptake of prepared
LHRHa-fluorescein-liposomes, and then cells were disrupted and the fluor-
escence intensity was determined with RF-5301 fluorescence spectrophoto-
meter while the total protein concentrations which represented the total
amount of the cells were determined using BCA assay. The ratio of the
fluorescence intensities to the total protein concentrations for each well
indicated the uptake efficiency.
Acknowledgement: Financial support for this work was obtained from the
National Basic Research Program of China (973 Program, No.
2007CB935801), National Science Foundation of China (No. 30672550),
and Program for New Century Excellent Talent in University.
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3.1. Materials
Docetaxel (Doc) was a gift from Mei Lian Co., Ltd (Chongqing P.R.C.).
LHRHa was purchased from Chinese Peptide Company (Shanghai, P.R.C.).
Authentic standards of docetaxel and paclitaxel (purity > 98%) were ob-
tained from the National Institute for Control of Pharmaceutical and Biolo-
gical Products (Beijing, P.R.C.). Egg yolk phospholipid (Lecithin) was pur-
chased from Shanghai Bio Life Science & Technology Co., Ltd (Shanghai,
PRC). The Doc injection was prepared in injection workshop in Shenghe
drug plant (Chengdu, P.R.C.). SKOV3, Hela, C26 and A549 cell lines
were provided by the Institute of Biochemistry and Cell Biology, Chinese
academy of sciences (Shanghai, P.R.C.). RPMI1640 Medium was pur-
chased from Gibco (USA). BCA assay was purchased from Pierce Chemi-
cal. All other chemicals were of analytical reagent grade and purchased
commercially.
In this study an Alltech HPLC (US) was used for the determination of
docetaxel in liposomes. A Bu¨chi R-114 rotary evaporator and a JY92-II
probe ultrasonic producer (Ningbo Scientz Bio-tech Co. LTD) were in-
volved in the preparation of liposomes. The fluorescence intensity was
determined according to a Shimadzu RF-5301 fluorescence spectrophoto-
meter and the phagocytosis pictures were taken with Carl Zeiss Axiovert
40 CFL phase difference/fluorescence/visible microscope.
3.2. Synthesis of monocholesterolsuccinate
Cholesterol 5.8 g and 1.5 g of succinic anhydride were added to 200 ml of
normal heptane and refluxed for 21 hours with 15 ml pyridine as catalyst
(see Scheme). The mixture was cooled and filtered and the resulting solid
was recrystallized from acetone and dried under vacuum to remove the
residual organic solvent (Wu et al. 2005).
3.3. Preparation of docetaxel loaded liposomes
Docetaxel, monocholesterolsuccinate and egg yolk phospholipids were dis-
solved in chloroform and then evaporated with a rotary evaporator under
438
Pharmazie 63 (2008) 6