Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified with cholesterol anchored transferrin and TAT

Article abstract of DOI:10.1016/j.ijpharm.2013.06.048

This study was mainly focused on developing a dual-ligand liposomal delivery system to enhance both targeting specificity and cellular uptake. The specific ligand transferrin (TF) and the cationic cell-penetrating peptide TAT were connected with cholesterol via a polyethylene glycol (PEG) spacer to prepare the dualligand liposomes (TAT/TF-PEG-LP). Then the in vitro cellular uptake by three kinds of cells that possessed different expressing levels of transferrin receptor (TFR) and the in vivo delivery efficiency were evaluated. Compared to the single-ligand TAT or TF modified liposomes (TAT-PEG-LP or TF-PEG-LP), TAT/TF-PEG-LP exhibited the enhanced cellular uptake and selectivity via the synergistic effect of both ligands in vitro. The ex vivo fluorescence imaging of tumors, the qualitative observation of tumor frozen section and the quantitative determination of cellular uptake in tumor tissues altogether showed the in vivo delivery efficiency of TAT/TF-PEG-LP was higher than that of other liposomes. In conclusion, the dual-ligand liposomes co-modified with TF and TAT possessed a strong capability for synergistic targeted delivery of payload into tumor cells both in vitro and in vivo.

Full text of DOI:10.1016/j.ijpharm.2013.06.048

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International Journal of Pharmaceutics xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
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Pharmaceutical nanotechnology
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Synergistic targeted delivery of payload into tumor cells by
dual-ligand liposomes co-modified with cholesterol anchored
transferrin and TAT
Jie Tanga,1, Li Zhanga,1, Yayuan Liua, Qianyu Zhanga, Yao Qina, Yujia Yina,
Q1
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Wenmin Yuana, Yuting Yanga, Yafei Xiea, Zhirong Zhanga, Qin Hea,b,∗
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a Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu,
Sichuan 610041, People’s Republic of China
b Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, Fudan University, Shanghai 201203, People’s Republic of China
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a r t i c l e i n f o
a b s t r a c t
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Article history:
This study was mainly focused on developing a dual-ligand liposomal delivery system to enhance both tar-
geting specificity and cellular uptake. The specific ligand transferrin (TF) and the cationic cell-penetrating
peptide TAT were connected with cholesterol via a polyethylene glycol (PEG) spacer to prepare the dual-
ligand liposomes (TAT/TF-PEG-LP). Then the in vitro cellular uptake by three kinds of cells that possessed
different expressing levels of transferrin receptor (TFR) and the in vivo delivery efficiency were evaluated.
Compared to the single-ligand TAT or TF modified liposomes (TAT-PEG-LP or TF-PEG-LP), TAT/TF-PEG-LP
exhibited the enhanced cellular uptake and selectivity via the synergistic effect of both ligands in vitro.
The ex vivo fluorescence imaging of tumors, the qualitative observation of tumor frozen section and the
quantitative determination of cellular uptake in tumor tissues altogether showed the in vivo delivery
efficiency of TAT/TF-PEG-LP was higher than that of other liposomes. In conclusion, the dual-ligand lipo-
somes co-modified with TF and TAT possessed a strong capability for synergistic targeted delivery of
payload into tumor cells both in vitro and in vivo.
Received 4 April 2013
Received in revised form 15 May 2013
Accepted 13 June 2013
Available online xxx
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Keywords:
Dual-ligand
Synergistic effect
Transferrin
Cell penetrating peptide
Liposomes
© 2013 Elsevier B.V. All rights reserved.
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1. Introduction
and could selectively recognize and bind to the specific receptor
over-expressed on tumor cells, resulting in increased targeting
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Cancer has become a horrible disease because of its dramatic
incidence, low recovery rate and high mortality. In the last two
decades, liposomal drug delivery systems hold extraordinary
potential for delivery of therapeutics to tumor, various strate-
gies have been used to improve their targeting specificity and
cellular uptake. PEGylation has been extensively employed to
enhance the accumulation of liposomes in tumor tissues through
enhanced permeability and retention (EPR) effects, which was the
passive form of targeting. In attempts to increase the specificity
of interaction between liposomes and tumor cells, recent efforts
in the liposome field have been focusing on the development of
the active tumor targeted liposomes, which were modified with
some specific ligands such as TF, folic acid, peptides or antibodies,
efficiency and less toxicity. Among various kinds of active targeting
moieties, TF is a very suitable candidate for cancer therapeutics. The
overexpression of TFR on many kinds of tumor cells makes this gly-
coprotein an effective and widely explored target for site-specific
delivery of anti-tumor drug to tumors (Singh, 1999; Anabousi
et al., 2006). Besides, as an endogenous blood glycoprotein (Suzuki
et al., 2007), the incorporation of TF in PEGylated liposomes would
not bring a detrimental impact on reticuloendothelial system
(RES) evasion in vivo compared with some other active targeting
moieties (McNeeley et al., 2007; Gabizon et al., 2003).
However, the presence of receptor-targeting moiety alone on
PEGylated liposomes limits the cellular uptake of liposomes due to
receptor saturation (Kibria et al., 2011; Sharma et al., 2012). Con-
sidering that an ideal tumor targeted drug delivery system should
not only selectively targeted delivery drugs to tumor, but also
deliver the drugs into tumor cells with high efficacy, the recep-
tor saturation needed to be overcame. In previous studies, the
cell-penetrating peptides (CPPs) conjugated to the surface of lipo-
somes have been widely investigated under in vitro conditions for
increasing the intracellular delivery of drugs (Wadia and Dowdy,
2002). And the cationic cell-penetrating peptide CPP (TAT) derived
∗
Corresponding author at: West China School of Pharmaceutics, Sichuan Uni-
versity, #17 Section 3, Southern Renmin Nan Road, Chengdu Municipality 610041,
Sichuan Province, People’s Republic of China. Tel.: +86 28 85502532;
fax: +86 28 85502532.
E-mail addresses: qinhe@scu.edu.cn, qinhe317@126.com (Q. He).
These authors contributed equally to this work.
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0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2013.06.048
Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
with cholesterol anchored transferrin and TAT. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.06.048

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from the HIV-1 protein TAT could facilitate the intracellular deliv-
ery of cargoes with various sizes and physicochemical properties
(Banks et al., 2005; Brooks et al., 2005; Gupta et al., 2005). Lipo-
somes modified with TAT could deliver the cargoes into cells with
high efficiency via an unsaturated and receptor/transporter inde-
pendent pathway (Torchilin et al., 2001). Here we followed a dual
mechanistic approach for targeting TFR on tumor cells and further
improving the cellular uptake of the targeted delivery vehicle. We
combined the receptor targeting property of TF with the enhanced
cell uptake effect of TAT to improve the transport of desired cargoes
to tumor.
In this study, cholesterol, an important component in liposomal
formulations, was used as a lipid anchor to connect with PEG to
form the cholesterol derivative lipids (CHO-PEG). To develop the
dual-ligand liposome modified with TAT and TF, the active target-
ing ligand TF was covalently conjugated with the CHO-PEG3500, and
TAT was attached to the distal end of a shorter CHO-PEG2000. As we
all known, TAT is a nonspecific functional molecule, which pene-
trates any cells (Torchilin et al., 2001), besides, the positive charge of
TAT would increase the instability and toxicity of liposomes in vivo.
These drawbacks limit the use of TAT in systemic administration.
But here the PEG chain length difference of two functional materials
in liposomal formulations could ensure TAT to be shielded dur-
ing circulation, then overcame the non-specificity, instability and
toxicity of liposomes caused by TAT in vivo. In this liposomal formu-
lation, TF with a longer PEG chain could help targeting tumor cells,
and at the same time mask the non-specificity of TAT, after binding
to target cells, TAT could mightily enhance cellular uptake, which
resulted in increased targeting specificity and cellular uptake effi-
ciency. To verify the synergistic effect of dual-ligand liposomes on
cellular uptake, we assessed its cellular uptake efficiency compared
with PEG-LP and single-ligand liposomes (TAT-PEG-LP and TF-PEG-
LP) in vitro. And to further validate the cell specificity, we compared
the differences of synergistic effect between three kinds of cells that
possessed different expressing levels of TFR. For the in vivo study,
we firstly investigated the distribution in tumors via ex vivo fluo-
rescence imaging, then the capability for the synergistic targeted
delivery of payload into tumor cells was further qualitatively eval-
uated via confocal laser scanning microscopy and quantitatively
determined via flow cytometer.
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2. Materials and methods
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2.1. Materials
Soybean phospholipids (SPC) were purchased from Shang-
hai Taiwei Chemical Company (Shanghai, China), and cholesterol
(CHO) was purchased from Chengdu Kelong Chemical Com-
pany (Chengdu, China). The PEG functional materials with
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different chain lengths (NHS-PEG2000/3500-MAL and mPEG2000
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NHS) were all purchased from JENKEM Technology (Bei-
jing, China). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-
(carboxyfluorescein) (CFPE) was purchased from Avanti Lipids
(USA). TAT peptide with terminal cysteine (Cys-AYGRKKRRQRRR)
was synthesized according to the standard solid phase peptide syn-
thesis by Chengdu KaiJie Bio-pharmaceutical Co., Ltd. (Chengdu,
China). 1,1ꢀ-Dioctadecyl-3,3,3ꢀ,3ꢀ-tetramethylindodicarbocyanine
4-chlorobenzenesulfonate salt (DiD) was purchased from Biotium
(USA). Holo-Transferrin human was purchased from Sigma (USA).
Fluorescein isothiocyanate (FITC) labeled anti-CD71 antibodies was
purchased from Beckman Coulter Inc. (USA). Collagenase type IV
and DNase I were purchased from Biosharp. The other chemicals
were obtained from commercial sources.
2.2. Synthesis of CHO-PEG2000-TAT/CHO-PEG3500-TF
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2.2.1. Synthesis of compound 5
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The compound
5 was synthesized as outlined in Fig. 1.
Cholesterol 1 and p-toluenesulfonyl chloride (molar ratio = 1:2)
in anhydrous pyridine was stirred for 16 h at room temperature.
After purification by a silica-gel chromatography column with
CHCl3/EtOAc (9:1) as the eluant, the cholesterol p-toluenesulfonate
was obtained. Then product 2 and diglycol (DEG) (molar ratio = 1:8)
were dissolved in dry 1,4-dioxane. The reaction mixture was
Fig. 1. Schematic of synthesis of the compounds 5 and 7. The synthesis was confirmed by 1H NMR, mass spectroscopy.
Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
with cholesterol anchored transferrin and TAT. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.06.048

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refluxed overnight to give the compound 3. After that, to the
solution of 3 (1 equiv.) in dry DMF, phthalimide (5 equiv.), triph-
enylphosphine (5 equiv.), and diethylazodicarboxylate-toluene
(5 equiv.) was added one by one, the mixture was stirred at room
temperature for 4 h. The crude product was purified on a silica-gel
chromatography column to get 4. Then the prepared 4 (1 equiv.)
in pyridine was added to the solution of hydrazine monohydrate
(5 equiv.), and the mixture was stirred at room temperature for
24 h to give the desired compound 5. The chemical structure was
verified by 1H NMR (Shohda and Sugawara, 2006; Pan et al.,
2007).
2.4. Size and zeta potential measurements
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The size and zeta potential of the liposomes were determined
using a Malvern Zetasizer Nano ZS90 instrument (Malvern instru-
ments Ltd., UK). Prior to measurement, 100 ␮l of the sample (lipid
concentration 2.1 mg/ml) was diluted to 1 ml using the same buffer.
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2.5. Cellular uptake in vitro
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2.5.1. Cell culture
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HepG2 cells, A2780 cells and HUVECs were grown in RPMI-1640
medium (GIBCO), DMEM medium (GIBCO) and DMEM medium
(GIBCO), respectively, which contained 10% FBS, 100 ␮g/ml strepto-
mycin, and 100 U/ml penicillin. The cells were maintained at 37 ◦C
in a humidified incubator with 5% CO2.
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2.2.2. Synthesis of CHO-PEG2000-TAT (compound 7)
The compound 5 was reacted with NHS-PEG2000-MAL (molar
ratio = 2:1) in dry dichloromethane (DCM) at room temperature
under argon in the presence of triethylamine for about 4 h. After the
thin layer chromatography (TLC) (DCM/MeOH/H2O = 3:0.5:0.001)
showed the disappearance of NHS-PEG2000-Mal, the reaction mix-
ture was filtered and the filtrate was evaporated under vacuum.
Excess 5 was removed by adding 5 ml of acetonitrile to precipitate
it, and the mixture was kept at 4 ◦C overnight. Then it was cen-
trifuged at 5000 rpm for 10 min. After that, the supernatant was
collected and evaporated again under vacuum to get the dry 6
(CHO-PEG2000-MAL).
The CHO-PEG2000-MAL and Cys-TAT (molar ratio = 1:1.5) were
reacted in the mixture of CHCl3/MeOH (V:V = 2:1) with gentle stir-
ring at room temperature for about 30 h (Qin et al., 2011). After
TLC (DCM/MeOH/H2O = 3:0.75:0.12) showed the disappearance of
CHO-PEG2000-MAL, the mixture was evaporated under vacuum,
the slight excess of Cys-TAT was removed by adding a small
volume of CHCl3, the insoluble material was filtered, and the super-
natant was evaporated again under vacuum to afford compound 7
(Fig. 1).
2.5.2. Evaluation of the expression of TF receptors
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HepG2 cells, A2780 cells and HUVECs were washed with PBS
(pH 7.4) and detached by treatment with trypsin, respectively.
Detached cells were incubated with FITC labeled anti-CD71 anti-
bodies for 30 min at 37 ◦C. Ten thousand cells per sample were
analyzed using a BDFACSAriaTM II flow cytometer (BD, San Jose,
CA, USA).
2.5.3. Quantitative evaluation of cellular uptake by confocal laser
scanning microscopy (CLSM)
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Liposomes labeled with CFPE were prepared as described above,
with the probe CFPE added to the lipid materials at first. HepG2
cells, A2780 cells and HUVECs were plated on gelatin-coated cover
slips in 6-well culture plates and cultured for 48 h. Different for-
mulations of liposomes were added to the plates with a total lipid
concentration of 0.21 mg/ml. After incubation for 4 h at 37 ◦C under
5% CO2, the cells were washed three times with cold PBS (pH
7.4) and 2 ml 2 ␮g/ml DAPI was added for 5 min, then cells were
washed again and fixed using 4% paraformaldehyde. Cover slips
were mounted cell-side down with slides and viewed using a Leica
TCS SP5 AOBS confocal microscopy system (Leica, Germany).
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2.2.3. Synthesis of CHO-PEG3500-TF
After the compound 6 (CHO-PEG3500-MAL) was well prepared as
given in Section 2.2.2, we synthesized CHO-PEG3500-TF as described
previously (Yang et al., 2008; Hatakeyama et al., 2004; Chiu et al.,
2006) with some modification. Briefly, TF in phosphate balanced
solution (PBS pH 8) reacted with 5× Traut’s reagent to yield TF-
SH. Free Traut’s reagent was removed by passing the Sephadex
G50 column. CHO-PEG3500-MAL was made into micelles with a
concentration of 1 mg/ml. Then TF-SH was coupled to micelles of
CHO-PEG3500-MAL at a protein-to-lipid molar ratio of 1:10 for 4 h
at 25 ◦C. Lastly, 10 mg/ml MEA was added to the mixture to end the
reaction.
2.5.4. Quantitative evaluation of cellular uptake by flow
cytometer
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Liposomes labeled with CFPE were prepared as described above.
HepG2 cells, A2780 cells and HUVECs were plated on gelatin-coated
cover slips in 6-well culture plates and cultured for 48 h. Different
formulationsof liposomes were added to the plates with a total lipid
concentration of 0.21 mg/ml. After incubation at 37 ◦C under 5% CO2
for 4 h, the cells were detached by treatment with trypsin for five
minutes, washed three times with cold PBS and finally resuspended
in 0.5 ml PBS for flow cytometry measurement.
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2.3. Preparation of liposomes
2.6. Evaluation of delivery efficiency in vivo
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TAT modified liposomes (TAT-PEG-LP) and PEGylated liposomes
(PEG-LP) were prepared by the thin film hydration methods as
described previously (Qin et al., 2011). Briefly, various amounts of
SPC/CHO/CHO-PEG2000/CHO-PEG2000-TAT (see Table 1) were dis-
solved in chloroform. Chloroform was then removed by rotary
evaporation. The obtained thin film was kept in vacuum for over
6 h to completely remove the residual organic solvent. The thin film
was hydrated in PBS (pH 7.4) for 1 h at 37 ◦C. Then it was further
intermittently sonicated by a probe sonicator at 100 W for 50 s. And
the TF modified liposomes (TF-PEG-LP) were prepared according to
2.6.1. Tumor-bearing mice models
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Nude mice weighing 20–25 g were purchased from Experiment
Animal Center of Sichuan University (PR China). All the animal
experiments adhered to the principles of care and use of laboratory
animals and were approved by the Experiment Animal Administra-
tive Committee of Sichuan University.
Tumor-bearing mice were established as following method.
Briefly, nude mice were inoculated subcutaneously with 1 × 106
HepG2 cells in the left flank. These models can be used for experi-
ment when the diameter of tumor reached about 10 mm.
a post-insertion method (Yang et al., 2008) in which CHO-PEG3500
-
TF micelles was prepared in advance using the above method and
then incubated with the matrix liposomes (PEG-LP) for 1 h at 37 ◦C,
As shown in Fig. 2, the dual-ligand liposomes (TAT/TF-PEG-LP) were
prepared according to the aforesaid post-insertion method with the
matrix liposomes PEG-LP replaced by TAT-PEG-LP.
2.6.2. Ex vivo DiD dye fluorescence imaging
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To evaluate the bio-distribution of liposomes in tumor bearing
mice, DiD was used as a fluorescent probe for ex vivo fluorescence
imaging (Ntziachristos et al., 2003; Zhang et al., 2010). And the
Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
with cholesterol anchored transferrin and TAT. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.06.048

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Table 1
Composition of liposomes (mol%) and their size and zeta potential.
Abbreviations
Corresponding compositions
Physical properties
SPC (%)
65.0
CHO (%)
29.0
Functional cholesterol lipids composition
Size (nm)
PDI
Zeta (mV)
4.64 0.42
TAT/TF-PEG-LP
4%CHO-PEG2000/1% CHO-PEG2000-TAT/1%
CHO-PEG3500-MAL
130.17 8.32
0.191 0.028
TAT-PEG-LP
TF-PEG-LP
PEG-LP
65.0
65.0
65.0
29.0
29.0
29.0
5%CHO-PEG2000/1% CHO-PEG3500-TAT
5%CHO-PEG2000/1% CHO-PEG2000-MAL
6% CHO-PEG2000
122.60 3.66
127.10 5.30
112.00 5.37
0.209 0.025
0.188 0.022
0.158 0.009
9.04 0.92
0.547 0.19
−0.424 0.37
The data represented the mean SD (n = 3).
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DiD-loaded liposomes were prepared by the method described
above, with DiD added to the lipid materials at first. The DiD-loaded
TAT/TF-PEG-LP, TAT-PEG-LP, TF-PEG-LP and PEG-LP were injected
into HepG2 tumor-bearing nude mice at a dose of 10 mg lipids/kg
via the tail vein. 24 h later, the mice were executed by cervical dis-
location. The whole tumors were removed and washed with cold
PBS. Then the images were captured by the CCD camera (Quick View
3000, Bio-Real, Austria).
liposomes were injected into mice for 24 h, tumors were excised
and cut into small pieces. Then the dissociation solution [Colla-
genase type IV (1 mg/ml) and DNase I (30 ␮g/ml)] was added at
37 ◦C for 1 h, following the process of sieving (70-␮m mesh), cen-
trifugation and washing with PBS for three times. Cells were finally
resuspended in 0.5 ml PBS for flow cytometry measurement. And
cells from tumor-bearing mice injected with PBS were served as
blank.
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2.7. Statistical analysis
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2.6.3. Qualitative analysis of delivery efficiency
To qualitatively investigate the delivery profiles of liposomes in
vivo, The DiD-loaded TAT/TF-PEG-LP, TAT-PEG-LP, TF-PEG-LP and
PEG-LP were injected into HepG2 tumor-bearing nude mice at a
dose of 10 mg lipids/kg via the tail vein. 24 h later, the mice were
executed by cervical dislocation, and tumors were excised and put
in liquid nitrogen immediately. Then tumors were frozen sectioned
(4 ␮m in thickness). Sections were stained with DAPI (2 ␮g/ml) for
5 min, washed three times with cold PBS, and then observed via
CLSM.
Analysis of variance (ANOVA) was used to test the statistical sig-
nificance of differences among groups. Statistical significance was
evaluated by using Student’s t-test or Dunnett’s test for the single
or multiple comparisons of experimental groups, respectively.
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3. Results
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3.1. Synthesis of CHO-PEG2000-TAT
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2.6.4. Quantitative determination of delivery efficiency
To prepare the TAT modified cholesterol derivate (CHO-PEG2000
-
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The quantitative determination by flow cytometry was per-
formed to further evaluate the delivery efficiency of liposomes
into tumors. The manner was similar to the previously described
methods with some modification (Kirpotin et al., 2006). After
TAT), we first synthesized CHO-PEG2000-MAL by conjugating the
PEG with a maleimide group (-MAL) to the compound 5, then linked
TAT to the distal end of CHO-PEG2000-MAL via a thioether bond. The
structure of CHO-PEG2000-MAL was verified by 1H NMR (400 MHz,
Fig. 2. Schematic illustration of the preparation method of dual-ligand liposomes.
Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
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Fig. 3. Expression levels of TFR. The expression of TFR on HepG2 cells (A), A2780 cells (B) and HUVECs (C) was confirmed by flow cytometer as described in materials and
methods. Black lines indicate non-treatment, and red lines indicate results obtained for the anti-CD71 antibody treatment. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
Q2
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CDCl3, ␦ppm): 6.64 (s, 2H), 5.25 (s, 1H), 3.82–3.45 (br, m, PEG pro-
tons, ∼181 H), 3.15–3.07 (m, 2H), 2.56 (s, 2H), 2.44 (t, 1H), 2.29–2.26
(m, 1H), 2.15–2.13 (m, 1H), 1.88–0.83 (m, CHO protons), with 0.675
(s, 3H), 0.89 (d, 6H), 0.91 (d, 3H,), 1.02 (s, 3H). TOF MS ES+ confirmed
the formation of CHO-PEG2000-TAT (Mw calculated = 4255 Da, Mw
observed = 4356 Da). 1H NMR was also used to confirm the struc-
ture of CHO-PEG2000-TAT, whose PEG and TAT backbone could be
found in 1H NMR. In addition, the disappearance of double bond of
MAL (6.64 (s, 2H)) also signified the form of thioether bond between
CHO-PEG2000-MAL and TAT (see supplementary material).
uptake difference of TAT/TF-PEG-LP and TAT-PEG-LP was between
HepG2 cells and HUVECs.
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3.5. Quantitative evaluation of cellular uptake in vitro by flow
cytometer
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Then the cellular uptake of different liposomes (see Table 1)
was quantitatively evaluated using flow cytometer. As shown in
Fig. 5a, compared to the control liposome PEG-LP, the cellular
uptake of TAT-PEG-LP, TF-PEG-LP and TAT/TF-PEG-LP was respec-
tively increased by 2.33, 2.33 and 22.17 times in HepG2 cells. In this
type of cell line, which expressed substantial TFR on surface, the cel-
lular uptake amount of TAT/TF-PEG-LP was far more than that of
TAT-PEG-LP plus TF-PEG-LP, so the synergistic effect of both ligands
on cellular uptake was obvious. However in the case of HUVECs
(Fig. 5c), whose TFR expression levels were lower, there was not
synergetic effect of TAT/TF-PEG-LP on cellular uptake, the uptake
amount of TAT-PEG-LP, TF-PEG-LP and TAT/TF-PEG-LP was 1.05,
9.42 and 8.32-fold higher than that of control liposome PEG-LP. And
in A2780 cells (Fig. 5b), the synergetic effect of TAT/TF-PEG-LP on
cellular uptake was between HepG2 cells and HUVECs. The uptake
amount of TAT-PEG-LP, TF-PEG-LP and TAT/TF-PEG-LP was 4.88,
1.48 and 11.85-fold higher than that of control liposome PEG-LP.
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3.2. Characteristics of liposomes
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Liposome size measurements showed that the sizes of all the
liposomes were mainly about 120 nm; the zeta potential of TF-PEG-
LP and PEG-LP was nearly neutral, while TAT-PEG-LP was obviously
positive charged, and the positive charge of TAT/TF-PEG-LP was
lower than that of TAT-PEG-LP (Table 1).
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3.3. Detection of TF receptor expression
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To evaluate the expression of the TF receptors on different cell
surfaces, flow cytometric analyses of HepG2 cells, A2780 cells and
HUVECs were carried out using FITC labeled anti-CD71 antibod-
ies (Fig. 3). The ratios of fluorescence intensity (cells incubated
with antibodies to non-treated cells) which reflected the expres-
sion level of TFR were 13.45 (HepG2 cells), 4.93 (A2780 cells) and
4.76 (HUVECs), respectively. It revealed that HepG2 cells expressed
substantial TFR, while fewer on the surface of A2780 cells and
HUVECs.
3.6. Ex vivo DiD dye fluorescence imaging
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Ex vivo NIR fluorescence imaging was performed on excised
mice tumors. As shown in Fig. 6, a strong NIR fluorescent signal
from the tumors of the mice injected intravenously with the DiD-
loaded TAT/TF-PEG-LP and TF-PEG-LP were observed 24 h after
injection. Tumors from mice treated with TAT-PEG-LP and PEG-
LP had weaker signal compared to TAT/TF-PEG-LP and TF-PEG-LP.
Control animal injected with saline solution produced no back-
ground signal.
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3.4. Qualitative evaluation of cellular uptake in vitro by confocal
laser scanning microscopy (CLSM)
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The cellular uptake of different liposomes (see Table 1) in HepG2
cells, A2780 cells and HUVECs were investigated by CLSM, as shown
in Fig. 4a. The dual-ligand liposomes (TAT/TF-PEG-LP) resulted
in stronger fluorescence signals inside the HepG2 cells than the
single-ligand liposomes (TAT-PEG-LP or TF-PEG-LP), indicating that
TAT/TF-PEG-LP was efficiently internalized by the HepG2 cells
under the synergistic effect of both ligands. However, in HUVECs,
whose TFR expression levels were lower, the difference of cellular
uptake between TAT/TF-PEG-LP and TAT-PEG-LP was not as obvi-
ous as that in HepG2 cells (Fig. 4c). And in A2780 cells, the cellular
3.7. Qualitative analysis of delivery efficiency in vivo
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The tumor sections from HepG2 tumor-bearing mice that
received DiD loaded PEG-LP, TAT-PEG-LP, TF-PEG-LP and TAT/TF-
PEG-LP were qualitatively analyzed using confocal laser scanning
microscopy. As illustrated in Fig. 7, the tumor frozen section of
TAT/TF-PEG-LP group showed strongest red fluorescence (fluores-
cence of DiD). TAT-PEG-LP group showed some red fluorescence but
weaker than TAT/TF-PEG-LP group. TF-PEG-LP and PEG-LP groups
showed little red fluorescence and had no obvious difference. The
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Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
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Fig. 4. CLSM images of HepG2 cells (a), A2780 cells (b) and HUVECs (c) incubated with different formulations of CFPE-labeled liposomes at 37 ◦C for 4 h. A, TAT/TF-PEG-LP; B,
TAT-PEG-LP; C, TF-PEG-LP; D, PEG-LP. The row I was the bright field. The blue fluorescence exhibited in row II was due to DAPI-staining of the nuclei. The green fluorescence
exhibited in row III was due to the CFPE-labeled liposomes. The forth row was the overlay sight. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
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result indicated that efficient targeted delivery of DiD was achieved
by the dual-ligand modified liposomes.
4. Discussion
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The ability of carriers to specifically targeting delivery cargoes
to tumors is important to effective cancer therapy. The active
tumor targeted liposomes, which were modified with some spe-
cific ligands such as transferrin, folic acid, peptides or antibodies,
could selectively recognize and bind to the specific receptor
over-expressed on tumor cells, then result in increased targeting
efficiency and less toxicity. However, the presence of receptor-
targeting moiety alone on PEGylated liposomes limits the cellular
uptake of liposomes due to receptor saturation (Sharma et al.,
2012); Harashima et al. also indicated that some kind of the ligand
modified carriers enter cells via the receptor mediated endocytosis
which is a saturated pathway, the saturation phenomenon might
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3.8. Quantitative determination of delivery efficiency in vivo
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The cytometric data allowed for quantitative analysis of the
results was obtained from treated HepG2 tumor-bearing mice
(Fig. 8). The fluorescent intensity represented cellular uptake
amount of different liposomes in tumor tissues. The fluorescent
intensity of the group receiving TAT/TF-PEG-LP was 1.31, 1.37 and
1.45-fold higher than that of the groups receiving TAT-PEG-LP, TF-
PEG-LP and PEG-LP, respectively. The result further demonstrated
that an efficient targeted delivery of DiD could be achieved by the
dual-ligand modified liposomes in vivo.
Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
with cholesterol anchored transferrin and TAT. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.06.048

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Fig. 5. Fluorescence intensity of HepG2 cells (a), A2780 cells (b) and HUVECs (c) measured by flow cytometer after incubated with different formulations of CFPE labeled
liposomes at 37 ◦C for 4 h. Blank (red lines), TAT/TF-PEG-LP (green lines), TAT-PEG-LP (blue lines), TF-PEG-LP (brown lines) and PEG-LP (purple lines). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)
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limit the cellular uptake of ligand modified liposomes (Kibria
et al., 2011). To generate further therapeutic efficacy, TAT which
could promote carriers to enter cells effectively via an unsaturated
and receptor/transporter independent pathway (Torchilin et al.,
2001) was applied to develop the dual-ligand liposomes (Fig. 2).
In this liposomal formulation, the receptor targeting property of
transferrin was combined with the enhanced cell uptake effect
of TAT to improve the transport of desired cargoes to tumor. In
previous studies, phospholipids have been extensively employed
as an anchor for both PEGylation and ligand modifying in liposomal
formulations. However, DSPE introduced a negative charge to the
liposomes surface, which might lead to additional plasma protein
binding (Zhao et al., 2007). In contrast, cholesterol was electrically
neutral. Besides, cholesterol is more chemically stable and much
cheaper than DSPE. Thus, we presumed cholesterol anchor may
have many advantages over DSPE anchor. Furthermore, to ensure
that the functional cholesterol derivative lipids were stable during
circulation, we chose the ether linkage to connect the cholesterol
anchor with PEG or ligands instead of the commonly used ester
bond which could be hydrolyzed by the esterase in the plasma (Xu
et al., 2008; Heyes et al., 2006). In this study, we aimed to develop
a dual-ligand drug delivery system (TAT/TF-PEG-LP) to enhance
the targeting selectivity and cellular uptake efficiency both in vitro
and in vivo, in which TAT and TF were steadily connected with the
cholesterol anchor via a stable ether linkage. The size data (Table 1)
and the transmission electron micrographs (see supplementary
material) showed that the dual-ligand liposomes kept the perfect
liposome structure and dispersed uniformly.
In previous studies, cationic macromolecules have been
reported to trigger thrombosis, embolization and hemolysis in vivo
(Antohi and Brumfeld, 1984; Zhu et al., 2007). Therefore, the pres-
ence of cationic peptides such as TAT on the liposomal surface
can induce some undesirable side effect. The presence of cationic
peptides TAT on liposomes can induce interactions with the ery-
throcyte membrane causing cell lysis and release of hemoglobin
(see supplementary material). Furthermore, the TAT was known
as a nonspecific molecule, which penetrates any cells. And in this
study, we utilized the PEG chain length difference of two functional
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Fig. 6. Ex vivo imaging of tumors given different liposomes via tail vein. A, TAT/TF-
PEG-LP; B, TAT-PEG-LP; C, TF-PEG-LP; D, PEG-LP; Blank, saline (n = 4).
Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
with cholesterol anchored transferrin and TAT. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.06.048

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Fig. 7. CLSM images of HepG2 tumor frozen sections from tumor-bearing mice receiving different formulations of DiD loaded liposomes. A, blank; B, TAT/TF-PEG-LP; C,
TAT-PEG-LP; D, TF-PEG-LP; E, PEG-LP. The row I was the bright field. The blue fluorescence exhibited in row II was due to DAPI-staining of the nuclei. The red fluorescence
exhibited in row III was due to the DiD loaded liposomes. The forth row was the overlay sight. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
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materials (CHO-PEG-TF and CHO-PEG-TAT) in the dual-ligand lipo-
somal formulations to ensure that TAT could be shielded during
circulation. To make sure the dual-ligand liposomes possess the
strongest synergistic effect on cellular uptake and proper masking
effect to the non-specific TAT, the PEG chain length of ligand which
could affect the cell binding and cellular uptake should be first iden-
tified (see supplementary material). The result showed that the PEG
chain length combinations of CHO-PEG2000-TAT/CHO-PEG3500-TF
could make the dual-ligand liposomes achieve the most effective
synergistic effect on cellular uptake, and in this condition, as shown
in Table 1, the positive charge of TAT/TF-PEG-LP was lower than that
of TAT-PEG-LP, which indicated that the TAT could be masked by
the longer PEG chain of CHO-PEG3500-TF to some extent. Then the
hemolytic toxicity of TAT/TF-PEG-LP was much lower than that of
TAT-PEG-LP in the hemolysis assay (see supplementary material).
So that we speculated the non-specificity, toxicity and instability
of liposomes caused by TAT could be overcame in vivo, which need
to be further proved in the future research.
In this study, we evaluated the synergetic effect of both ligands
and verified the targeting specificity of dual-ligand liposomes via
cellular uptake by three kinds of cells which possessed different
expression levels of TFR on their surface (Fig. 3). As shown in Fig. 5,
compared to the control liposome PEG-LP (purple lines), although
the single-ligand liposomes TAT-PEG-LP (blue lines) increased the
cellular uptake amount to some degree, it exhibited no regular-
ity in three different cells lines due to the non-specificity of TAT,
while the other single-ligand liposomes TF-PEG-LP (brown lines)
only showed a slight enhancement on cellular uptake in HepG2
cells and A2780 cells due to receptor saturation. However, the dual-
ligand liposomes TAT/TF-PEG-LP exhibited the enhanced cellular
uptake and selectivity at the same time. As shown in Fig. 5a, the
cellular uptake amount of TAT/TF-PEG-LP was far more than that of
PEG-LP, TAT-PEG-LP and TF-PEG-LP in HepG2 cells which expressed
substantial TFR on surface, and the synergistic effect of TAT/TF-
PEG-LP on cellular uptake varied from the cells to cells, when the
expression level of TFR on cell surface was lower (such as HUVECs),
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Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
with cholesterol anchored transferrin and TAT. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.06.048

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could partially mask the positive charge of TAT, thus increasing
the stability of liposomes in vivo. These results demonstrated that
an efficient targeted delivery of payload could be achieved by the
dual-ligand modified liposomes in vivo.
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5. Conclusions
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In this study, we successfully developed the dual-ligand lipo-
somes modified with the specific ligand TF motif and non-specific
TAT. This liposomal delivery system possessed increased cellular
uptake efficiency and targeting specificity in the cells whose TFR
expression levels were high, and achieved an efficient synergistic
targeted delivery of payload into tumor cells in HepG2 tumor-
bearing nude mice.
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Fig. 8. Cytometric quantitation of cell suspensions from HepG2 tumor-bearing nude
mice receiving different formulations of DiD loaded liposomes. Data represented the
mean SD. *P < 0.05; **P < 0.01, ***P < 0.001, versus TAT/TF-PEG-LP.
Acknowledgments
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The work was funded by the National Natural Science Founda-
tion of China (81072599), the National Basic Research Program of
China (973 Program, 2013CB932504) and the School of Pharmacy,
Fudan University & the Open Project Program of Key Lab of Smart
Drug Delivery, Ministry of Education & PLA, China. The authors
thank Professor Zhenlei Song for providing help on the synthesis
of cholesterol derivative lipids.
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TAT/TF-PEG-LP could not recognize and bind with target cell by
the TF motif efficiently, hence the cellular uptake of TAT/TF-PEG-LP
was mainly mediated by TAT and almost the same as that of TAT-
PEG-LP (Fig. 5c). The result indicated that only when the amount
of TFR on cells reached a certain point would the synergetic effect
between TAT and TF motif start to appear. It is reported that many
tumor cells expressed much more TFR than normal cells, so the
dual-ligand liposomes in our study were expected to achieve the
effective synergistic targeted delivery. In this condition, the dual-
ligand liposomes attached to TFR on the tumor cell surface via the
specific ligand TF, then penetrated into tumor cells with high effi-
ciency predominantly mediated by TAT (Fig. 2). Consistently, the
CLSM analysis also confirmed the significant synergetic effect of
dual-ligand liposomes on cellular uptake in HepG2 cells (Fig. 4a)
and it was more obvious than in A2780 cells and HUVECs (Fig. 4b
and c), strengthening the point of view that the dual-ligand lipo-
somes possessed increased cellular uptake efficiency and target
specificity in vitro.
The in vivo experiments were performed on the HepG2 tumor-
bearing nude mice, which had higher TFR expressed on the cell
surface in tumor tissue. Fig. 6 exhibited that the tumor distribu-
tions of TAT/TF-PEG-LP and TF-PEG-LP were higher than that of
PEG-LP owing to the active targeting effect of TF motif. We could
also see that the signal from tumors of the mice treated with TAT-
PEG-LP was fairly weak due to the non-specificity and instability of
liposomes caused by TAT. However, the ex vivo imaging of tumors
only reflected the accumulation capacity of liposomes in tumor
tissues but not the ability to enter cells. Then there was not clear
signal difference between TF-PEG-LP and TAT/TF-PEG-LP in Fig. 6
because the targeting ability of liposomes largely depends upon
the passive targeting of PEGylation and the active targeting of TF
motif. Although TAT/TF-PEG-LP and TF-PEG-LP had the similar
distributions in tumors, after they arrive at tumor tissues, with the
help of TAT, the ability for delivery of payload into cells of TAT/TF-
PEG-LP was much stronger than that of TF-PEG-LP according to the
qualitative (Fig. 7) and quantitative evaluations (Fig. 8). As shown
in Fig. 7 the tumor frozen sections of TF-PEG-LP and PEG-LP groups
had little red fluorescence (fluorescence of DiD), indicating that
although TF modified liposome could achieve the active targeting
in vivo according to the previous studies (Hong et al., 2010), it
could not more efficiently target DiD into tumor cells in vivo due
to receptor saturation. This point was further certified by the
cytometric data in vivo (Fig. 8). And the cellular uptake amount of
TAT/TF-PEG-LP in tumor tissues was higher than that of the three
other liposomes (Figs. 7 and 8). The results might be induced by
the increased targeting specificity mediated by TF motif and the
enhanced cellular uptake efficiency predominantly mediated by
TAT. Besides, the aqueous layer of CHO-PEG3500-TF of liposomes
Appendix A. Supplementary data
533
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ijpharm.
2013.06.048.
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References
537
Anabousi, S., Bakowsky, U., Schneider, M., Huwer, H., Lehr, C.M., Ehrhardt, C., 2006.
In vitro assessment of transferrin-conjugated liposomes as drug delivery sys-
tems for inhalation therapy of lung cancer. Eur. J. Pharm. Sci. 29, 367–374.
Antohi, S., Brumfeld, V., 1984. Polycation-cell surface interactions and plasma mem-
brane compartments in mammals. Interference of oligocation with polycationic
condensation. Z. Naturforsch. C 39, 767–775.
Banks, W.A., Robinson, S.M., Nath, A., 2005. Permeability of the blood-brain barrier
to HIV-1 TAT. Exp. Neurol. 193, 218–227.
Brooks, H., Lebleu, B., Vive‘s, E., 2005. Tat peptide-mediated cellular delivery: back
to basics. Adv. Drug Deliv. Rev. 57, 559–577.
Chiu, S-J., Liu, S., Perrotti, D., Marcucci, G., Lee, R.J., 2006. Efficient delivery of a Bcl-
2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-
targeted liposomes. J. Control Release 112, 199–207.
Gabizon, A., Horowitz, A.T., Goren, D., Tzemach, D., Shmeeda, H., Zalipsky, S., 2003.
In vivo fate of folate-targeted polyethylene-glycol liposomes in tumor-bearing
mice. Clin. Cancer Res. 9, 6551–6559.
Gupta, B., Levchenko, T.S., Torchilin, V.P., 2005. Intracellular delivery of large
molecules and small particles by cell-penetrating proteins and peptides. Adv.
Drug Deliv. Rev. 57, 637–651.
Hatakeyama, H., Akita, H., Maruyama, K., Suhara, T., Harashima, H., 2004. Factors
governing the in vivo tissue uptake of transferrin-coupled polyethylene glycol
liposomes in vivo. Int. J. Pharm. 281, 25–33.
Heyes, J., Hall, K., Tailor, V., Lenz, R., MacLachlan, I., 2006. Synthesis and character-
ization of novel poly(ethylene glycol)–lipid conjugates suitable for use in drug
delivery. J. Control Release 112, 280–290.
Hong, M., Zhu, S., Jiang, Y., Tang, G., Sun, C., Fang, C., Shi, B., Pei, Y., 2010. Novel anti-
tumor strategy: PEG–hydroxycamptothecin conjugate loaded transferrin–PEG-
nanoparticles. J. Control Release 141, 22–29.
Kibria, G., Hatakeyama, H., Ohga, N., Hida, K., Harashima, H., 2011. Dual-ligand mod-
ification of PEGylated liposomes shows better cell selectivity and efficient gene
delivery. J. Control Release 153, 141–148.
Kirpotin, D.B., Drummond, D.C., Shao, Y., Shalaby, M.R., Hong, K., Nielsen, U.B., Marks,
J.D., Benz, C.C., Park, J.W., 2006. Antibody targeting of long-circulating lipidic
nanoparticles does not increase tumor localization but does increase internal-
ization in animal models. J. Cancer Res. 66, 6732–6740.
McNeeley, K.M., Annapragada, A., Bellamkonda, R.V., 2007. Decreased circulation
time offsets increased efficacy of PEGylated nanocarriers targeting folate recep-
tors of glioma. Nanotechnology 18, 385101 (11 pp).
Ntziachristos, V., Bremer, C., Weissleder, R., 2003. Fluorescence imaging with
near-infrared light: new technological advances that enable in vivo molecular
imaging. Eur. Radiol. 13, 195–208.
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
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567
568
569
570
571
572
573
574
575
576
577
578
Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
with cholesterol anchored transferrin and TAT. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.06.048

GModel
IJP134361–10
ARTICLE IN PRESS
10
J. Tang et al. / International Journal of Pharmaceutics xxx (2013) xxx–xxx
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
Pan, X., Wu, G., Yang, W., Barth, R.F., Tjarks, W., Lee, R.J., 2007. Synthesis of cetuximab-
immunoliposomes via a cholesterol-based membrane anchor for targeting of
EGFR. Bioconj. Chem. 18, 101–108.
Qin, Y., Chen, H., Yuan, W., Kuai, R., Zhang, Q., Xie, F., Zhang, L., Zhang, Z., Liu, J., He,
Q., 2011. Liposome formulated with TAT-modified cholesterol for enhancing the
brain delivery. Int. J. Pharm. 419, 85–95.
Sharma, G., Modgil, A., Sun, C., Singh, J., 2012. Grafting of cell-penetrating pep-
tide to receptor-targeted liposomes improves their transfection efficiency and
transport across blood-brain barrier model. J. Pharm. Sci. 101, 2468–2478.
Shohda, K.-i., Sugawara, T., 2006. DNA polymerization on the inner surface of a giant
liposome for synthesizing an artificial cell model. R. Soc. Chem. 2, 402–408.
Singh, M., 1999. Transferrin as a targeting ligand for liposomes and anticancer drugs.
Curr. Pharm. Des. 5, 443–451.
Suzuki, R., Takizawa, T., Kuwata, Y., Mutoh, M., Ishiguro, N., Utoguchi, N., Shinohara,
A., Eriguchi, M., Yanagie, H., Maruyama, K., 2007. Effective anti-tumor activity
of oxaliplatin encapsulated in transferrin–PEG-liposome. Pharm. Nanotechnol.
346, 143–150.
Wadia, J.S., Dowdy, S.F., 2002. Protein transduction technology. Curr. Opin. Biotech-
nol. 13, 52–56.
Xu, H., Deng, Y., Chen, D., Hong, W., Lu, Y., Dong, X., 2008. Esterase-catalyzed dePE-
Gylation of pH-sensitive vesicles modified with cleavable PEG-lipid derivatives.
J. Control Release 130, 238–245.
Yang, X., Koh, C.G., Liu, S., Pan, X., Santhanam, R., Yu, B., Peng, Y., Pang, J., Golan,
S., Talmon, Y., Jin, Y., Muthusamy, N., Byrd, J.C., Chan, K.K., Lee, L.J., Marcucci,
G., Lee, R.J., 2008. Transferrin receptor-targeted lipid nanoparticles for deliv-
ery of an antisense oligodeoxyribonucleotide against Bcl-2. Mol. Pharm. 6,
221–230.
Zhang, C., Liu, T., Su, Y., Luo, S., Zhu, Y., Tana, X., Fan, S., Zhang, L.L., Zhou, Y., Cheng,
T.M., Shi, C.M., 2010. A near-infrared fluorescent heptamethine indocyanine
dye with preferential tumor accumulation for in vivo imaging. Biomaterials 31,
6612–6617.
Zhao, X., Muthusamy, N., Byrd, J.C., Lee, R.J., 2007. Cholesterol as a bilayer anchor for
PEGylation and targeting ligand in folate-receptor-targeted liposomes. J. Pharm.
Sci. 96, 2424–2435.
Zhu, S., Qian, F., Zhang, Y., Tang, C., Yin, C., 2007. Synthesis and characterization
of PEG modified N-trimethyl amino ethyl methacrylate chitosan nanoparticles.
Eur. Polym. J. 43, 2244–2253.
599
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601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
Torchilin, V.P., Rammohan, R., Weissig, V., Levchenko, T.S., 2001. TAT peptide on
the surface of liposomes affords their efficient intracellular delivery even at low
temperature and in the presence of metabolic inhibitors. Proc. Nat. Acad. Sci. 98,
8786–8791.
Please cite this article in press as: Tang, J., et al., Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified
with cholesterol anchored transferrin and TAT. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.06.048