Oestrone-targeted liposomes for mitoxantrone delivery via oestrogen receptor – synthesis, physicochemical characterization and in-vitro evaluation

Article abstract of DOI:10.1111/jphp.12736

Objectives: Targeted delivery of mitoxantrone (MTO, an anthraquinone drug with high antitumour effect) may be achieved using a novel nanoparticulate delivery system via binding the oestrogen receptor (ER, highly expressed in a variety of human tumours). Methods: A novel liposomal nanoparticle (NP) was developed using a conjugate derived from 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀-NH₂) and oestrone (ES, is known to bind the ER) to produce an ES-targeted PEGylated liposome (ES-SSL). The resulting targeted NP was loaded with MTO to produce a targeted liposome-MTO formulation (ES-SSL-MTO). Key findings: The targeted formulation (~140 nm, 1.5 mV) achieved over 95% drug encapsulation efficiency and a favourable stability at 4, 25 and 37 °C up to 48 h. The flow cytometric data indicated that cellular uptake of ES-SSL into human leukaemia HL-60 cells was mediated via binding the oestrogen receptor. In addition, the ES-SSL-MTO significantly reduced the growth of HL-60 cells. Conclusions: Our results provide a proof of principle that ES-modified PEGylated liposomes can target the ER, thereby potentially improving the therapeutic benefits in ER-overexpressed tumours.

Full text of DOI:10.1111/jphp.12736

Research Paper
Oestrone-targeted liposomes for mitoxantrone delivery via
oestrogen receptor – synthesis, physicochemical
characterization and in-vitro evaluation
a
a
a
a
a
a
a
a
Qiang Hao , Guoxing Xu , Yue Yang , Yuxin Sun , Dengli Cong , Hongrui Li , Xin Liu , Zeng Wang ,
a
a
a
a
a
a
a
a
Zheng Zhang , Jinglin Chen , Yao Li , Xue Luan , Lin Wang , Lin Tian , Kun Liu , Yan Li ,
b
a
Qianru Jiao and Jin Pei
a
b
School of Pharmaceutical Sciences, Jilin University, Changchun, Jilin and School of Clinical Medicine, Fujian Medical University, Fuzhou,
Fujian, China
Keywords
Abstract
cancer targeting; chemotherapy; liposomal
nanoparticles; oestrogen receptor
Objectives Targeted delivery of mitoxantrone (MTO, an anthraquinone drug
with high antitumour effect) may be achieved using a novel nanoparticulate
delivery system via binding the oestrogen receptor (ER, highly expressed in a
variety of human tumours).
Correspondence
Jin Pei, School of Pharmaceutical Sciences,
Jilin University, Changchun 130012, Jilin,
China.
Methods A novel liposomal nanoparticle (NP) was developed using a conjugate
derived from 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino
E-mail: peijin@jlu.edu.cn
(
polyethylene glycol)-2000] (DSPE-PEG2000-NH ) and oestrone (ES, is known to
2
Received December 20, 2016
Accepted March 26, 2017
bind the ER) to produce an ES-targeted PEGylated liposome (ES-SSL). The
resulting targeted NP was loaded with MTO to produce a targeted liposome-
MTO formulation (ES-SSL-MTO).
doi: 10.1111/jphp.12736
Key findings The targeted formulation (~140 nm, 1.5 mV) achieved over 95%
drug encapsulation efficiency and a favourable stability at 4, 25 and 37 °C up to
48 h. The flow cytometric data indicated that cellular uptake of ES-SSL into
human leukaemia HL-60 cells was mediated via binding the oestrogen receptor.
In addition, the ES-SSL-MTO significantly reduced the growth of HL-60 cells.
Conclusions Our results provide a proof of principle that ES-modified PEGy-
lated liposomes can target the ER, thereby potentially improving the therapeutic
benefits in ER-overexpressed tumours.
[
13]
reticuloendothelial system (RES),
grafted with polyethylene glycol (PEG) to improve the bio-
liposomes have been
Introduction
[
14–16]
Chemotherapy is one of the main treatments for advanced
logical stability.
Tumour cells commonly overexpress receptors such as
[1]
and metastatic cancer. Among chemotherapeutics, mitox-
antrone (MTO) is an anthraquinone drug with high antitu-
[
17]
[18,19]
epidermal growth factor receptor,
[20]
folate receptor,
[21]
[2]
mour effect on advanced breast cancer, acute myeloblastic
[
transferrin receptor,
[22]
fucose receptor,
[23]
tor,
this study, we have reported a method to attach oestrone
sigma recep-
[24]
3]
[4]
leukaemia and non-Hodgkin’s lymphomas. However,
MTO causes bone marrow suppression, moderate gastroin-
oestrogen receptor (ER)
and antigens
etc. In
[5–7]
[25]
testinal toxicity and cardiotoxicity,
suggesting that safe,
(ES, a ligand of ER) onto 1, 2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[amino (polyethylene glycol)-
effective and stable delivery systems are highly required for
the clinical use of MTO.
2000] (DSPE-PEG2000-NH ) to produce DSPE-PEG2000-ES
2
Liposomes have substantially been used as the drug
carrier due to the biocompatible and biodegradable
(Figure 1). DSPE-PEG2000-ES was incorporated into lipo-
somes using a ‘post-insertion’ method to produce the ES-
[
8–12]
[26]
characteristics.
However, liposomes cause low stability
grafted PEGylated liposomes (ES-SSL).
The resulting
in physiological media and therefore have a tendency for
liver/spleen accumulation. Due to the fact that PEGyla-
tion may significantly reduce the recognition by the
ES-SSL nanoparticles (NPs) were loaded with MTO to
achieve the targeted drug formulation (ES-SSL-MTO). ER
has been found on the plasma membrane of human acute
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
1

Targeted liposomes for chemotherapy
Qiang Hao et al.
Figure 1 (a) Synthesis of oestrone-COOH. (b) Synthesis of DSPE-PEG2000-oestrone.
myeloblastic leukaemia HL-60 cells and is expressed
throughout the cell cycle.
medium, penicillin–streptomycin and foetal bovine serum
(FBS) were purchased from Gibco Inc. (New York, NY,
USA). Mitoxantrone Hydrochloride (MTO), Amiloride
Hydrochloride and Verapamil Hydrochloride were bought
from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Cell
counting kit-8 (CCK-8) kits were purchased from Beyotime
Biotechnology Co., Ltd., Shanghai, China. All reagents and
solvents unless specified were either of analytical or HPLC
grade.
[27]
Therefore, in-vitro cellular
uptake and antitumor effect were assessed using HL-60 cells
for the capacity of targeted liposomes to deliver MTO for
treatment of ER-positive tumours. To our knowledge, this
study provides (for the first time) a proof of principle that
ES-modified PEGylated liposomes can selectively deliver
MTO into cancer cells via targeting the ER.
Materials and Methods
Cell culture
Materials
Human acute myeloblastic leukaemia HL-60 cells were pur-
chased from Procell Life Science Co., Ltd. (Wuhan, China)
and cultured in RPMI 1640 medium containing 12% FBS
and 1% penicillin–streptomycin antibiotics. Cells were cul-
tured at 37 °C in a humidified atmosphere with 5% CO2.
Phosphatidylcholine (PC), 1, 2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[amino (polyethylene glycol)-2000]
(
DSPE-PEG2000-NH ) and 1, 2-distearoyl-sn- glycero-3-
2
phosphoethanolamine-N-[methoxy (polyethylene glycol)-
000] (DSPE-PEG2000) were purchased from Nanocs Inc.
New York, NY, USA). Oestrone was purchased from
2
(
Synthesis of DSPE-PEG2000-ES
TCI Development Co., Ltd. (Shanghai, China). Succinic
anhydride (SAA) and trimethylamine (TEA) were pur-
chased from Tianjin Fu Yu Fine Chemical Co., Ltd.
To a solution of ES (500 mg, 1.85 mmol), TEA (56 mg,
5.56 mmol) and DMAP (23 mg, 0.185 mmol) in dry THF
(4 ml), succinic anhydride (370 mg, 3.7 mmol) was added
and stirred for 42 h at room temperature. The resulting
mixture was concentrated by vacuum centrifugation and
dissolved in 10 ml water. The pH of the solution was
adjusted to 9–10 with K CO and to pH 1 with 12 N HCl.
(
Tianjing, China). Dimethylaminopyridine (DMAP) was
procured from Beijing Ouhe Technology Co., Ltd. (Bei-
jing, China). Tetrahydrofuran (THF), carbonyldiimidazole
(CDI), dichloromethane (CH Cl ) and ether were obtained
2 2
from Beijing Chemical Factory (Beijing, China). RPMI 1640
2
3
2
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Qiang Hao et al.
Targeted liposomes for chemotherapy
The solution was undisturbed to precipitate out the
deposit, following which the content was filtered to obtain
ES-COOH, 674 mg, 98% yield (Figure 1a).
The SSL-MTO was prepared as mentioned above with-
out DSPE-PEG2000-ES; the ES-L-MTO was prepared as
mentioned above without DSPE-PEG2000-NH ; the L-MTO
2
To a solution of ES-COOH (50 mg, 0.135 mmol) in
dichloromethane (2 ml), carbonyldiimidazole (CDI)
was prepared as mentioned above without DSPE-PEG2000-
NH and DSPE-PEG2000-ES.
2
(
21.9 mg, 0.135 mmol) was added and stirred for 1 h at
room temperature. The resulting mixture was added with
DSPE-PEG2000-NH2 (27 mg, 0.0135 mmol) and stirred
overnight. Subsequently, the mixture was filtered and con-
centrated to 0.5 ml. Diethyl ether (10 ml) was then added
dropwise and the deposit was precipitated out in 2 h at a
stationary position. The mixture was filtered to produce
ES-CO-NH-PEG2000-DSPE (thereafter referred as DSPE-
PEG2000-ES), 46 mg, 72% yield (Figure 1b). The structure
Characterization of liposomes
Entrapment efficiency and drug loading capacity
Free MTO was separated from the encapsulated drug by
dialysis (PBS, pH 7.4). Subsequently, the encapsulated
MTO was decomplexed from liposomes using 3% Triton-X
100 and the drug concentration was determined at 655 nm
using a UV-visible spectrophotometer (UV-visible spec-
trophotometer UV2800; Unico (Shanghai) Instrument Co.,
Ltd., Shanghai, China). The MTO concentration was calcu-
lated from the calibration curve over the linear range from 1
to 24 g/ml. The Entrapment efficiency (EE) was calculated
using the formula:
1
of DSPE-PEG2000-ES was confirmed using NMR and
matrix-assisted laser desorption/ionization time of flight
mass spectroscopy (MALDI-TOF) (Autoflex speed TOF/
[28]
TOF, Bruker, Germany).
Preparation of liposomes
EE ¼ ðdrug concentration after dialysis=
drug concentration before dialysisÞ ꢀ 100%:
Drug lading was calculated as the percentage of the mass
of MTO encapsulated in liposomes to the mass of the
MTO-loaded liposomes.
Mitoxantrone-loaded liposomes were prepared using the
[29]
ammonium sulphate gradient method.
cholesterol and DSPE-PEG2000 in the molar ratio of
1 : 9 : 1 were dissolved in chloroform and dried to a thin
Briefly, PC,
1
lipid film at 40 °C by rotary vacuum evaporation (Rotary
Evaporator R201D, Yukang, China). The lipid film was rehy-
drated with 300 mM ammonium sulphate solution for 4 h,
which was followed by a vortex oscillation for 5 min and a
sonication in an ice-bath for 10 min to produce liposomes.
The liposomes were extruded 5 times through 0.22 lm
polycarbonate membrane and 5 times through 0.08 lm poly-
Particle size and zeta potential
The particle size and zeta potential of liposomes containing
MTO were measured using the dynamic light scanning
(Zetasizer Nano ZS; Malvern Instruments, Worcestershire,
UK). The concentration of MTO was fixed at 1 mg/ml.
[30]
carbonate membrane, respectively.
Subsequently, the
DSPE-PEG2000-ES solution was added to the liposome solu-
tion at the molar ratio of 1 : 200 (DSPE-PEG2000-ES : lipids)
and incubated at 37 °C for 1 h. The resulting targeted lipo-
somes (ES-SSL) were further incubated with MTO at 37 °C
under different conditions (Table 1). Free MTO was
removed from the encapsulated drug (ES-SSL-MTO) by dial-
ysis against phosphate-buffered saline (PBS, pH 7.4).
Transmission electron microscopy
The morphology of liposomes was examined using trans-
mission electron microscopy (TEM) (JEM-2100F; JEOL
Ltd., Tokyo, Japan). For TEM sample preparation, ES-SSL-
MTO (95 lg/ml of MTO) was placed on a copper grid
coated with carbon film and stained with 1% phospho-
Table 1 Entrapment efficiency and drug loading of oestrone-SSL-
mitoxantrone prepared at 37 °C under different conditions (N.T. = not
tested) (Mean ꢁ SD, n = 3)
[
31]
tungstic acid.
Volume of
Stability study
ammonium Incubation
Drug:lipid
sulphate
Time with
The ES-SSL-MTO was stored at 4 and 25 °C in PBS (pH
7.4) up to 48 h and the particle sizes were then measured
using Malvern Zetasizer Nano ZS. In addition, the EE was
measured using the method described above to determine
the release of MTO from liposomes during the storage.
In addition, the ES-SSL-MTO was incubated in 50% (v/v)
FBS at 37 °C and the size distribution was analysed using
Malvern Zetasizer Nano ZS.
(molar ratio) solution (ml) MTO (h)
EE (%)
DL (%)
1:20
10
1
1
1
1
2
4
80.22 ꢁ 0.63 2.22 ꢁ 0.01
64.46 ꢁ 0.65 1.82 ꢁ 0.03
82.89 ꢁ 0.73 2.33 ꢁ 0.02
84.26 ꢁ 0.26 3.13 ꢁ 0.04
95.47 ꢁ 0.41 4.28 ꢁ 0.03
77.88 ꢁ 1.35 2.91 ꢁ 0.03
1
2
5
0
1:15
20
2
2
0
0
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
3

Targeted liposomes for chemotherapy
Qiang Hao et al.
incubated with serum-free medium containing coumarin-
Competitive binding study
6-(4 lg/ml)loaded ES-SSL for 2 h. Cells were harvested at
Liposomes containing coumarin-6 (a lipophilic fluorescent
[
1000 rpm for 5 min, washed three times with ice-cold PBS
(pH 7.4) and resuspended in 500 ll PBS for flow cytometry
(Cytomics FC 500; Beckman Coulter Ltd.).
32,33]
probe, entrapped inside the bilayers of liposomes
)
TM
were prepared by the methods described above, in which
coumarin-6 was dissolved in chloroform with lipids before
drying to a thin lipid film. Free coumarin-6 was removed
using dialysis against PBS (pH 7.4).
Statistical analyses
HL-60 cells were seeded in 12-well plates at a density of
5
Data are represented as the mean ꢁ standard deviation of
at least three independent experiments. Statistical compar-
isons were performed using one-way analysis of variance
with SPSS19.0 software (SPSS Inc.). P < 0.05 was consid-
ered significant.
5
9 10 cells/well and incubated overnight at 37 °C. For
the competitive binding study, cells were added with or
without 0.15 mM ES and incubated for 30 min. Free ES was
removed at 113g for 5 min. After this, cells were added with
SSL and ES-SSL formulations containing Coumarin-6
(
4 lg/ml) in serum-free medium and incubation for 4 h.
Following incubation, cells were harvested (1000 rpm for
min), washed thrice with ice-cold PBS (pH 7.4), and
Results
5
Characterization of DSPE-PEG2000-ES
resuspended in 500 ll ice-cold PBS (pH 7.4) for flow
cytometry (Cytomics FC 500; Beckman Coulter Ltd.,India-
TM
The structure of ES, ES-COOH, DSPE-PEG2 -NH and
000
1
DSPE-PEG2000-ES was confirmed by H NMR spectra (Var-
2
napolis IN, USA). 10 000 cells were measured for each sam-
ple. Coumarin-6 was excited at 488 nm and detected at
[38]
ian-300 MHz NMR, Varian, USA) (Figures S1 and 2).
1
The typical H NMR spectrum (300 MHz, CDCl ) of
5
25 nm.
3
DSPE-PEG2000-ES showed peaks at d 9.38 (m, 2H, 2 9
H-Ar), 8.20 (m, 1H, H-Ar) (Figure 2b).
Antiproliferative effect
In addition, MALDI-TOF MS showed that the molecular
weight (M ) of DSPE-PEG -NH is 2175 (Figure 2c).
DSPE-PEG2000-NH2 was reacted with ES-COOH
The antiproliferative effect of MTO alone and MTO-loaded
liposomes was assessed using HL-60 cells with CCK-8
w
2000
2
[
34]
4
Cells were seeded at 1 9 10 cells/well in 96-well
kits.
plates at 37 °C overnight. After this, cells were added with
MTO at 0.05, 0.25, 0.5, 2.5, 5 and 25 ng/ml or their liposo-
(M = 370) to form DSPE-PEG -ES (one H O molecule
w
2000
2
was removed to form the amide group, see Figure S2), so
the theoretical Mw of DSPE-PEG2000-ES is 2527. Data of
MALDI-TOF MS in Figure 2d showed that the Mw of
[35]
mal formulations and incubated for 48 h.
incubation, 10 ll CCK-8 were added to each well. After
h, the results were measured at 450 nm using a micro-
Following
DSPE-PEG2000-ES is 2521. The difference in M of DSPE-
w
4
PEG2000-ES between the theoretical value (2527) and the
value obtained from MALDI-TOF MS (2521) is most likely
plate reader (DNM-9602; Beijing Perlong New Technoiogy
Co., Ltd, Beijing, China). The relative cell viability (%) was
calculated as follows:
due to the polydispersity of PEG2000 (M of PEG
w
ranges
2000
from 1800 to 2200 in accordance with the manufacturer’s
instructions). Therefore, results in Figure 2 confirmed suc-
cessful synthesis of the DSPE-PEG2000-ES conjugate.
Cellviabilityð%Þ¼ðAsample ꢂAblankÞ=ðAcontrol ꢂAblankÞꢀ100;
where Acontrol and Asample are the absorbances in the
absence and presence of sample treatment, respectively.
Ablank is the background absorbance of the medium. The
IC50 of different groups was calculated using SPSS 19.0
Characterization of liposomes
Encapsulation efficiency and drug lading capacity
(
SPSS Inc., Chicago, IL, USA).
The EE of drug formulations was determined under differ-
ent conditions including the molar ratio of MTO and lipo-
somes, volume of ammonium sulphate solution and
incubation time with MTO (Table 1).
Internalization mechanisms of ES-SSL-MTO
HL-60 cells were seeded in 12-well plates at a density of
5
When the volume of ammonium sulphate solution was
20 mL and the incubation time was 1 h, the EE was 82.89%,
84.26% and 75.49% (data not shown in Table 1) at a drug:
lipid molar ratio of 1 : 20, 1 : 15 and 1 : 10 (drug: lipids),
respectively. The results indicated that ES-SSL achieved bet-
ter EE at smaller drug: lipid molar ratio (Table 1).
5
were then added with inhibitory agents
9 10 cells/well and incubated overnight at 37 °C. Cells
[36,37]
including
sucrose (0.45 M), genistein (0.2 M) and amiloride
hydrochloride (0.01 M) and incubated for 30 min. Follow-
ing incubation, cells were washed with growth medium and
4
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Qiang Hao et al.
Targeted liposomes for chemotherapy
1
1
Figure 2 (a) H-NMR spectrum of DSPE-PEG2000-NH
2
. (b) H-NMR spectrum of DSPE-PEG2000-ES. (c) MALDI-TOF MS of DSPE-PEG2000-NH
2
(M
w
= 2175). (d) MALDI-TOF MS of DSPE-PEG2000-ES (M
w
= 2521).
At the molar ratio of 1 : 20 and 1 h incubation time, the
Stability study
EE was 80.22%, 64.46%, and 82.89% with 10, 15 and 20 ml
ammonium sulphate solution, respectively (Table 1).
At the molar ratio of 1 : 20 and 20 ml ammonium sul-
phate solution, the EE was 84.26%, 95.47% and 77.88%
corresponding to the hydration times of 1, 2 and 4 h,
respectively (Table 1).
The particle sizes of ES-SSL-MTO were 120 and 150 nm
when stored at 4 and 25 °C in PBS (pH 7.4) for 48 h,
respectively, which were not significantly changed in com-
parison with the data of Table S1 and Figure 3a. In addi-
tion, the leakage of MTO from liposomes was also
determined. As shown in Figure 4a, less than 8% drugs
were released from SSL-MTO and ES-SSL-MTO (PEGy-
lated formulations) at 4 °C within 48 h, respectively (Fig-
ure 4a). In addition, less than 15% drugs were released
from SSL-MTO and ES-SSL-MTO at 25 °C within 48 h
As shown in Table 1, the effects on DL of ES-SSL under
different conditions were similar to those recorded in EE of
ES-SSL. Therefore, ES-SSL-MTO that were prepared at the
molar ratio of 1 : 15 when incubated with MTO for 2 h
within 20 mL ammonium sulphate solution (EE = 95.47%
and DL = 4.28%), were used in following studies.
(
Figure 4b). The drug leakage of PEGylated formulations
was significantly less than the results from L-MTO and ES-
L-MTO (non-PEGylated counterparts) (Figure 4a and 4b).
In addition, the particle size distribution results indicated
that no significant aggregation occurred when ES-SSL-MTO
was incubated in 50% FBS at 37 °C for 24 h (Figure 4c),
Particle size and zeta potential
The particle size of liposome-MTO formulations was in the
range of 110–140 nm (Table S1). The particle size distribu-
tion of ES-SSL-MTO was shown in Figure 3a. The particle
size and zeta potential of ES-SSL-MTO were 139.3 ꢁ
[
39]
which was similar to data previously recorded. However,
slight non-specific binding of serum proteins onto liposomes
took place at 48 h under the same conditions (Figure 4d).
1
.15 nm (PDI = 0.12 ꢁ 0.008) and 1.54 ꢁ 0.53 mV,
respectively (Table S1).
Cellular uptake
Transmission electron microscopy
A flow cytometry analysis was used to determine the cellular
uptake of coumarin-6-loaded SSL and ES-SSL. Results
showed that the number of fluorescence-positive cells
treated with coumarin-6-loaded ES-SSL (Figure 5a) was 17-
fold higher than that of coumarin-6-loaded SSL (Figure 5b).
The TEM image presented a homogenous and spherical
nature of ES-SSL-MTO (Figure 3b). In addition, TEM con-
firmed the particle size (~140 nm) of ES-SSL-MTO
obtained by dynamic light scanning.
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
5

Targeted liposomes for chemotherapy
Qiang Hao et al.
Figure 3 (a) Particle size distribution of oestrone-SSL-mitoxantrone. (b) transmission electron microscopy of oestrone-SSL-mitoxantrone.
In addition, ES as a competitive inhibitor was used to
block the ER, resulting in a significant cellular uptake
Discussion
reduction (~4 folds) of ES-SSL compared to ES-SSL
without the treatment of ES (Figure 5b and 5d). In con-
trast, cellular uptake of SSL was not significantly changed
with or without pretreatment of ES (Figure 5a and 5c).
These results indicated that the internalization of targeted
In this study, we have developed an ES-targeted sterically
stabilized liposome NP for MTO delivery. ES as a ligand of
ER was covalently coupled to DSPE-PEG2000-NH (DSPE-
2
PEG2000-ES). The DSPE-PEG2000-ES was transferred into
the monolayer of preformed liposomes (ES-SSL-MTO)
[
26]
formulations was achieved in
manner.
a
receptor-dependent
using a ‘post-insertion’ method.
Results in Table 1 indi-
cated that the EE and DL were significantly influenced by
the molar ratio of MTO and lipid, volume of ammonium
sulphate solution and incubation time with MTO. As a
result, ~95% EE and ~4% DL were achieved at the molar
ratio of 1 : 15 (drug to lipids) when incubated with MTO
at 37 °C for 2 h within 20 ml ammonium sulphate solu-
tion (Table 1). Optimization of the targeted formulations
under different conditions, to improve the EE and DL, will
be the focus of future work.
Antiproliferative effect
HL-60 cells were incubated with MTO (0.05, 0.25, 0.5, 2.5,
and 25 ng/ml) and MTO-loaded liposomal formulations
5
for 48 h. As shown in Figure 6, the ES-SSL-MTO had a sig-
nificantly stronger antiproliferative effect (P < 0.05) rela-
tive to MTO, L-MTO, ES-L-MTO and SSL-MTO. In
addition, the IC50 values were 29, 13, 7, 2.5 and 0.7 ng/ml
for MTO, L-MTO, ES-L-MTO, SSL-MTO and ES-SSL-
MTO, respectively.
Using dynamic light scattering at the aforementioned
conditions, the diameters of the non-PEGylated liposome,
non-targeted liposome and targeted liposome NPs and
their formulations loaded with MTO (L-MTO, SSL-MTO
and ES-SSL-MTO) were in the range of 110–140 nm
Uptake inhibition in present of different
inhibitors
(
Table S1). It is known that tumour blood vessels have
leaky vasculature gaps, which may result in the enhanced
[
40]
Results in Figure 7 presented that the internalization of ES-
SSL in HL-60 cells was significantly reduced using sucrose
permeability and retention (EPR) effect.
can be utilized for improved penetration and accumulation
The EPR effect
[
40]
(
an inhibitory agent for clathrin-mediated endocytosis),
of NPs with a diameter <200 nm.
The particle size of
genistein (an inhibitory agent for caveolae-mediated endo-
cytosis) and amiloride (an inhibitory agent for
macropinocytosis). Although there is an evidence of the
receptor-mediated internalization of targeted formulations
in HL-60 cells (Figure 5), results in Figure 7 indicated that
ES-SSL also induced non-specific or receptor-independent
uptake.
ES-SSL-MTO was ~140 nm (Figure 3a and Table S1), sug-
gesting that ES-SSL-MTO may accumulate inside the
[
41]
tumour area via the EPR effect.
When NPs are intravenously administered they may
non-specifically bind serum proteins causing instability in
the blood. It is well established that PEGylation has been
[41]
substantially used to improve the stability of NPs.
6
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Qiang Hao et al.
Targeted liposomes for chemotherapy
Figure 4 (a) Drug released from oestrone-SSL at 4 °C (n = 3). (b) Drug released from oestrone-SSL at 25 °C (n = 3). (c) Particle size distribution
of oestrone-SSL-mitoxantrone incubated in 50% (v/v) foetal bovine serum at 37 °C for 24 h. (d) Particle size distribution of oestrone-SSL-mitoxan-
trone incubated in 50% (v/v) foetal bovine serum at 37 °C for 48 h.
Indeed, the particle sizes of ES-SSL-MTO were not signifi-
cantly changed when incubated at 4 and 25 °C. In addi-
tion, results of the ES-SSL-MTO formulation integrity in
Figure 4a and 4b indicated that ~20% MTO was released
from non-PEGylated liposomal formulations (L-MTO and
ES-L-MTO) when incubated at 4 and 25 °C up to 48 h; in
contrast, slight drug leakage (<8%) was evident with
PEGylated counterparts (SSL-MTO and ES-SSL-MTO) at
same conditions, confirming that PEGylation may enhance
the stability of liposomal formulations. It is also worth
noting that ES-SSL-MTO did not non-specifically bind
serum proteins when incubated with FBS at 37 °C up to
significantly reduced by pretreatment with free ES (Fig-
ure 5d). These results indicate that cellular uptake of
ES-SSL was mediated by the ER, implying that ES-SSL-
MTO may specifically deliver MTO into HL-60 cells.
Recently,
a
novel anisamide-targeted amphiphilic
cyclodextrin (CD) nanoparticle (NP) has developed by
Evans et al. that the anisamide (4-methoxybenzamide, a
hydrophobic molecule) was introduced into DSPE-
PEG5000-Maleimide to form DSPE-PEG5000-anisamide and
DSPE-PEG5000-anisamide was subsequently incorporated
into the lipidic bilayer structure of the amphiphilic CD NPs
[
42]
following the postinsertion approach.
As a result, the
24 h (Figure 4c), which was similar to data previously
recorded.
anisamide-targeted NPs achieved specific uptake via the
ligand–receptor binding, suggesting that the anisamide is
placed at the distal end of the targeted CD NPs with DSPE-
PEG5000 in between. Therefore, results in Figure 5c and 5d
suggest that the oestrone (a hydrophobic molecule) is most
likely placed at the distal end of the targeted liposomes with
DSPE-PEG2000 in between (see the schematic of ES-SSL-
MTO in Figure 8).
[
39]
These results suggest that PEGylation may
provide a sterically stabilized effect for ES-SSL-MTO,
potentially leading to the long blood circulation and more
opportunity to accumulate in tumours following the EPR
[
41]
effect.
this hypothesis.
Future in-vivo work will be required to confirm
The ER is overexpressed on many human tumours. In
this study, HL-60 cells that are known to highly express the
ER, were used as a reference cell line for in-vitro investiga-
Indeed, ES-SSL-MTO significantly reduced the cell
growth (<5% cell viability at 48 h) relative to MTO,
L-MTO, ES-L-MTO and SSL-MTO (Figure 6). In addition,
blank liposomes (ES-SSL) as a control did not cause any
cell death, suggesting that the antiproliferative effect of
ES-SSL-MTO was not due to the cytotoxicity from ES-SSL
[
27]
tions.
Results in Figure 5a and 5b presented that ES-SSL
achieved significantly higher cellular uptake (~74% fluores-
cein-positive cells) relative to non-targeted SSL (~4%). In
addition, cellular internalization of ES-SSL-MTO was
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
7

Targeted liposomes for chemotherapy
Qiang Hao et al.
Figure 5 Fluorescence-positive HL-60 cells (%, mean ꢁ SD, n = 3) after transfection of coumarin-6-loaded liposomes with or without 2 h
pretreatment of 0.15 mM oestrone (*P < 0.05). (a) Coumarin-6-loaded SSL without pretreatment. (b) Coumarin-6-loaded oestrone-SSL without
pretreatment. (c) Coumarin-6-loaded SSL with pretreatment. (d) Coumarin-6-loaded oestrone-SSL with pretreatment.
Figure 6 Viability of HL-60 cells treated with mitoxantrone alone
and mitoxantrone-loaded liposomal formulations for 48 h. Control
Figure 7 Effect of endocytosis inhibitors on cellular uptake of
means empty oestrone-SSL. Data were presented as mean ꢁ SD
oestrone-SSL in HL-60 cells. (*P < 0.001, compared with oestrone-
(
n = 5). (*P < 0.001 vs oestrone-SSL-mitoxantrone).
SSL, mean ꢁ SD, n = 3).
but due to the capacity of ES-SSL to deliver MTO into
ER-positive cells.
significantly less efficient than ES-SSL-MTO (Figure 6).
Therefore, the internalization mechanisms of these liposo-
mal NPs were further assessed using endocytosis inhibitory
It is interesting to note that L-MTO, ES-L-MTO and
SSL-MTO formulations also reduced cell growth, although
8
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Qiang Hao et al.
Targeted liposomes for chemotherapy
Figure 8 Targeted delivery of mitoxantrone using oestrone-targeted liposomal nanoparticles via the oestrogen receptor. These ligand-incorpo-
rated nanoparticles may guide the mitoxantrone delivery into oestrogen receptor-positive cancer cells, mainly through the ligand–receptor-
mediated endocytosis pathway. When mitoxantrone is released from nanoparticles it plays a role as a type II topoisomerase inhibitor to disrupt the
[2]
[3]
[4]
synthesis and repair of DNA in cancer cells (i.e. breast cancer, acute myeloblastic leukaemia and non-Hodgkin’s lymphomas ).
agents (Figure 7). It is known that endocytosis occurs
mainly in four mechanisms: clathrin-mediated endocyto-
sis, caveolae, micropinocytosis and clathrin- and caveolae-
via the oestrogen receptor. The resulting ES-SSL-MTO
formulation showed favourable physicochemical features
in terms of particle size and stability, which are com-
patible to requirements for prolonged circulation in the
blood. Specific cellular uptake of ES-SSL-MTO was
achieved via the ligand–receptor-mediated pathway,
leading to efficient antiproliferative effect in ER-positive
HL-60 cells. In order to optimize the targeted drug
formulations in the treatment of ER-positive cancer,
the grafting density of PEG with lipids will be investi-
gated to reduce non-specific internalization in future
work.
[37,43]
independent endocytosis.
mediated endocytosis was assessed using sucrose,
The effect of clathrin-
a type
[44]
of clathrin-coated pit formation blocking agent. Genistein,
an inhibitory agent disrupting caveolae, was used to evalu-
[
45]
ate the effect of caveolae-mediated endocytosis.
addition, amiloride as a microtubule-disrupting agent
In
46]
[
was employed to assess the effect of macropinocytosis.
Results of Figure 7 indicated that cellular uptake of ES-
SSL was significantly reduced with pretreatment of
sucrose, genistein and amiloride, which was similar to
results obtained from ES-L and SSL (data not shown),
suggesting that the internalization of these liposomal for-
mulations were mediated via clathrin-mediated endocyto-
sis, caveolae and micropinocytosis. However, such non-
specific cellular uptake may cause unwanted cytotoxicity.
It has been reported that PEGylation may decrease cellular
Declarations
Conflict of interests
All Authors declare no conflict of interests.
[47,48]
uptake due to the steric hindrance.
The effect of the
Acknowledgements
density of PEG on inhibition of non-specific cellular
uptake and the subsequent in-vitro and in-vivo activity of
ES-SSL-MTO will be investigated in future studies.
This work was funded by Science and Technology Develop-
ment Project of Jilin Science and Technology Department
(
20130727054YY, 20060410-3), and Jilin Province Develop-
ment and Reform Commission (2014N149). We would like
to thank Professor Jianfeng Guo for helpful comments on
the paper.
Conclusions
In this study, an oestrone-targeted PEGylated liposomal
nanoparticle has been prepared to deliver mitoxantrone
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
9

Targeted liposomes for chemotherapy
Qiang Hao et al.
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