Article

Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX pubs.acs.org/molecularpharmaceutics

Development of a Liposomal Formulation of Acetyltanshinone IIA

for Breast Cancer Therapy
Qi Wang,† Man Luo,† Na Wei,† Alex Chang,‡ and Kathy Qian Luo*,§
†
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457
‡
Department of Oncology, Johns Hopkins Singapore, Singapore 308433
§
Faculty of Health Sciences, University of Macau, Taipa, Macau, China
*
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ABSTRACT: Acetyltanshinone IIA (ATA), synthesized in our group exhibiting

good anti-breast cancer effects, is expected to replace the commonly used anti-ER+
breast cancer (breast cancer cells overexpressing the estrogen receptor) drug
tamoxifen. To promote the clinical progress of ATA, polyethylene glycol (PEG)-
modified liposomes were used to encapsulate ATA along with improving its
bioavailability and in vivo anticancer efficiency. The resulting liposomal ATA
exhibited a spherical shape with an average size of 188.5 nm. In vitro evaluations
showed that liposomal ATA retained the anti-breast cancer efficacy of ATA while
exerting much less cytotoxicity toward noncancerous cells. Significantly,
pharmacokinetics analysis showed that the AUC0−24h of liposomal ATA was 59
times higher than that of free ATA, demonstrating increased bioavailability of ATA.
Preclinical experiments demonstrated that liposomal ATA reduced the growth of
ER-positive human breast tumor xenografts by 73% in nude mice, and the
liposomal ATA exhibited a much lower level of toxicity than that of free ATA with
respect to zebrafish larval mortality, body formation, and heart function during development. Moreover, 7-day and 21-day tissue
toxicity levels were determined in mice by intravenous administration of a maximum dosage of liposomal ATA (120 mg/kg).
The results showed no obvious tissue damage in major organs, including the heart, liver, spleen, kidney, and brain. In summary,
we have developed a clinical formulation of liposomal ATA with the high bioavailability and potent efficacy for the treatment of
ER-positive breast cancer.
KEYWORDS: anti-breast cancer drugs, acetyltanshinone IIA (ATA), mPEG-liposomes, bioavailability, pharmacokinetics,
toxicity study

1. INTRODUCTION Encapsulated by poly(ethylene glycol) methyl ether-block-

Breast cancer has become a major public health problem in the
current society and suffered from the resistance of frequently
used anti-breast cancer drugs including tamoxifen. Therefore, a
new chemotherapeutic agent for more effective breast cancer
treatment has been urgently needed recently. Tanshinone IIA
(TIIA), one of the major hydrophobic components of
Danshen,1−3 has received major attention from the scientists
for the high anticancer activity.4 Deriving from TIIA,
acetyltanshinone IIA (ATA) was synthesized in our group
and was further approved to have high anti-breast cancer
efficacy.5 Specifically, ATA displayed a strong growth-
inhibitory effect on estrogen receptor-positive (ER+) breast
cancer cells through degrading the ERα protein.6 More
significantly, ATA produced a more potent growth inhibitory
activity toward ER+ breast cancer cells than that of tamoxifen,
which is a commonly used anti-ER+ breast cancer drug.6
In light of improved therapeutic effects, it is of great
potential to develop ATA into a new anti-breast cancer drug.
However, our initial pharmacokinetic study showed that ATA
was quickly removed from the plasma in both mouse and rats.
poly(lactide-co-glycolide) (mPEG-PLGA), the 24 h blood-
circulation time of ATA was extended to 10-fold that of
unbound ATA.7 However, the majority of the ATA was
trapped in red blood cells rather than in plasma.
Herein, it is urgent to develop ATA into a clinical
formulation to promote its clinical process. In this report,
Federal Drug Administration-approved liposomes have at-
tracted our attention that the liposome-based formulations
represented excellent biocompatibility and improved bioavail-
ability in vivo.8−12 Also, a conventional liposome is a mature
and widely used intravenous dosage form among all the
contemporary formulations,8−12 which ensures the further
clinical evaluation of liposomal ATA. In this report, the main
constituents of liposomal ATA are phospholipids and
cholesterol, which are components of the mammalian cell
Received: May 6, 2019
Revised: July 24, 2019
Accepted: July 26, 2019

© XXXX American Chemical Society A DOI: 10.1021/acs.molpharmaceut.9b00493

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Molecular Pharmaceutics Article

Figure 1. (A) Structural features of liposomal ATA. ATA molecules were encapsulated in the lipid bilayer of the liposome. The PEG chain is
located on the surface of the liposome. (B) SEM images. The liposomal ATA was uniformly distributed. (C) Size distributions. The average size of
liposomal ATA was 188.5 nm based on intensity. (D) Zeta potential. The zeta potential of liposomal ATA was −31.0 mV.

membrane, making them compatible with biological systems.
On the other hand, poly(ethylene glycol) methyl ether (PEG)-
modified phospholipids were utilized to prevent the engulf-
ment of liposomes by blood cells (Figure 1A). Also, PEG
chains are expected to increase the circulation time of
liposomal ATA in vivo, thus improving its bioavailability.
Furthermore, based on previous reports, we hypothesized that
the manufacture of liposomal ATA with a smaller particle size
might facilitate its ability to reach tumor tissues through an
enhanced permeability and retention (EPR) effect, resulting in
an enhanced antitumor effect.13−16
The liposomal ATA was prepared using a reverse-
evaporation method and was optimized through an orthogonal
array test generating the drug loading efficiency of 12.35% with
the size of 188.5 nm. The liposomal ATA formulation could be
stably stored at 4 °C and diluted in 5% glucose in clinical
storage and use. Specifically, important preclinical experiments
were conducted in this report including pharmacokinetic
profiles, anti-breast cancer efficacy, cytotoxicity, and small-
animal toxicity to pave the way for clinical use of liposomal
ATA. The information obtained from this study can be used to
B DOI: 10.1021/acs.molpharmaceut.9b00493
guide the future manufacturing and development of liposomal
ATA into a new anti-breast cancer drug.
2. MATERIALS AND METHODS
2.1. Materials. Pure phosphatidylcholine (PC3) and 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-
(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG)
were obtained from LIPOID Pte. Ltd. (Singapore). Choles-
terol, sodium dodecyl sulfonate (SDS), Tween 80, dialysis
sacks (Mw cutoff of 12,000 kDa), and 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased
from Sigma-Aldrich (St. Louis, MO). Chloroform (CHCl3)
and methanol (MeOH) were obtained from Merck Pte. Ltd.
(Singapore).
Dulbecco’s Minimum Essential Medium (DMEM), pen-
icillin/streptomycin (PS), and trypsin were purchased from
GIBCO. Fetal bovine serum (FBS) was purchased from
HyClone. Human umbilical vein endothelial cells (HUVECs)
and MCF-7 breast cancer cells were obtained from the ATCC
(Manassas, VA).
2.2. Preparation and Characterization of ATA-Loaded
PEG-Liposomes (Liposomal ATA). A reverse-evaporation
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Molecular Pharmaceutics
method was used to prepare the liposomal ATA. First, PC3
(120.8 mg), ATA (29.9 mg), DSPE-PEG (39.9 mg), and
cholesterol (52.8 mg) were dissolved in chloroform (CHCl3, 5
mL). Distilled (DI) H2O (1.5 mL) was then added to the
solution and treated with ultrasound (37% strength) for 3 min.
CHCl3 was then removed by rotary evaporation. Finally, DI
H2O (10 mL) was added to rehydrate it to obtain liposomal
ATA, and the liposomal ATA was ultrasonicated for 8 min to
produce a smaller particle size. Blank liposomes were prepared
using the same method but without the addition of ATA to the
formulation.
The size distribution and zeta potential of the liposomes
were determined using a laser electrophoretic light scatter
analyzer (Malvern Nano-ZS Particle Sizer). The morphology
was determined by scanning electron microscopy (SEM)
(JSM6700-FESEM, JEOL). Briefly, liposome samples were
spread onto the surface of a carbon tape and then sputter-
coated with gold under a vacuum. Afterward, the prepared
samples were observed under a scanning electron microscope
using magnification ranges of 5000−20000 with an electron
beam energy of 20 kV.
2.3. Determination of Encapsulation Efficiency and
the Drug Loading Rate. A solution of liposomal ATA (1
mL) was centrifuged at a low speed of 500 rpm at 28 °C for 5
min to remove large particles. A fixed amount of the
supernatant containing liposomal ATA was diluted with
methanol, which will dissolve the liposome structure to release
ATA to the desired concentration, and the concentration of
ATA in the supernatant was referred to as the concentration of
the encapsulated drug. The concentration of ATA was
measured simultaneously by RP-HPLC analysis (Shimadzu
LC-20AT pump liquid chromatograph; chromatographic
column: Kromasil 100-5C8, 250 mm × 4.6 mm, 5 μm). A
mobile phase system consisting of methanol−H2O (80:20, v:v)
was pumped at a flow rate of 1 mL/min with a column
temperature of 30 °C, and the column eluents were monitored
at a wavelength of 254 nm.7,17,18 The drug loading rate (DL%)
and percentage of encapsulation efficiency (EE%) were
calculated as follows:
DL% = amount of drug in liposomes
/amount of feeding materials
EE% = amount of drug in liposomes
/amount of feeding drug
2.4. Thermal Analyses of Liposomal ATA. Thermal
analyses that include thermogravimetric (TG) investigation
and differential thermal analysis (DTA) were performed using
Diamond TG/DTA (PerkinElmer instrument).19−21 The
thermal decomposition was conducted on free ATA, PC3,
cholesterol, DSPE-PEG, a physical mixture of the four
components, and freeze-dried liposomal ATA. Approximately
2 mg of a vacuum-dried coal sample with an average particle
diameter less than 200 μm was placed in the sample crucible.
The sample was heated in pure N2 from 30 to 400 °C at a
heating rate of 10 °C/min. The melting points of the samples
were determined from thermal graphs.
2.5. Storage Stability of Liposomal ATA. The storage
stability of liposomal ATA was investigated by measuring the
size, zeta potential, and EE% of liposomal ATA during the
experiment. Liposomal ATA was diluted using phosphate-
C DOI: 10.1021/acs.molpharmaceut.9b00493
Article
buffered saline (PBS, pH 7.4) and incubated at 4 or 37 °C for
1−7 days. Under all conditions, 1 mL of the suspension was
collected at designated intervals; the size and zeta potential
were detected using a laser electrophoretic light scatter
analyzer (Malvern Nano-ZS Particle Sizer), and the EE% was
measured using HPLC.
2.6. Drug Release from Liposomes. The release of ATA
from liposomes was investigated using dialysis and compared
with the release of free ATA in the release medium (ultrapure
water with 1% SDS). Six milliliters of the release medium
containing an equivalent amount of liposomal ATA or free
ATA was placed in a dialysis sack (MWCO 12,000, Sigma).
The sack was then immersed in 50 mL of the release medium
with gentle stirring at 37 °C. The samples (2 mL) were
removed from the sack at designated time intervals, and an
equal volume of the release medium was replenished at the
same time.22−25 The amount of released drug was determined
by RP-HPLC as described in Section 2.3: “Determination of
Encapsulation Efficiency and the Drug Loading Rate”. The
similarity of the release profiles between the free ATA and
liposomal ATA was evaluated by the similarity factor (f 2)
shown below
l ÄÅ ÉÑ−0.5 |
o
o Å ÑÑ o
o
oÅÅÅ o
2Ñ
Ñ
f 2 = 50logm ÅÅ1 + ∑ t t ÑÑ Ñ }
o
o Å o
o
n
oÅÇ Å Ñ
ÑÖ o
1
n ~
( R − T ) × 100
n t=1
where n is the number of time points, Rt is the release value of
liposomal ATA at time t, and Tt is the release value of free
ATA.26,27
2.7. Determination of the Pharmacokinetic Profile of
Liposomal ATA in Rats. Female SD rats (INVIVOS,
Singapore) were randomly divided into two groups (n = 6):
free ATA and liposomal ATA. Free ATA was suspended in a
solution of PEG300:ethanol:Tween 80 (60:25:15, v/v/v). The
free ATA suspension or liposomal ATA was administered at an
equivalent dose of ATA at 20 mg/kg body weight via a single
intravenous injection in the tail vain. At different time points
(15 min, 30 min, and 1, 2, 3, 4, 5, 6, 8, and 24 h), blood
samples (0.3 mL) were collected from the suborbital vein and
placed in heparinized tubes. The blood samples were
centrifuged at 3000 rpm at 4 °C for 10 min to collect plasma.
The samples were then pretreated and injected into RP-HPLC
for detection of ATA as described in a previous work.6
Microsoft Excel PKsolver was used to analyze the pharmaco-
kinetic data.
2.8. Cell Culture. HUVECs and MCF-7 cells were cultured
in a DMEM-based culture medium supplemented with 1%
penicillin/streptomycin and 10% FBS. Cell cultures were
maintained at 37 °C in a humidified incubator supplemented
with 5% CO2.
2.9. Determination of the Anticancer Efficacy and
Cytotoxicity of Liposomal ATA in Vitro. The MTT
reduction ability was determined as an index of the metabolic
activity of the mitochondria, which can indicate cell viability.
Briefly, MCF-7 cells or HUVECs were seeded in 96-well plates
and cultured overnight. Free ATA (dissolved in DMSO diluted
in DMEM), liposomal ATA, and blank liposomes were added
at different concentrations, and the cells were incubated for 24
h. MTT solution (10 μL; 5 mg/mL) was then added into each
well, and they were incubated for 4 h at 37 °C in the CO2
incubator. One hundred microliters of the solubilization
solution containing 1% SDS and 0.1% hydrochloric acid was
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Molecular Pharmaceutics
added to each well to dissolve the formazan crystals. After 8 h,
the absorbance of the solubilized MTT formazan product was
measured using a spectrophotometer at an absorbance
wavelength of 595 nm, and the cell viability was calculated
from the optical density (OD) value of the tested sample and
the vehicle control (untreated cells) using the following
equation: cell viability (%) = OD tested sample/OD vehicle control ×
100. Nine sets of data obtained in triplicate from three
independent experiments were averaged to generate the final
data with the standard deviation, which are presented as the
mean ± SD.
2.10. Determination of the Antitumor Effect of
Liposomal ATA in Vivo. This mouse experiment was
conducted in accordance with the protocol approved by the
Institutional Animal Care and Use Committee, Nanyang
Technological University, Singapore. To ensure that the
estrogen receptor-positive breast cancer MCF-7 cells could
grow into solid tumors in female mice, a tablet of 1.5 mg β-
estradiol 17-acetate was implanted subcutaneously seven days
prior to tumor inoculation into female nude mice (18−22 g,
6−8 weeks of age). MCF-7 cells (5 × 106) were mixed with
Matrigel in a final volume of 0.2 mL and subcutaneously
injected into the armpit of the mouse.
After the tumor reached a minimum volume of 25 mm3, an
aliquot (200 μL) of PBS, blank liposomes, free ATA, or
liposomal ATA (20 mg ATA/kg) was intravenously injected
into the tumor-bearing nude mice (n = 5 per group) every
other day. The tumor size and the weight of the nude mouse
were measured and recorded during the treatment period. The
tumor volume was determined by measuring its length and
width and calculated using the following formula: V = (L ×
W2)/2 where L is the longest dimension parallel to the skin
surface, and W is the dimension perpendicular to L and parallel
to the surface. After the treatment period, the tumors and
major organs were harvested.
2.11. Toxicity Evaluation of Liposomal ATA in
Zebrafish. 2.11.1. Zebrafish Maintenance and Fish Embryo
Generation. Male and female zebrafish were bred in separate
tanks. The temperature of the water in the tanks was
maintained at 28.5 °C, and the pH value was within 6.8.
Adult zebrafish were placed in the upper level of a spawning
tank the evening prior to mating at a ratio of female/male =
2:1. The next morning after mating, the embryos were
collected from the lower level of the spawning tank, washed,
and kept in fresh E3 medium (13.7 mmol/L NaCl, 0.54 mmol/
L KCl, 0.025 mmol/L Na2HPO4, 0.044 mmol/L KH2PO4,
0.42 mmol/L NaHCO3, 1.3 mmol/L CaCl2, and 1.0 mmol/L
MgSO4, and the pH was adjusted to 7.2 before usage).
2.11.2. Toxicity Evaluation of Liposomal ATA in Zebrafish
Larvae. The chorion surrounding the embryo was removed
enzymatically at 4 h post fertilization (hpf) following the
procedures described in the literature.28,29 At 6 hpf, the
embryos were transferred to 96-well plates at one embryo per
well with 500 μL of E3 medium containing different
compositions: E3 medium alone (negative control), E3
medium containing blank liposomes, various concentrations
of liposomal ATA, or free ATA dissolved in DMSO.
Unexposed embryos (embryos raised in E3 medium) were
also incubated to monitor the natural quality of the embryos.
Throughout the experiment, incubation solutions with
freshly added ATA were replaced daily. Images of the zebrafish
were captured using a dissecting microscope. The heart rate
was also recorded as described previously.30,31 Twenty
D DOI: 10.1021/acs.molpharmaceut.9b00493
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zebrafish were used for each group in each experiment, and
the zebrafish experiments were carried out at least four times.
2.11.3. Malformation and Mortality Rates of Zebrafish
Embryos Exposed to Liposomal ATA. To examine the effects
of free ATA and liposomal ATA on embryos during
development, several exposure durations were chosen. These
durations included 6−10 hpf (gastrula period), 14−24 h
(important organ formation stage), 10−24 hpf (somite stage),
24−48 hpf (pharyngeal phase), and 48−72 hpf (the incubation
period). During each period, embryos were exposed to free
ATA, liposomal ATA, or blank liposomes at the equivalent
concentration of 5 μM. At 96 hpf, larvae or embryos were
microscopically examined to determine the malformation and
mortality rates. The heart rate was also recorded at 24, 72, and
96 hpf.28−31 N = 20 fish/group.
2.12. Toxicity Evaluation of Liposomal ATA on Mice.
BALB/C mice purchased from (INVIVOS, Singapore) were
used to evaluate the toxicity of ATA and liposomal ATA in
vivo. The maximum dosage of liposomal ATA was determined
to be 120 mg/kg body weight as this is the maximum amount
of liposomal ATA that could be prepared in a 0.2 mL volume
for intravenous injection. Twenty mice (10 female and 10
male) were used in this study. A solution of 0.2 mL liposomal
ATA at a dose of 120 mg/kg body weight was intravenously
injected into the tail vain of each mouse. The behaviors of the
mice were observed, and the body weight was recorded after
administration every other day. The mice were then sacrificed
after 7 days (5 female and 5 male) or 21 days (5 female and 5
male), and different organs such as the heart, liver, spleen,
lung, kidney, and brain were collected, fixed in 4%
paraformaldehyde, and processed for H&E staining. The
damage in each organ was assessed and imaged to record any
pathological changes.
The maximal dosage of free ATA [dissolved in PEG300:e-
thanol:tween 80 = 60:25:15 (v/v/v) as a stock solution then
diluted using 0.9% NaCl at a volume ratio of 1:9 before
injection] at 30 mg/kg body weight was also injected into
BALB/C mice to evaluate the toxicity of free ATA as a
control.32
2.13. Statistical Analysis. Statistical analysis was
performed using SPSS 10.0 software. Descriptive data were
expressed as the arithmetic mean value plus or minus the
standard deviation. Statistics were performed for all compar-
isons. All quantitative results were obtained from at least
triplicate samples. A t-test was applied to detect differences
between groups. In all evaluations, *p < 0.05 was considered
statistically significant.
3. RESULTS AND DISCUSSION
3.1. Preparation and Characterization of Liposomal
ATA. To increase the water solubility and bioavailability of
hydrophobic ATA, liposomes were utilized to encapsulate
ATA. Three components were utilized to make liposomes:
PC3, DSPE-PEG, and cholesterol. PC3 and cholesterol possess
excellent biocompatibility in vivo. DSPE-PEG allows a longer
circulation in vivo for this drug delivery system, protects ATA
from degradation within the circulation system, and enhances
the delivery of ATA to tumor tissues (Figure 1A).
To determine an optimal condition to formulate liposomal
ATA, an orthogonal array test was designed with nine
experiments, [L9(34)]. This array test allowed us to examine
four critical experimental factors (A: ratio of PC3:cholesterol,
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Molecular Pharmaceutics Article

B: ratio of DSPE-PEG:ATA, C: ultrasonic time, and D:

homogeneous ultrasonic time) at three levels (Table S1).33,34
The reverse-phase evaporation method was employed to
prepare liposomal ATA. HPLC was used to measure the
concentration of ATA in each experiment from which the
percentage of encapsulation efficiency (EE%) and rate of drug
loading (DL%) were calculated (Table S2). The results of
array tests were further analyzed, and the optimal conditions to
formulate liposomal ATA were determined after comparing the
calculated parameters (Table S3): PC3:cholesterol = 9:4,
DSPE-PEG:ATA = 7, ultrasonic time = 3 min, and
homogeneous ultrasonic time w = 8 min. Under these
conditions, the EE% and DL% of liposomal ATA were found
to be 98.94% and 12.35%, respectively.
This high level of EE should prevent a burst release of
encapsulated ATA and, thus, is expected to produce a smooth
and steady drug release profile. In contrast, a moderate 12%
drug loading rate would satisfy the requirement for decreased
liposome usage, reduced costs, and improved compliance of
patients.
The functional performance of nanoparticle-based delivery
systems depends on the physicochemical properties of the
particles, such as size, morphology, charge, and physical state.
SEM analysis showed that the liposomal ATA was spherical
and not aggregated (Figure 1B). The average particle size of
liposomal ATA was found to be 188.5 nm with a relatively
narrow size distribution (Figure 1C). The nonaggregative
dispersion and relatively narrow size distribution of liposomal
ATA may ensure its smooth and steady drug release properties.
In addition, a small size is supposed to allow the easy
dissemination of liposomal ATA through the tumor neo-
vascularization. Consequently, the passive targeting ability of
liposomal ATA for tumors may be achieved. The zeta potential Figure 2. TG/DTA profiles of liposomal ATA and the materials used
to formulate ATA in an individual or mixed form. Four characteristic
was −31.0 mV, which suggests the stability of the liposomal absorption peaks appeared in the DTA curve when ATA, cholesterol,
ATA (Figure 1D). Likewise, due to the PEG chain on the DSPE-PEG, and PC3 were physically mixed together, while only one
surface of liposomes, an enhanced circulation period could be absorption peak appeared in the DTA curve in the formulated
obtained. Greater accumulation in the tumor tissue may be liposomal ATA.
anticipated for liposomal ATA, potentially further facilitating
its therapeutic effects and reducing the off-target effects on The mass loss of PC3 was complicated, occurring in the range
normal tissues. from 250 to 350 °C. The phase transition peak of PC3 was
3.2. Using Thermal Analysis (TG/DTA) to Study the 109.16 °C (Figure 2).
Properties of Liposomal ATA. The thermodynamic study of The individual phase transition peak of cholesterol, DSPE-
liposomal ATA was conducted to achieve three objectives: (1) PEG, PC3, and ATA can be found by analyzing the TG/DTA
to confirm the existence of ATA in liposomes, (2) to evaluate curves obtained from the four physically mixed components.
the stability of drug-loaded liposomes, and (3) to validate These four DTA peaks were found at 143.84 °C, 54.72 °C,
whether the drug-loaded liposomes were successfully prepared. 105.24 °C, and 165.23 °C, which corresponded to phase
The thermal analysis of liposomal ATA was investigated using transition temperatures of cholesterol (148.96 °C in the
TG/DTA. The results of pure and formulated components are cholesterol curve), DSPE-PEG (54.90 °C in the DSPE-PEG
shown in Figure 2. curve), PC3 (109.16 °C in the PC3 curve), and ATA (175.72
The TG/DTA curve of pure ATA showed that the mass loss °C in the ATA curve). In opposition to the physical mixture,
of ATA occurred in just one step in the temperature range of the curve for liposomal ATA showed only one phase transition
220−310 °C. This step consisted of an accelerated mass loss temperature (65.04 °C), which suggested that ATA was
reaching ∼100% that was associated with a small DTA molecularly dispersed within liposomes (Figure 2).
endothermic peak at 175.72 °C, which was presumably caused The blank liposomes and the physical mixture of liposomal
by the phase transition of ATA. The materials used to prepare materials of cholesterol, DSPE-PEG, and PC3 were also
liposomal ATA showed different TG/DTA profiles compared analyzed by TG/DTA. From the enlarged DTA curve, three
with ATA. For example, the mass loss of cholesterol occurred DTA peaks were detected from the components of liposome
in the temperature range from 220 to 330 °C that was materials. In contrast, only one DTA peak at 72.03 °C was
associated with a DTA endothermic peak after 300 °C. The detected from the blank liposomes (Figure S1), which is quite
phase transition peak of cholesterol was 148.96 °C. The mass different from the DTA peak at 65.04 °C from liposomal ATA
loss of DSPE-PEG occurred in the temperature range of 300− (Figure 2).
400 °C associated with the DTA endothermic peak after 350 The thermodynamic results indicated that the physical
°C. The phase transition peak of DSPE-PEG was 54.90 °C. mixture of four components of liposomal ATA would not affect
E DOI: 10.1021/acs.molpharmaceut.9b00493
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the state of their existence because each melting point of these
components could be easily found in the thermodynamic curve
of the physical mixture. In contrast, when these components
were molecularly integrated in the liposomal ATA, only one
melting point was detected, suggesting that the ATA molecules
were dissolved in the lipid bilayer of liposomes. This result
confirmed that ATA was successfully and stably carried by the
PEG-liposomes.
3.3. Evaluating the Storage Stability of Liposomal
ATA. The size distribution, surface charge, and EE% of
liposomal ATA were measured during a storage period of 7
days to assess their storage stability. This information is
important for developing liposomal ATA into a clinical
formulation.
Figure 3A,B shows that the size and zeta potential of the
liposomal ATA remained constant at 4 °C during the 7 days of
storage. In addition, the EE% remained at a very high level
(>95%) in the first 4 days at 4 °C and was slightly reduced to
>80% from day 5 to 7 (Figure 3C). In contrast, different
Figure 3. Storage stability of liposomal ATA. Liposomal ATA was
stored in PBS at 4 or 37 °C for 7 days. (A) Size changes in liposomal
ATA. The size of liposomal ATA was more stable at 4 °C. (B) Zeta
potential of liposomal ATA. The zeta potential of liposomal ATA was
more stable at 4 °C. (C) EE% change in liposomal ATA. *p < 0.05,
***p < 0.001 compared with day 0. (D) Drug release profile.
Liposomal ATA displayed a gradual and prominent release profile
compared with free ATA. f 2 < 50 between free ATA and the
liposomal ATA group.
F DOI: 10.1021/acs.molpharmaceut.9b00493
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stability features were observed at 37 °C. The size and zeta
potential did not significantly change in the first 5 days (Figure
3A,B). However, smaller liposomes started to aggregate to
form larger liposomes after 5 days, resulting in a significant
increase in average particle size from ∼190 to ∼380 nm and a
significant reduction of the average zeta potential from −42
mV to nearly −68 mV. We do not expect that this aggregation
problem will affect the in vivo efficacy of liposomal ATA as
they should be metabolized in the body in less than 3 days.
The EE% of liposomal ATA did not decrease at 37 °C in the
first 4 days and was slightly reduced to ∼80% after 7 days
(Figure 3C). In conclusion, liposomal ATA should be stored at
4 °C to maintain its small particle size and high encapsulation
efficiency.
The compatibility and stability of liposomal ATA with two
commonly used intravenous dosing media, saline (0.9% NaCl)
and isotonic glucose solution (5% glucose), were evaluated.
After being incubated with 5% glucose at room temperature for
24 h, the particle size of the liposomal ATA was reduced by 15
nm, and the zeta potential of liposomal ATA was reduced by 6
mV. In contrast, incubation with 0.9% NaCl resulted in a much
greater variation of the particle size and zeta potential of
liposomal ATA at 50 nm and 38 mV, respectively (Figure
S2A,B). More importantly, these changes occurred within the
first several hours of incubation. A significant reduction of EE%
was also detected when liposomal ATA was incubated with
0.9% NaCl for 12 h, while no reduction was observed when
liposomal ATA was incubated with 5% glucose for 12 h
(Figure S2C). These results indicated that liposomal ATA was
more stable in 5% glucose than in 0.9% NaCl, and therefore
liposomal ATA should be diluted with 5% glucose for
intravenous injection in future clinical applications.
The release profiles of ATA were compared between free
ATA and liposomal ATA in aqueous medium. The graph in
Figure 3D shows that most free ATA could not be released
from the dialysis bag into the surrounding medium, likely
because of its poor solubility, which could not produce a
sufficient concentration gradient between the two sides of the
dialysis membrane for the diffusion of ATA. However, ATA
exhibited a better release profile when it was encapsulated with
liposomes. For example, ∼78% of ATA was released from
liposomal ATA, while only ∼38% was released from free ATA
after 72 h. More interestingly, unlike the flat release profile of
free ATA, a steady increase in ATA release was observed over
the 72 h.
This result not only supported the conclusion that ATA
molecules were successfully encapsulated within liposomes, but
it also demonstrated that encapsulating ATA within liposomes
could prolong its release. Other important information
obtained from this study included the finding that the
liposomal ATA formulation could be more stably stored at 4
°C and diluted in 5% glucose for intravenous injection or
diffusion.
3.4. Determination of the Pharmacokinetic Profile of
Liposomal ATA in Rats. The most important objective of
this study was to develop a clinical formulation of ATA with
improved bioavailability. To assess whether the formulated
ATA could meet this goal, free and liposomal ATA at 20 mg/
kg were administered to rats via a single intravenous injection.
At different time points, blood samples were obtained from
rats, and the concentrations of ATA in plasma and whole
blood were determined using a well-validated HPLC method.6
The pharmacokinetic profiles shown in Figure 4 revealed that
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Molecular Pharmaceutics Article

Figure 4. Pharmacokinetic (PK) profiles of free and liposomal ATA from plasma and blood of rats after intravenous injection. N = 6 rats/group.

Table 1. Pharmacokinetic Parameters of Free ATA and Liposomal ATA in Rat Plasma and Whole Blooda
free ATA liposomal ATA ratio (lipo/free-ATA)
parameter plasma blood plasma blood plasma blood
CL{[(mg/kg)/μM]/min} 0.24 25 0.004 2672 0.02 106
AUC0−inf (μM·min) 85 882 5009 27,912 59 32
a
CL: clearance; AUC0−inf: area under the plasma concentration-time curve from time zero to infinity.

Figure 5. The cytotoxicity of free and liposomal ATA was evaluated by the MTT assay. (A) Liposomal ATA displayed a good growth inhibitory
effect in breast cancer MCF-7 cells. (B) Liposomal ATA was not toxic to normal endothelial HUVECs at concentrations ≤5 μM. *p < 0.05, **p <
0.01, ***p < 0.001 between tested samples and the control group. Data were collected from triplicate wells in each experiment repeated three
times.

the concentrations of ATA in plasma and whole blood from
the free ATA-treated group were at a very low level at 15 min.
Compared with free ATA, liposomal ATA had significantly
higher concentrations of ATA in both plasma and whole blood.
We noticed that the concentration of ATA is higher in the
whole blood group than in the plasma group, which may be
due to the higher internalization of liposomal ATA by the
blood cells. This distribution of liposomal ATA in the blood
cells may increase its storage ability and contribute to a longer
circulation time of ATA in the blood stream.
The pharmacokinetic parameters of ATA were then
calculated using Excel PKsolver. According to the requirements
for selecting the pharmacokinetic calculation model, the values
of Akaike’s information criterion and residual sum of squares
(Re) were minimized, and the value of the determinate
coefficient (r) was maximized. The free ATA and liposomal
ATA belonged to the two-chamber model in whole blood. Free
ATA belonged to the one-compartment model in plasma, and
liposomal ATA belonged to the two-chamber model in plasma.
The mean retention time was calculated using the statistical
moment. The main pharmacokinetic parameters of ATA in rat
plasma and whole blood are listed in Table 1 and Tables S4
and S5.
As shown in Table 1, the free ATA and liposomal ATA-
treated groups exhibited significantly different pharmacokinetic
G DOI: 10.1021/acs.molpharmaceut.9b00493
parameters. Compared with free ATA, the 24 h area under the
curve (AUC0−24h) of liposomal ATA was increased 59-fold in
plasma and 32-fold in whole blood, whereas the clearance rate
was reduced 50-fold in plasma and increased 106-fold in whole
blood. These results demonstrated that liposomal ATA
significantly extended the retention time and enhanced
bioavailability of ATA in vivo.
3.5. In Vitro Determination of the Anticancer Efficacy
and General Cytotoxicity of Liposomal ATA. As liposomal
ATA was designed for intravenous injection, we wanted to
investigate whether liposomal ATA could retain its anticancer
effect without damaging endothelial cells. To assess this
possibility, the viabilities of human breast cancer MCF-7 cells
and human umbilical vein endothelial cells (HUVECs) were
determined by the MTT assay after the cells were incubated
with various concentrations of free ATA, liposomal ATA, or
blank liposomes for 24 h.
The MTT results showed that, at 0.5 μM, liposomal ATA
significantly reduced the viability of MCF-7 cells to ∼60%
compared with the control group, while free ATA did not
affect the viability of MCF-7 cells (Figure 5A), demonstrating
that formulating ATA with liposomes has increased its
cytotoxicity on breast cancer cells. Importantly, 0.5 μM
liposomal ATA did not reduce the viability of endothelial
HUVECs (Figure 5B), suggesting that it has little toxicity
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Molecular Pharmaceutics Article

toward noncancerous cells. At elevated concentration ranges of After the MCF-7 cells had grown into solid tumors,
1−20 μM, both liposomal and free ATA produced a much liposomal ATA (20 mg/kg) was injected into the tumor-
stronger growth inhibitory effect on MCF-7 cells than that of bearing nude mice through the tail vein every other day for 3
the control group (Figure 5A). weeks. During the treatment period, both the tumor size and
We also calculated the concentration that could reduce 50% body weight were measured. As shown in Figure 6A, tumors
of the cell viability after a 24 h incubation. The 24 h IC50 grew rapidly in the control group, whereas in the free ATA-
values showed that liposomal ATA had a lower (0.4-fold) IC50 treated group, tumor growth was significantly reduced. More
value than that of free ATA in breast cancer MCF-7 cells and a importantly, liposomal ATA exerted an even better tumor
higher (1.5-fold) IC50 value in endothelial cells (Table 2). In growth inhibitory effect (73%) than that of free ATA (61%)
summary, these results suggested that liposomal ATA had a (Table 3).
higher anticancer efficacy than that of free ATA and was less
toxic to HUVECs in comparison to its free form. Table 3. Tumor Growth Inhibition Ratio
sample tumor size (mm3) inhibition ratio (%) p value
Table 2. IC50 Values of Liposomal ATA vs Free ATA
control 1,611 ± 612 0
ratio of IC50 ratio of IC50 blank liposomes 897 ± 360 44 0.023 (*)
24 h IC50 breast cancer (lipo/free endothelial (lipo/free
values MCF-7 cells ATA) HUVECs ATA) free ATA 625 ± 301 61 0.009 (**)
liposomal ATA 432 ± 428 73 0.007 (**)
liposomal 1.0 μM 0.4-fold 22 μM 1.5-fold
ATA
free ATA 2.3 μM 14 μM No significant body weight differences were found between
the treatment groups (free ATA, blank liposomes, and
liposomal ATA) and the control group injected with PBS
3.6. Evaluating the in Vivo Antitumor Effect of during the experiment, which suggested that liposomal ATA
Liposomal ATA. As ATA has been previously shown to did not cause serious side effects in nude mice (Figure 6B).
effectively inhibit the growth of ER+ breast cancer MCF-7 At the end of the animal experiment, tumor tissues were
cells, in this study, MCF-7 cells were utilized to establish removed and photographed. The images in Figure 6C revealed
human xenograft tumors in nude mice. A tablet of 1.5 mg β- that three out of four tumors from the liposomal ATA-treated
estradiol 17-acetate was first implanted dermatologically into mice had a much smaller size than the tumors from free ATA-
female mice, and seven days later, MCF-7 cells (5 × 106) were treated mice. We propose that the following reasons
mixed with Matrigel and subcutaneously injected into the contributed to a higher antitumor efficacy of liposomal ATA.
armpit of the mice. First, the encapsulation of ATA with liposomes significantly

Figure 6. Determination of the antitumor effect of liposomal ATA in a human breast tumor xenograft model. (A) Graphs of the tumor size during
the treatment period. Tumor growth was effectively suppressed after the treatment of liposomal ATA. (B) Body weight of nude mice during the
treatment. No apparent body weight loss was found in all four groups. (C) Pictures of harvested tumors at the end of the experiment. The
liposomal ATA treatment group had the smallest tumor size. N = 5 mice/group. *p < 0.05, **p < 0.01 between samples and the control group.

H DOI: 10.1021/acs.molpharmaceut.9b00493
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Molecular Pharmaceutics Article

Figure 7. Toxicity evaluation of liposomal ATA in zebrafish embryos. (A) Types of induced malformations in zebrafish embryos at 96 hpf: (a)
normal development, (b) yolk sac absorption delay, (c) pericardial edema, and a (d, e) bent body axis. (B) Deformity and mortality rates of
zebrafish at 96 hpf. Less deformity and mortality were observed in zebrafish in the liposomal ATA-treated group compared with the free ATA-
treated group under the same treatment concentration. (C) Heartbeat of zebrafish. *p < 0.05, **p < 0.01 between samples and the control group.
N = 20 fish/group, and each experiment was performed at least four times.

increased their plasma concentration. Second, the average
particle size of liposomal ATA was 188.5 nm, which should
facilitate its exit from the leaky tumor vascular system to reach
tumor cells and enhance its therapeutic effect.
On a separate note, we found that blank liposomes exhibited
a minor tumor growth inhibitory effect (Figure 6A). The
results of the MTT assay also showed that the blank liposome
reduced the viability of MCF-7 cells (Figure 5A). These two
observations suggested that internalized liposomes might affect
the membrane integrity of tumor cells, which reduced cell
viability and tumor growth. Further studies will be conducted
to understand why blank liposomes produced a minor tumor
growth inhibitory effect, which can also help demonstrate that
the enhanced anticancer activity is due to the encapsulation of
ATA with liposomes but not due to the additive effect of blank
liposomes plus free ATA.
I DOI: 10.1021/acs.molpharmaceut.9b00493
3.7. Evaluation of the Potential Toxicity of Liposomal
ATA on Zebrafish. To date, our data have shown that the
newly formulated liposomal ATA displayed good antitumor
effects with little toxicity to human endothelial cells at ≤5 μM
and did not reduce the body weight of tumor-bearing mice at a
dose of 20 mg/kg. As our ultimate goal for liposomal ATA is to
use it to treat cancer, it is necessary to evaluate its potential
toxicity in vivo. We first utilized zebrafish because they are
cheap and easy to acquire in large quantities and because of the
similarities of the zebrafish genome to the human genome and
statuses of both fish and humans as vertebrates.
In this experiment, zebrafish larvae at the age of 6 h post
fertilization (hpf) were exposed to different concentrations of
free or liposomal ATA, and the morphological changes were
observed at 96 hpf. Moreover, the heart rates were measured at
48, 72, and 96 hpf. During the treatment with free or liposomal
ATA, various kinds and degrees of toxicity were observed over
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Molecular Pharmaceutics Article

Figure 8. Toxicity study of 5 μM free or liposomal ATA during zebrafish development. (A) Zebrafish embryos at different stages. (B) Deformity
and mortality rate of zebrafish at different periods during development. (C) Heartbeat of zebrafish at different developmental stages. *p < 0.05, **p
< 0.01 between samples and the control group.

the course of zebrafish development. At high concentrations,
ATA resulted in death or malformations in the early
developmental period. Images of malformations observed in
this experiment are shown in Figure 7A, including pericardial
edema, yolk sac absorption delay, and curvature of the spine.
The overall percentages of deformity and mortality are
shown in Figure 7B. A 5% mortality and no deformity were
observed in the control group. Blank liposomes did not cause
significant deformities or mortalities under the tested
concentrations. The data indicated that free ATA caused
extensive death or body deformation in zebrafish at high
concentrations (5−20 μM). In particular, 100% mortality was
recorded in zebrafish treated with 10 μM and 20 μM free ATA,
and the total amount of deformity and death rate exceeded
J DOI: 10.1021/acs.molpharmaceut.9b00493
90% at 5 μM. The combined rate of deformity and mortality
was 40% at 2.5 μM. These results suggested that free ATA at 5
μM was toxic to zebrafish development.
In contrast, liposomal ATA showed much lower toxicity
toward zebrafish than that of free ATA. The sum percentage of
mortality and deformity caused by liposomal ATA was lower
than that of free ATA. Importantly, no toxicity was observed at
a low concentration of 1 μM, and 10% mortality was detected
at 2.5 μM. In addition, the sum percentage of mortality and
deformity did not exceed 50% at high concentrations of 10−20
μM.
Additionally, the heart rate (heartbeats per minute) of
zebrafish was measured to determine heart function (Figure
7C). In the control group, the heartbeat gradually increased
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Molecular Pharmaceutics
from 48 hpf to 96 hpf, which resulted from normal heart
growth during zebrafish development. Similar to the control
group, blank liposomes also formed this normal pattern at
most liposome concentrations. Free ATA at 1−5 μM caused
significant changes in the heartbeat compared with the control
group. No heartbeats could be detected in the zebrafish treated
with high concentrations of free ATA (10−20 μM) as they had
all been killed. Compared with free ATA, lower toxicity was
observed in the liposomal-ATA treated group. No signs of
cardiotoxicity were observed in fish larvae treated with
liposomal ATA at 1−10 μM at 96 hpf. Although the heartbeat
of 20 μM at 48 hpf was significantly increased compared with
the control group, this elevation was not observed at 72 hpf
and 96 hpf, which suggested that this side effect could be
overcome during fish development.
3.8. Determining the Effects of 5 μM Liposomal ATA
on the Malformation and Mortality of Zebrafish
Embryos during Early Development. The above zebrafish
toxicity evaluation showed that 5 μM liposomal ATA did not
affect heart function and had much less toxicity than that of
free ATA. In this experiment, we further compared the
differential toxicity between free and liposomal ATA (5 μM)
during five major periods of zebrafish development (Figure
8A).
These five time periods are the (1) gastrula period (6−10
hpf, morphogenetic movements of involution, convergence,
and extension form the epiblast, hypoblast, and embryonic
axis; through the end of epiboly), (2) somite stage (10−24 hpf,
somite, pharyngeal arch primordium, and neuromere develop-
ment; primary organogenesis; earliest movement; tail appear-
ance), (3) important organ formation stage (14−24 h, primary
organogenesis; earliest movement; the tail appears), (4)
pharyngeal phase (24−48 hpf, phylotypic-stage embryo;
body axis straightens from its early curvature around the
yolk sac; circulation, pigmentation, and fins begin to develop),
and (5) hatching period (48−72 hpf, completion of rapid
morphogenesis of primary organ systems; cartilage develop-
ment in the head and pectoral fin; hatching occurs
asynchronously) (Figure 8A). Mortality, abnormality, and
heart function were also assessed in each developmental period
of zebrafish.
Figure 8B shows the overall deformity and mortality rates of
zebrafish after the treatment of various samples in different
exposure periods. The sensitivity window was determined
according to the statistical results. No deformity or mortality
was observed in the control group during all developmental
stages. The blank liposomes did not produce significant
toxicity in zebrafish, and less than 18% mortality or deformity
was observed in all tested periods.
Free ATA caused the highest mortality (100%) during early
development at 6−10 hpf and resulted in lower mortality
(53.85%) in the later developmental periods at 10−24 hpf.
Even lower mortality rates of 11.76% and 14.29% were
detected in other periods of 14−24 hpf and 24−48 hpf,
respectively. These results suggested that free ATA mainly
affected the gastrula period and early somite stage of zebrafish
development. In contrast, liposomal ATA significantly
decreased the toxicity of ATA in zebrafish. For example,
much lower mortality rates (29−31%) were recorded at
exposure times of 6−10 and 10−24 hpf in comparison to free
ATA.
In addition to evaluating larval mortality and abnormality,
zebrafish heart function was also examined. As shown in Figure
K DOI: 10.1021/acs.molpharmaceut.9b00493
Article
8C, blank liposomes and liposomal ATA did not cause obvious
changes in the heartbeat compared with the control group in
all treatment periods. Free ATA did not cause notable changes
in the heartbeat in the 6−10 hpf and 14−24 hpf exposure
periods but significantly increased the heartbeat in the periods
at 10−24, 24−48, and 48−72 hpf compared with the control
group. These results further confirmed that liposomal ATA was
safer than free ATA.
Although encapsulating ATA with liposomes reduced its
toxicity, liposomal ATA still showed a negative impact during
early embryonic development. This finding suggested that
liposomal ATA should not be administered to breast cancer
patients who are pregnant.
3.9. Evaluating the Toxicity of Liposomal ATA in
Mice. The results of the toxicity study in zebrafish showed that
liposomal ATA had much lower toxicity than that of free ATA.
In this study, the acute toxicity evaluation was conducted in
mice, which is a required process for the development of
liposomal ATA into a therapeutic agent. Acute toxicity refers
to the adverse effects of a substance caused by either a single
exposure or multiple exposures in a short period of time
(usually less than 24 h). Because liposomal ATA did not cause
any mice to die at the dose of 120 mg/kg, which is the highest
dose that could be prepared, this maximum dose was used in
this study. Equal numbers of female and male mice (n = 10)
were administered with 120 mg/kg liposomal ATA via an
intravenous injection. The saline group and blank liposomes
were used as controls.
After injection, the mice were monitored for 21 days in
terms of behavioral changes related to the following:
respiratory system, motor system, convulsive reaction, reflex
system, eyes, cardiovascular system, excretory system, and
muscle tension (Table S6). The results revealed no death in
either female or male mice after dosing, and the following
abnormal behaviors were observed. First, all control, liposomal
ATA, and blank liposome-treated groups produced reflex
irritation and were sensitive to noise and touch, suggesting
neuromuscular toxicity. This response quickly recovered,
implying that the injection process induced a transient stress
response. Second, liposomal ATA and blank liposomes
increased spontaneous activities in mice, which could be
quickly recovered, indicating the occurrence of a minor
recoverable motor malfunction. Third, liposomal ATA resulted
in transient salivation, which was related to disturbed
autonomic functions. In summary, liposomal ATA may have
toxic effects on motor function and autonomic function in
mice. Liposomal ATA did not cause strong acute toxicity in
male or female mice, and all the aforementioned symptoms
could be rapidly recovered (i.e., in 1 day), which suggested that
liposomal ATA was quite safe in mice.
As the highest dose that could be prepared for free ATA is
30 mg/kg, we used this dose to conduct the toxicity study of
free ATA. Mice appeared to have inspiratory difficulties and
produced a wheezing sound when they breathed. These
disorders involved malfunctions in pulmonary edema, respira-
tory secretion accumulation, and enhanced cholinergic
function. However, at 120 mg/kg, which is 4 times higher
than free ATA, liposomal ATA did not cause such problems.
These results further confirmed our previous findings in
zebrafish: liposomal ATA is safer than free ATA.
In addition to observing behavioral changes, the body
weights of the injected mice were measured at multiple time
points over 21 days. The graphs in Figure S3 show that the
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Molecular Pharmaceutics
weights of male and female mice increased slightly and that no
significant changes were observed between the liposomal ATA
and saline control group. These results showed that a single
intravenous dose of liposomal ATA at 120 mg/kg would not
affect the normal increment of mouse body weight.
Histopathology analysis was conducted for important
organs, including the heart, liver, spleen, lung, kidney, and
brain at 7 and 21 days post injection by H&E staining. The
images in Figures 9 and 10 showed no obvious necrosis or
Figure 9. Short effect of liposomal ATA on various organs of BALB/
C mice (female). Blank liposomes and liposomal ATA (at an
equivalent dosage of 120 mg/kg ATA) were injected into mice via a
single intravenous injection. Images of H&E staining show no obvious
necrosis in the heart, liver, spleen, kidney, and brain 7 days after
injection (50×). More red blood cells were found in lung tissues of
the liposomal ATA group. N = 5 mice/group.
significant inflammation in all tissues excluding the lung,
indicating the relatively low toxicity of liposomal ATA. The
lung tissue of the liposomal ATA-treated group appeared
firmer and showed slight congestive phenomena compared
with the control group, indicating weak toxicity to the lung
tissue (Figures 9 and 10). A similar phenomenon was observed
in the tissue sections of male mice (Figures S4 and S5).
4. CONCLUSIONS
How to construct a clinical formulation of anti-breast cancer
chemotherapeutic agent ATA with improved bioavailability is a
difficult challenge to meet during drug development. In this
study, by using PEG-modified liposomes, we were able to
L DOI: 10.1021/acs.molpharmaceut.9b00493
Article
Figure 10. Long effect of liposomal ATA on various organs in BALB/
C mice (female) 21 days after injection. Images of H&E staining show
that liposomal ATA caused light congestion in the lung (50×). N = 5
mice/group.
encapsulate a promising anticancer agent ATA into nano-
particles with a suitable size and further evaluate the preclinical
properties. Encapsulating ATA into liposomes significantly
increased its release capacity in vitro and increased the ATA
plasma concentration 59-fold in rats. The formulation of
liposomal ATA was stable for storage at 4 °C for at least 4 days
and after being diluted with 5% glucose for intravenous
administration for 12 h. Liposomal ATA displayed strong
potency toward ER+ breast cancer cells by inhibiting their
proliferation in vitro and by reducing tumor growth by 73% in
nude mice. At the maximum dose of 120 mg/kg, in 21 days,
liposomal ATA did not show obvious toxicity, such as death,
reduced body weight or damage to major organs. However,
liposomal ATA showed minor toxicity in zebrafish larvae
during their early development and resulted in a slight
abnormality in the lung tissue in mice. Thus, liposomal ATA
should not be given to cancer patients who are pregnant, and
more clinical studies are needed to determine whether ATA
should be given to cancer patients with lung disease. The
successful development of ATA into a clinical formulation with
improved bioavailability and better anticancer efficacy
provided an example for drug formulation. Furthermore, the
information obtained from this study can be used to guide the
future manufacturing and development of liposomal ATA into
a new anti-breast cancer drug.
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Molecular Pharmaceutics
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.molpharma-
ceut.9b00493.
Orthogonal array test design with four critical factors at
three variable levels, results of the orthogonal array tests,
analysis of orthogonal array tests, pharmacokinetic
parameters of free and liposomal ATA in rat whole
blood after iv administration, pharmacokinetic parame-
ters of free and liposomal ATA in rat plasma after iv
administration, behavioral changes in mice after
injection of the maximum dose of liposomal ATA and
blank liposomes, TG/DTA profiles of the mixture of
ATA, cholesterol, and DSPE-PEG, blank liposomes,
compatibility and stability of liposomal ATA, weight
changes in female and male mice after injection of the
maximum dose of liposomal ATA and blank liposomes,
toxicity of liposomal ATA in male mice after injection of
the maximum dose of liposomal ATA and blank
liposomes, recovery of male mice after injection of the
maximum dose of liposomal ATA and blank liposomes
(PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: kluo@um.edu.mo. Phone: 853-8822-4233. Fax: 853-
8822-2314.
ORCID
Qi Wang: 0000-0003-3694-5826
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This study was supported by grants awarded to K.Q.L. from
the Johns Hopkins Singapore Research Fund, the Start-Up
Fund from the Faculty of Health Sciences, and the Start-Up
Research Grant (no. SRG2016-00068-FHS) from the Uni-
versity of Macau. We would like to thank Prof. Sierin Lim for
her kind help in managing the JHS Research Fund.
■
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N DOI: 10.1021/acs.molpharmaceut.9b00493
Mol. Pharmaceutics XXXX, XXX, XXX−XXX