Membrane Loaded Copper Oleate PEGylated Liposome Combined with Disulfiram for Improving Synergistic Antitumor Effect In Vivo

Article abstract of DOI:10.1007/s11095-018-2414-5

Purpose: This work aims to create a novel Cu²⁺ liposome with excellent loading stability and develop synergistic effect with disulfiram (DSF) for the treatment of tumor. Methods: Copper oleate was incorporated into the liposome membrane via alc

Full text of DOI:10.1007/s11095-018-2414-5

Pharm Res (2018) 35:147
https://doi.org/10.1007/s11095-018-2414-5
RESEARCH PAPER
Membrane Loaded Copper Oleate PEGylated Liposome
Combined with Disulfiram for Improving Synergistic Antitumor
Effect In Vivo
Lingli Zhou1 & Liu Yang1 & Chulei Yang1 & Yi Liu1 & Qiuyue Chen2 & Wenli Pan3 & Qing Cai1 & Lifeng Luo1 & Lu Liu1 & Shan Jiang1
Haibing He1 & Yu Zhang1 & Tian Yin4 & Xing Tang1
&
Received: 9 March 2018 /Accepted: 19 April 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
.
.
.
KEY WORDS antitumor efficacy copper disulfiram
ABSTRACT
Purpose This work aims to create a novel Cu2+ liposome
with excellent loading stability and develop synergistic effect
with disulfiram (DSF) for the treatment of tumor.
liposome synergistic effect
.
Methods Copper oleate was incorporated into the liposome
membrane via alcohol injection method in this work. In vitro
release test was applied to evaluate the release profile of the
liposomes. Pharmacokinetic studies were performed in rats
and the antitumor efficacy was assessed in mice bearing hep-
atoma xenografts.
ABBREVIATIONS
5-Fu
5-fluorouracil
AUC
CTR1
Area under concentration time curve
Copper transporter 1
Cu(DTC)2 bis(Dithyldithiocarbamate)-copper
Cu(OI)2-L Copper oleate liposome
Results The copper oleate liposome (Cu(OI)2-L) was formu-
lated and the loading efficiency were more than 85%. TEM
images confirmed that the Cu(OI)2-L had a spherical mor-
phology with an average diameter of 100 nm. Cu(OI)2-L
displayed a biphasic release profile, with >70% retained drug
over 8 h incubation in PBS at pH 7.4. Pharmacokinetic stud-
ies demonstrated that Cu(OI)2-L had a prolonged circulation
time and increased AUC when compared to the injection of
copper oleate solution. The antitumor efficacy test demon-
strated an enhanced tumor inhibition rate with the treatment
of Cu(OI)2-L and DSF nanoparticles, indicating an improved
synergistic antitumor effect.
Cu(OI)2-S Copper oleate solution
DDTC-Na Sodium diethyldithiocarbamate
DSF
EPR
IR
Disulfiram
Enhanced permeability and retention
Infrared
MPS
MRT
NMR
NPs
NS
PBS
PDI
PK
Mononuclear phagocyte system
Mean residence time
Nuclear magnetic resonance
Nanoparticles
Normal saline solution
Phosphate buffer saline
Polydispersity index
Pharmacokinetics
Conclusions The Cu(OI)2-L was suitable to be employed in
combination with disulfiram for tumor treatment and can also
open up opportunities for targeted delivery of copper.
TEM
TIR
UV
Transmission electron microscope
Tumor inhibition rate
Ultraviolet
* Xing Tang
tanglab@126.com
INTRODUCTION
1
Department of Pharmaceutics, School of Pharmacy, Shenyang
Pharmaceutical University Shenyang 110016, China
Nanoparticle (NP)-based drug delivery systems have long
been demonstrated to enhance the therapeutic efficacy and
reduce systemic toxicity of antitumor drugs through targeted
delivery to tumor sites (1). Prolonged pharmacokinetics can be
achieved via surface modification of the nano-carrier with
polyethylene glycol (PEG) chains, providing a steric effect that
can avoid recognition and phagocytosis by the mononuclear
2
Wuya College of Innovation, Shenyang Pharmaceutical University
Shenyang 110016, China
3
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University Shenyang 110016, China
4
School of Functional food and Wine, Shenyang Pharmaceutical University
Shenyang 110016, China

147 Page 2 of 11
Zhou et al. (2018) 35:147
phagocyte system (MPS) (2–4). Furthermore, nano-carriers
with appropriate sizes can achieve increased accumulation
in many types of solid tumors via the enhanced permeability
and retention (EPR) effect (5,6). Among those nano-particles
investigated in the clinic, the liposome is widely used due to its
excellent biocompatibility, and the ability to incorporate both
hydrophobic and hydrophilic drugs into its internal cores or
lipid membrane, depending on the properties of the drug (7).
Disulfiram (DSF), a conventional anti-alcohol substance,
manifests potent effects in cancer treatment in recent studies
(8). Moreover, increasing evidence suggests that the anticancer
mechanism proceeds in an Cu2+ dependent manner (9–11).
DSF, as a bivalent metal ion chelator, can form complexes
with Cu2+ and enhance the intracellular transport of Cu2+. As
Cu2+ is involved in a wide range of cellular physiological pro-
cesses, this is speculated to be the main cause of the excess
reactive oxygen species (ROS) generation in several solid tu-
mors (12–14). In comparison to DSF and Cu2+ alone,
DSF/Cu2+ complexes exhibit a much stronger ROS induc-
tion activity, causing a strong but transient killing effect on
tumor cells. As well, the final product Cu(DTC)2 exhibits a
delayed but long-lasting cytotoxic effect.
The molecular target of Cu(DTC)2 has not been elucidated
clearly until a recent study reveals that it binds and immobi-
lizes NPL4, which is an essential component of p97-NPL4-
UFD segregase complex, thereby inhibiting the p97 pathway.
(15) As a upstream pathway of proteasome-ubiquitin system,
the p97 pathway inhibition triggered a series of cellular reac-
tions, including blocking of protein degradation, accumula-
tion of Poly-Ub protein, inhibition of NF-κB, consequently
leading to the apoptosis and necrosis of the cancer cell.
However, due to the strict regulation of copper systems
in vivo, traditional oral copper preparations are ineffective at
enhancing cellular copper content (16,17). Dietary copper
absorbed from the intestinal epithelial cells is reduced to
Cu+ and imported into the liver by copper transporter 1
[CTR1]. Once the cellular copper content rises above the
normal value, the genetic expression of CTR1 will be down-
regulated, and only limited amounts of exogenous copper will
be transferred intracellularly. At the same time, ATP7A and
ATP7B, two Cu-transporting P-type ATPases, which are
known to take charge of cellular copper efflux in mammals,
translocate cellular copper to the membrane to excrete it out
of the cell.
However, when the highly water soluble cupric salts are
employed for a liposome formulation, heavy leakage is prone
to occur. To solve this problem, a new drug loading method
for copper loading was reported in Jai Woong Seo’s study (21),
in which a 64Cu-loaded liposome was prepared by incorpo-
rating a copper chelator-lipid conjugate in the lipid mem-
brane, and the resultant formulation was stable in serum
and exhibited long-circulating PK in vivo. This implies that
loading a lipophilic form of copper into the membrane may
provide more possibilities for in vivo copper delivery.
Based on this, the objective of this work was to develop a
novel Cu2+-loaded liposome formulation with great serum
stability, for a more effective synergy therapy with DSF nano-
particles. Here, to improve the loading stability and encapsu-
lation efficacy, an oil soluble salt form of copper, copper ole-
ate, was synthesized and formulated in the liposome. Unlike
the conventional water soluble form of copper, copper oleate
is highly lipophilic, and thus a high encapsulation efficiency of
more than 84% could be achieved by incorporating it into the
lipid membrane. The liposome exhibits negative surface
charge at pH 7 and hence, excellent liposome stability.
Additionally, a negative surface charge of the liposome is pro-
posed to exert a Bfix effect^ of copper ions through electro-
static forces. Drug release kinetics of the final formulation
were evaluated in vitro, and pharmacokinetic studies were per-
formed to assess the serum stability. Finally, the syner-
gistic effects of the liposome formulation with DSF
nanoparticles (NPs) (22) towards tumor treatment was
evaluated, and the therapeutic efficiency was compared
with the treatment of DSF NPs plus oral copper gluco-
nate and DSF NPs alone (Fig. 1).
MATERIALS AND METHODS
Materials
Lipoid@ E80 and DSPE-PEG2000 were purchased from
Lipoid (Ludwigshafen, Germany). The sodium oleate and
copper chloride were purchased from Sinopharm Chemical
Reagent (Shanghai, China). Chloroform was purchased from
Shandong Yu Wang Industrial Co., Ltd. (Shandong, China).
Cholesterol was purchased from J&K Chemical Technology
(Beijing., China). All other reagents were purchased from
Concord Technology (Tianjin, China).
Since conventional oral copper formulations are not effec-
tive at raising cellular copper content, the approach of formu-
lating copper into liposomes has been considered. Several
approaches have previously been proposed to load copper
into liposomes, including entrapping soluble copper salts in-
side the aqueous liposomal core (18), chelating copper with
other anticancer drugs to form stable copper-drug complexes
in the internal core of the vesicles (19) and attaching copper
onto the surface of liposomes via a chelation method (20,21).
Synthesis and Characterization of Copper Oleate
Copper oleate was synthesized by an ion exchange method as
reported previously (23) and shown in Scheme 1. Based on the
distinct dissolving properties, the final product was separated
from the reaction mixture (a mixture of NaCl, CuCl2, sodium
oleate, copper oleate) by a liquid-separation operation. The

Cu2+ Liposome Combined with DSF for Improving Synergistic Effect (2018) 35:147
Page 3 of 11 147
Fig. 1 Anti-tumor mechanisms of combined utilization of Cu2+ and disulfiram.
organic phase was washed by sterile water 3 times to purify the
copper oleate. The successful synthesis of copper oleate via the
ion exchange method was confirmed by IR, H-NMR and
Briefly, all lipid materials including lecithin, cholesterol and
copper oleate were dissolved in an appropriate amount of
ethanol. Next, a constant volume of the ethanol solution was
added to sterile water under stirring, and the mixture was
heated to remove the ethanol. After further evaporation of
the residual ethanol, the liposome suspension was filtered
through first 200 nm, then 100 nm polycarbonate filters, to
obtain liposomes with the desired size distribution.
In order to improve the stability of the liposomes and pre-
vent oxidation, freeze-drying technology was applied to solid-
ify the liposomal suspension. Influencing factor tests and long-
term tests were performed, and the results indicated a high
storage stability of the lyo-product (Scheme 2).
1
TGA analysis, and the results are shown in Fig. 2a, b.
Solubility tests of copper oleate in different aqueous solutions
were carried out, which showed that the solubility of copper
oleate was pH-dependent in aqueous solution, as shown in
Fig. 2c. The oil-water partition coefficient was evaluated in a
n-octanol-water system, and the lgP was approximately 2.7. In
order to determine the copper content, sodium
diethyldithiocarbamate (DDTC-Na) was utilized to form
DDTC-Cu complexes with copper oleate. The ultraviolet ab-
sorption of the complexes was measured by UV at 456 nm,
and the results were calculated against the absorption of
DDTC-Cu formed by a copper standard solution.
Characterization of Copper Oleate Liposomes
Preparation of Cu(OI)2?-L
The measurement of particle size distribution and zeta poten-
tial was carried out by a ZETASIZER NANO-ZSE dynamic
light scattering particle size / zeta potential analyzer (Malvern
Instruments Ltd). Morphological observations were
Cu(OI)2-L was prepared by an ethanol injection method, and
the size was controlled via a membrane extrusion method.
Scheme 1 Synthesis of copper oleate.

147 Page 4 of 11
Zhou et al. (2018) 35:147
Fig. 2 (a) Infrared spectroscopic spectra for oleic acid (a), sodium oleate (b) and copper oleate (c). In comparison to the IR spectra of sodium oleate, a new peak
at 1510.1 cm−1 appears, due to the larger radius of the copper atom affecting the asymmetric stretching vibration of carboxyl groups, leading to the increased
wave number of the asymmetric stretching vibration and reducing the symmetrical stretching vibration frequency. (b) The 1H-NMR spectra for Sodium oleate (a)
and copper oleate (b). The NMR spectra of oleic acid and copper oleate demonstrated that the oleic acid group retained integrity during the synthesis process. (c)
The solubility profile of copper oleate in aqueous solutions at different pH values.
performed using a JEM-2100 transmission electron micro-
scope (TEM) (Japan Electron Optics Laboratory, Japan). In
order to determine the encapsulation efficiency, free copper
oleate was removed by gel filtration on a Sephadex G-50
(Henghui Biotechnology, Beijing, China) column. The eluted
liposome fraction was disrupted by isopropyl alcohol for ana-
lyzing of the drug content. The copper was quantified by the
UV method as described in BSynthesis and Characterization
of Copper Oleate^ Section. The encapsulation efficiency was
calculated as [ACu (after filtration)]/[ACu (before filtration)] ×
100%.]
through a 0.45 μm microporous membrane and analyzed
by the UV method as describes in BSynthesis and
Characterization of Copper Oleate^ Section to calculate
copper(II) release. The cumulative release of each sampling
point was calculated according to the following formula.
ꢀ
ꢁ
n
Q % ¼ CnV 0 þ ∑ Cn−1V =Mt  100%
i¼1
Where Q is the quantity of released drug,Cn is the drug
concentration measured for the n time, V is the sampling
volume, V0 is the total volume of the release medium, and
Mt represents the total drug content.
In Vitro Release of Copper Oleate Liposomes
Pharmacokinetic Study
The in vitro release performance of the copper oleate liposome
was studied via a dialysis method at a physiological tempera-
ture of 37°C. Phosphate buffer saline (PBS) of pH 5 and
pH 7.4 containing 0.8% SDS (w/v) was chosen as the release
medium. The samples were prepared in quadruplicate for
each pH condition. 2 mL of the medium was sampled at
0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h, and the same volume of
fresh medium was added each time. Samples were filtered
A copper oleate solution was prepared by diluting the copper
oleate with normal saline solution containing 2% (w/v)
Tween80 and 15% (w/v) PEG400 as the solubilizing com-
pound. The dose of copper was 0.4 mg·kg−1 in both groups.
10 healthy male SD rats (Experimental Animal Center,
Shenyang Pharmaceutical University), weighing approxi-
mately 200 g were fasted overnight and randomly divided into
Scheme 2 Preparation of membrane-loaded copper oleate liposome via ethanol injection method.

Cu2+ Liposome Combined with DSF for Improving Synergistic Effect (2018) 35:147
Page 5 of 11 147
2 groups (n = 5). The rats from each group were administrated
with copper oleate injection (Cu (OI) 2-S) or copper oleate
liposomes (Cu(OI)2-L) via tail vein injection. At 3 min, 8 min,
15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h after
administration, 0.5 mL of blood was sampled through the
orbital vein and quickly transferred to 1.5 mL EP tubes pre-
treated with heparin. The plasma was subjected to a wet di-
gestion method to release the copper, and then assayed using
an atomic absorption spectrophotometer.
RESULTS
Preparation and Characterization of the Liposomes
The final liposome formulation was composed of phospholip-
id E80: cholesterol: DSPE-mPEG2000 with a molar ratio of
20:8:1, and the successful incorporation of copper oleate into
the liposomes was confirmed by transmission electron micros-
copy (TEM). The TEM image in Fig. 3a shows that the
Cu(OI)2-L had a spherical morphology, with an average di-
ameter of approximately 100 nm. The dynamic light scatter-
ing measurements further validated this result, showing a
mean diameter of 109.6 nm. The size distribution of the lipo-
somes, along with the PDI of 0.044, indicates a homogenous
formulation. Cu(OI)2-L exhibited a negative zeta potential of
−24.91 mV. The optimal encapsulation efficiency of copper
was found to be 84% at a drug/lipid ratio of 1:40 (w/w), and
displayed a pH–dependence, whereby the encapsulation in-
creased by more than 30% when the pH value of the aqueous
phase changed from 5.0 to 8.0.
Antitumor Efficacy Study in Combination Therapy
with DSF
KunMing mice (5–6 weeks of age) were purchased from the
Experimental Animal Center, Shenyang Pharmaceutical
University. Mice ascites tumor H-22 cells (Professor Deng
Yihui’s laboratory, Shenyang Pharmaceutical University) in
the logarithmic growth phase were diluted with normal saline
solution (NS) to approximately 1.5 × 107cells/ mL. 0.1 mL of
the cell suspension was inoculated into the peritoneal cavity of
the mice. The ascetic fluid was extracted 2 weeks after inocu-
lation, and was diluted with saline solution to a final cell con-
centration of 1.5 × 107 mL−1. 0.1 mL of the H-22 cells (1.5 ×
107 cells/ mL) suspension were s.c. inoculated into the left
axilla of the mice. When the mouse hepatoma model was well
established and the tumor volume reached about 100 mm3,
the rats were evenly divided into 5 groups according to the
principle of consistency (n = 6), receiving normal saline
(10 mL· kg−1), 5-FU solution (20 mg· kg−1), DSF NPs (3 mg·
mL−1) plus normal saline (10 mL· kg-1), DSF NPs(40 mg· kg-
1) plus oral Cu(OI)2-S (copper oleate solution) (0.3 mg·kg−1)
or DSF NPs (40 mg· kg−1) plus Cu(OI)2-L(0.3 mg·kg−1) via tail
vein injection on days 1, 4, 7, 10, 13. The DSF nanoparticles
(NPs) used here were formulated by our group in a previous
study (22). The body weight of the mice were monitored, and
the morphology of the tumor was recorded to evaluate effica-
cy and safety of the treatment. The tumor inhibition rate
(TIR) used as the evaluation criteria for antitumor efficiency
was calculated by the following equation.
In Vitro Release Profile of Cu(OI)2?-L
To determine the drug retention ability of the carrier in dif-
ferent pH conditions, in vitro release assays were performed in
pH 5.0 or 7.4 PBS containing 0.8% SDS at 37°C for 48 h. As
shown in Fig. 4a, the release of Cu(OI)2-L was rapid in the
initial 8 h, and gradually slowed down over time in both me-
dium. The release rate was affected by the pH conditions of
the release medium; the liposomes incubated in the pH 7.4
medium retained more than 70% copper oleate, while only
50% was retained in the pH 5.0 medium after 8 h of incuba-
tion, confirming pH sensitivity of the formulation.
Pharmacokinetic (PK) Study
Pharmacokinetic (PK) studies were performed and the results
were calculated using the software DAS 02.1A (Drug and
Statistics, China). The data was fitted for a two-
compartment model. As shown in Fig. 5, copper oleate solu-
tion was cleared rapidly from plasma, likely due to distribution
to other tissues or accumulation in the liver for excretion.
However, significant differences in the pharmacokinetic pro-
file were observed when a liposome formulation was used to
deliver the drug. Cu(OI)2-L exhibited a plasma elimination
half-life (t1/2 β) of 2.99 1.78 h, which was nearly 2-fold that
of the Cu(OI)2-S, and consistently, the mean residence time
(MRT) of the Cu(OI)2-L was ~2 times longer compared with
that of the Cu(OI)2-S. Additionally, a much higher bioavail-
ability was achieved by the Cu(OI)2-L, demonstrating an area
under concentration time curve (AUC) of 2.94 1.13 mg·L−1
·h, which was approximately 3-fold greater than that of the
Cu(OI)2-S.
TIRn ¼ ðVNS−VnÞ=VNS⋅100%
Where TIRn represents the average tumor inhibition rate
in group n, VNS represents the mean tumor volume of the
saline group, and Vn represents the mean tumor volume of
certain administration group.
Statistical Analysis
All mean values
SD expressed in the results were
compared by two-tailed unpaired t-tests for 2-group
comparison or one way ANOVA. P values <0.05 were
considered statistically significant.

147 Page 6 of 11
Zhou et al. (2018) 35:147

Cu2+ Liposome Combined with DSF for Improving Synergistic Effect (2018) 35:147
Page 7 of 11 147
4.5
RFig. 3 Characterization of the liposomes (a) TEM image for the Cu(OI)2-L.
Cu(OI)2-L
Cu(OI)2-S
(b) Schematic diagram of Cu(OI)2-L. (c) The influence of drug/lipid ratio (w/w)
4.0
on encapsulation efficiency and copper content in the liposome suspension.
(d) The influence of mass ratio of cholesterol/Lipoid@E80 on encapsulation
3.5
efficiency and copper content. (e) The influence of pH of the aqueous phase
3.0
on encapsulation efficiency and copper content. (The concentration of lipid
material is fixed at 30 mg·mL−1). (f) The particle size and (g) zeta potential of
2.5
the Cu(OI)-L.
2.0
1.5
1.0
Anti-Tumor Effect
0.5
0.0
The antitumor efficacy of Cu(OI)2-L in combination of DSF
NPs was evaluated using KunMing mice bearing H-22 cells
-0.5
0
2
4
6
8
hepatoma xenografts. The body weight and the tumor volume
of each animal was recorded during the experiment, and the
data was plotted against time(days) and shown in Fig. 6a, b.
The mice were sacrificed 14 days after the treatment and then
the tumor was excised and weighted, and the morphology of
the tumors is shown in Fig. 6c. It can be seen in Fig. 6b that all
the treatments showed a tumor reduction effect compared
Time (h)
Fig. 5 The average plasma concentration of copper against time after intra-
venous administration of Cu(IO)2-S and Cu(OI)2-L in rats (n = 5).
with the negative control group. The tumor inhibition rate
(TIR) was calculated to compare the effects of each treatment,
and results showed a tumor reduction effect in the order of
Fig. 4 (a) The release profiles of Cu(OI)2-L in PBS of pH 5 and pH 7.4. (b) The schematic diagram for the accelerated release profile of Cu2+ in low pH
conditions.

147 Page 8 of 11
Zhou et al. (2018) 35:147
Fig. 6 (a) The relative body weight and (b) the tumor volume of mice in different groups (n = 6). (c) Photograph of the tumor in different groups.
DSF NPs alone < DSF NPs plus oral Cu(OI)2-S < 5-Fu <
Cu(OI)2-L plus DSF NPs, with TIRs of 26.8%, 35.5%,
47.4% and 50.3%, respectively.
lecithin and cholesterol. By taking advantage of their unique
structure, both hydrophilic and hydrophobic substances can
be formulated into liposomal preparations. Several anti-
tumor drugs such as doxorubicin, and vincristine have been
formulated into liposomes and been clinically approved due to
their considerable chemotherapeutic effects (24–26).
Unlike hydrophilic agents, which dissolve in the aqueous
core, hydrophobic drugs are located within the membrane
bilayer. Thus, the maximum solubility of the drug in the mem-
brane may be the main factor that influences the loading
efficiency of lipophilic drugs. Theoretically, based on different
affinities between the drug and the lipid membrane, the drug/
lipid ratio can be raised from 1: 100 to 1: 1, particularly when
the drug is similar to the structure of the membrane compo-
nent. However, excess membrane solubility may lead to
slower drug release rate. In this work, copper oleate was syn-
thesized and shown to be lipophilic, with a partition coefficient
(lgP) of 2.7, and as a result an optimal loading efficiency of
DSF: Disulfiram nanoparticles. NS: Normal saline, as a
negative control. 5-Fu: 5-fluorouracil, as a positive control.
Cu(OI)2-S: Administration of copper oleate solution per oral.
Cu(OI)2-L: Administration of copper oleate liposome via tail
vein injection. (D) The reaction of disulfiram with Cu2 +
.
DISCUSSION
Since Bangham from the United Kingdom first proposed the
concept of liposomes, liposomal drug delivery systems have
made significant progress in the fields of anti-tumor and
anti-infection treatments in the last 5 decades. Liposomes gen-
erally present as spherical vesicles, in which aqueous cores are
enclosed by one or more concentric bilayers formed by

Cu2+ Liposome Combined with DSF for Improving Synergistic Effect (2018) 35:147
Page 9 of 11 147
84% was obtained at a drug/lipid ratio of 1:40, indicating
copper oleate is suitable for incorporation in the liposome
membrane. If the drug binding capacity of a membrane is
limited, excess drugs may damage the stability of the mem-
brane and even lead to disruption. As shown in Fig. 4c, an
obvious decline of encapsulation efficiency was observed when
the drug/lipid ratio was increased from 1:40 to 1:24. It was
also noted that alterations in the lipid composition (increasing
the mass ratio of cholesterol) did not affect the encapsulation
efficiency significantly, as shown in Fig. 3d, and thus it is as-
sumed that the incorporation of copper oleate was mainly due
to hydrophobic interactions, rather than interactions(such as
hydrogen-bond interactions, Van der Waals, electrostatic in-
teractions, etc.)between the drug and the lipid materials. This
assumption was confirmed by the pH-reliant encapsulation
efficiency and the pH-reliant water solubility, as shown in
Figs. 2c and 3e, where low pH values led to increased solubil-
ity of copper oleate in the aqueous solution, and thus reduced
the incorporation in the membrane.
The liposomes prepared in this study exhibited negative
zeta potential in physiological pH conditions, and this may
be due to the use of the negatively charged lipids Lipoid@
E80 and DSPE-mPEG (27). This inter-particle electrostatic
repulsion caused by the negative surface charge likely contrib-
uted to stability of the liposomes by avoiding aggregation. The
opposite charges between Cu2+ and the liposome surface are
assumed to exert a Bfix effect^ on free copper ions, increasing
the loading stability, as shown in Fig. 4b. As well, the variable
spatial structure of PEG chains should provide a steric hin-
drance on the surface, furthering protecting the Cu2+
located near the external aqueous phase by burying
within the PEG shell.
It is well known that only drug released from the carrier
produces a therapeutic effect, and the drug-delivery efficacy of
a liposomal formulation is determined by how the drug re-
leases and where it is released. The in vitro release kinetics were
investigated to assess the implications for in vivo delivery effi-
ciency. As discussed earlier, hydrophobic drugs are trapped in
the lipid membrane through interactions with membrane ma-
terials and hydrophobic effects (28), and thus the drug can be
released only after these interactions are disrupted or weak-
ened by changes in the external environment. When tested in
aqueous medium, as shown in Fig. 4a, more than 70% of the
encapsulated copper oleate was retained over 8 h incubation
in the simulated physiological medium of pH 7.4, confirming
excellent copper retaining ability while in circulation.
Additionally, pH-sensitive release kinetics were also observed,
and three mechanisms are proposed to explain this pH-
sensitivity of the liposomes. (1) Under acidic conditions, cop-
per oleate present in the outer leaflet is exposed to excess
hydrogen ions and contacts to form a more stable oleic acid
molecule, leading to the release of Cu2+; moreover, one cop-
per oleate molecule breaks down into two oleic acid molecules
in the ion exchange reaction, and this results in increments in
degrees of freedom in the membrane. (2) In acidic conditions,
increased surface charge of the liposome eliminates the Bfix
effect^ on Cu2 +, as shown in Fig. 4b, leading to the escape of
the copper(II) ions. (3) The Lipoid@ E80 used in this study
contains a small amount of acidic lipid. When the acid phos-
pholipids are protonated by excessive hydrogen ions in the
acidic medium, the size of polar head group increases and
the charge changes, resulting in reduced Van der Waals
interactions and increased electrostatic repulsion between
acyl chains. All of the above mechanisms were hypothesized
to destroy the stability of bilayer structure, and accelerating
the release of encapsulated drugs in acidic conditions. In
addition, a typical biphasic release profile (29) was also ob-
served, and this phenomenon is particularly obvious in the
acidic release medium. The release profile may also be ex-
plained by the distribution of copper oleate in the liposome
membrane. Any copper oleate incorporated in the periphery
of the liposome membrane may undergo the following de-
composition reaction when in contact with the aqueous solu-
tion, thereby releasing copper(II) ions.
CuðOIÞ2⇄Cu2þ þ 2OI−; OI− þ Hþ⇄HOI:
Thus, free drug in the outer aqueous phase, the drug phys-
ically adsorbed on the liposome surface and the drug wrapped
in the outer layer of the phospholipid bilayer will be preferen-
tially released. The Cu2+ wrapped in the lipid layer closed to
the internal water phase could be released only by diffusion
through the lipid matrix or upon rupturing of the liposomes.
In the pharmacokinetics study, the half-life of Cu(OI)2-L
and Cu(OI)2-S was found to be 2.99 h and 1.84 h respectively.
A much higher bioavailability was also observed from the
Cu(OI)2-L, with the concentration time curve (AUC) of
2.94 mg·L−1·h, which was approximately 3-fold greater than
that of the Cu(OI)2-S. The results confirmed that the nano-
particles modified with PEG possess the ability to avoid rec-
ognition by mononuclear phagocyte system (MPS) (30), and
provide an improved pharmacokinetic profile compared with
the copper oleate solution. A steep slope in the blood concen-
tration curve in the initial 8 min was observed, after adminis-
tration of both the liposomal formulations and copper oleate
solution, as shown in Fig. 5. This indicates an initial sharp
decrease of blood copper content after injection, followed by
a gentler decline. This phenomenon is consistent with the
pharmacokinetics of most endogenous substances in vivo. As
it is a trace element involved in many physiological and path-
ologic activities in mammals, copper content in vivo is strictly
controlled by a regulatory system. Free copper ions in the
blood circulation can quickly accumulate in the liver after
injection. In a high copper environment, ATP7A and
ATP7B, two Cu-transporting P-type ATPase, which are
known to take charge of cellular copper efflux in mammals,

147 Page 10 of 11
Zhou et al. (2018) 35:147
translocate cellular Cu2+ to the membrane for clearance and
promote copper excretion through the biliary duct. As time
passes, the proteins involved in copper transportation are
gradually saturated, and thus the clearance rate of copper is
reduced. In this case, with the liposome formulation, the rapid
decline in the plasma concentration curve may be explained
by the initial burst release profile of Cu(OI)2-L as discussed
earlier. Assuming that PEGylated liposomes loaded Cu2+ can
entirely escape the capture of the MPS, then only the
copper(II) ions released from the vesicle can be cleared.
Therefore, those copper oleate molecules that are well
protected in the inner leaflet of the bilayer will bear the im-
portant task of tumor-targeting delivery and developing the
synergistic effect with DSF.
CONCLUSION
In order to improve the antitumor effect of disulfiram, a
PEGylated liposome copper preparation was formulated in
this study. The encapsulation efficiency of the liposomes was
improved by the synthesis of copper oleate, which also over-
came the common leakage problems faced when water-
soluble copper is used to incorporate in a liposome. The cop-
per ions and disulfiram could accumulate in tumor cells when
Cu(OI)2-L was injected combined with disulfiram nanoparti-
cles. The properties of the copper oleate liposomes resulted in
drug release in acidic media, made the vesicles more likely to
accumulate in tumor tissue, and improved the efficacy and
safety while reducing side effects of the preparation. The ves-
icle size of about 100 nm is more easily able to accumulate in
the tumor through the EPR effect. Compared with the tradi-
tional oral copper preparations, copper liposomes exhibited
excellent in vitro and in vivo stability, and were able to extend
the circulation time and improve the bioavailability, and thus
were more suitable to be employed in combination with di-
sulfiram for tumor treatment. The strategy of incorporating
copper in a liposome can also be applied to treat other copper-
deficiency diseases, and open up opportunities for targeted
delivery of copper.
Generally, vesicles with a particle size around 100 nm are
more likely to penetrate and accumulate into solid tumors via
the enhanced penetration effect (EPR) effect (31). Moreover,
the pH sensitivity of liposomes increases the potential for the
drug to be released in the tumor. The poorly-developed vas-
culature in a tumor is often unable to provide enough nutri-
tion to the rapid dividing cells, resulting in lack of oxygen and
nutrients in the tumor cells. Lactic acid produced by the an-
aerobic metabolism activity can lead to an acidic pH in many
solid tumors. Although the acidic tumor environment may
contribute to tumor invasion in some cases, it also provides
an opportunity for tumor-targeted delivery systems (32,33). As
mentioned above, the liposome prepared in this work
displayed considerable pH sensitivity, which may help prevent
drug leakage in the blood circulation, but promote the release
of the copper in tumor tissue. As the liposomes loaded with
Cu2+ reach tumor tissues, the copper ions encapsulated in the
liposomes may transport into tumor cells by the following
two ways: (1) by colliding with the cell membrane, follow-
ed by transference of the copper ions by a concentration
gradient or lipid exchange (34), without being limited by
the numbers of Ctr1, or (2) the free Cu2+ released in the
tumor tissue can be chelated by DSF and this process can
facilitate the internalization of copper in a CTR1 inde-
pendent manner, leading to an excessive ROS reaction,
and final product Cu(DTC)2 further providing a lasting
antitumor effect. The tumor-targeted release of copper
ions from the liposome was validated in the tumor thera-
py results. When tested in mice bearing hepatoma xeno-
grafts, the Cu(OI)2-L demonstrated a significant synergis-
tic effect efficacy with the DSF NPs. As shown in Fig. 6c,
the tumor growth is effectively inhibited by the Cu(OI)2-
L- DSF NPs combination therapy with a TIR of 50.3%,
while DSF NPs plus oral copper supplementation exhib-
ited a much weaker antitumor effect with a TIR of
35.5%, which is similar to the therapy of DSF NPs alone.
The efficacy test data indicated that Cu(OI)2-L is able to
increase the cellular copper level effectively and develop
synergism with DSF.
ACKNOWLEDGMENTS AND DISCLOSURES
Amanda Pearce is gratefully thanked for correcting English of
the manuscript. We would like to thank the reviewers for their
constructive comments to improve the research. There is no
conflict of interest with the subject matter or materials
discussed in the manuscript.
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