Highly Stable, Coordinated Polymeric Nanoparticles Loading Copper(II) Diethyldithiocarbamate for Combinational Chemo/Chemodynamic Therapy of Cancer

Article abstract of DOI:10.1021/acs.biomac.9b00367

Disulfiram (DSF) has excellent in vitro anticancer activity in the presence of Cu(II). The anticancer mechanism studies have demonstrated that copper(II) diethyldithiocarbamate, Cu(DDC)₂, is the crucial DSF's metabolite exhibiting anticancer ac

Full text of DOI:10.1021/acs.biomac.9b00367

Article
pubs.acs.org/Biomac
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Highly Stable, Coordinated Polymeric Nanoparticles Loading
Copper(II) Diethyldithiocarbamate for Combinational Chemo/
Chemodynamic Therapy of Cancer
Xinyu Peng,† Qingqing Pan,† Boya Zhang,† Shiyu Wan,‡ Sai Li,‡ Kui Luo,§ Yuji Pu,*,† and Bin He†
†National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
‡School of Chemical Engineering, Sichuan University, Chengdu 610065, China
§Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital, Sichuan University, Chengdu 610041,
China
S
* Supporting Information
ABSTRACT: Disulfiram (DSF) has excellent in vitro anticancer
activity in the presence of Cu(II). The anticancer mechanism
studies have demonstrated that copper(II) diethyldithiocarbamate,
Cu(DDC)2, is the crucial DSF’s metabolite exhibiting anticancer
activity. In this paper, highly stable polymeric nanoparticles were
fabricated via a coordination strategy between Cu(II) and
carboxylic groups in poly(ethylene glycol)-b-poly(ester-carbonate)
(PEC) for efficient loading of Cu(DDC)2, which was generated by
the in situ reaction of DSF and Cu(II). The properties of
nanoparticles such as drug loading contents, sizes, and
morphologies could be tuned by varying the feeding ratios of
DSF, Cu(II), and PEC. These Cu(II)/DDC-loaded nanoparticles
showed excellent stability in both neutral and weak acidic solutions
and under dilution. In vitro anticancer study established that
Cu(II)/DDC-loaded nanoparticles could enable a combination therapy of Cu(DDC)2-based chemotherapy and chemodynamic
therapy mediated by bioavailable Cu(II) that was not in the form of Cu(DDC)2. The in vivo antitumor results demonstrated
that the Cu(II)/DDC-loaded nanoparticles showed superior antitumor efficacy to DSF/Cu(II). Our study provided a facile and
effective strategy of highly stable coordination-mediated polymeric nanoparticles for combinational therapy of cancer.
Despite the excellent in vitro anticancer activity, DSF still has
some hurdles for its further in vivo cancer therapy. The main
shortcoming of DSF is the rapid metabolism in blood,12,13 and
the enzymatic metabolites of DSF in liver are nontoxic to
cancer cells.14 To address the issue of extreme instability in
blood, polymeric drug delivery systems (DDSs) have been
developed to encapsulate DSF, alleviating the metabolite
rate.15−18 However, due to the intrinsic nonpolar structure and
instability, the drug loading contents (DLCs) of DSF in these
polymeric micelles were very low (usually 2−5%).17,19,20
Cu(II) is a vital factor for the high anticancer efficacy of
DSF; however, in many previous research works, only DSF was
loaded in DDSs and Cu(II) was neither loaded nor
supplemented simultaneously. Although the copper concen-
tration in many human tumor tissues is higher than that in
normal tissues, the absolute Cu(II) concentration in tumor is
still too low to inspire the therapeutic activity.21 DDSs loading
DSF alone without Cu(II) supplementation showed very
1. INTRODUCTION
Cancer is a global threat to human lives and drug resistance has
been found during chemotherapy using clinical therapeutics.
Since exploring new anticancer drugs encounters high costs
and long testing periods, drug repurposing that screens the
approved drugs for various diseases as candidate antineoplastic
drugs is now a promising approach to address these issues.1
Disulfiram (DSF) has been widely used as an antialcoholism
drug for over 60 years. Recently, DSF is found to exhibit
superb in vitro anticancer activity especially in the presence of
Cu(II).2,3 The anticancer mechanism of DSF is still being
explored, and current study has established that DSF can target
ALDH-positive cancer cells like cancer stem cells,4,5 suppress
proteasomes’ activities to inhibit NF-κB, PI3K, and TGF-β
pathways,6,7 and induce the apoptosis of the tumor cells
through a redox-related process.8 The anticancer activity of
DSF is Cu(II)-dependent.9,10 A recent study has revealed that
Cu(DDC)2, the anticancer metabolite of DSF, could inhibit
cancer by targeting the p-97-NPL4-UFDI protein complex,
inducing accumulation of ubiquitinated proteins and a heat
shock response.11
Received: March 14, 2019
Revised: May 15, 2019
© XXXX American Chemical Society
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Scheme 1. (A) Illustration of Cu(II)/DDC-Loaded Nanoparticle Preparation and the Proposed Interchain Coordination
Effects for Efficient Loading of Cu(DDC)2; (B) Anticancer Mechanism Illustration of Cu(II)/DDC-Loaded Nanoparticles
a
a
After internalization into cancer cells, Cu(DDC)2 was released to exert chemotherapy and Cu(II) that was available by GSH could induce ROS
generation and apoptosis to fulfill chemodynamic therapy.
limited in vivo antitumor efficacy.17,22 Therefore, exogenous
Cu(II), such as copper gluconate, is orally taken to increase the
copper concentration in tumor in clinical after DSF is
intravenously administrated.11 Although copper is an essential
trace metal for human beings, uptake of large amounts of
copper would lead to systemic toxicity. Besides, delivering DSF
and Cu(II) simultaneously to tumor tissues with high
concentrations via different ways is a challenge. Therefore,
the strategy of delivering Cu(DDC)2 instead of DSF had been
developed recently.23−25 Compared with the DSF-based
DDSs, the delivery of Cu(DDC)2 averted the inefficiency of
DSF and additional supplementation of Cu(II).
The Cu(DDC)2 complex was obtained by the reaction of
Cu(II) and DSF (or sodium diethyldithiocarbamate, DDC).
Chen et al. developed Cu(DDC)2-loaded polymeric micelles
loaded via the reaction of Cu(II) and DDC.25 The micelles
showed excellent inhibition to drug-resistant prostate cancer
cells (DU145-TXR) in a manner of paraptosis. Although
complexation of Cu(II) and oxygen atoms in poly(L-lactide)
(PLA) or poly(ethylene glycol) (PEG) could stabilize
Cu(DDC)2, we postulated other stronger Cu(II) ligands
containing polymer would not only codeliver Cu(II) and DSF
but also enhance the stability of Cu(DDC)2-loaded polymeric
DDS.
(PEC), was designed to form the stable nanoparticles by the
coordination effects of Cu(II) and carboxylic groups in the
polymer side chains, which could afford a core-cross-link-like
architecture to enhance the stability of polymeric nanoparticles
(Scheme 1A). The nanoparticles’ properties and DLCs of
Cu(DDC)2 could be tuned by varying the feeding ratios of
DSF, Cu(II), and PEC. These Cu(II)/DDC-loaded nano-
particles exhibited excellent stability in neutral and weak acidic
conditions and against dilution. The anticancer mechanism
study established that Cu(II)-mediated chemodynamic
therapy26−28 and Cu(DDC)2-based chemotherapy were
fulfilled in these Cu(II)/DDC-loaded nanoparticles (Scheme
1B). Benefiting from the excellent stability and combinational
chemo/chemodynamic therapy, the Cu(II)/DDC-loaded
nanoparticles showed superior in vivo antitumor effect to
DSF/Cu(II).
2. EXPERIMENTAL SECTION
2.1. Materials. Methoxy poly(ethylene glycol) (mPEG, Mn
=
2000 Da) was purchased from Sigma-Aldrich Co. and used as
received. All the other chemical reagents were from Tansoole
Platform (Shanghai Titan Technology Co. Ltd.) and used as received.
The solvents were obtained from Kelong Chemical Co. (Chengdu,
China) and purified before use. Roswell Park Memorial Institute
(RPMI) 1640 medium (HyClone), fetal bovine serum (FBS,
HyClone), penicillin−streptomycin (HyClone), thiazolyl blue
tetrazolium bromide (MTT, Energy Chemical), Annexin V-FITC/
PI apoptosis detection kit (Dojindo Co.), PI/RNase staining buffer
In this study, we developed a highly stable coordinated
nanoparticles to load Cu(DDC)2 that was generated by the in
situ reaction of Cu(II) and DSF. A carboxylic group containing
amphiphilic copolymer, mPEG-block-poly(ester-carbonate)
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solution (BD Pharmingen), and reactive oxygen species (ROS) assay
kit (Beyotime Biotechnology) were used for cell study.
weight of DDC (or Cu) in NPs
weight of NPs
DLCs (%) =
DLEs (%) =
× 100%
2.2. Characterization. The chemical structures of the synthetic
1
compounds and polymers were identified by H NMR (Bruker 400
weight of DDC (or Cu) in NPs
weight of the feeding DSF (or Cu)
× 100%
MHz) and Fourier-transform infrared (FTIR) spectroscopy (Thermo
Scientific Nicolet iS10). The molecular weights of polymers were
characterized by gel permeation chromatography (GPC, Waters), and
dimethylformamide (DMF) was used as the mobile phase. The sizes
and ζ-potentials of nanoparticles were measured by dynamic light
scattering (DLS, Malven ZetasizerNano ZS). The size and
morphology of nanoparticles were observed with transmission
electron microscopy (TEM, JEOL JSM-6490LA, 100 kV, without
negatively staining).
2.3. Synthesis of Compound 1. 2,2-Bis(hydroxymethyl)-
propionic acid (50 g, 372 mmol) and potassium hydroxide (20.9 g,
373 mmol) were dissolved in DMF (200 mL). Benzyl bromide (76.51
g, 0.447 mmol) was added dropwise, and the mixture was stirred at
100 °C for 12 h. The solution was concentrated under vacuum after
cooling and filtration. The residue was diluted with ethyl acetate (100
mL), washed with deionized water (3 × 100 mL) and brine, and dried
with anhydrous Na2SO4. The product was obtained as a colorless
crystal after recrystallization in toluene for three times (47.6 g, yield
57%).
2.4. Synthesis of Compound 2. To a solution of compound 1
(15.0 g, 66.0 mmol) and ethyl chloroformate (14.5 g, 133 mmol) in
anhydrous THF (200 mL) was added trimethylamine (13.8 g, 136
mmol) dropwise in the solution in an ice bath. The solution was
stirred at room temperature for 2 h. The white precipitate was filtered,
and the solution was concentrated. The product was obtained as a
colorless crystal after recrystallization in ethyl acetate for three times
(12.7 g, yield 76%).
2.5. Synthesis of PEC-Bn. PEC-Bn was synthesized by a ring-
opening polymerization using mPEG (Mn 2000 Da) as a macro-
initiator and stannous octoate (Sn(Oct)2) as catalyst. mPEG (1.0 g,
0.5 mmol) was dried under vacuum at 100 °C for 4 h. Under argon
atmosphere, L-lactide (0.866 g, 6.0 mmol), compound 2 (1.62 g, 6.5
mmol), and Sn(Oct)2 (7.0 mg, 0.017 mmol) were added. Then the
mixture was heated at 125 °C under vacuum for 24 h. The reaction
was quenched by immersing the reaction tube into an ice bath. The
crude product was dissolved in dichloromethane and precipitated in
cold ethyl ether for three times. The product was dried under vacuum
(3.45 g, yield 95%).
2.9. Stability Study of Nanoparticles. Cu(II) or Cu(II)/DDC-
loaded nanoparticles were resuspended in different Acetate Buffered
Saline (ABS buffers, pH 5.0, 6.8, and 7.4) at room temperature. At
predetermined time intervals, the sizes and ζ-potentials of nano-
particles were measured by DLS. Cu(II) or Cu(II)/DDC-loaded
nanoparticles resuspended solutions (0.5 mg/mL) were diluted with
PBS by 50 (0.01 mg/mL) and 500 times (0.001 mg/mL), and their
sizes and ζ-potentials at 0 and 24 h were measured at room
temperature. The stability of the nanoparticles in the presence of
proteins was studied in PBS (pH 7.4) with 10% FBS. The sizes of
nanoparticles (0.5 mg/mL) were measured by DLS at 0 and 24 h.
2.10. Drug Release. The Cu(II)-loaded nanoparticles were
dispersed in 1 mL of pH 7.4 PBS buffer with or without GSH (5
mM). Then the mixture was transferred into a dialysis bag (MWCO 1
kDa) and immersed in 25 mL of PBS (pH 7.4) with different GSH
concentrations in the vials. The vials were placed in a shaking bed at
37 °C. At a predetermined time, 1 mL of the release media was taken
out and fresh media was supplemented. The released Cu(II) content
was measured by ICP-AES.
2.11. Cell Culture. The human nonsmall cell lung cancer cell line,
A549, was cultured in RPMI 1640 medium with 10% FBS, 100 U/mL
penicillin, and 100 U/mL streptomycin, and the solution was
maintained at 37 °C in a humidified incubator with 5% CO2.
2.12. Cytotoxicity Test. A549 cells were seeded in 96-well plates
at a density of 4 × 103 cells per well and cultured for 12 h. The cells
were then cultured in the medium containing DSF, DSF/Cu(II), or
Cu(II)/DDC-loaded nanoparticles at different DDC concentrations
for 48 h. Cells were then treated with MTT containing serum-free
medium (0.5 mg/mL, 100 μL) for 4 h. The purple formazan crystals
were dissolved in dimethyl sulfoxide (DMSO, 150 μL) and the
absorbance at 490 nm was measured on a microplate reader (Thermo
Scientific MK3). The cytotoxicities of the polymer PEC and Cu(II)-
loaded nanoparticles were tested similarly.
2.13. Cellular Uptake of RhB-Loaded Nanoparticles. Rhod-
amine B-loaded PEC nanoparticles were prepared according to our
previous study.29 The cellular uptake of the nanoparticles was studied
by confocal laser scanning microscopy (CLSM).
2.14. Intracellular Copper Content Test. A549 cells were
seeded in 6-well plates (5 × 105 cells per well) and cultured for 12 h.
Afterward, cells were treated with medium containing CuCl2, Cu(II)/
DSF, Cu(II)-loaded nanoparticles, or different Cu(II)/DDC-loaded
nanoparticles (Cu(II) concentration: 2 μg/mL) for 4 h. Cells were
harvested and counted, washed twice by PBS, and digested by the
digestion solution containing 65% nitric acid and 15% H2O2 overnight
at 65 °C. The lysate was diluted to a final volume of 10 mL, and Cu
content was measured by ICP-AES. The Cu(II) levels in cells were
expressed as Cu content (ng) per 104 cells.
2.15. Apoptosis Study. A549 cells were seeded in 6-well plates
(2 mL per well, 2.5 × 105 cells) and cultured for 12 h. The culture
medium was removed, and the cells were treated with medium
containing DSF, different Cu(II)/DDC loaded nanoparticles, or
Cu(II)/DSF (DSF concentration: 20 ng/mL) for 48 h. Cells were
harvested and washed twice by PBS. For the apoptosis study, cells
were then suspended in a binding buffer provided by the Annexin V-
FITC/PI apoptosis detection kit as the manufacture’s protocols. The
cells were then mixed with Annexin V-FITC and PI, incubated in the
dark at RT for 15 min, and analyzed by a BD Accuri C6 flow
cytometer.
2.6. Synthesis of PEC. The mixture of PEC-Bn (3.0 g, 0.43
mmol) and Pd/C (0.3 g, 10%w/w) in dry DMF was stirred under H2
atmosphere for 48 h. The hydrogen gas was freshly prepared by
reaction of zinc and hydrochloric acid. Pd/C was filtered, and the
solution was concentrated to obtain PEC (2.45 g, yield 98%).
2.7. Preparation of Cu(II)/DDC-Loaded Nanoparticles. A
series of Cu(II) or Cu(II)/DDC-loaded nanoparticles were prepared
by varying feeding ratios of COOH:DSF:Cu(II). Taking nanoparticle
M2 as an example, the solution of PEC (10 mg, 20 μmol COOH),
CuCl2 (2.25 mg, 13.2 μmol), and DSF (1.95 mg, 6.6 μmol) in DMF
(0.4 mL) was ultrasonicated for 2 h. The solution was then dropwise
added into stirring ultrapure water (700 r/min). Afterward, the
mixture was stirred for 12 h, dialyzed against ultrapure water for 24 h.
The precipitate was removed by centrifugation and the supernatant
was lyophilized to obtain M2. Cu(II)-loaded nanoparticles and other
Cu(II)/DDC-loaded nanoparticles were prepared similarly.
2.8. Drug Loading Contents and Drug Loading Efficiencies.
The drug loading contents (DLCs) and drug loading efficiencies
(DLEs) of DDC in nanoparticles were measured by high-performance
liquid chromatography (HPLC) with a reverse-phase C-18 column
(Agilent) and a UV detector at 265 nm using mixed solvent of
methanol and pH 7.0 PBS (80/20, V/V) as the mobile phase. The
flow rate was 1.0 mL/min. The content of copper was measured by
inductively coupled plasma atomic emission spectroscopy (ICP-AES).
The DLCs and DLEs of Cu(II) or DDC were calculated by the
following equations:
2.16. Intracellular ROS Detection by DCFH-DA. A549 cells
were seeded in glass bottom dishes (1 mL per dish, 1 × 105 cells) and
cultured for 24 h. The cells were then treated with a medium
containing Cu(II)/DDC-loaded nanoparticles, DSF/Cu(II) (DSF
concentration: 10 ng/mL), Cu(II)-loaded nanoparticles, or CuCl2
(Cu(II) concentration: 3 μg/mL) for different times. Thereafter, cells
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were incubated with DCFA-DA (10 μM) for 20 min and washed with
PBS for three times. The intracellular ROS signal was observed by
CLSM.
benzyl groups in PEC-Bn were deprotected by Pd/C to give
PEC that contains free carboxylic groups.
The structures of the carbonate monomer and copolymers
2.17. In Vivo Antitumor Efficacy. All animal experiments were
performed in accordance with the Guidelines for Care and Use of
Laboratory Animals of West China Hospital, Sichuan University and
approved by the Animal Ethics Committee of China.30 Female Balb/
C nude mice (4−5 weeks of age, 18−22 g) were purchased from
Chengdu Dashuo Experiment Animal Co. Ltd. 1.0 × 106 A549 cells
were inoculated at the right flank of mice to build the A549 tumor
model. After tumor growth reached about 100 mm3, the mice were
randomly divided into 3 groups (n = 6) and treated with saline (by
intravenous injection, i.v.), DSF/Cu(II) (DSF, 10 mg/kg, i.v.; copper
gluconate, 1.5 mg/kg, oral administration), and M3 (1 mg/kg in DSF
dosage, i.v.) on days 0, 4, 8, 12. The body weights and tumor volumes
of tumor-bearing mice were recorded every 4 days. The tumor
volumes and tumor inhibition rates were calculated according to our
previous study.31 The H&E staining study of tumor and normal
organs was carried out according to our previous literature.32
were characterized by 1H NMR and FTIR (Figures 1 and S1−
2.18. Statistical Analysis. All mean values
SD reported in
results section were compared by Student’s t test. P values of no more
than 0.05 were considered significant differences.
3. RESULTS AND DISCUSSION
1
Figure 1. H NMR spectra of PEC-Bn and PEC in CDCl3.
3.1. Polymer Synthesis and Design. The biodegradable
polymer mPEG-b-poly(ester-carbonate), PEC, was synthesized
as shown in Scheme 2. Polyesters and polycarbonates are
S2). The monomer composition in PEC was calculated by
NMR; the degrees of polymerization of 2 and LA were 11 and
22, respectively (Table S1). The molecular weight of PEC-Bn
was determined by GPC (Figure S3 and Table S1) and the
molecular weight was ∼5 kDa. The successful deprotection of
PEC-Bn was verified by NMR. As shown in Figure 1, the peaks
that are corresponding to the benzyl groups at 7.33 ppm
disappeared in the NMR spectrum of PEC, which was
consistent with the results in the previous literature.36,37
3.2. Stability of Coordination-Mediated Cu(II)-Loaded
Polymeric Nanoparticles. We first studied the Cu(II)
loading efficiency of carboxyl-containing PEC by coordination
effects. We found that PEC alone could not self-assemble into
nanoparticles (data not shown here), which were probably due
to the negative charge repulsion of carboxylic groups and poor
hydrophobicity of PEC. However, nanoparticles were formed
after the addition of Cu(II), which was verified by the
detection of nanoparticles by DLS. The driving force of
nanoparticle formation was mainly attributed to the
intermolecular coordination effects of carboxylic groups and
Cu(II) (Figure 2A).38 Although the complexation of Cu(II)
and oxygen atoms in PEC could facilitate Cu(II) loading,25 we
would attribute the nanoparticle formation mainly to the
coordination effects owing to the excellent nanoparticle under
dilution, which will be discussed later.
Scheme 2. Synthesis of Monomer (A) and PEG-b-
poly(ester-carbonate) PEC (B)
A series of Cu(II)-loaded polymeric nanoparticles were
prepared by varying the feeding molar ratios of carboxylic
groups (COOHs) in PEC to Cu(II) to study the Cu(II)
loading efficiency. The successful loading of Cu(II) was
confirmed and quantitively determined by ICP-AES, and the
Cu(II) concentrations in these nanoparticles were in the range
0.19−0.23 mM (Table S2). The copper content showed an
increase trend with increasing feeding ratio of Cu(II) to
COOH when the ratio was less than 1:1 (drug loading
contents (DLCs) ranges from 2.66% to 3.10%, Figure 2B).
When the feeding ratio of Cu(II)/COOH was 1:1, the Cu(II)
concentration was the highest (DLC of 3.10%, 0.23 mM) in
the Cu(II)-loaded polymeric nanoparticles. However, a further
increase of Cu(II)/COOH ratio lead to a decrease of DLC of
Cu(II) (2.68%). The trend of drug loading efficiencies (DLEs)
employed because they are biodegradable and biocompatible
polymers and extensively studied in various biomedical
applications.33−35 In addition, PLA was used to endow
hydrophobicity to efficiently load hydrophobic Cu(DDC)2;
polycarbonate was designed to load Cu(II) mainly by
coordination interactions between carboxylic groups in
polycarbonate and Cu(II). A cyclic carbonate monomer,
compound 2 in Scheme 2A, was first synthesized by the
cyclization of compound 1, which was obtained by a benzyl
protection reaction of 2,2-bis(hydroxymethyl)propionic acid.
Compound 2 and L-lactide (LA) were then copolymerized by
ring-opening polymerization using mPEG (Mn 2000 Da) as a
macroinitiator and Sn(Oct)2 as a catalyst to give PEC-Bn. The
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Figure 2. Preparation and characterization of the Cu(II)-loaded polymeric nanoparticles. (A) The illustration of the Cu(II)-loaded nanoparticle
preparation by coordination effects. The impact of Cu(II)/COOH feeding molar ratios on DLCs and DLEs (B), and the sizes and ζ-potentials (C)
of Cu(II)-loaded nanoparticles. (D) TEM image of nanoparticles M1 (the Cu(II)/COOH feeding molar ratio was 2:3); the inset shows the size
results by DLS. (E) The changes of Z-average sizes of M1 over time in different pH conditions. (F) The size changes of M1 after dilution for 50
and 500 times at 0 and 24 h.
was similar to that of DLCs in the Cu(II)-loaded nanoparticles,
and the DLEs were in the range 17.9%−71.8%. The effect of
Cu(II)/COOH ratios on the sizes and ζ-potentials of Cu(II)-
loaded nanoparticles was also studied (Figure 2C). We found
that the feeding Cu(II)/COOH ratios had a subtle effect on
the nanoparticles’ sizes and ζ-potentials. The nanoparticles
showed average Z-average sizes of 120−135 nm, and ζ-
potentials were in the range −17.6 to −20.5 mV.
The typical TEM image of Cu(II)-loaded nanoparticle M1
(Cu(II)/COOH = 2:3) is shown in Figure 2D. Obviously, M1
showed a core−shell structure with a size less than 200 nm,
which was consistent with the DLS result (the inset in Figure
2D). The core in the TEM image was ascribed to Cu(II) and
poly(ester-carbonate) blocks of PEC, and the shell was a
hydrophilic PEG layer.
The stability of the Cu(II)-loaded nanoparticles was first
investigated by studying the changes of nanoparticles’ sizes and
ζ-potentials at predetermined time intervals in different pH
conditions (Figure 2E and Figure S8A). The sizes and ζ-
potentials of M1 showed negligible changes over 72 h in all pH
conditions (pH 7.4, 6.8, and 5.0), indicating the high stability
of Cu(II)-coordinated polymeric nanoparticles. We then
studied the stability of Cu(II)-loaded nanoparticles against
dilution.39 As shown in Figure 2F, the sizes showed weak
variations at 0 and 24 h after 50- or 500-times dilution (the
initial concentration of M1 was 0.5 mg/mL). Together, these
results established that Cu(II)-coordinated polymeric nano-
particles exhibited excellent stability.
3.3. Drug Loading Properties of Coordination-
Mediated Cu(II)/DDC-Coloaded Polymeric Nanopar-
ticles. Inspired by the excellent stability of Cu(II)-coordinated
PEC nanoparticles and the intrinsic anticancer effect of
Cu(DDC)2, we then evaluated the capacity of PEC to coload
Cu(II) and DSF. Because the reaction between DSF and
Cu(II) was very rapid (within several minutes, Figures S4 and
S5), Cu(DDC)2 could be generated and loaded in situ during
the nanoparticle formation, which was mainly driven by the
coordination effects between COOH and Cu(II) as well as
hydrophobic interactions between PEC and Cu(DDC)2
(Scheme 1A). The obtained nanoparticles were denoted as
Cu(II)/DDC-loaded nanoparticles. The as-prepared typical
nanoparticles’ suspensions are shown in Figure S6, and
precipitation of Cu(DDC)2 was not observed. The colors of
the nanoparticle suspensions were more brown and darker
with increased DSF/Cu(II) feeding molar ratios, suggesting a
higher loading content of Cu(DDC)2.
A series of Cu(II)/DDC-loaded nanoparticles were
fabricated by varying the feeding ratios of DSF/Cu(II)/
COOH (Table S2). The drug loading contents (DLCs) and
the drug loading efficiencies (DLEs) of DDC, Cu(DDC)2, and
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Cu(II) in the Cu(II)/DDC-loaded nanoparticles are summar-
ized in Table S2 and Figure 3. When the Cu(II)/COOH ratio
precipitation of Cu(DDC)2 during nanoparticle preparation.
The highest DLCs of Cu(II) and Cu(DDC)2 in these
nanoparticles were about 4.55% and 10.0%, respectively.
The effect of the feeding molar ratios on the sizes and ζ-
potentials of Cu(II)/DDC-loaded nanoparticles was also
studied. As shown in Figure 3, the Z-average sizes of
nanoparticles increased with the increasing DSF/Cu(II) ratios
from about 100 to 300 nm, except that the nanoparticles
prepared with a feeding DSF/Cu(II)/COOH ratio of 3:2:1.5,
which showed smaller size because of its extremely low DLCs
of Cu(II) and DDC that was induced by large amount of
precipitation of Cu(DDC)2. The ζ-potentials of Cu(II)/DDC-
loaded nanoparticles showed no obvious change patterns and
they were in the range from −18 to −25 mV. We then chose
Cu(II)/DDC-loaded nanoparticles (M2−M4) that were
prepared with a feeding Cu(II)/COOH ratio of 2:3 for further
study due to the highest Cu(DDC)2 loading content in M4
and the obvious differences of Cu(DDC)2 loading contents in
three nanoparticles.
Figures 4A1, B1, and C1 show the TEM images of Cu(II)/
DDC-loaded nanoparticles M2, M3, and M4, respectively.
Obviously, Cu(II)/DDC-loaded nanoparticles (M2−M4)
were distinct from the corresponding Cu(II)-loaded nano-
particle M1 in size and morphology (Figure 2D). The
nanoparticles sizes by TEM increased with increasing feeding
ratio of DSF/Cu(II), which was consistent with the DLS
results (Figure 3). Nanoparticles M2−M4 showed different
morphologies due to the different Cu(II) and Cu(DDC)2
loading contents. The morphologies of Cu(II)/DDC-loaded
nanoparticles changed from spherical to irregular with
increasing DSF/Cu(II) ratios. The successful loading of
Figure 3. Impact of feeding molar ratios of DSF/Cu(II)/COOH on
the nanoparticle properties (Z-average sizes, ζ-potentials, Cu(II) and
DDC concentrations) of Cu(II)/DDC-loaded nanoparticles. Gen-
erally, when the feeding molar ratio of Cu(II)/COOH was fixed,
higher Cu(II) and DDC loading contents could be obtained when
DSF/Cu(II) was 1:1. (M1−M4 represent the nanoparticles prepared
by fixed feeding Cu(II)/COOH molar ratio of 2:3 and different
feeding amounts of DSF).
was fixed, the DLCs of Cu(II) and DDC would first increase
and then decrease with the increasing DSF/Cu(II) ratios from
0:1 to 3:2 (0 to 1.5 in Figure 3). The low DLCs of Cu(II) and
DDC in the high DSF/Cu(II) ratio of 1.5 (Cu(II)/COOH
was 1:1 or 4:3) were ascribed to that excessive DSF resulted in
Figure 4. TEM images of nanoparticles M2 (A1), M3 (B1), and M4 (C1); the insets are the corresponding DLS results. Size changes of M2−M4
in different pH conditions (A2, B2, and C2) and under dilution (A3, B3, and C3).
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Figure 5. (A) Cell viabilities of A549 cells treated with DSF, DSF/Cu(II) (DSF and Cu(II) in equimolar amount), and Cu(II)/DDC-loaded
nanoparticles M2−M4 for 48 h. (B) Intracellular Cu contents of A549 cells treated with different formulations at a Cu(II) concentration of 2 μg/
mL for 4 h (n = 3).
Cu(II) and Cu(DDC)2 were further confirmed by scanning
transmission electron microscopy (Figure S7).
showed different release behavior in the presence and absence
of 10 mM GSH (Figure S10). In the presence of GSH, a burst
release of Cu(II) was observed after 8 h, suggesting the GSH-
sensitiveness of the Cu(II)-coordinated polymeric nano-
particles.
3.4. In Vitro Anticancer Efficacy of Cu(II)/DDC-Loaded
Nanoparticles. Before the in vitro anticancer study of Cu(II)/
DDC-loaded PEC nanoparticles, the cytotoxicity of PEC
against normal cells was studied by an MTT assay to evaluate
its cytocompatibility. NIH 3T3 cells were treated with cell
culture medium with varying concentrations of PEC for 48 h.
As shown in Figure S11, the viability of NIH 3T3 cells was
PEC dose-dependent; 85.0% and 70.7% cells were alive after
the treatment with PEC at polymer concentrations of 100 and
250 μg/mL, respectively. The cytotoxicity results suggested
that PEC showed good cytocompatibility.
The in vitro anticancer activity of DSF, DSF/Cu(II), and
Cu(II)/DDC-loaded nanoparticles M2−M4 in A549 cells was
then evaluated by an MTT assay. As shown in Figure 5A, DSF
alone showed poor anticancer activity due to the shortage of
extracellular Cu(II). However, DSF/Cu(II) showed signifi-
cantly enhanced anticancer activity (IC50:21.0 ng/mL) due to
the presence of the same molar amount of extracellular Cu(II),
consistent with many previous reports that DSF showed
superb in vitro anticancer efficacy in the presence of sufficient
Cu(II). M4 showed comparable anticancer activity to DSF/
Cu(II) with an IC50 value of 21.7 ng/mL. Interestingly, M2
and M3 with less DLC of Cu(DDC)2 than M4 showed lower
IC50 values of 9.4 and 10.7 ng/mL, respectively, about half of
the IC50 value of DSF/Cu(II), suggesting better anticancer
activity than DSF/Cu(II). The best anticancer activity of M2
was aberrant to its lowest DLC of Cu(DDC)2. Therefore, we
envisaged that high DLC of Cu(II) could fulfill chemodynamic
therapy since the cytotoxicity of PEC was low.
Cu(II) is an essential element of life; however, deficient or
excessive copper could lead to corresponding diseases.
Elevated copper contents have been found in various human
tumor tissues relative to normal tissues. The high copper
concentration in tumor tissue is essential to Cu(II)-involved
enzymatic activity and angiogenesis.21 In addition, excessive
Cu(II) that acts as oxidant could lead to oxidative stress in
cells.40 For example, Ma et al. had recently reported that
Cu(II)-amino acid nanoparticles could efficiently inhibit
cancer cells by Cu(II)-mediated chemodynamic therapy
where Cu(II) reacted with intracellular GSH and H2O2
Nanoparticle stability plays an important role in further long
blood circulation, which is the key to its in vivo antitumor
efficiency. Inspired by the excellent stability of the afore-
mentioned coordination-mediated Cu(II)-loaded polymeric
nanoparticles, we studied the stability of these Cu(II)/DDC-
loaded nanoparticles in different pH conditions. Nanoparticles
M2−M4 were treated with PBS at different pH (pH 7.4, 6.8,
and 5.0) and their sizes and ζ-potentials were measured at
different time points (Figures 4A2, B2, C2, and Figure S8B−
D). Notably, all nanoparticles showed excellent stability in all
pH conditions over 72 h, except that M4 showed a slight
increase in size at 1 h and M2−M4 showed slight increases in
ζ-potential at 1−4 h. Despite a slight disturbance in sizes and
ζ-potentials in the initial stage, the nanoparticles were stable in
sizes and ζ-potentials over 72 h. The dilution study also
established that Cu(II)/DDC-loaded nanoparticles were stable
after even 50- to 500-times dilution after 24 h (the initial
nanoparticles concentrations were 0.5 mg/mL, Figures 4A3,
B3, and C3).
The stability of Cu(II)/DDC-loaded nanoparticles under
10% FBS condition was also carried out (Figure S9). After a 24
h incubation, M3 and M4 showed slight size increase and were
still monodisperse, suggesting excellent stability in the presence
of 10% FBS. In contrast, M1 and M2 showed poorer stability
in the condition of 10% FBS and the sizes were polydisperse at
24 h. The poorer stability of M1 and M2 in FBS conditions
were probably ascribed to the Cu(II)-mediated interaction
with FBS. In M3 and M4, however, hydrophobic interactions
between Cu(DDC)2 and polymers may stabilize the nano-
particle and hinder the Cu(II)-FBS interactions. Collectively,
these results suggested that Cu(II)/DDC-loaded nanoparticles
were stable in neutral, weak acidic environments and under
dilution. M3 and M4 showed excellent stability even in the
presence of serum.
Given the excellent stability of Cu(II)/DDC-loaded nano-
particles, we assumed that their in vitro drug release behaviors
in different pH conditions may be very slow. The especially
low concentration of DDC is hard to be detected by HPLC,
therefore, the drug release of DDC was not shown here. Since
Cu(II) is an oxidant and GSH acts reducing agents to balance
the redox homeostasis and has much higher concentration in
cancer cells, we then studied whether GSH could affect the
release of Cu(II). Notably, the Cu(II)-loaded nanoparticles
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Figure 6. (A) CLSM images of A549 cells treated with CuCl2 and M1 at the same concentrations for 6 and 24 h. (B, C) Apoptosis rates of A549
cells treated with CuCl2 and M1 at a Cu(II) concentration of 3 μg/mL for 48 h.
successively to lead to upregulation of ROS that induces cancer
cell apoptosis (Scheme 1B).41
Cu(II)-mediated chemodynamic therapy exerted anticancer
activity by generating highly cytotoxic ROS like ·OH that
could destroy proteins, lipids, and DNA, inducing cell
apoptosis (Scheme 1B).41,43,44 The intracellular ROS gen-
eration could be detected by a probe 2′,7′-dichlorodihydro-
fluorescein diacetate (DCFH-DA), a small molecule that could
be taken by cells and the followed oxidation by intracellular
ROS could give 2′,7′-dichlorofluorescein (DCF) with green
fluorescence.45,46 Cells treated with M1 showed stronger
fluorescence intensity than those treated with CuCl2 at 6 and
24 h (Figure 6A), demonstrating the more ROS generation
induced by higher cellular uptake of Cu(II). An FITC-PI assay
was carried out to assess the M1-induced apoptosis. As shown
in Figure 6B,C, M1 and CuCl2 induced comparable cell
apoptosis (23.8% vs 24.2%) at the same Cu(II) concentration.
However, cells treated with M1 were more in the stage of late
apoptosis (21.2% vs 19.0%), confirming its higher cytotoxicity.
3.6. Cu(II)/DDC-Loaded Nanoparticles Inhibit Cancer
Cells via a Chemo/Chemodynamic Combinational
Therapy. We studied the anticancer mechanism of Cu(II)/
DDC-loaded nanoparticles by investigating the ROS gener-
ation induced by DSF/Cu(II) and Cu(II)/DDC-loaded
nanoparticles. The CLSM results showed that M2 treated
cells had the strongest green fluorescence at 6 and 24 h (Figure
7A), demonstrating that the most ROS was generated. Similar
results were obtained in the flow cytometry (Figure S14).
The apoptosis study showed that the apoptosis rates of DSF,
DSF/Cu(II), M2, M3, and M4 were 12.2%, 16.9%, 17.6%,
13.2%, and 4.9%, respectively (Figure 7B). Notably, M4, which
had the highest DLC of DDC and thereby very low DLC of
Cu(II) that are not in the form of Cu(DDC)2 because DDC
was a strong chelator of Cu(II), induced very low and
comparable apoptosis rate relative to control group (3.7%),
indicating that Cu(DDC)2 per se cannot generate intracellular
ROS, which was consistent with the previous report by Wang
et al.14 Since DDC was a strong chelator of Cu(II), Cu(DDC)2
could not react with GSH. Therefore, Cu(DDC)2 alone could
not induce ROS generation in cells, and M4 with the highest
The cellular uptake behaviors of the coordination-mediated
nanoparticles were first studied by CLSM. Because of the very
low fluorescence of Cu(II)/DDC-loaded nanoparticles, Rhod-
amine B-loaded PEC nanoparticles was prepared for CLSM
study. Like other polymeric nanoparticles internalized by
endocytosis, the Rhodamine B-loaded nanoparticles showed a
time-dependent internalization (Figure S12), demonstrating
the efficient cellular uptake of the polymeric nanoparticles.
Second, the intracellular copper contents of A549 cells treated
with M2−M4 as well as DSF and DSF/Cu(II) were studied;
the results are shown in Figure 5B. DSF/Cu(II) showed
significantly enhanced Cu(II) uptake relative to CuCl2,
suggesting that DSF facilitated the cellular uptake of Cu(II).42
M2 showed the highest Cu(II) uptake due to its high Cu(II)
loading content, and the intracellular copper contents of these
groups were in the order of M2 > M3 ≈ DSF/Cu(II) > M1 ≈
M4 > CuCl2. The high intracellular copper contents induced
by M2 and M3 were consistent with their excellent in vitro
anticancer activity, confirming our envisagement that high
DLC of Cu(II) could inhibit cancer cells.
3.5. Cu(II)-Loaded Nanoparticles Induce Intracellular
ROS Generation and Cancer Cell Apoptosis. To
demonstrate the chemodynamic therapy induced by Cu(II)
uptake, we first studied the cytotoxicity of Cu(II)-loaded
nanoparticles M1 as well as CuCl2 against A549 cells. As
shown in Figure S13, CuCl2 showed a dose-dependent toxicity
to A549 cells, indicating the toxicity of high concentration of
Cu(II). M1 showed significantly enhanced cancer cell
inhibition relative to CuCl2, suggesting the PEC-enhanced
Cu(II)-mediated toxicity. The intracellular Cu content study
demonstrated that M1 showed elevated cellular uptake of
Cu(II) to that of CuCl2 (Figure 5B), implying the enhanced
nanoparticle-mediated cellular uptake of Cu(II). Considering
the low cytotoxicity of PEC, the enhanced cytotoxicity of M1
was mainly ascribed to the higher Cu(II) uptake of M1.
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Figure 7. (A) CLSM images of A549 cells treated with DSF/Cu(II) and nanoparticles M2−M4 at a DDC concentration of 10 ng/mL for 6 and 24
h. (B, C) Apoptosis rates of A549 cells treated by DSF, DSF/Cu(II), nanoparticles M2−M4 at a DDC concentration of 20 ng/mL for 48 h.
Figure 8. Body weights (A) and tumor volumes (B) of tumor-bearing mice treated with saline (control), DSF/Cu(II) (10/1.5 mg/kg), and M3 (1
mg/kg in DSF) on days 0, 4, 8, and 12.
DLC of Cu(DDC)2 showed the least apoptosis. In contrast,
M2 and M3 with less DLC of Cu(DDC)2 induced much more
apoptosis than M4, which was ascribed to the more
internalization of Cu(II) that was in the form of GSH-available
Cu(II) rather than in the form of Cu(DDC)2. Cu(II) that
coordinated with PEC could react with GSH and H2O2
successively to give ROS that induced apoptosis.
The more apoptosis induced by M2 and M3 was also
confirmed by the cell morphology results. Figure S15 displays
the microphotographs of A549 cells that were treated with
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different formulations (Cu(II) concentrations were 2 μg/mL)
for 4 h. Cells treated with M2 and M3 showed obvious
shrinkage of cell structures, demonstrating higher apoptosis
rates. Overall, we had established that Cu(DDC)2 and the
Cu(II) that was not in the form of Cu(DDC)2 in our copper-
coordinated nanoparticles enabled chemotherapy and chemo-
dynamic therapy, respectively.
AUTHOR INFORMATION
■
Corresponding Author
*Y. Pu. E-mail: yjpu@scu.edu.cn.
ORCID
Kui Luo: 0000-0002-3536-1485
Yuji Pu: 0000-0002-4465-0262
Bin He: 0000-0002-0939-9869
3.7. In Vivo Antitumor Efficacy of Cu(II)/DDC-Loaded
Nanoparticles. The in vivo antitumor efficacy of M3 and
DSF/Cu(II) was then studied in A549 xenograft nude mice. In
the DSF/Cu(II) group, DSF was administrated by intravenous
injection at a dose of 10 mg/kg, and Cu(II) was supplemented
by oral administration at a dose of 1.5 mg/kg. Given the highly
cytotoxic nature of Cu(DDC)2, M3 was intravenously injected
with a low dose (1 mg/kg in DSF). The body weights of
tumor-bearing mice were monitored, and the results are shown
in Figure 8A. There was no significant weight loss in three
groups, suggesting the low systemic toxicity of M3. Figure 8B
shows the changes of tumor volumes after treatment. DSF/
Cu(II) group showed limited antitumor efficacy due to the
rapid metabolism of DSF in the blood. In contrast, M3 showed
much better antitumor efficacy than saline and DSF/Cu(II)
groups considering that the dose of M3 was only equivalent to
10% of the DSF/Cu(II) group. The tumor inhibition rates of
DSF/Cu(II) and M3 on day 28 were 16.6% and 51.6%,
respectively. The in vivo antitumor study demonstrated that
our coordination-mediated Cu(II)/DDC-loaded polymeric
nanoparticles could efficiently improve the antitumor efficacy
of DSF/Cu(II) even at a very low dose. The H&E histological
analysis showed that M3 induced more cancer cell necrosis and
had negligible toxicity to normal tissues (Figure S16).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the financial support of National Natural
Science Foundation of China (No. 51773130 and 51573111),
Fundamental Research Funds for the Central Universities
(YJ201764), and Jiangsu international cooperation project
(BZ2017062). Dr. Xi Wu in the Analytical & Testing Center,
Sichuan University is thanked for his help with ICP-AES
testing.
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We reported a coordination strategy for fabrication of Cu(II)-
and Cu(II)/DDC-loaded polymeric nanoparticles with ex-
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.9b00367.
NMR, FTIR, GPC, and other molecular properties of
polymers; drug loading contents of Cu(II) and DDC in
nanoparticles; other cellular experimental results; H&E
results of normal organs and tumors (PDF)
J
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Biomacromolecules XXXX, XXX, XXX−XXX