Platinum covalent shell cross-linked micelles designed to deliver doxorubicin for synergistic combination cancer therapy

Article abstract of DOI:10.2147/IJN.S130938

The preparation of polymer therapeutics capable of controlled release of multiple chemotherapeutic drugs has remained a tough problem in synergistic combination cancer therapy. Herein, a novel dual-drug co-delivery system carrying doxorubicin (DOX) and platinum(IV) (Pt[IV]) was developed. An amphiphilic diblock copolymer, PCL-b-P(OEGMA-co-AzPMA), was synthesized and used as a nanoscale drug carrier in which DOX and Pt(IV) could be packaged together. The copolymers were shell cross-linked by Pt(IV) prodrug via a click reaction. Studies on the in vitro drug release and cellular uptake of the dual-drug co-delivery system showed that the micelles were effectively taken up by the cells and simultaneously released drugs in the cells. Futhermore, the co-delivery polymer nanoparticles caused much higher cell death in HeLa and A357 tumor cells than either the free drugs or single-drug-loaded micelles at the same dosage, exhibiting a synergistic combination of DOX and Pt(IV). The results obtained with the shell cross-linked micelles based on an anticancer drug used as a cross-linking linkage suggested a promising application of the micelles for multidrug delivery in combination cancer therapy.

Full text of DOI:10.2147/IJN.S130938

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O r I g I N a l r e s e a r c h
Platinum covalent shell cross-linked micelles
designed to deliver doxorubicin for synergistic
combination cancer therapy
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
12 May 2017
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caiying Zhu1,*
Jingjing Xiao1,*
Ming Tang2
Abstract: The preparation of polymer therapeutics capable of controlled release of multiple
chemotherapeutic drugs has remained a tough problem in synergistic combination cancer therapy.
Herein, a novel dual-drug co-delivery system carrying doxorubicin (DOX) and platinum(IV)
(Pt[IV]) was developed. An amphiphilic diblock copolymer, PCL-b-P(OEGMA-co-AzPMA),
was synthesized and used as a nanoscale drug carrier in which DOX and Pt(IV) could be packaged
together. The copolymers were shell cross-linked by Pt(IV) prodrug via a click reaction. Studies
on the in vitro drug release and cellular uptake of the dual-drug co-delivery system showed that
the micelles were effectively taken up by the cells and simultaneously released drugs in the cells.
Futhermore, the co-delivery polymer nanoparticles caused much higher cell death in HeLa and
A357 tumor cells than either the free drugs or single-drug-loaded micelles at the same dosage,
exhibiting a synergistic combination of DOX and Pt(IV). The results obtained with the shell
cross-linked micelles based on an anticancer drug used as a cross-linking linkage suggested a
promising application of the micelles for multidrug delivery in combination cancer therapy.
Keywords: dual-drug co-delivery system, amphiphilic diblock copolymer, shell cross-linked
micelles, synergistic combination cancer therapy
hua Feng1
Wulian chen3
Ming Du1
1Medical center of Diagnosis and
Treatment for cervical Diseases,
Obstetrics and gynecology hospital,
shanghai Medical college, Fudan
University, shanghai, 2Department
of Otorhinolaryngology-head and
Neck surgery, Ningbo Medical
center, li huli hospital, Ningbo,
3state Key laboratory of Molecular
engineering of Polymers, Department
of Macromolecular science, Fudan
University, shanghai, china
*These authors contributed equally
to this work
Abbreviations
ATRP, atom transfer radical polymerization; AzPMA, 3-azidopropyl methacrylate;
CL, caprolactone; DAPI, 4′,6-diamidino-2-phenylindole; DDSs, controlled drug
delivery systems; DLS, dynamic light scattering; DMF, dimethylformamide; DOX,
doxorubicin; DP, degree of polymerization; EE, encapsulation efficiency; FT-IR, Fourier
1
transform infrared; GPC, gel permeation chromatography; H NMR, proton nuclear
magnetic resonance; HBMP, 2-hydroxyethyl 2-bromo-2-methylpropanoate; LE, loading
efficiency; MDR, multidrug resistance; Mn, average number molecular weight; MWCO,
molecular weight cut-off; NCL, non-cross-linked; NHS, N-hydroxysuccinimide;
OEGMA, oligo(ethylene glycol) ethyl methacrylate; PAzPMA, poly(AzPMA); PBS,
phosphate-buffered saline; PCL, polycaprolactone; PDI, polydispersity index; PEG,
poly(ethylene glycol); PMDETA, N,N,N′,N″,N′″-pentamethyldiethylenetriamine;
POEGMA, poly(OEGMA); Pt(IV), platinum(IV); PTX, paclitaxel; ROP, ring-opening
polymerization; SCL, shell cross-linked; TEM, transmission electron microscopy; TGA,
thermogravimetric analysis; THF, tetrahydrofuran; ZnPc, zinc-phthalocyanine.
correspondence: Ming Du
Obstetrics and gynecology hospital,
shanghai Medical college, Fudan
University, Da lin road, No 358,
shanghai 200011, china
Tel/fax +86 21 6345 0944
email duming0111@126.com
Introduction
The use of most chemotherapeutic drugs is limited by severe side effects, low therapeu-
tic index and development of drug resistance.1–3 Driven by an urgent need for practical
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applications in chemotherapy, effective DDSs have been to be stable against disassociation under high dilution, and
developed. Over the past few decades, DDSs have success- their aggregation is independent of external influences
fully improved the therapeutic efficiency of chemotherapeutic such as changes in pH values and other interference.26–33
drugsbyimprovingdrugsolubility, reducingsystemictoxicity, Recently, Liao et al designed core cross-linked polymer
increasing circulation time in the blood and controlling drug micelles carrying DOX, camptothecin and cisplatin for
release profile.4–7 However, due to the ability of the tumor cells single-nanoparticle combination cancer therapy, but the
to develop MDR, traditional DDSs used for the delivery of system was difficult to synthesize.34 As compared to core
single chemotherapeutic drug cannot prevent MDR, which ulti- cross-linked micelles, SCL micelles are facile to load drug
mately leads to treatment failure in most cancer therapy.8–10
via physical entrapment.35–38 If chemotherapeutic drugs are
In principle, chemotherapeutic drugs with different employed as the cross-linker in micelles, SCL micelles can
activities delivered simultaneously to the same cancer cells easily achieve co-delivery of multiple drugs. Thus, this new
can maximize cytotoxicity and reduce dosage of each drug.11,12 co-delivery drug system has two different drug-loading
Furthermore, synergistic combination therapy can also mini- methods, physical entrapment and covalent loading of drug,
mize the chance of developing resistance to any one drug by and thus exhibits different drug release rates. To the best of
the cells. Thus, the use of nanoparticle-based multidrug co- knowledge, the strategy of using chemotherapeutic drug as
delivery systems has been proposed to overcome undesirable a cross-linker in SCL micelles to construct novel multidrug
toxicity and MDR in the modern clinical cancer therapy.13–15 co-delivery systems is rarely reported. Therefore, it is still a
To date, among the amounts of drug carriers, polymeric promising challenge in combination cancer therapy.
micelles have been widely employed in the development
It is worth noting that the drugs chosen for combination
of multidrug co-delivery systems due to many advantages, therapy should have nonoverlapping toxicity profiles.39–41
such as core–shell nanostructures, excellent biocompat- The side effects of DOX mainly arise from cardiotoxicity,
ibility, biodegradability and versatile chemistry for further while those of cisplatin result from neurotoxicity. Thus,
functionalization.16,17 In addition, they are of nanoscopic size the combination of DOX and cisplatin could show a highly
and stealthy property. Thus, polymeric micelles usually lead synergistic potency, and suppress the growth of tumor cells
to a prolonged blood circulation and passive accumulation more efficiently than a single drug.41–43 In this study, chemo-
in solid tumor through the enhanced permeability and reten- therapeutic drug was utilized as a cross-linker to construct
tion effect.18–20 These features make polymeric micelles the a novel dual-drug co-delivery system for the combined
preferred choice for drug delivery purposes. For example, delivery of DOX and Pt(IV). The amphiphilic diblock copo-
Wang et al have reported that an amphiphilic copolymer lymer PCL-b-P(OEGMA-co-AzPMA) was synthesized,
PEG-poly(lactide-co-glycolide) could co-deliver hydrophilic and then DOX was encapsulated in the hydrophobic core
DOX and hydrophobic PTX by improved double-emulsion of the micelles. Incorporation of Pt(IV) prodrug and shell
method, showing a synergistic effect and enhanced antitumor cross-linking were simultaneously carried out via a click
efficacy.21 Conte et al developed a dual carrier system for reaction of the azide group in the shell of the micelles with
co-delivery of DOX and photosensitizer ZnPc based on a alkyne groups in Pt(IV) prodrug (Scheme 1). The unique
biodegradable amphiphilic block copolymer, PEG-b-PCL.22 SCL micelles were well characterized by FT-IR, GPC, TEM
The ZnPc/DOX-loaded nanoparticles suppressed the growth and DLS. Studies on the drug release and cellular uptake
of tumor cells more efficiently than nanoparticles loaded only of the SCL micelles were carefully performed to evaluate
with DOX, thus indicating a combined effect of DOX and the drug release behavior of SCL micelles. Meanwhile, an
ZnPc. Although these non-covalent drug-loaded polymeric enhanced therapeutic efficacy was observed in comparison
micelles are easy to fabricate, micelle structural integrity and to a single drug, especially at a significantly lower dose.
drug encapsulation stability are of concern. Upon intravenous
Materials and methods
administration, polymeric micelles will be subjected to a
high dilution, ionic strength, and large shear forces. When Materials
structural stability of the micelles is affected, the drug burst DOX and cisplatin were purchased from Beijing Huafeng
release is inevitable during blood circulation, leading to United Technology Co., Ltd. OEGMA (Mn =480 g/mol, 99%)
undesirable toxicity and a low therapeutic index.23–25
purchased from Sigma-Aldrich and Co was passed through a
To solve the above-mentioned stability issue of poly- neutral alumina column to remove the inhibitor prior to use.
meric micelles, shell or core cross-linked micelles have Toluene (99%; Aldrich) was refluxed with sodium beads/
been proposed. After micelles cross-linking, they are found benzophenone complex and distilled until the solution turned
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Platinum covalent shell cross-linked micelles for doxorubicin delivery
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Scheme 1 synthesis routes employed for the preparation of dual-drug-loaded shell cross-linked micelles.
Abbreviations: azPMa, 3-azidopropyl methacrylate; DOX, doxorubicin; OegMa, oligo(ethylene glycol) ethyl methacrylate; PMDeTa, N,N,N′,N″,N′″-
pentamethyldiethylenetriamine; Pt, platinum; Tea, triethyl alcohol; TOl, toluene.
purple. Copper(I) bromide (99.999%; Aldrich), PMDETA was obtained by drying in vacuo overnight. The molecular
(98%; Aldrich), hydrogen peroxide (30%; J&K CHEMICA), weight and molecular weight distribution of PCL-Br were
sodium vitamin (VcNa; 99%; Aladdin) and succinic anhy- determined by GPC using THF as the eluent, revealing an
dride (99%; Aladdin) were used as received without further Mn of 7.19 kDa and a PDI of 1.09 (Figure 1).
purification. AzPMA and the difunctional initiator, HBMP,
were synthesized according to literature procedures.44,45
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A Pt(IV)-containing difunctional alkyne was prepared from
cisplatin. The experimental procedure is presented in Supple-
mentary materials.
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Preparation of Pcl-Br macroinitiator and
Pcl-b-P(OegMa-co-azPMa)
PCL-Br macroinitiator was obtained via the ROP of CL
using the difunctional initiator HBMP. In a typical procedure,
a dry 50 mL Schlenk flask (flame-dried under vacuum prior
to use) was charged with HBMP (0.15 mL, 0.8 mmol), CL
(5 mL, 47 mmol) and dry toluene (10 mL). Then, 0.45 mL
solution of Sn(Oct)2 in toluene (30 mg/mL) was added under
nitrogen protection. The polymerization lasted for 5 h in
an oil bath at 130°C and was terminated by immersing the
flask into liquid N2. The mixture was diluted with THF,
and precipitated into an excess of cold diethyl ether. After
repeated purification by dissolving in THF and precipitating
in cold diethyl ether, a white solid (4.3 g, yield of 81.1%)
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Figure 1 comparison of gel permeation chromatograms of (A) Pcl-Br, (B) Pcl-b-
P(OegMa-co-azPMa) with tetrahydrofuran used as the elute and (C) shell cross-
linked micelles with dimethylformamide used as the elute.
Abbreviations: azPMa, 3-azidopropyl methacrylate; M, molecular weight; Mn, aver-
age number molecular weight; OegMa, oligo(ethylene glycol) ethyl methacrylate;
Pcl, polycaprolactone; PDI, polydispersity index.
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PCL-b-P(OEGMA-co-AzPMA), an amphiphilic diblock deionized water for 3 days (MWCO =3,500 g/mol), and
copolymer, was prepared by ATRP of AzPMA and OEGMA the deionized water was exchanged every 6 h. Finally, the
using PCL-Br as a macroinitiator and CuBr/PMDETA as a SCL micelles loaded with DOX and Pt(IV) were obtained
catalytic system. Typically, a dry 50 mL Schlenk flask (flame- by freeze-drying. The size distribution of the micelles was
dried under vacuum prior to use) was charged with PCL-Br measured by DLS using a Malvern autosizer 4700 instrument.
(1.25 g, 0.2 mmol) and CuBr (28.6 mg, 0.2 mmol). Then, TEM images were obtained on a JEOL 1230 transmission
OEGMA (5.3 mL, 12 mmol), AzPMA (0.5 g, 3 mmol), DMF electron microscope, and samples for TEM measurements
(15 mL) and PMDETA (0.08 mL, 0.4 mmol) were introduced were prepared by casting a drop of the sample solution on
via a gastight syringe. The mixture was degassed with three carbon-coated copper grids. The LE of Pt(IV) was determined
freeze–evacuate–thaw cycles followed by immersing into by TGA. TGA was performed on a Perkin-Elmer Pyris 1
an oil bath at 70°C. The polymerization lasted 12 h and was TGA apparatus at a heating rate of 20°C/min from 100 to
terminated by immersing the flask into liquid N2. The mixture 800°C under a flow of air. To determine the LE of DOX,
was diluted with THF and passed through a short neutral the SCL micelle (5 mg) was dispersed in 10 mL of 1 M HCl
alumina column to remove the residual copper catalyst. The with 10 mM VcNa and stirred for 3 days at 37°C. Then, the
solution was concentrated under vacuum and precipitated solution was diluted to the concentration of 50 µg/mL, and
into cold diethyl ether. The above dissolution–precipitation UV–vis spectroscopy was performed to determine the DOX
cycle was repeated three times. The final product was dried concentration (maximum absorption at 480 nm), wherein a
in vacuo overnight, yielding a white viscous solid (4.2 g, calibration curve was plotted for DOX/1 M HCl containing
yield of 67.2%). The molecular weight and molecular 10 mM VcNa solutions with different DOX concentrations.
weight distribution were determined by GPC. The GPC UV–vis absorbance spectra were measured with a Perkin-
was performed at 35°C using THF with an elution rate Elmer Lambda 35 spectrophotometer. The LE (%) was
of 1.0 mL/min on Agilent 1100 equipped with a G1310A calculated by the following equation:
pump, a G1362A refractive index detector and a G1314A
Amount of drug present in nanoparticles
Amount of nanoparticles
variable wavelength detector. Polystyrene standard samples
LE (%) =
×100%
were employed for the GPC calibration (Mn =31,000 g/mol,
1
PDI =1.19). H NMR spectra were recorded on a Bruker
The EE was calculated by the following equation:
Avance 400 spectrometer using CDCl3 as solvent, and trim-
ethylsilane was used as internal standard. 1H NMR (CDCl3):
0.77–0.95 (3H, CH3CCH2 of POEGMA and PAzPMA),
1.38 (2H, OCH2CH2CH2CH2CH2CO of PCL), 1.64 (4H,
OCH2CH2CH2CH2CH2CO of PCL), 1.95–1.80 (2H, CH2CCH3
of POEGMA and PAzPMA), 2.09 (2H, CH2CH2N3), 2.30
(2H, OCH2CH2CH2CH2CH2CO of PCL), 3.37 (5H, CH3O of
POEGMA and CH2CH2N3 of PAzPMA), 3.64 (4H, OCH2CH2
of POEGMA), 4.06 (2H, OCH2CH2CH2CH2CH2CO of PCL).
FT-IR spectra were obtained by a NEXUX-470 spectrom-
EE (%) =
Amount of drug present in nanoparticles
×100%
Initial amount of drug used to prepare nanoparticles
According to the above formula, the LE and EE of DOX
were 8.5% and 41.0%, respectively, while the LE and EE of
Pt(IV) were 8.5% and 35.6%, respectively.
eter. FT-IR: 2,100 cm−1 (νN ).
In vitro drug release from scl micelles
The release of DOX and Pt(IV) from SCL micelles was
studied at 37°C in four different medias: (a) PBS of pH 7.4,
(b) PBS of pH 7.4 with 5 mM VcNa, (c) PBS of pH 5.5 and
3
Preparation of scl micelles loaded with
DOX and Pt(IV)
PCL-b-P(OEGMA-co-AzPMA) (80 mg) and DOX (20 mg) (d) PBS of pH 5.5 with 5 mM VcNa. Typically, 20 mg of
were dissolved in dimethylsulfoxide/DMF (V:V =1:1), SCL micelles was dispersed in 4 mL deionized water and was
and then deionized water was added dropwise under vig- transferred to a dialysis bag with an MWCO of 3,500 g/mol
orous stirring. After another 4 h under stirring, the Pt(IV) and then immersed in 40 mL of four different media at 37°C
complex (20 mg, 0.03 mmol), CuSO4 (5.3 mg, 0.03 mmol) with constant shaking. At various time intervals, 10 mL
and VcNa (5.94 mg, 0.03 mmol) were added. The reaction of external medium was withdrawn and replaced with the
mixture was stirred for 24 h at room temperature under N2 same volume of fresh buffer solution. Then, an inductively
atmosphere. Subsequently, the solution was dialyzed against coupled plasma-mass spectrometer was used to determine
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Platinum covalent shell cross-linked micelles for doxorubicin delivery
the platinum concentration, and UV–vis spectroscopy was Results and discussion
performed to determine the DOX concentration.
Preparation of copolymer and scl
micelles loaded with DOX and Pt(IV)
The synthesis route of PCL-b-P(OEGMA-co-AzPMA)
amphiphilic diblock copolymer is illustrated in Scheme 1.
In vitro cellular uptake and cell assay
The cellular uptake and intracellular release behaviors of
SCL micelles loaded with DOX and Pt(IV) were determined Utilizing the bifunctional initiator HBMP, ATRP macro-
using confocal fluorescence microscopy. The HeLa cells
were seeded onto glass-bottom petri dishes and incubated at
37°C in an atmosphere containing 5% CO2 for 24 h to allow
cell attachment. The medium was then replaced with 2 mL
culture serum-free medium containing SCL micelles sample.
After incubation for 6 h, the cells were washed three times
with PBS and fixed with paraformaldehyde for 30 min at 4°C,
and nuclei were stained with DAPI. Fluorescence images of
cells were obtained using confocal fluorescence microscope.
The cellular images were acquired with a fluorescence micro-
scope (BX51; Olympus), and the pictures were analyzed
using Image-Pro Plus.
initiator PCL-Br was synthesized by the ROP of CL. GPC
analysis revealed an Mn of 7.19 kDa and a PDI of 1.09. The
DP of PCL-Br was determined to be 55, from the ratio of
the integration area of the peak at 4.06 ppm (methylene
protons adjacent to ester) and that of the peak at 1.95 ppm
(methyl protons adjacent to the bromine terminal) in 1H NMR
spectrum (Figure 2A). Thus, the polymer was denoted as
PCL55-Br and is abbreviated as PCL-Br in this paper. Then,
PCL-b-P(OEGMA-co-AzPMA) amphiphilic diblock copo-
lymer was synthesized via ATRP of OEGMA and AzPMA
using PCL-Br as a macroinitiator and CuBr/PMDETA as
a catalytic system in DMF at 70°C. As seen from the GPC
trace in Figure 1, a monomodal elution peak and a clear shift
to the higher molecular weight side were noted, compared
with PCL-Br macroinitiator. The chemical structure of PCL-
HeLa (human cervical carcinoma cells) and A357 (human
malignant melanoma cells) were chosen to evaluate the
in vitro cytotoxicity of SCL micelles loaded with DOX and
Pt(IV) by CCK-8 kit assays. The CCK-8 kit was purchased
from Dojindo (Shanghai, China). The cells were seeded in
96-well plates at a density of 1×104 cells/well and incubated
at 37°C in an atmosphere containing 5% CO2 for 24 h to
allow cell attachment. Then, the medium was replaced with a
fresh medium containing the indicated concentration of SCL
micelles. After incubation for 48 and 72 h, the medium was
aspirated and replaced by 100 µL of fresh medium containing
10 µL of CCK-8. The cells were incubated for another 2 h
at 37°C in dark. Afterward, the absorbance of each well was
measured at a wavelength of 450 nm using a microplate
reader. The relative cell viability (%) was determined by the
following equation:
1
b-P(OEGMA-co-AzPMA) was analyzed by H NMR. As
shown in Figure 2B, characteristic signals of PCL, OEGMA
and AzPMA were all present. The DP of POEGMA segment
1
was determined to be 40 as indicated by H NMR analy-
sis from the signal integration ratio of peaks at 3.64 ppm
(methylene protons of OEGMA units) relative to peak at
4.06 ppm (methylene protons adjacent to ester of PCL block).
In the same way, the overall DP of P(OEGMA-co-AzPMA)
was calculated to be 53 by 1H NMR analysis. The polymer
was denoted as PCL-b-P(OEGMA0.75-co-AzPMA0.25)53 and
had an Mn,NMR of 27.6 kDa, and is abbreviated as PCL-b-
P(OEGMA-co-AzPMA) in this paper. Moreover, a peak at
2,100 cm−1 characteristic of the azide vibration and a peak at
1,110 cm−1 for ether vibration appeared, and the intensity of
ester bond at 1,725 cm−1 and alkane at 2,950–2,860 cm−1 also
greatly increased after polymerization (Figure 3), indicating
the presence of azide group and PEG side chain. These results
confirmed that the amphiphilic diblock copolymer PCL-b-
P(OEGMA-co-AzPMA) was successfully synthesized.
To prevent drug burst release and achieve co-delivery of
DOX and Pt(IV) after intravenous injection, cross-linking
of PCL-b-P(OEGMA-co-AzPMA) shell was carried out
using Pt(IV) complex containing bifunctional alkyne, which
could react with pendent azide groups via azide–alkyne click
chemistrytogeneratePt(IV)linkageswithinthehydrophilicshell.
The obtained SCL micelles were then characterized by FT-IR,
Isample − Iblank
Cell viability (%) =
Icontrol − Iblank
where Isample, Icontrol and Iblank represent the absorbance intensity,
at 450 nm, of cells treated with different samples, control
cells (nontreated) and blank well without cells, respectively.
The same process was applied to determine the cytotoxicity
of PCL-b-P(OEGMA-co-AzPMA) copolymer against
HeLa, A357 and HEK-293 (human embryonic kidney)
cells. The HeLa, A357 and HEK-293 cell lines were pur-
chased from the Cell Bank of Chinese Academy of Sciences
(Shanghai, China).
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Abbreviations: azPMa, 3-azidopropyl methacrylate; OegMa, oligo(ethylene glycol) ethyl methacrylate; Pcl, polycaprolactone.
GPC, TEM and DLS. As revealed by the FT-IR spectra in and after shell cross-linking. As seen from TEM images
Figure 3, it was clearly found that the peak at 2,100 cm−1 char- in Figure 4, under a drying state, both micelles revealed
acteristic of the azide vibration almost disappeared, whereas the formation of fairly monodisperse and spherical-shaped
three new peaks at 3,440, 1,634 and 1,560 cm−1 ascribed to nanoparticles. The number-average diameters of SCL
Pt(IV) prodrug appeared after the shell cross-linking reac- micelles and NCL micelles were determined to be 30 and
tion, indicating the reaction of azide in the hydrophilic shell 50 nm, respectively. The hydrophilic shell of SCL micelles
of copolymer with dialkyne cross-linker. Moreover, the SCL was covalently cross-linked, and the molecular chains were
micelles were characterized by GPC with DMF as the elute. packed. Thus, the diameter of SCL micelles was smaller
The GPC trace of SCL micelles is shown in Figure 1C. The than that of NCL micelles. As revealed by DLS results, it
molecular weight was shown to increase from 31,000 g/mol was also found that the average diameter of SCL micelles
for PCL-b-P(OEGMA-co-AzPMA) to 491,000 g/mol for SCL was smaller than that of NCL micelles after the shell cross-
micelles, providing another evidence for the successful cross- linking reaction, decreasing from 239.9 to 280.3 nm. The
linking of PCL-b-P(OEGMA-co-AzPMA) shell.
results revealed by DLS were in reasonable agreement with
To further confirm the success of shell cross-linking, that obtained from TEM measurement. Both the results
TEM and DLS were employed to investigate the morphology indicated that the diameter of SCL micelles was smaller than
and size of amphiphilic diblock copolymer micelles before that of NCL micelles. It is worth noting that TEM technique
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Platinum covalent shell cross-linked micelles for doxorubicin delivery
As described above, there were alkyne groups in Pt(IV)
prodrug and azide groups in the shell of micelles. Then, shell
cross-linking was carried out via the click reaction of azide
groups in the shell of micelles with alkyne groups in Pt(IV)
prodrug. Therefore, the SCL micelles could be utilized to
achieve co-delivery of two different chemotherapeutic drugs;
that is, DOX was loaded in the hydrophobic core of micelles
via physical entrapment, while Pt(IV) prodrug was loaded
in the hydrophilic shell of micelles via azide–alkyne click
reaction. Thus, the obtained SCL micelles had two different
drug-loading methods, physical entrapment and covalent
loading of drug, exhibiting different drug release rates.
To determine the LE and EE of DOX, a calibration curve was
plotted for DOX/1 M HCl containing 10 mM VcNa solutions
with different DOX concentrations, and then the content of
DOX was determined according to the calibration curve.
The amount of Pt(IV) prodrug was determined by TGA. As
known, Pt is a thermally stable compound, while polymers
typically decompose at temperatures ,400°C. As shown in
Figure 5, it was found that cisplatin underwent a total weight
loss of 34.7% at 500°C, indicating that only elemental plati-
num remained after 500°C. During the thermal analysis of
SCL micelles, platinum of 5.5 wt % remained, which corre-
sponds to about 8.5 wt % of cisplatin in the SCL micelles.
Due to the abundance of cellular reducing agents and
low pH in endosomes/lysosomes, the cross-linker, Pt(IV)
prodrug, can release cytotoxic Pt(II) species after the cellular
uptake of SCL micelles loaded with DOX and Pt(IV),46,47
thus leading to micelles degradation and the release of
DOX. Therefore, the drug release profile was investigated
under mildly acidic and reducing environments in vitro
(Figure 6). It was found that only 12.2% of Pt(IV) and 13.4%
of DOX were released from SCL micelles within 72 h at
pH 7.4. Then, in mildly acidic environments (pH 5.5), the
release of Pt(IV) and DOX was relatively accelerated, and
the amount of released Pt(IV) and DOX reached 24.3% and
40.0%, respectively. When sodium ascorbate was used as a
reductant in solution, more significant acceleration of drug
release was observed, and the most rapid release rate was
observed at pH 5.5 with 5 mM sodium ascorbate. In the
acidic and reducing condition, 60.4% of Pt(IV) and 92.7%
of DOX were released within 72 h. It was worth noting that
the amount of DOX released was higher than that of Pt(IV)
prodrug; this was because DOX was loaded in the core of
micelles via physical entrapment, while Pt(IV) prodrug was
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Figure 3 Fourier transform infrared spectra of (A) Pcl-Br, (B) Pcl-b-P(OegMa-
co-azPMa) and (C) shell cross-linked micelles.
Abbreviations: azPMa, 3-azidopropyl methacrylate; OegMa, oligo(ethylene
glycol) ethyl methacrylate; Pcl, polycaprolactone.
determines nanoparticles dimension in the dry state, whereas
DLS reports intensity-average dimension in solution, leading
to results showing different sizes. Moreover, the hydrophilic
shell of micelles was POEGMA, and was invisible under
TEM measurement.45 Meanwhile, to compare the structural
stability of SCL micelles and NCL micelles, DMF, a good
solvent for copolymer, was used in DLS measurements
(Figure 4D). After both micelles were added in DMF, the
NCL micelles dissociated into unimers and did not give any
reading, whereas the SCL micelles still maintained the struc-
tural integrity, thus confirming the stabilization of micelles
by shell cross-linking. The average diameter of SCL micelles
was larger in DMF because the solvent DMF could swell
SCL micelles by penetrating. Based on the above results, the
obtained SCL micelles were successfully fabricated, and the
structural stability was also confirmed.
In vitro drug release and cell uptake
In aqueous solution, the amphiphilic diblock copolymer loaded in the shell of micelles via covalent cross-linking.
could self-assemble into micelles with hydrophobic PCL as The above results indicated that the Pt(IV) cross-linking shell
core and hydrophilic P(OEGMA-co-AzPMA) as shell. Thus, could act as a diffusion barrier and block DOX burst release
DOX was encapsulated in the hydrophobic core of micelles. during blood circulation (pH 7.4 and low level of reducing
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in DMF (D).
Abbreviations: DMF, dimethylformamide; Ncl, non-cross-linked; scl, shell cross-linked.
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pH in endosomes/lysosomes, Pt(IV) cross-linking shell
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fluorescence microscopy study was conducted. As seen in
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Figure 7, the strong red fluorescence of DOX was observed
within the cell nuclei, which contained blue DAPI. The cell
nuclei turned red because DOX was bound to DNA. It was
also found that there was little red fluorescence inside the
cytoplasm. This is because the cross-linking shell of micelles
was degraded under the mildly acidic pH inside endosomes
and lysosomes and the highly reducing microenvironment in
the cytosol.34,38,45 These results indicated that SCL micelles
could effectively release DOX inside cells, and DOX was
accumulated inside the cell nuclei.
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Figure 5 Thermogravimetric analysis curve of cisplatin, Pcl-b-P(OegMa-co-azPMa)
copolymer and Pt(IV) prodrug-loaded Pcl-b-P(OegMa-co-azPMa) copolymer.
Abbreviations: azPMa, 3-azidopropyl methacrylate; OegMa, oligo(ethylene
glycol) ethyl methacrylate; Pcl, polycaprolactone; Pt, platinum.
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Platinum covalent shell cross-linked micelles for doxorubicin delivery
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Figure 6 In vitro (A) Pt and (B) DOX release profiles of Pt(IV)- and DOX-loaded shell cross-linked micelles in aqueous solution at () ph 7.4, () ph 5.5, () ph 7.4 with
5 mM sodium ascorbate and () ph 5.5 with 5 mM sodium ascorbate.
Abbreviations: DOX, doxorubicin; Pt, platinum.
polymeric materials before they are applied as an effective
In vitro cell assay
The materials used as drug carriers in DDSs should be
nontoxic, nonimmunogenic and preferably biodegradable.
drug carrier for the therapeutic treatment of tumors. When
the drug carriers are applied to the body, they should be
It is very important to evaluate the potential toxicity of low or nontoxic to the normal tissue cells. In this study, the
Figure 7 Fluorescence microscopy images of hela cells after incubation with DOX- and Pt(IV)-loaded shell cross-linked micelles for 6 h: (A) DOX (red), (B) cell nuclei
stained by 4′,6-diamidino-2-phenylindole (blue), (C) bright field and (D) overlay of (A–C).
Abbreviations: DOX, doxorubicin; Pt, platinum.
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Zhu et al
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cytotoxicity of PCL-b-P(OEGMA-co-AzPMA) amphiphilic demonstrating that the drug-loaded micelles could efficiently
diblock copolymer against HeLa, A357 and HEK-293 cells release drugs under acidic pH in endosomes/lysosomes and
was carefully investigated by the CCK-8 assays. As seen intracellular reduction. However, in contrast with free single
from Figure 8, PCL-b-P(OEGMA-co-AzPMA) showed drug and single-drug-loaded micelles, SCL micelles loaded
no obvious cytotoxic effect on HeLa and A357 cells at a with DOX and Pt(IV) showed higher cytotoxicity toward
concentration range of 0.05–1.0 mg/mL. Even at the con- both A357 and HeLa cells, especially at low concentration.
centration of up to 5.0 mg/mL, the cell viability was above It was found that the cell viabilities of A357 and HeLa cells
80% after incubation for 72 h. In HEK-293 normal cells were 6.4% and 21.0%, respectively, after treatment with
(Figure 8), although PCL-b-P(OEGMA-co-AzPMA) caused 1.0 µM of co-delivery drugs for 48 h, which were much
a slight decrease in the cell viability at the concentration of lower than that of free single drug and single-drug-loaded
1.0 mg/mL when the incubation time increased to 72 h, the micelles. It was worth noting that SCL micelles loaded with
cell viability was still nearly 70%. These results suggest that DOX and Pt(IV) containing 0.1 µM of drug concentration
PCL-b-P(OEGMA-co-AzPMA) copolymers exhibit a good also showed an effective antitumor activity toward A357 and
biocompatibility, and are suitable to be used as a platform HeLa cells, exhibiting a robust enhancement of combination
for anticancer drugs.
potency. Furthermore, with the increase in incubation time
The antitumor activity and synergistic effect of SCL to 72 h, SCL micelles loaded with DOX and Pt(IV) showed
micelles loaded with DOX and Pt(IV) at different drug more potent abilities of cellular growth inhibition against
concentrations and incubation times were studied in vitro A357 and HeLa cells, which indicated that more Pt(IV)
using the CCK-8 assays. A357 and HeLa cells were first diesters released cytotoxic Pt(II) species under intracellular
separately seeded in 96-well plates. After incubating for reduction and DOX could be further released from SCL
24 h to allow cell attachment, the cells were treated with micelles over time. Meanwhile, the distinguishing antitumor
the free DOX, free PTX, DOX-loaded micelles, Pt(IV)- activity of single drugs and SCL micelles loaded with DOX
loaded micelles and SCL micelles loaded with both DOX and Pt(IV) could be seen from the IC50 values. As shown in
and Pt(IV) containing different drug concentrations for 48 Table 1, the IC50 values of SCL micelles loaded with DOX
and 72 h. As shown in Figure 9, after 48 h incubation, all and Pt(IV) against A357 and HeLa cells at 48 h were 0.08 and
samples showed growth inhibition abilities against A357 0.24 µM, respectively, which reduced to 0.02 and 0.09 µM
and HeLa cells, and the cytotoxicity increased along with at 72 h. Compared with free single drugs and single-drug-
the increasing concentrations of drugs. Free single drug and loaded micelles, SCL micelles loaded with DOX and Pt(IV)
single-drug-loaded micelles induced similar cytotoxicity, showed a much lower IC50, indicating highest antitumor
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Figure 8 cell cytotoxicity of Pcl-b-P(OegMa-co-azPMa) against a357, hela and heK-293 cells with different incubation times of (A) 48 and (B) 72 h.
Abbreviations: azPMa, 3-azidopropyl methacrylate; OegMa, oligo(ethylene glycol) ethyl methacrylate; Pcl, polycaprolactone.
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Platinum covalent shell cross-linked micelles for doxorubicin delivery
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Figure 9 In vitro cell viability of a357 and hela cells against single drugs and shell cross-linked micelles loaded with DOX and Pt(IV) at different concentrations and
incubation times. *P,0.05 compared with diblock-DOX-Pt.
Abbreviations: DOX, doxorubicin; Pt, platinum.
activity. From the results discussed above, it is understood micelles. These novel SCL micelles might provide a promis-
that SCL micelles loaded with DOX and Pt(IV) could signifi- ing platform for co-delivery of multiple antitumor drugs and
cantly reduce cell viability even at very low concentrations, might have an important practical application in synergistic
and outperform free single drugs and single-drug-loaded combination drug chemotherapy.
Table 1 Ic50 values of single drugs and shell cross-linked micelles loaded with DOX and Pt(IV) corresponding to a357 and hela cells
at different incubation times
Incubation
time
Cells
IC50 (µM)
Free Pt
Free DOX
Pt(IV)-loaded
micelles
DOX-loaded
micelles
DOX- and Pt(IV)-
loaded micelles
48 h
72 h
48 h
72 h
a357
a357
hela
hela
7.19
5.37
2.16
0.41
0.24
0.13
0.50
0.21
8.46
7.19
2.33
0.93
0.81
0.14
1.09
0.25
0.08
0.02
0.24
0.09
Abbreviations: DOX, doxorubicin; Pt, platinum.
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8. Gottesman MM. Mechanisms of cancer drug resistance. Annu Rev Med.
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9. Woodcock J, Griffin JP, Behrman RE. Development of novel combina-
tion therapies. N Engl J Med. 2011;364:985–987.
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Conclusion
In summary, SCL micelles prepared via the click reaction
of azide group in the shell of micelles with alkyne groups in
the Pt(IV) prodrug were employed as carriers to co-deliver
DOX and Pt(IV) for combination cancer therapy. The results
of FT-IR, GPC, TEM and DLS demonstrated that the struc-
tures of SCL micelles had hydrophilic PEG chains as shell
and hydrophobic PCL as core encapsulating DOX. Studies
on drug release showed that the drug release behavior of
SCL micelles was pH- and reduction-sensitive, overcoming
the drug burst release and achieving controlled drug release.
The results of cellular uptake of the co-delivery SCL micelles
further demonstrated that the micelles were effectively taken
up by the cells and released drugs in the cells. The in vitro cell
assay indicated that the diblock copolymer exerted no obvious
cytotoxicity, and is suitable for use as drug carrier. The co-
delivery SCL micelles caused much higher cell death in both
HeLa and A357 tumor cells than free drug or single-drug-
loaded micelles at the same dosage, exhibiting a synergistic
combination of two drugs. The strategy of using anticancer
drug as cross-linking linkage is a versatile approach to con-
struct multidrug delivery systems, which might result in more
efficient and patient-compliant cancer therapy.
18. Yu J, Deng H, Xie F, et al. The potential of pH-responsive PEG-
hyperbranched polyacylhydrazone micelles for cancer therapy.
Biomaterials. 2014;35:3132–3144.
19. Tomcin S, Kelsch A, Staff H, et al. HPMA-based block copolymers
promote differential drug delivery kinetics for hydrophobic and
amphiphilic molecules. Acta Biomater. 2016;35:12–22.
20. Kataoka K, Harada A, Nagasaki Y. Block copolymer micelles for drug
delivery: design, characterization and biological significance. Adv Drug
Deliv Rev. 2001;47:113–131.
21. Wang H, Zhao Y, Wu Y, et al. Enhanced anti-tumor efficacy by
co-delivery of doxorubicin and paclitaxel with amphiphilic meth-
oxy PEG-PLGA copolymer nanoparticles. Biomaterials. 2011;32:
8281–8290.
Acknowledgments
The authors acknowledge the financial support from the
National Natural Science Foundation of China (81372796)
and the Shanghai Scientific and Technological Innovation
Project (124119a2400).
Disclosure
The authors report no conflicts of interest in this work.
22. Conte C, Ungaro F, Maglio G, et al. Biodegradable core-shell nano-
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Supplementary materials
Preparation of platinum(IV) (Pt[IV])
1.2 mmol) and N-hydroxysuccinimide (NHS) (138 mg,
1.2 mmol) was added to a solution of Pt(IV) compound. The
reaction mixture was stirred at room temperature overnight,
and then propargylamine (110 mg, 2 mmol) was added. After
another 24 h, the solution was concentrated under vacuum and
precipitated into cold diethyl ether. After filtration, a Pt(IV)
prodrug soluble in water (yield 180 mg, 75.0%) containing
difunctional alkyne was obtained. Proton nuclear magnetic
resonance (δ, ppm, 400 MHz, dimethylsulfoxide-d6):
6.12-5.80 (6H, NH3), 3.23 (2H, CH2CCH), 2.60 (2H,
OOCCH2), 2.39 (2H, NHCOCH2), 2.13 (1H, CCH).
prodrug containing difunctional alkyne
Pt(IV) prodrug containing difunctional alkyne was prepared
according to the referenced methods.1–4 In the first procedure,
cisplatin (1.0 g, 3.05 mmol) was added to 3.5 mL of H2O2
30 w/v. The mixture was stirred at 70°C for 5 h and then at
room temperature overnight. After filtration and washing
with ice cold water, ethanol, and diethyl ether, the bright
yellow powder oxoplatin (Pt(NH3)2Cl2(OH)2) was obtained
(yield 0.85 g, 83.4%).
In a second procedure, succinic anhydride (684 mg,
6.834 mmol) was added to a suspension of oxoplatin (600 mg,
1.8 mmol) in dimethylformamide (15 mL). The mixture
was stirred at 70°C for 24 h. The solution was concentrated
under vacuum and precipitated into cold diethyl ether. After
filtration, the product (Pt(NH3)2Cl2(OOCCH2CH2COOH)2)
was dried in vacuo overnight, yielding a pale-yellow solid
(431.6 mg, yield of 93.0%).
References
1. Shi Y, Liu SA, Kerwood DJ, Goodisman J, Dabrowiak JC. Pt(IV) com-
plexes as prodrugs for cisplatin. J Inorg Biochem. 2012;107(1):6–14.
2. Rieter WJ, Pott KM, Taylo KM, Lin W. Nanoscale coordination poly-
mers for platinum-based anticancer drug delivery. J Am Chem Soc.
2008;130(35):11584–11585.
3. Dhar S, Daniel WL, Giljohann DA, Mirkin CA, Lippard SJ. Polyvalent
oligonucleotide gold nanoparticle conjugates as delivery vehicles for
platinum(IV) warheads. J Am Chem Soc. 2009;131(41):14652–14653.
4. Duong HT, Huynh VT, de Souza P, Stenzel MH. Core-cross-linked
micelles synthesized by clicking bifunctional Pt(IV) anticancer drugs
to isocyanates. Biomacromolecules. 2010;11( 9):2290–2299.
In a third procedure, (Pt(NH3)2Cl2(OOCCH2CH2COOH)2)
(217 mg, 0.5 mmol) in dimethylformamide, DIC (247 mg,
H
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Figure S1 1h NMr spectra of Pt(IV) containing difunctional alkyne.
Abbreviation: 1h NMr, proton nuclear magnetic resonance.
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