Enhanced Antimetastatic Activity of the Ruthenium Anticancer Drug RAPTA-C Delivered in Fructose-Coated Micelles

Article abstract of DOI:10.1002/mabi.201600513

The ruthenium complex—dichlororuthenium (II) (p-cymene) (1,3,5-triaza-7-phosphaadamantane) (RAPTA-C)—has shown to be remarkably effective at suppressing the growth of solid tumor metastases. However, poor delivery efficacy and the lack of targeting ability of the common drug delivery system pose significant obstacles to maximize the therapeutic benefit of RAPTA-C. Inspired by the overexpression of GLUT5 transporter on the surface of breast cancer tissues but not the healthy mammary tissues, the use of d-fructose as the targeting moiety of the drug carrier can significantly improve the cellular uptake of nanoparticles, thus further enhancing the therapeutic efficiency of RAPTA-C. In this work, fructose-micelles and 2-hydroxyethyl acrylate (HEA)-micelles are prepared to investigate the difference in cellular uptake. It is found that glycopolymer leads to an increased uptake by breast cancer cells, while the HEA-micelles show less uptake. This behavior is also reflected by the slightly faster movement of fructose-coated micelles in MCF-7 tumor spheroid models using light sheet microscopy as analytical tool. The incorporation of RAPTA-C into micelles can enhance the inhibitory effect of the ruthenium drug demonstrated using invasion, chemotaxis, and haptotaxis assays. As a result, fructose-coated nanoparticles can be a promising drug delivery platform of RAPTA-C for the treatment of metastatic breast cancer. (Figure presented.).

Full text of DOI:10.1002/mabi.201600513

Macromolecular
Bioscience
Full Paper
Enhanced Antimetastatic Activity of the
Ruthenium Anticancer Drug RAPTA-C
Delivered in Fructose-Coated Micelles
Mingxia Lu, Fan Chen, Janina-Miriam Noy, Hongxu Lu, Martina H. Stenzel*
The ruthenium complex—dichlororuthenium (II) (p-cymene) (1,3,5-triaza-7-phosphaadaman-
tane) (RAPTA-C)—has shown to be remarkably effective at suppressing the growth of solid
tumor metastases. However, poor delivery efficacy and the lack of targeting ability of the
common drug delivery system pose significant obstacles to maximize the therapeutic ben-
efit of RAPTA-C. Inspired by the overexpression of GLUT5 transporter on the surface of breast
cancer tissues but not the healthy mammary tissues, the use of d-fructose as the targeting
moiety of the drug carrier can significantly improve the cellular uptake of nanoparticles, thus
further enhancing the therapeutic efficiency of RAPTA-C. In this work, fructose-micelles and
2-hydroxyethyl acrylate (HEA)-micelles are prepared to investigate the difference in cellular
uptake. It is found that glycopolymer leads to an increased uptake by breast cancer cells,
while the HEA-micelles show less uptake. This behavior is also reflected by the slightly faster
movement of fructose-coated micelles in MCF-7 tumor sphe-
roid models using light sheet microscopy as analytical tool.
The incorporation of RAPTA-C into micelles can enhance the
inhibitory effect of the ruthenium drug demonstrated using
invasion, chemotaxis, and haptotaxis assays. As a result, fruc-
tose-coated nanoparticles can be a promising drug delivery
platform of RAPTA-C for the treatment of metastatic breast
cancer.
1. Introduction
(HER-2) has been utilized as a targeting site for the Trastu-
zumab drug delivery system. Trastuzumab is a humanized
monoclonal antibody and has been widely used for HER2-
positive metastatic breast cancer.[2] In the case of triple-
negative breast cancer,[3] which is lacking the expression
of progesterone receptor (PR), estrogen receptor (ER), and
human epidermal growth factor receptor 2 (HER-2/neu),
other targeting mechanism are sought after. Interestingly,
an overexpression of fructose transporter GLUT5 could be
observed in cell lines such as MCF-7 and MDA-MB-231.[4]
Glucose transporters (GLUTs) are transmembrane proteins,
which participate in the cellular uptake of carbohydrates
in order to provide energy for cellular metabolism.[5] This
possible targeting mechanism has been investigated by
Zhang et al. who have successfully applied fructose pen-
dants on phosphorescent metal complexes to increase the
cellular uptakes of the complexes.[6]
Breast cancer is the second most common cancer world-
wide after the lung cancer; it is also the main cause of
cancer death in women. The number of people who are
suffering from breast cancer is increasing at a high rate.[1]
Current research has identified different surface character-
istics between normal and breast cancer cells. These unique
breast cancer sites are therefore great candidates for active
targeting. For instance, human epidermal receptor-2
M. Lu, F. Chen, J.-M. Noy, H. Lu, M. H. Stenzel
Centre for Advanced Macromolecular Design (CAMD)
School of Chemistry
University of New South Wales
Sydney NSW 2052, Australia
E-mail: M.stenzel@unsw.edu.au
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Recently, significant effort has been devoted to
the investigation of ruthenium complexes in med-
ical chemistry, due to the fact that their chemothera-
peutics exhibit minimal side effects and overcome
drug resistance.[7] Unlike the most commonly used
metal-based drugs such as cisplatin, which displays
severe toxicity and drug resistance when applied in
treatment of cancer,[8] ruthenium drugs have been
shown to possess anticancer and antimetastatic prop-
erties.[9] Furthermore, the ruthenium drugs possess
the multiple oxidation states in biological fluids, effec-
tive ligand exchange kinetics,[10] as well as multiple
cytotoxic routes that can heighten the response of the
cell toward the drug.[11] In light of these properties, two
promising anticancer ruthenium complexes: NAMI-A
(imidazolium trans-[tetrachlorido(1H-imidazole)(S-dime-
thyl sulfoxide)ruthenate(III)]) and KP1019 (indazolium
trans-[tetrachloridobis(1H-indazole)ruthenate(III)]), have
been on clinical trials.[7] It was reported that NAMI-A seems
not effective against primary tumor and nontoxic in vitro,
even though it has remarkable selectivity against metas-
tases.[12] KP1019 is more cytotoxic after the reduction of
Ru(III) and suggested to be delivered to the tumor cells
by protein mediated pathway. However, ruthenium(III)
compounds can easily involve in ligand exchange reac-
tion in aqueous medium which may affect its medic-
inal properties. Dichlororuthenium (II) (p-cymene)
(1,3,5-triaza-7-phosphaadamantane) (RAPTA-C), the
ruthenium compound which has a different metal-
lodrug scaffold, was found to be very selective and
efficient on metastatic cancer cells in vivo, but weakly
cytotoxic in vitro.[13] Specifically, RAPTA-C comprises
a Ru(II) center, an arene ligand, a phosphine ligand
and chlorides ligands. The arene ligand is associ-
ated with stabilizing the Ru(II) center and providing
lipophilic properties. Moreover, the size of the arene
ligand influences the degree of DNA damage and cyto-
toxicity. The presence of the chloride ligands is crucial
as they quickly replace with water in order to activate
the drug. Moreover, the phosphine ligand increases the
water-solubility of the compound.[14] RAPTA-C was first
observed to inhibit pH-dependent DNA damage.[15] It
was suggested that RAPTA-C could be applied to selec-
tively damage DNA in cancer cells. Basically, RAPTA-C
coordinates DNA via the loss of labile chloride ligands,
further the single-stranded oligomers wrap around
the metal center forming multiple coordination bonds
which eventually distort the oligonucleotide.[16] A later
study revealed that RAPTA-C shows strong antiangio-
genic activity against tumor tissue that significantly
decreases the density of microvessels. In addition to
this, it also exhibits fast renal excretion without accu-
mulating in vital organs.[17] At last, it was also observed
that RAPTA-C inhibits cell proliferation in vitro by
trigging arrest in the G2/M phase of the cell cycle, as
well as cancer cell apoptosis.[18]
However, RAPTA-C has its disadvantages, as most met-
allodrugs, it presents low solubility and rapid degrada-
tion prior to reaching the target.[18] Encapsulation of
ruthenium drugs in nanoparticles such as micelles and
liposomes have already proven to be a successful strategy
to combat the shortcomings of the drug.[11] Drug carrier
can not only protect the drug from premature inactiva-
tion, but can also enhance drug uptake by tumors while
targeting groups on the surface can aid the retention of
the drug-loaded nanoparticle in the tumor. Specifically,
ruthenium drugs delivered in micelles was reported
to enhance the antimetastatic effect in vitro.[19] In this
paper, we demonstrate how the use of fructose-decorated
nanoparticles can enhance the favorable properties fur-
ther. Glycopolymer with pendant fructose groups were
shown to display an enhanced cellular uptake by can-
cerous cells.[20] Polymers based on fructose were found to
be more efficient than other glycopolymers in their ability
to enhance cellular uptake.[21] Inspired by our recent work
using ruthenium drug loaded micelles based on poly(2-
hydroxyethyl acrylate) (PHEA) and poly(d,l-lactic acid)
PLA, we have investigated the effect of fructose-coated
micelles on the delivery of RAPTA-C. Therefore, two
types of block copolymers were prepared as depicted in
Scheme 1. After conjugation of RAPTA-C and micelle for-
mation, the biological activity was investigated to eluci-
date the effect of fructose on the overall activity.
2. Experimental Section
All chemicals were reagent grade and used as received, unless
otherwise specified. 1,3,5-triaza-7-phosphaadamantane (PTA;
97%, Aldrich), methanol (HPLC grade, APS), dichlororuthenium(II)
(p-cymene) dimer (RuCl2(p-cymene) dimer; 97%, Aldrich), dichlo-
romethane (DCM; >99.8%, Aldrich), n-hexane (95%, Ajax Fine-
chem), 2-chloroethanol (>99%, Aldrich), sodium iodide (NaI;
>99.5%, Aldrich), tin(II) 2-ethyl hexanoate (SnOct2; 95%, Aldrich),
and 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide; Aldrich), toluene
(99%, Ajax), acryloyl chloride (97%, Lancaster), 2-chloroethanol
(>99%, Aldrich), triethylamine (Et3N; >99%, Aldrich), N,N-dimeth-
ylacetamide (DMAc; 99.9%, Aldrich), N,N-dimethylformamide
(DMF; 99%, Aldrich), d-fructose (99%, Aldrich), Sulfuric acid (95%–
98%, Ajax Finechem), silica gel (60 A, 70–230 mesh, Aldrich),
Nile Red (Aldrich), acetone (HPLC grade, Aldrich), sodium
hydroxide (98%, Aldrich), deuterated dimethylsulfoxide-d6
(DMSO-d6; Cambridge Isotope Laboratories), ethyl acetate (EtOAc;
95%, Ajax Finechem), diethyl ether (99%, Ajax Finechem), anhy-
drous acetone (0.0075% H2O, Merck Millipore), were used without
any further purification. 2-Hydroxyethyl acrylate (HEA; 96%,
Aldrich) was destabilized by passing it over a column of basic
alumina. 2,2-Azobis(isobutyronitrile) (AIBN; 98%, Fluka) was
purified by recrystallization from methanol. The reversible addi-
tion-fragmentation transfer (RAFT) agent benzyl (2-hydroxyethyl)
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O
OH
HO
OH
HO
OH
Acetone
ric Acid
5%Sulfu
O
O
O
OH
O
O
O
O
Cl
O
O
O
OH
HEA
O
O
O
O
1-O-AiPrFru
O
A
I
B
C
o
O
N
0
7
,
D
Cl
t
CEA
M
a
,
A
O
c
c
A
,
a
M
t
O
D
7
,
0
o
C
N
B
O
S
S
I
A
HO
O
PLA macro RAFT
O
S
n
TFA/H2O=5:1(v/v)
O
O
O
S
S
O
S
S
y
y
x
HO
O
HO
O
n
x
n
O
S
O
S
O
O
O
O
O
O
O
O
O
OH
HO
HO
OH
OH
Cl
Cl
PLA-b-P[(1-O-AFru)-s-CEA]
PLA- -P[HEA- -CEA]
b s
Drug conjugation
Scheme 1. Synthesis of the fructose monomer, amphiphilic polymer of PLA-b-P [1-O-AFru-s-CEA] and PLA-b-P [HEA-s-CEA] via RAFT polymeri-
zation and subsequent conjugation of RAPTA-C by (1) Finkelstein reaction, (2) attachment of PTA, and (3) RAPTA-C complexation.
carbonotrithioate (BHCT) was synthesized according to literature
procedures.[22]
(3 g, 21 μmol) and BHCT (0.05 g, 0.21 μmol) were mixed and
stirred under vacuum at 120 °C for 12 h. After purging with
nitrogen, SnOct2 (0.01 g, 0.027 μmol, solution in 1 mL toluene)
was added. The temperature was increased to 140 °C and the
mixture was left to react for 5 h. After cooling, dichloromethane
was added to dissolve the cooled polymer mixture. Under stir-
ring, the polymer solution was added dropwise to an excess of
chilled methanol. The precipitated product was dried under
vacuum.
2.1. Synthesis of RAPTA-C
RAPTA-C was synthesized according to the previous report.[14]
RuCl2 (p-cymene) dimer (50 mg, 82 μmol), and PTA (26 mg,
160 μmol) were dissolved in 35 mL of anhydrous methanol. The
reaction was refluxed under nitrogen for 5 h. Then the solution
was filtered, and methanol was removed under reduced pressure.
Red–orange crystals were formed using a DCM-hexane mixture
and the compound was confirmed by NMR.
2.3. Synthesis of 2-Chloroethyl Acrylate
Acryoyl chloride (6 g, 660 μmol) was dissolved in 30 mL of DCM
and added dropwise into a mixture of 2-chloroethanol (2.9 g,
730 μmol) and Et3N (10.1 mL) combined in 70 mL of DCM, and
reacted overnight in an ice-bath. The product was washed with
Milli-Q water and extracted with DCM, and the solvent was then
2.2. Synthesis of PLA MacroRAFT Agent
The synthesis of the PLA MacroRAFT agent was described
in detail previously.[23] 3,6-Dimethyl-1,4-dioxane-2,5-dione
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removed under reduced pressure, to give a pale yellow liquid.
1H NMR (300 MHz, CDCl3): δ (ppm) = 6.45 (d, 1H), 6.17 (dd, 1H),
5.84 (d, 1H), 4.38 (t, 1H), 3.69 (t, 1H).
[MacroRAFT]/[AIBN] = 120:30:1:0.5. The solution was deoxygen-
ated by purging with nitrogen for 45 min, and then polymer-
ized at 70 °C. The polymer was precipitated into cold methanol,
and dried under vacuum to give polylactide-b-(poly[1-O-acry-
loyl-2,3:4,5-di-O-isopropylidene-β-d-fructopyranose-co-2-chlo-
2.4. Polymerization of HEA and CEA with PLA
MacroRAFT
roethyl acrylate]) (PLA-P[1-O-AiPrFru-CEA]) as
a pale yellow
solid.
HEA, 2-chloroethyl acrylate (CEA), PLA MacroRAFT, and AIBN as
initiator were combined and dissolved in DMAc, to give a ratio
of [HEA]/[CEA]/[MacroRAFT]/[AIBN] = 120:30:1:0.1. The solution
was deoxygenated by purging with nitrogen for 45 min. The
flask was then placed in an oil bath at 70 °C. The polymer was
precipitated into cold diethyl ether, and dried under vacuum to
give polylactide-b-(poly[2-hydroxyethyl acrylate-co-2-chloroethyl
acrylate]) (PLA-P[HEA-CEA]) as a pale yellow solid.
2.7. Deprotection of the Copolymer
The polymer PLA-P (1-O-AiPrFru-CEA) was mixed with 2 mL
of TFA/H2O (5:1 v/v) at room temperature and stirred for
40 min. The solution was dialyzed against Milli-Q water (MWCO =
3500 g mol−1) for 2 d and freeze-dried to give a pale white powder
(denoted as PLA-P [1-O-AFru-CEA]).
2.5. Synthesis of 1-O-acryloyl-2,3:4,5-di-
2.8. Conjugation of RAPTA-C to Polymer
O-isopropylidene-β-d-fructopyranose (1-O-AiPrFru)
2.8.1. Finkelstein Reaction
2,3:4,5-di-O-isopropylidene-β-d-fructopyranose was synthe-
sized by the following procedure. d-fructose (24 g, 133 μmol)
was dried under vacuum for 30 min, and was then added to
a cooled solution of conc. sulfuric acid (23.33 mL) in acetone
(466.67 mL) in a 2 L round bottom flask. The mixture was stirred
at room temperature for 1.5 h. The mixture was then chilled
before a cold solution of sodium hydroxide (73.33 g) in Milli-
Q water (333.3 mL) was added under stirring. Then, acetone
was removed under reduced pressure, and the resulting solu-
tion was extracted with DCM (3 × 200 mL). The organic phases
were combined, subsequently washed with three portions
of Milli-Q water (3 × 200 mL), dried with Na2SO4, and filtered,
and the solvent was then removed under reduced pressure to
obtain a crystalline solid. The crude product was recrystallized
by dissolving it in boiling diethyl ether (5 mL g−1), cooling and
adding hexane (5 mL g−1), to give 18.9 g (54.5%) of pure product.
Synthesis of 1-O-AiPrFru followed this method. 2,3:4,5-di-O-
isopropylidene-β-d-fructopyranose (5 g, 20 μmol) and Et3N
(5.5 mL, 40 μmol) were dissolved in 140 mL of anhydrous DCM
and the solution was cooled to 0 °C under nitrogen atmos-
phere. Then a solution of acryloyl chloride (2.5 mL, 29.6 μmol)
in 60 mL of anhydrous DCM was added dropwise. The resulting
solution was stirred for 2 h. The resulting mixture was added
onto 300 mL of crushed ice, and extracted with ethyl acetate
(3 × 100 mL). The combined organic phases were washed with
saturated NaHCO3 and water (3 × 100 mL); and dried with
Na2SO4. The solvent was evaporated under a reduced pressure
to give a crude viscous liquid. It was purified by flash chro-
matography, eluting with 2/1 hexane/ethyl acetate, and was
obtained as a colorless sticky liquid. 1H NMR (300 MHz, CDCl3):
δ (ppm) = 6.51 (d, 1H,), 6.19 (dd, 1H), 5.88 (d, 1H), 4.64 (dd, 1H),
4.53 (d, 1H), 4.38 (d, 1H), 4.27 (dd, 1H), 4.16 (m, 1H), 3.87 (m, 1H),
1.57, 1.51, 1.41, 1.37 (4s, 12H).
The polymer and sodium iodide were dried under nitrogen for
30 min before dissolving in 10 mL of anhydrous acetone in a
Schlenk flask. The solution was refluxed at 70 °C for 7 d. The solu-
tion was dialyzed against Milli-Q water (molecular weight cut-off
(MWCO) = 3500 g mol−1) for 2 d and freeze-dried to give a pale
yellow solid.
2.8.2. Attachment of PTA to Copolymer
The polymer and PTA were combined in a 10 mL Schlenk flask,
evacuated and filled with nitrogen before degassed DMSO-d6
(1 mL) was added. The mixture was reacted for 7 d.
2.8.3. RAPTA-C Complexation
The ruthenium dimer was subsequently dissolved in the DMSO-
PTA copolymer solution for 4 d to obtain an orange solution,
which was analyzed via NMR without further purification.
2.8.4. Micellization of Drug-Loaded Copolymer with Nile
Red Encapsulated
Typically, 0.25 mL of the RAPTA-C copolymer DMSO solution,
where c(polymer) = 20 mg mL−1, was diluted with 0.75 mL of
the Nile Red (0.05 mg) DMF solution. Then the mixture was
added to 1.75 mL of Milli-Q water by using a syringe pump, at
a rate of 0.05 mL h−1. The free RAPTA-C, Nile Red, and the sol-
vent were removed by dialyzing against Milli-Q water (MWCO =
3500 g mol−1), which was subsequently diluted to 10 mL.
2.8.5. Micellization of Drug-Loaded copolymer
Typically, 0.25 mL of the RAPTA-C copolymer DMSO solution,
where c(polymer) = 20 mg mL−1, was diluted with 0.75 mL of
DMF. Then the mixture was added to 1.75 mL of Milli-Q water
by using a syringe pump, at a rate of 0.05 mL h−1. The deep red-
orange solution was subsequently dialyzed against Milli-Q water
(MWCO = 3500 g mol−1) to give a pale orange solution, which was
diluted to 7 mL.
2.6. Polymerization of 1-O-AiPrFru and CEA with PLA
MacroRAFT
1-O-AiPrFru, CEA, PLA MacroRAFT, and AIBN as initiator were
dissolved in DMAc, to give
a ratio of [1-O-AiPrFru]/[CEA]/
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ruthenium. Samples were analyzed with prior digestion with
2.8.6. Nuclear Magnetic Resonance (NMR) Spectrometry
Aqua regia solution at 80 °C for 1 h.
NMR was conducted using a Bruker Avance III 300, 5 mm BBFO
probe (1H: 300.17 MHz, 13C: 75.48 MHz). Further characteriza-
tion was conducted using a Bruker DMX 600 MHz spectrometer
(1H, 600.13 MHz) with a QNP probe. NMR spectra were processed
using either the Bruker TOPSPIN 3.2 software. Samples were ana-
lyzed in the solvents DMSO-d6. All chemical shifts are stated in
ppm (δ) relative to tetramethylsilane (δ = 0 ppm), referenced to
the chemical shifts of residual solvent resonances (1H and 13C).
31P spectra were calibrated to H3PO4 (85%; δ = 0 ppm).
2.8.12. In Vitro Cell Culture
Human breast carcinoma MDA-MB-231 cells, human breast car-
cinoma MCF-7 cells, and mouse macrophages RAW264.7 were
cultured in T25 cell culture flasks with 5% CO2 at 37 °C. The cells
were cultured in Dulbecco's modified Eagle medium (DMEM) sup-
plemented with 10% fetal bovine serum, 4 × 10−3 m glutamine,
and 100 U mL−1 penicillin. After reaching 70% confluence, the cells
were washed with phosphate buffered saline (PBS) and collected
by trypsin/ethylenediaminetetraacetic acid (EDTA) treatment.
2.8.7. Size Exclusion Chromatography (SEC)
SEC was determined using a Shimadzu modular system con-
taining a DGU-12A degasser, LC-10AT pump, SIL-10AD auto-
matic injector, CTO-10A column oven, RID-10A refractive index
detector. A 50 × 7.8 mm guard column and four 300 × 7.8 mm
linear columns (500, 103, 104, and 105 A pore size, 5 μm particle
size) were used for the analyses. N,N-Dimethylacetamide (DMAc,
HPLC grade, 0.05% w/v of 2,6- dibutyl-4-methylphenol, 0.03%
w/v of LiBr) with a flow rate of 1 mL min−1 used for analyses.
The injection volume was 50 μL. The samples were prepared by
dissolving 2–3 mg mL−1 of the analyte in DMAc, followed by fil-
tration through a 0.45 μm filter. The unit was calibrated using
commercially available linear polymethyl methacrylate stand-
ards (0.5–1000 kDa, Polymer Laboratories). Chromatograms were
processed using Cirrus 2.0 software (Polymer Laboratories).
2.8.13. Flow Cytometry
Cellular uptake was measured using a flow cytometer (BD FAC-
Sort). Cells (MCF-7, MDA-MB-231, RAW264.7) were seeded in
6-well tissue culture polystyrene plates at a density of 5 × 105 cells
per well with 3 mL DMEM cell culture medium at 37 °C with
5% CO2 for 1 d prior to micelle treatment. Micelles were loaded
to the cells at a working concentration of 125 μg mL−1 and then
incubated for 2 h. Nontreated cells were used as controls. The cell
monolayer was washed three times with cold PBS and treated
with trypsin/EDTA to detach the cells. The cells were collected,
centrifuged, and resuspended in cold Hank's balanced salt solu-
tion. The cell suspensions were used for flow cytometry analysis
on BD FACSCanto II Analyzer (BDBiosciences, San Jose, USA), col-
lecting results from at least 50 000 events.
2.8.8. Dynamic Light Scattering (DLS)
2.8.14. Laser Scanning Confocal Microscopy
The measurements conducted using a Malvern Zetasizer Nano ZS
instrument with a 4 mV He–Ne laser operating at λ = 632 nm and
noninvasive backscatter detection at 173°. Measurements were
carried out in a disposable cuvette with a solution of 1 mg mL−1
polymer in distilled water at 25 °C. Three measurements with the
number of runs, attenuator, and path length being automatically
adjusted by the instrument, depending on the sample quality.
MCF-7 cells were seeded in 35 mm Fluorodishes at a density of
1 × 105 per dish with 3 mL culture medium and cultured for 1 d.
Cells were then incubated with micelles (125 μg mL−1) at 37 °C for
2 h. After incubation, the cells were washed three times with PBS.
Then the dishes were mounted in PBS and observed under a laser
scanning confocal microscope system (Zeiss LSM 780) equipped
with a Diode 405-30 laser, an argon laser and a DPSS 561-10 laser
connected to a Zeiss Axio Observer.Z1 inverted microscope. The
cellular uptake was observed with a 100 × 1.4 NA oil objective.
The ZEN2012 imaging software (Zeiss) was used for image acqui-
sition and processing.
2.8.9. Transmission Electron Microscopy (TEM)
The TEM images were obtained using a JEOL1400 TEM, consisting
of a beam voltage of 100 kV and a Gatan Charge-coupled device
(CCD) facilitating the acquisition of digital images. Samples were
prepared by casting a 0.5 mg mL−1 polymeric micelle solution
onto a copper grid. The grids were air-dried and then negatively
stained with uranylacetate (UA) for 2 min and air-dried again.
2.8.15. Light Sheet Microscopy
The multicellular tumor spheroids (MCTs) were obtained using
liquid overlay method. MCF-7 cells were suspended in DMEM.
200 μL of the suspension with 2 × 103 cells was seeded into each
well of an ultralow attachment 96-well plate (Corning). The
plate was then centrifuged for 5 min at 500 × g and incubated
at 37 °C with 5% CO2 for 4 d. After the incubation, cells were then
incubated with micelles (125 μg mL−1) at 37 °C for 2 h. The MCTs
observed under a light sheet microscopy (Zeiss lightsheet Z.1).
2.8.10. Fluorescence Spectroscopy
Fluorescence spectra of samples were obtained on Cary Eclipse
Fluorescence Spectrophotometer (Aglient Technologies). The
excitation and emission wavelength were 490 and 512 nm.
2.8.11. Inductively Coupled Plasma Optical Emission
Spectrometry (ICPOES)
2.8.16. Cytotoxicity Assay
The cytotoxicity of free RAPTA-C and fructose-micelles was
measured by WST-1 assay, which is a colorimetric assay for the
The Perkin-Elmer Optima DV7300 ICPOES (Perkin-Elmer, Nor-
walk, CT, USA) was used for quantitative determinations of
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quantification of the cell viability and proliferation. It is based
on the cleavage of a tetrazolium salt (WST-1) by mitochondrial
dehydrogenases in viable cells. Increased enzyme activity leads
to increase in the amount of formazan dye, which is measured
with a microplate reader. MDA-MB-231 cells were used for assay.
The cells were seeded at a density of 8 × 103 cells per well in
96-well cell culture plates, and incubated at 37 °C in a 5% CO2 for
1 d. Micelle solutions were sterilized by UV irradiation for 15 min
in a biosafety cabinet, then serially diluted (2 × dilution) with
sterile Milli-Q water. The cell culture medium was discarded and
replaced with 100 μL 2× concentrated DMEM serum medium.
Samples were added into the plate at 100 μL per well and incu-
bated for 48 h. The plates were washed once with PBS. Each well
was then treated with 100 μL of fresh warm medium along with
5 μL of WST-1. The plates were then incubated for 2 h. Then the
absorbance of the sample against the background control on
a Benchmark Microplate Reader (Bio-Rad) was read at a wave-
length of 450 nm with a reference wavelength of 655 nm. The
means and standard deviations were calculated by four wells
under each condition in the measurement.
were incubated and allowed to migrate for 24 h. Then the cells
on the upper surface of the filters were removed with a cotton
swab. The invading cells in the lower surface were determined
by crystal violet assay.
2.8.20. Crystal Violet Assay
The method of crystal violet assay was according to the reported
paper.[12] The cells presented on the lower surface of the filter
were fixed with a 0.5 mL 1.1% w/v glutaraldehyde solution for
15 min. After the fixation the inserts were washed with distilled
water 0.5 mL three times and air-dried. The cells then stained
for 20 min with 0.5 mL 0.1% w/v crystal violet prepared in
200 × 10−3 m boric acid, pH 9.0. Then inserts were washed with
distilled water 0.5 mL three times and air-dried prior to dissolve
the dye with 0.5 mL 10% acetic acid solution. The optical den-
sity was read at 596 nm with Benchmark Microplate Reader
(Bio-Rad).
2.8.21. Statistical Analysis
A two-way analysis of variance (ANOVA) was performed to
compare multiple groups, and a t-test was used to compare sta-
tistical difference between two groups. Results at p < 0.05 were
considered as significance. Experiments were performed in
triplicate.
2.8.17. Metastasis Assay with Cell Lines
The cells were seeded in 6-well cell culture plates at a density
of 1.5 × 105 per well with 4 mL culture medium and cultured for
1 d. Cells were then treated with fructose-micelles or RAPTA-C at
37 °C for 2 h. After incubation, the cells were detached by trypsin
and collected for the following experiments.
3. Results and Discussion
2.8.18. Invasion Assay
3.1. Synthesis of Block Copolymers with Different Shells
The invasion assay was performed according to the reported
paper.[19] 24-well Millicell hanging cell culture inserts (8 μm pore
size) were coated with 50 μL of 1 mg mL−1 Matrigel (diluted in
serum-free DMEM; BD Sciences) and air-dried overnight at room
temperature. The filters were then reconstituted with serum free
medium immediately before use. 0.5 × 105 cells in 0.2 mL serum
free medium (with 0.1% bovine serum albumin (BSA)) were added
to the upper chamber and the lower compartment was filled with
normal medium. The cells were incubated for 72 h to allow to
invasion through the matrix. Then the cells on the upper surface
of the inserts were removed with cotton swabs. The invading
cells in the lower surface were determined by crystal violet assay.
PLA was employed as hydrophobic block due to its biode-
gradable property which allows the efficient release of the
macromolecule drug upon hydrolysis. PLA was obtained by
ring-opening polymerization of lactide[23] initiated with a
hydroxyl containing RAFT agent, to yield a polymer with a
dispersity of Ð = 1.33 from SEC. According to NMR the ratio
between PLA repeating unit and RAFT end functionality was
calculated to be 247, which can provide some indication on
the chain length but cannot be used to confidently elucidate
the molecular weight.[24] The PLA MacroRAFT was chain
extended with HEA and CEA, which forms the hydrophilic
block and provides the reactive sites for further RAPTA-C
conjugation, respectively (Scheme 1). The resulting polymer
was characterized by NMR (Figure S1, Supporting Informa-
tion) and SEC (Table 1). The sugar containing polymer (PLA-
b-P [1-O-AiPrFru-s-CEA]) was synthesized with the same
procedure as the HEA-polymer, followed by the deprotection
of the sugar moiety with TFA/H2O (v:v = 5:1) and dialysis
against Milli-Q water to remove the acid, affording the final
fructose-polymer (PLA-b-P [1-O-AFru-s-CEA]). The product
2.8.19. Chemotaxis and Haptotaxis Assay
The migrating assay was performed according to the reported
paper.[19] In the chemotaxis assay, the inserts were used without
coating. In the haptotaxis assay, the lower surface of filter was
coated with 10 μg mL−1 fibronectin and left at 4 °C overnight.
Then the insert was rinsed with PBS before cell seeding. After
the cells were treated with the samples for 1 h, then washed
with PBS three times, collected by trypsin treatment and cen-
trifugation, and resuspended in serum free medium with 0.1%
BSA. 0.5 × 105 cells in 0.2 mL serum free medium (with 0.1%
BSA) were added to the upper compartment of each insert. For
chemotaxis, the lower compartment was filled with normal
medium. For haptotaxis, the lower compartment was filled with
serum free medium supplemented with 0.1% w/v BSA. The cells
1
was confirmed by H-NMR (Figure S2, Supporting Informa-
tion) and the characterization is shown in Table 1. Both the
HEA-polymer and sugar polymer have comparable repeating
units of CEA and hydrophilic monomers, which enables
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Table 1. Reaction conditions and characterization of polymers.
Polymer
Reactants concentration
[mol L−1
Conv. [%] of
monomer
]
HEA/
AiPrFru
CEA PLA RAFT Solvent Temp. Time
HEA/
AiPrFru
CEA
Mn,theor
[g mol−1
Mn,SEC
[g mol−1
]
Ð
agent
[°C]
[h]
]
PLA247-b-P[HEA96
-
1.0
0.25
0.24
0.0084
DMAc
DMAc
70
2
80
80
32 200
38 260
1.26
1.39
s-CEA24
PLA247-b-P[1-O-
AFru90-s-CEA24
]
0.98
0.0082
70
1
75
80
43 500
23 070
]
the investigation of the effect of sugar units on cellular
uptake.
of the RAPTA-C polymer (Figure S5 bottom, Supporting
Information).
3.2. RAPTA-C Conjugation
3.3. Synthesis and Characterization of the
Polymeric Micelles
The method of RAPTA-C conjugation was previously
described in literature.[18] The conjugation of RAPTA-C
to the polymer was monitored by 31P NMR.[18] The chlo-
ride residue of CEA was first converted to iodide using
Finkelstein reaction to provide a better leaving group for
further substitution reactions. The completion of this hal-
The micelles were formed by the dropwise addition of
polymer solution into Milli-Q water, followed by the
removal of the organic solvent by dialysis against Milli-
Q water. The DLS results indicate that the hydrodynamic
diameter of HEA-micelles and fructose-micelles were
157 and 131 nm, respectively. The Nile Red encapsulated
micelles were formed by adding a mixture of polymer
and Nile Red solution into Milli-Q water. Nile Red was
applied as fluorescence probe to monitor the internali-
zation of micelles and cellular uptake. The diameters of
each nanoparticles type increased slightly after encapsu-
lating Nile Red in the hydrophobic core (Table 2). Also, the
zeta potentials of all the particles were positively charged
owing to the presence of the positive charge drug in the
shell. The TEM images revealed spherical morphology of
all micelles (Figure S6, Supporting Information). Aqua
regia digested all the organic parts of the micelles, leaving
only the ruthenium in the solution. The concentration
of ruthenium in each micelle solution was then quanti-
fied by ICPOES. The results are displayed in Table 2. HEA-
ogen exchange reaction for HEA-polymer was confirmed
1

by the H NMR signal shift from 3.80 ppm (CH2 Cl) to

3.40 ppm (CH2 I) (Figure S4, Supporting Information).
However, due to the peak overlaps in the 1H NMR spec-
trum of the fructose-polymer, HSQC 2D NMR was used to
monitor this reaction. As shown in Figure S2 (Supporting
Information), after the reaction, the peak at 3.82 ppm,

which correlates to the protons of CH2 Cl of CEA, shifted

to 3.35 ppm (CH2 I). The iodide moiety was subsequently
substituted with PTA at room temperature for 7 d. The
substitution was verified by 31P NMR spectra spectros-
copy: The phosphorous peak of PTA shifted from −103 to
−89 ppm after the successful substitution of PTA to the
polymer chain (Figure S5 top, Supporting Information).
Furthermore, after the conjugation of the ruthenium
dimer to the PTA-polymer, a downfield shift to −20 ppm of
the phosphorous peak confirmed the successful synthesis
micelles contained more ruthenium (3.7 × 10−2 mmol L−1
)
than fructose-micelles (2.4 × 10−2 mmol L−1) and the lower
Table 2. Characterization of micelles by DLS and ICPOES.
Micelles
Polymers
Size
[nm]
Ð
Zeta
[mV]
[Ru]a)
[mmol L−1
]
3.7 × 10−2
3.7 × 10−2
HEA-micelles
PLA247-b-P[HEA96-s-CEA24
]
]
157
203
0.33
0.44
36.7
39.3
HEA-micelles (with Nile
Red encapsulated)
PLA247-b-P[HEA96-s-CEA24
2.4 × 10−2
2.4 × 10−2
Fructose-micelles
PLA247-b-P[1-O-AFru90-s-CEA24
]
]
131
168
0.22
0.38
25.9
26.7
Fructose-micelles (with
Nile Red encapsulated)
PLA247-b-P[1-O-AFru90-s-CEA24
a)ICPOES results in mg/L were converted to mmol/L using the molecular weight of Ru = 101.07 g/mol.
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Figure 1. Cellular uptake of HEA and fructose-micelles measured by confocal microscopy. Micelles were loaded with Nile Red (red). Cell nuclei
were stained as blue using Hoechst 33342. Scale bas is 20 μm.
ruthenium content in fructose-micelles can be explained
by the poorer accessibility of iodide due to the steric hin-
drance of fructose-moieties.
the breast cancer cells, which is in agreement with the
previous work by Zhao et al.[20b] Moreover, the uptakes
of fructose-coated micelles was slightly higher than HEA-
coated micelles in all three cell lines. While the higher
uptake by the breast cancer cell lines can be explained
with the presences of GLUT5 transporter, macrophages do
not overexpress this transporter. The higher uptake of the
fructose-micelles compared to the HEA micelle could be
the result of the increased metabolism of the cells thanks
to the presence of sugar as energy source.
3.4. Micelles Internalization
The internalization of HEA-micelles and fructose-micelles
was monitored via laser scanning confocal microscopy.
The micelles were incubated with MCF-7 cells for 2 h.
The nuclei of the cells were stained with Hoechst 33342
and shown blue in Figure 1. The red fluorescence within
the cytosol demonstrated the successful internalization
of HEA-micelles and fructose-micelles, which have been
loaded with Nile Red (Figure 1).
The quantitative uptake of micelles by MDA-MB-231,
MCF-7, and RAW 264.7 cells was determined by flow
cytometry (Figure 2). HEA-micelles and fructose-micelles
were exposed to these cell lines for 2 h using similar
polymer concentration. The fluorescence intensity of
each type of micelles was normalized[20b] according to
values obtained from fluorospectrometry prior to the flow
cytometry test. This was necessary as the encapsulation
efficiency of Nile Red differed between the two micelles
(Tables S1 and S2, Supporting Information). As depicted
in Figure 2 the breast cancer cells took up significantly
more fructose-micelles than the macrophages, due to the
overexpression of GLUT5 transporters on the surface of
Figure 2. Cellular uptake of HEA and fructose-micelles deter-
mined by flow cytometry.
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RAPTA-C delivered in the fructose-
micelles. Matrigel was used as a base-
ment membrane matrix in the invasion
assay. MDA-MB-231 was chosen due
to its invasive ability. Specifically, the
cells with the serum free medium were
loaded in the insert while the lower
surface of the insert was submerged
in normal medium which was pre-
ferred by the cells. Hence, the invasive
cells would intend to migrate from the
Matrigel to the lower surface. Subse-
quently, RAPTA-C or the RAPTA-C loaded
micelles were added into the assay. The
micelles formed by the polymer without
RAPTA-C showed no inhibitory effects
on the metastasis of MDA-MB-231,
according to the previous study from
our group.[19] The working concentration
of ruthenium in the fructose-micelles
(95 × 10−6 m determined by ICPOES) and
RAPTA-C (160 × 10−6 m) were both lower
than their IC50 values which meant the
cell viability was unaffected by the drug
at this concentration. After the cells
Figure 3. Light sheet images of MCF-7 spheroids treated with A) HEA-micelles and B)
fructose-micelles. C,D) The fluorescence intensity distribution of HEA-micelles and fruc-
tose-micelles, respectively. Scale bar is 100 μm.
3.5. Micelles Penetration of MCTs
incubated with fructose-micelles or free RAPTA-C, the
internalization of ruthenium compounds will reduce the
migration. As shown in Figure 4B, both the micelles and
RAPTA-C can inhibit cell invasion, while the fructose-
micelles showed better inhibiting ability with even lower
concentration of ruthenium. The chemotaxis can promote
cell migration by chemical stimulus and haptotaxis by a
contact stimulus. Both the fructose-micelles showed signif-
icant effect on inhibiting cell migration (Figure 4D,F). These
migration inhibitory assays demonstrated that the incor-
poration of RAPTA-C into micelles could enhance the inhib-
itory effect of the ruthenium drug. The better inhibiting
effect of fructose-micelles may be due to the enhanced
internalization ability of nanoparticles in cancer cells.[28]
The enhanced inhibition of fructose-coated micelles are
in good agreement with earlier reports on PHEA-based
micelles that showed similar enhanced activity of the
loaded RAPTA-C drug.[19]
Better cellular uptake can influence how micelles behave
in tumor tissue. It has been reported that enhanced cel-
lular uptake can directly lead to faster penetration in
tumor spheroid models.[25] While the penetration into
spheroid is more complicated with the size of the nano-
particle playing a pivotal role,[26] the penetration is usu-
ally enhanced in the presence of targeting ligands.[27] The
penetration graph of the MCTs (Figure 3) indicates that
the micelles penetrated into the spheroids successfully
and released the encapsulated Nile Red which can dif-
fuse deeper into the inner core. The higher fluorescence
intensity, which was quantified using light sheet micros-
copy (Figure 3C,D) means that slightly more Nile Red from
fructose-coated micelles internalized in the spheroids and
distributed evenly in the whole spheroid after the micelles
were incubated for 2 h.
3.6. Antimetastatic Effect of Micelles
To evaluate the performance of a drug carrier for ruthe-
nium drugs, the use of assays that measure antimetastatic
activity is more telling than traditional cell proliferation
assays. Although ruthenium drugs display some cytotox-
icity, often with IC50 vales ranging well above 500 × 10−6 m,
the key performance indicator is the ability to prevent the
formation of metastasis. Therefore, three different assays,
invasion, chemotaxis, and haptotaxis assays (Figure 4),
were used to evaluate the migration inhabitation of
4. Conclusions
A fructose-polymer and an HEA-polymer were synthe-
sized using RAFT polymerization, and the self-assembled
micelles were investigated for the cellular uptake. HEA-
micelles are taken up by nonspecific endocytosis, while
fructose-micelles are taken up by transporter mediated
endocytosis which was proven to be more efficient on
internalizing this type of nanoparticle. Fructose-coated
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Supporting Information
Supporting Information is available from the Wiley Online
Library or from the author.
Acknowledgements: M.L. acknowledges the Chinese Scholarship
Council (CSC) for scholarship support. M.H.S. thanks the
Australian Research Council (ARC) for support.
Received: December 9, 2016; Revised: January 20, 2017;
Published online: ; DOI: 10.1002/mabi.201600513
Keywords: micelles; multicellular tumor spheroids; ruthenium
drugs; tumor metastasis
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