Cholesterol-based nucleolipid-ruthenium complex stabilized by lipid aggregates for antineoplastic therapy

Article abstract of DOI:10.1021/bc200565v

A novel ruthenium complex, linked to a cholesterol-containing nucleolipid (named ToThyCholRu), stabilized by lipid aggregates for antineoplastic therapy is presented. In order to retard the degradation kinetics typically observed for several ruthenium-based antineoplastic agents, ToThyCholRu is incorporated into a liposome bilayer formed by POPC. The resulting nanoaggregates contain up to 15% in moles of the ruthenium complex, and are shown to be stable for several weeks. The liposomes host the ruthenium - nucleolipid complex with the metal ion surrounded by POPC lipid headgroups and the steroid moiety inserted in the more external acyl chain region. These rutheniumcontaining liposomes are more effective in inhibiting the growth of cancer cells than a model NAMI-A-like ruthenium complex, prepared for a direct evaluation of their antiproliferative activity. These results introduce new perspectives in the design of innovative transition-metal-based supramolecular systems for anticancer drug vectorization. (Graph Presented)

Full text of DOI:10.1021/bc200565v

Article
pubs.acs.org/bc
Cholesterol-Based Nucleolipid-Ruthenium Complex Stabilized by
Lipid Aggregates for Antineoplastic Therapy
Luca Simeone,† Gaetano Mangiapia,‡,∥ Giuseppe Vitiello,‡,∥ Carlo Irace,§ Alfredo Colonna,§
Ornella Ortona,‡,∥ Daniela Montesarchio,*,† and Luigi Paduano*,‡,∥
‡
†Dipartimento di Chimica Organica e Biochimica, Dipartimento di Chimica “Paolo Corradini”, Universita
̀
“Federico II” di Napoli,
Complesso Universitario di Monte S. Angelo, via Cintia, 4, 80126 Naples, Italy
∥CSGI − Consorzio interuniversitario per lo sviluppo dei Sistemi a Grande Interfase
§Dipartimento di Farmacologia Sperimentale, Universita
̀
‘‘Federico II’’ di Napoli, via D. Montesano 49, 80131 Naples, Italy
S
* Supporting Information
ABSTRACT: A novel ruthenium complex, linked to a
cholesterol-containing nucleolipid (named ToThyCholRu),
stabilized by lipid aggregates for antineoplastic therapy is
presented. In order to retard the degradation kinetics typically
observed for several ruthenium-based antineoplastic agents,
ToThyCholRu is incorporated into a liposome bilayer formed
by POPC. The resulting nanoaggregates contain up to 15% in
moles of the ruthenium complex, and are shown to be stable
for several weeks. The liposomes host the ruthenium−
nucleolipid complex with the metal ion surrounded by
POPC lipid headgroups and the steroid moiety inserted in
the more external acyl chain region. These ruthenium-
containing liposomes are more effective in inhibiting the
growth of cancer cells than a model NAMI-A-like ruthenium
complex, prepared for a direct evaluation of their anti-
proliferative activity. These results introduce new perspectives
in the design of innovative transition-metal-based supra-
molecular systems for anticancer drug vectorization.
along with KP1019 and RAPTA-C, has reached human clinical
testing and has completed Phase I clinical trials.5
INTRODUCTION
■
In the past three decades, a number of ruthenium-based drugs
have been proposed as antineoplastic agents, and some of them
have been successfully tested for potential antitumor activity.1
In several cases, the design of these compounds was largely
inspired by well-known Pt complexes. Indeed, Cisplatin is still
the most widely used chemotherapeutic drug for the treatment
of several malignancies,2,3 though its use is associated with
severe side effects, and limited in many anticancer therapeutic
protocols by primary and acquired resistance to it. These
drawbacks have led to exploration of other transition metal
complexes as efficient alternatives to Pt-based agents. Osmium,
ruthenium, and titanium complexes offer some very interesting
examples.4
As confirmed by the large number of papers appearing in the
literature in the last two decades, ruthenium complexes have
emerged as the most promising alternatives to Pt-based
antineoplastic agents in biological screenings. Since the early
1980s, Sava and co-workers have been pioneers in studying the
perspectives of transition-metal complexes, developing, among
others, the ruthenium complex named NAMI-A, found to be a
very active anticancer agent in in vitro assays. This compound,
Even if a large number of studies on Ru complexes have been
published in recent years, to the best of our knowledge the
unique example of nanovector-carrying ruthenium complexes
for anticancer therapy has been reported by some of us only.6−8
As is well-known, amphiphilic self-assembly allows an
efficient bottom-up strategy in order to obtain nanosized
aggregates whose volume and shape are quite easily tunable.
The incorporation of appropriate complex structural motifs in
suitable amphiphilic building blocks, used as starting mono-
mers, may lead to functional aggregates of great interest in the
development of novel smart materials, with applications in both
nanotechnology and nanomedicine. In this framework, with the
aim of obtaining efficient delivery and controlled release of
anticancer agents, we recently synthesized a new nucleolipid-
based ruthenium complex that spontaneously incorporates into
the phospholipid membrane of a liposome. As an evolution of
Received: October 31, 2011
Revised: February 13, 2012
Published: February 28, 2012
© 2012 American Chemical Society
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our previous work, we describe here the synthesis, molecular,
and aggregation characterization along with bioactivity
investigations of a novel cholesterol-containing ruthenium
complex, baptized ToThyCholRu. This system has been
studied as such and in parallel, when lodged in the biomimetic
membrane formed by the naturally occurring lipid 1-palmitoyl-
2-oleoyl-sn-glycero-3-phosphocholine, POPC.
Our purposes are to develop highly biomimetic liposomes
able to preserve the bioactive ruthenium complex core from
degradation and to deliver a higher amount of antineoplastic
agent to the target cells, thus resulting into a more active
anticancer drug.
By exploiting the high affinity of cholesterol with
phospholipids, ToThyCholRu has been designed so to achieve
easy penetration through the cell membrane, thus facilitating
ruthenium complex internalization. This strategy has been
successfully applied in the antisense strategy for gene therapy
using oligonucleotides end-modified with a cholesterol motif.
In fact, it has been observed that cholesterol conjugation to
oligonucleotides results in high accumulation of the oligomers
in various liver9 and spleen cell types.10
received. TLC analyses were carried out on silica gel plates
from Merck (60, F254). Reaction products on TLC plates were
visualized by UV light and then by treatment with a 10%
Ce(SO4)2/H2SO4 aqueous solution. For column chromatog-
raphy, silica gel from Merck (Kieselgel 40, 0.063−0.200 mm)
was used.
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC,
from Avanti Polar and cholesterol, Chol, from Sigma, purity
>99%, were used as received. Water used for liposome
preparation was obtained by Millipore Elix 3 apparatus.
NMR spectra were recorded on Bruker WM-400, Varian
Gemini 200, and Varian Inova 500 spectrometers, as specified.
All the chemical shifts are expressed in ppm with respect to the
residual solvent signal. Peak assignments have been carried out
on the basis of standard H−H COSY and HSQC experiments.
For the ESI MS analyses, a Waters Micromass ZQ instru-
mentequipped with an electrospray sourcewas used in the
positive and/or negative mode. MALDI TOF mass spectro-
metric analyses were performed on a PerSeptive Biosystems
Voyager-De Pro MALDI mass spectrometer in the linear mode
using 3,4-dihydroxybenzoic acid as the matrix.
Regarding the NAMI-A ruthenium complex, in spite of the
quite encouraging clinical results, some questions are still open
about its in vivo applicability, mainly due to its low stability in
physiological conditions. In analogy with Cisplatin, the chloride
ligands of the ruthenium complex are replaced by hydroxide
ligands in a few hours. This leads to partial hydrolysis of the
complex and to poly-oxo species formation.11,12 Although it is
guessed that the formation of poly-oxo species does not really
alter the ruthenium activity, at least for some tumor cell lines,13
as far as Cisplatin is concerned it has been shown that its
premature aquation and hydrolysis in the extracellular medium
deactivate up to 98% of the Pt complex administered. Thus, in
order to drastically reduce the degradation process, our basic
idea was to move in vivo ToThyCholRu using POPC-based
liposomes as carrier. The ruthenium complex anchored to the
cholesteryl−thymidine nucleolipid is incorporated by force into
the liposome bilayer, in a region where the contact with water
or hydroxy groups is reduced, thus sensibly retarding the
degradation kinetics. The amphiphilic nanoaggregates de-
scribed here contain up to 15 mol % ruthenium complex,
which at this composition is stable for several weeks.
The study was carried out through the following steps: (i)
synthesis of the Ru(III) complex, built on a thymidine scaffold
incorporating the lipophilic cholesterol moiety and a hydro-
philic polyoxyethylene chain; (ii) structural characterization of
the aggregates generated by this complex, in the absence and in
the presence of POPC; (iii) control of the stability of the Ru
complexes in both forms; and (iv) antiproliferative in vitro
investigations of the formulation.
The self-aggregation properties were studied by dynamic
light scattering, DLS, and small-angle neutron scattering, SANS,
whereas the degradation process was studied by UV−vis
measurements and electron paramagnetic resonance, EPR. A
limited panel of human tumor cell lines (HeLa cervical cancer
cells, CaCo-2 and WiDr epithelial colorectal adenocarcinoma
cells, MCF-7 breast adenocarcinoma cells, SH-SY5Y neuro-
blastoma cells) have been used to evaluate the cytotoxic activity
of the Ru-containing aggregates.
The NMR and MS characterization of ToThyCholRu and
related synthetic intermediates are described in detail in the
Supporting Information.
Synthesis. Synthesis of 3-(4-Pyridylmethyl), 3′-O-Cho-
lesteroxyacetyl, 5′-O-(4,4′-Dimethoxytriphenyl-methyl)-
thymidine (4). 5′-O-(4,4′-Dimethoxytriphenylmethyl)-
thymidine 2 (396 mg, 0.73 mmol) was dissolved in 5 mL of
dry DMF. K2CO3 (302 mg, 2.18 mmol) and 4-(bromomethyl)-
pyridine hydrobromide (276 mg, 1.09 mmol) were sequentially
added to the reaction mixture, left under stirring at 60 °C. After
12 h, TLC analysis indicated the presence of one main product
in the reaction mixture. Alkylated compound 3 was not isolated,
but directly converted into its 3′-conjugated derivative 4 (see
Scheme 1). To this purpose, the solvent was removed and the
residual solid was taken up in DCM, washed with water, dried
over anhydrous Na2SO4, and concentrated in vacuo.
Successively, the residue was dissolved in 3 mL of dry DCM,
and then DMAP (32 mg, 0.266 mmol), cholesteroxy-acetic acid
8 (118 mg, 0.266 mmol), and DCC (55 mg, 0.266 mmol) were
sequentially added. The reaction mixture was stirred for 12 h at
room temperature, and then the solvent was removed in vacuo;
the crude product was purified by chromatography on a silica
gel column with n-hexane/AcOEt (1:1, v/v, containing 1% of
TEA) as eluent, yielding the desired compound 4 in 19% yields
for the two steps (144 mg, 0.14 mmol, unoptimized yield).
Synthesis of 3-(4-Pyridylmethyl), 3′-O-Cholesteroxyacetyl
Thymidine (5). 4 (140 mg, 0.13 mmol) was dissolved in 3 mL
of a 1% TCA solution in DCM and stirred for 1 h at room
temperature (see Scheme 1). The reaction mixture was then
concentrated in vacuo and purified by chromatography on a
silica gel column with CHCl3/CH3OH (99:1, v/v) as eluent,
giving the desired compound 5 in almost quantitative yields
(100 mg, 0.13 mmol).
Synthesis of 3-(4-Pyridylmethyl), 3′-O-Cholesteroxyacetyl,
5′-O-Monomethoxy-triethylene Glycol Acetyl Thymidine (6,
ToThyChol). 5 (96 mg, 0.13 mmol) was dissolved in 2 mL of
dry DCM, and then DMAP (46 mg, 0.38 mmol), carboxylic
acid 10 (41 mg, 0.18 mmol), and DCC (38 mg, 0.18 mmol)
were sequentially added (see Scheme 1). The reaction mixture
was stirred for 12 h at room temperature and the solvent was
then removed in vacuo; the crude product was purified by
chromatography on a silica gel column with n-hexane/acetone
MATERIALS AND METHODS
General Synthetic Methods. All the reagents were of the
highest commercially available quality and were used as
■
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Scheme 1. Synthetic Scheme for the Preparation of
Ruthenium Complex 1 (ToThyCholRu)
Scheme 2. Synthetic Scheme for the Preparation of
Cholesteroxy Acetic Acid (8)
a
a
a
Reaction conditions: (a) NaH, tert-butyl bromoacetate, THF, 12 h,
r.t.; (b) HCOOH, 2 h, r.t.
Scheme 3. Synthetic Scheme for the Preparation of CH3O-
a
TEG Acetic Acid (10)
a
Reaction conditions: (a) 4-(bromomethyl)pyridine, K2CO3, DMF, 12
h, 60 °C; (b) cholesteroxy acetic acid (8), DCC, DMAP, CH2Cl2, 12
h, r.t.; (c) 1% TCA in CH2Cl2, 1 h, r.t.; (d) CH3O-TEG acetic acid
(10), DCC, DMAP, CH2Cl2, 4 h, r.t.; (e) [trans-RuCl4(DMSO)2]−
Na+, CH3CN, 4 h, 50 °C.
a
Reaction conditions: (a) NaH, tert-butyl bromoacetate, THF, 12 h,
r.t.; (b) HCOOH, 2 h, r.t.
(1:1, v/v) as eluent, giving the desired compound 6 in 85%
yields (106 mg, 0.11 mmol).
was then purified by chromatography on a silica gel column
with CHCl3 as eluent, yielding the desired ester 9 in 98% yield
(2.5 g, 8.96 mmol).
Synthesis of (Monomethoxy)Triethylene Glycol Acetic Acid
(10). Ester 9 (630 mg, 2.26 mmol) was dissolved in 1.5 mL of
formic acid and stirred at room temperature for 2 h (see
Scheme 3). The solvent was then removed in vacuo, and the
residue was coevaporated three times with CHCl3 (3 × 3 mL),
yielding the desired carboxylic acid 10 in 99% yield (500 mg,
2.25 mmol).
Synthesis of Complex 1 (ToThyCholRu). 6 (32 mg, 0.033
mmol) was dissolved in 1 mL of dry acetonitrile and then
[trans-RuCl4(DMSO)2]− Na+ (14 mg, 0.033 mmol) was added.
The reaction mixture was stirred at 50 °C and the solvent was
then removed in vacuo (see Scheme 1). The reaction,
monitored by TLC on alumina, showed in 4 h the total
disappearance of 6 to give the desired product 1 in quantitative
yields.
Synthesis of Complex 11 (AziRu). In analogy to the
synthesis of 1, pyridine (3.0 μL, 0.037 mmol) was dissolved
in 0.3 mL of dry acetonitrile and then [trans-RuCl4(DMSO)2]−
Na+ (15.6 mg, 0.037 mmol) was added. The reaction mixture
was stirred at 50 °C for 2 h and the solvent was then removed
in vacuo, giving the desired product AziRu, identified on the
basis of its NMR and ESI-MS data (data not shown), in
quantitative yields.
Synthesis of tert-Butyl Cholesteroxy-Acetate (7). Choles-
terol (963 mg, 2.5 mmol) was dissolved in 4 mL of dry THF,
and NaH 60% p.p. (200 mg, 4.98 mmol) and then t-butyl
bromoacetate (920 μL, 6.22 mmol) were sequentially added to
the reaction mixture taken at 0 °C. After 24 h at room
temperature, the reaction was quenched by addition of CH3OH
(2 mL) and the solvent removed in vacuo (see Scheme 2). The
residual solid was taken up in DCM, washed with water, dried
over anhydrous Na2SO4, and concentrated in vacuo. The
residue was then purified by chromatography on a silica gel
column using n-hexane/AcOEt (9:1, v/v) as eluent, yielding the
desired compound 7 in 31% yield (392 mg, 0.77 mmol).
Synthesis of Cholesteroxy-Acetic Acid (8). Ester 7 (300 mg,
0.59 mmol) was dissolved in 3.0 mL of formic acid and stirred
at room temperature for 2 h. The solvent was then removed in
vacuo and the residue coevaporated three times with CHCl3 (3
× 3 mL), yielding the desired compound 8 in almost
quantitative yields (266 mg, 0.59 mmol) (see Scheme 2).
Synthesis of tert-Butyl(Monomethoxy)Triethylene Glycol
Acetate (9). Monomethoxytriethylene glycol (1.5 g, 9.13
mmol) was dissolved in 6.0 mL of dry THF, and NaH 60%
p.p. (730 mg, 18.3 mmol) and then t-butyl bromoacetate (3.37
mL, 22.8 mmol) were sequentially added (see Scheme 3). The
solution was stirred at room temperature for 12 h, then
CH3OH (1 mL) was added and the solvent removed in vacuo.
The crude product, dissolved in CHCl3 and filtered on Celite,
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Lipid-Based Aggregate Preparation. The samples contain-
ing ToThyChol and ToThyCholRu were dissolved in a
pseudophysiological solution. This latter solution was prepared
in double-distilled and degassed water dissolving appropriate
amounts of NaCl and KH2PO4 so that their final concen-
trations were 0.140 mol dm−3 and 0.010 mol dm−3,
respectively. pH was then adjusted to 7.4 by adding appropriate
amounts of KOH. This value was checked to be within 0.1 pH
units by means of a Radiometer pHM220 pH-meter equipped
with a combined glass electrode previously calibrated with
IUPAC standard buffer solutions.
For the samples containing POPC and ToThyCholRu, a
standard procedure to form vesicles was applied: weighed
amounts of ToThyCholRu and POPC were dissolved in
chloroform, favoring the process by a slight warming (∼40 °C)
and sonication treatment (∼5 min). Then, the solutions were
transferred in round-bottom glass tubes, and a thin film of the
solutes was obtained through evaporation of the solvent with
dry nitrogen and keeping the samples under vacuum for at least
24 h. The film was then hydrated with H2O or the
pseudophysiological solution.
The vesicle suspensions were slightly heated at 40 °C and
sonicated for 15 min at 59 kHz with a FALC LBS2 ultrasonic
bath and, finally, repeatedly extruded through polycarbonate
membranes of 100 nm pore size, for at least 11 times.
Samples for SANS measurements were prepared in heavy
water (D2O, isotopic enrichment >99.8%, molar mass 20.03 g
mol−1) purchased from Aldrich, in order to minimize the
incoherent contribution to the scattering cross sections arising
from the system. Samples to be analyzed by EPR also included
1% (w/w) of spin-labeled phosphatidylcholine (1-palmitoyl-2-
[n-(4,4-dimethyloxazolidine-N-oxyl)]stearoyl-sn-glycero-3-
phosphocholine, n-PCSL, n = 5, 7, 10, 14), purchased from
Avanti Polar Lipids and stored at −20 °C in ethanol solutions.
Lipid-Based Aggregate Characterization. The lipid-based
aggregate characterization has been achieved by different
experimental techniques such as DLS, to get information on
particle dimensions; SANS, to reach a deeper insight into the
aggregate structure; UV−vis measurements, to evaluate their
stability in time; and EPR, to confirm aggregate stability and
investigate the membrane microstructure.
Dynamic Light Scattering (DLS). Dynamic light scattering
investigations were performed with a setup composed of a
Photocor compact goniometer, a SMD 6000 Laser Quantum
50 mW light source operating at 5325 Å, and a PMT-120-OP/
B and a correlator (Flex02−01D) purchased from Correla-
tor.com. All the measurements were performed at (25.00
0.05) °C with the temperature controlled through the use of a
thermostat bath. In dynamic light scattering, the intensity
autocorrelation function g(2)(t) is measured and related to the
electric field autocorrelation function g(1)(t) by the Siegert
relation14
where τ = 1/Γ. Laplace transforms were performed using a
variation of CONTIN algorithm incorporated in Precision
Deconvolve software. From the relaxation rates, the z-average of
the diffusion coefficient D may be obtained as follows:15
Γ
D = lim
2
q→0 q
(3)
where q = (4πn0)/(λ sin(θ/2)) is the modulus of the scattering
vector, n0 the refractive index of the solution, λ the incident
wavelength, and θ the scattering angle. An example of Γ fit vs
q2, to get the slope of the straight line, is shown in Figure S1 of
the Supporting Information.
Provided that the solutions are quite dilute, the Stokes−
Einstein equation, which rigorously holds at infinite dilution for
non-interacting spherical species16 diffusing in a continuous
medium, may legitimately be used to evaluate the hydro-
dynamic radius RH of the aggregates:17,18
kT
6πηD
R
=
H
(4)
where k is the Boltzmann constant, T is the absolute
temperature, and η is the medium viscosity. In the case of
nonspherical particles, RH in eq 4 represents the radius of
equivalent spherical aggregates with the same diffusion
coefficient.
Small-Angle Neutron Scattering (SANS). Small angle
neutron scattering measurements were performed at 25 °C
with the KWS2 instrument located at the Heinz Maier-Leibnitz
neutron source of the Julich Centre for Neutron Science
̈
(Garching, Germany). Neutrons with an average wavelength of
7 Å and 19 Å and a wavelength spread Δλ/λ ≤ 0.2 were used. A
two-dimensional 128 × 128 array scintillation detector at three
different wavelength (W)/collimation (C)/sample-to-detector
(D) distances (W7ÅC8mD8m, W7ÅC8mD2m, and W19ÅC8mD8m)
was used. These configurations allowed collection of data in a
range of the scattering vector modulus q = 4π/λ sin(θ/2)
located between 0.002 and 0.25 Å−1. The obtained raw data
were corrected for background and empty cell scattering.
Detector efficiency corrections, radial average, and trans-
formation to absolute scattering cross sections were made
with a secondary plexiglass standard.19
Depending on the scattering intensity profiles, different
models were adopted to fit the collected date. For cylindrical
aggregates (as in the case of the ToThyCholRu aggregates, see
below), the following equation was used:
(2)
(1)
2
2 2 2
2 2
g
(t) = 1 + β |g (t)|
(1)
⎛
⎞
⎠
ϕ (1 − ϕ )π R Δ ρ
q R
cyl
cyl
q
dΣ
dΩ
⎜
⎟
⎟
=
exp −
⎜
⎝
4
where β is the coherence factor, which accounts for the
deviation from ideal correlation and depends on the
experimental geometry. The function g(1)(t) can be written as
the Laplace transform of the distribution of the relaxation rates,
Γ, used to calculate the translational diffusion coefficient, D
⎛
⎜
⎞
⎟
dΣ
dΩ
+
⎝
⎠
(5)
incoh
where R is the radius of the base, ϕcyl the cylinder volume
fraction, and Δρ the scattering length density difference
between the cylinders and the solvent.
+∞
⎛ ⎞
τA(τ) exp − d ln τ
t
τ
(1)
(t) =
⎜
⎟
g
∫
⎝
⎠
(2)
−∞
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In the presence of vesicles, the system was modeled as a
collection of unilamellar and multilamellar vesicles:20
washed with PBS buffer solution (Sigma), collected by trypsine
(Sigma), and then inoculated in a 96-microwell culture plate at
density of 104 cells/well. Cells were allowed to grow for 24 h;
then, the medium was replaced with fresh medium and cells
were treated for a further 72 h with a range of concentrations
(25−1000 μM) of (i) AziRu and POPC liposomes, and (ii) of
the nucleolipidic aggregates ToThyChol (6, Scheme 1),
ToThyCholRu (1, Scheme 1), and ToThyCholRu/POPC. In
more detail, 1 or 2 μL of physiological solutions containing
POPC, AziRu, ToThyChol, ToThyCholRu, and ToThyChol-
Ru/POPC were added to cell culture medium to give
concentrations ranging from 25 to 1000 μM.
Cell number and proliferation were determined by the trypan
blue dye exclusion test. After these treatments, the medium was
removed and the cells were washed twice with PBS buffer
solution (Sigma) and then incubated with a trypsin−EDTA
solution (Sigma) at 37 °C for 5 min. Trypsin was inactivated by
resuspending the cells in medium containing 10% FBS
(Cambrex). The cells were pelleted at 250g and resuspended
in PBS. Viable cells, cells that excluded 0.4% trypan blue
(Sigma), were then counted with a hemocytometer. Con-
currently, cell viability was evaluated with an MTT assay
procedure, which measures the level of mitochondrial
dehydrogenase activity using the yellow 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma)
as substrate.23 The assay was based on the redox ability of living
mitochondria to convert dissolved MTT into insoluble purple
formazan. Briefly, after the treatments the medium was
removed and the cells were incubated with 20 μL/well of an
MTT solution (5 mg/mL) for 1 h in a humidified 5% CO2
incubator at 37 °C. The incubation was stopped by removing
the MTT solution and by adding 100 μL/well of DMSO to
solubilize the purple formazan. Finally, the absorbance was
monitored at 530 nm by using a multiwell plate reader in a
Perkin-Elmer LS 55 Luminescence Spectrometer (Perkin-
Elmer Ltd., Beaconsfield, U.K.).24
2
⎛
⎝
⎞
⎠
⟨|f(q)|⟩
dΣ
dΩ
1
2
⎜
⎟
⎟
=
⟨|f(q)| ⟩ 1 +
(S(q) − 1)
⎜
2
2
q
⟨|f(q)| ⟩
⎛
⎞
⎟
dΣ
dΩ
⎜
⎝
+
⎠
(6)
incoh
In eq 6, f(q) is the form factor of a bilayer containing
information on the shape of the scattering objects, whereas
S(q) is the structure factor that takes into account the
interferences occurring among the bilayers belonging to a
single stack of lamellae. (dΣ/dΩ)incoh represents the incoherent
contribution to the cross section measured, mainly due to the
presence of hydrogenated molecules.
UV−vis Measurements. All the UV−vis measurements were
acquired on a Cary Varian 5000 spectrophotometer equipped
with a multicell holder. Temperature within the cell was
controlled using the Peltier thermocouple. Quartz cuvettes with
a path length of 1.0 cm were used for the measurements.
Wavelength scans were performed at 25 °C from 200 to 800
nm, with a 300 nm/min scan speed.
Electron Paramagnetic Resonance (EPR). EPR spectra were
recorded on a 9 GHz Bruker Elexys E-500 spectrometer
(Bruker, Rheinstetten, Germany). Capillaries containing the
samples were placed in a standard 4 mm quartz sample tube
containing light silicone oil for thermal stability. The temper-
ature of the sample was regulated at 25 °C and maintained
constant during measurement by blowing thermostatted
nitrogen gas through a quartz Dewar. The instrumental settings
were as follows: sweep width, 120 G; resolution, 1024 points;
modulation frequency, 100 kHz; modulation amplitude, 1.0 G;
time constant, 20.5 ms, incident power, 5.0 mW. Several scans,
typically 64, were accumulated to improve the signal-to-noise
ratio. Values of the outer hyperfine splitting, 2Amax, were
determined by measuring, through a homemade MATLAB-
based routine, the difference between the low-field maximum
and the high-field minimum. This parameter is a useful
empirical measure of the lipid chain dynamics and order in both
gel and fluid phases of lipid bilayers.21,22 The main source of
error on the 2Amax value is the uncertainty in composition of
samples prepared by mixing few microliters of mother
solutions. For this reason, reproducibility of 2Amax determi-
nation was estimated by evaluating its value for selected
independently prepared samples with the same nominal
composition. It was found that the uncertainty was 0.2 G.
Cell Cultures, Proliferation, and Viability. Bioactivity and
cytotoxicity of ruthenium-containing nucleolipidic nanopar-
ticles and of AziRu was investigated using a limited panel of
human tumor cell lines (HeLa cervical cancer cells, CaCo-2 and
WiDr epithelial colorectal adenocarcinoma cells, MCF-7 breast
adenocarcinoma cells, SH-SY5Y neuroblastoma cells), all
purchased from ATCC (American Type Culture Collection,
Manassas, Virginia, USA). Cells were cultured at 37 °C in a
humidified 5% CO2 atmosphere and were grown in Dulbecco’s
modified Eagle’s medium (DMEM, Invitrogen, Paisley, U.K.)
containing high glucose (4.5 g/L) or in RPMI medium
(Invitrogen, Paisley, U.K.). Media were supplemented with
10% fetal bovine serum (FBS, Cambrex, Verviers, Belgium), L-
glutamine (2 mM, Sigma, Milan, Italy), penicillin (100 units/
mL, Sigma), and streptomycin (100 μg/mL, Sigma), according
to ATCC recommendations. For cytotoxicity studies, cells were
The calculation of the concentration required to inhibit the
net increase in the cell number and viability by 50% (IC50) is
based on plots of data carried out in triplicate and repeated
three times. IC50 values were obtained using a dose response
curve by nonlinear regression using a curve fitting program,
GraphPad Prism 5.0, and are expressed as mean SEM.
RESULTS AND DISCUSSION
■
Synthesis. Thymidine-based ruthenium complex 1 (Figure
1a)following the design of a uridine-based system previously
described by some of us6was prepared in five straightforward
steps as depicted in Scheme 1.
As a privileged ligand for transition metal complexes, one
pyridine residue, attached through a methylene linker at the N-
3 position of thymidine, was selected to provide the functional
group useful for ruthenium trapping. Its insertion on the
pyrimidine nucleobase was achieved in almost quantitative
yields by treatment of the 5′-DMT protected nucleoside 2 with
4-(bromomethyl)pyridine in DMF using K2CO3 as the base
(Scheme 1).
In order to obtain the target amphiphilic nucleolipid, the
ribosidic hydroxy groups of alkylated nucleoside 3 were
conjugated with hydrophilic and lipophilic chains. Ester
linkages were chosen to attach these components to hydroxy
moieties, and thus obtain effective pro-drugs, on the basis of
several advantages. First of all, they can be realized by simple
and high-yielding couplings of suitable carboxylic acids; second,
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very broad signals, which can be considered diagnostic of the
effective complex formation.26,27
Dynamic Light Scattering (DLS). ToThyChol (6, Scheme
1) exhibits a quite high solubility in water (>1−2 mmol/kg)
with respect to the almost insoluble pure cholesterol.
Furthermore, it shows self-aggregation properties due to its
amphiphilic characteristics. DLS measurements carried out on
ToThyChol at increasing sample concentration in pseudophy-
siological conditions reveal the presence of a monomodal radii
distribution centered around 150 nm in the whole concen-
tration range studied (see Figure 2 graph a). The dimensions of
ToThyChol aggregates are shown in SI Table S1.
Figure 1. Molecular structure of (a): cholesterol-based nucleolipid-
ruthenium complex 1 (ToThyCholRu); (b) pyridine-based ruthenium
complex 11 (AziRu).
esters are stable covalent bonds in neutral and extracellular
media, but are easily reversible once inside the cells, being
rapidly degraded by esterases.
The desired nucleolipid 6 was synthesized by sequential
insertion on 3 of two diverse appendages: one hydrophilic
oligo(ethylene glycol) chain at the 5′-OH moiety, and one
cholesterol residue, as the lipophilic component, attached at the
3′-OH position.
Cholesterol, selected here as the lipophilic decoration of the
target functional nucleolipid in consideration of both its affinity
with lipid membrane constituents and its easy availability, was
first converted into ester 7 by reaction with tert-butyl
bromoacetate and NaH in THF, followed by treatment with
formic acid, assuring tert-butyl removal and recovery of target
acid 8 in almost quantitative yields (Scheme 2).
A similar synthetic scheme was adopted to obtain the
selected hydrophilic component, i.e., monomethoxy-triethylene
glycol (TEG), in the form of carboxylic acid. This oligoether
was first converted into tert-butyl ester 9, then subjected to
formic acid-promoted cleavage, giving oligoether-derivatized
acetic acid 10 (Scheme 3).
Figure 2. Example of size distribution of the ToThyChol and
ToThyCholRu aggregates at 90°: (a) ToThyChol in pseudophysio-
logical conditions, (b) ToThyCholRu in water, (c) ToThyCholRu in
pseudophysiological conditions, (d) ToThyCholRu-POPC 15:85, (e)
pure POPC in pseudophysiological conditions.
ToThyCholRu (1, Scheme 1) shows ability in nano-
structuring, though its stability is limited in time. In fact,
depending on the environmental conditions, we observed a
clear change of the Ru-complex properties in a few hours
(pseudophysiological conditions) or a few days (pure water).
DLS measurements performed on ToThyCholRu fresh
solution both in water and in pseudophysiological conditions
show a bimodal relaxation time distribution corresponding to
the presence of two different aggregates whose mean
hydrodynamic radii are ∼10 and ∼30 nm, respectively (see
Figure 2 graphs b, c). However, the aggregate size progressively
tends to increase, leading to the formation of small dark
particles after 78 h in water and after 4−6 h in
pseudophysiological conditions.
Reaction of 3 with cholesterol derivative 8, first, followed by
detritylation and condensation of the resulting 5 with
monomethoxy-TEG acetic acid 10, with both couplings carried
out using DCC as the activating agent, led to target nucleolipid
6. Successive complexation with ruthenium was achieved by
mixing 6 with the salt [trans-RuCl4(DMSO)2]−Na+, prepared
following literature procedures,25 in CH3CN at 50 °C for 4 h.
All the intermediate compounds were purified by silica gel
column chromatography and characterized by 1H and 13C
NMR spectroscopy and MS analysis. Target salt 1, purified on
alumina, was identified by ESI-MS spectrometry in the negative
mode, showing m/z values in accordance with the expected
mass. More difficult was the NMR characterization of the
complex, with the 1H and 13C NMR spectra showing
broadened signals, due to the presence of the paramagnetic
UV−vis. As in the case of NAMI-A,11,12,28 ToThyCholRu
complex in aqueous solution also suffers the replacement of its
four chloride ligands by hydroxide ions, water molecules, or
even buffer components, resulting in degradation of the initial
complex. This effect is shown by the time evolution of the UV−
vis spectra of ToThyCholRu both in pure water and in
pseudophysiological conditions (see Figure 3 a, b).
Small-Angle Neutron Scattering (SANS). Information on
the aggregate structure can be achieved by SANS measure-
ments. The morphology of the aggregates has been obtained by
means of small-angle neutron scattering. As shown in Figure 4a,
the scattering profile of ToThyCholRu in pure heavy water
1
Ru(III) nucleus. In the H NMR spectrum in CDCl3 of salt 1,
this also induced a dramatic upfield shift of the pyridine
protons and of the methyl protons of the DMSO ligand, here
found at δ = −1.87 ppm and δ = −12.66 ppm, respectively, as
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Figure 3. Absorbance evolution with time of 0.1 mmol kg−1 ToThyCholRu solution in (a) water (72 h), (b) pseudophysiological conditions (4 h).
Two isosbestic points are present at 274 and 358 nm, respectively. The dashed lines refer to the formation of dark particles in solution, and the
arrows indicate the direction of evolution with time. The inset represents the absorbance evolution with time of NAMI-A solution (reproduced with
permission of International Journal of Pharmaceutics, Elsevier Limited, The Boulevard, Langford Lane Kidlington, Oxford, OX5 1GB, U.K. License
number 2757030300954 released on Sep 27, 2011).
Supporting Information. The simulation suggests that a quite
satisfactory reproduction of the experimental SANS data is only
possible by admitting that the cylindrical objects represent
more than 99% of the total volume of the aggregates, so that it
is likely that in the ToThyCholRu systems the dominating
population of aggregates presents a cylindrical shape.
On this basis, the experimental scattering data were modeled
through eq 5. Model fit allows the estimation of the micelle
radius R that results as (3.1 0.5) nm. Unfortunately, the lack
of the Guinier regime29 in the scattering profile does not
provide the possibility to calculate the length of the cylindrical
micelles, l. Nonetheless, an estimation of its value can be
obtained from the knowledge of the DLS hydrodynamic radius
RH that for a cylinder is given by30
3
4
2
2
⎡
3
R
=
R l 1.0304 + 0.0193x + 0.06229x
H
⎣
Figure 4. Scattering cross-section profiles for (a) ToThyCholRu in
pure D2O, (b) ToThyCholRu-POPC 5:95, and (c) ToThyCholRu-
POPC 15:85 (in pseudophysiological conditions). The curves
correspond to the appropriate fit equations. For a better visualization,
data have been multiplied by a scale factor, as indicated.
3
4
−7
7
⎤
⎦
+ 0.00476x + 0.00166x + 2.66·10
x
)
(
(7)
where x = ln[l/(2R)] and R is the cylinder radius.
In order to avoid an overestimation of the experimental data,
the above equation has been applied only to the larger
aggregates in Figure 2. Upon application of eq 7 to
ToThyCholRu aggregates, and on the basis of the hydro-
dynamic radius obtained by DLS, namely, 85−120 nm, it is
possible to estimate that the cylinder length ranges between
1000 and 1400 nm.
Stabilization of ToThyCholRu. In order to increase the
stability of ToThyCholRu in aqueous solution, the complex was
lodged into POPC phospholipid bilayers.
Several samples of the system ToThyCholRu-POPC at
different molar ratio (5:95, 10:90, 15:85, 20:80) in
pseudophysiological solutions have been prepared, and their
evolution with time has been monitored by UV−vis measure-
ments.
As for the pure ToThyCholRu solutions, all the systems were
light yellow immediately after preparation, giving super-
imposable spectra. However, while 5:95, 10:90, and 15:85
samples remained substantially unchanged for weeks, the 20:80
sample turned brown after 5−6 days. An example of the UV−
shows a power law decay dΣ/dΩ ∝ q−1. The slope −1 in a
double log representation suggests the presence of cylindrical
or near-cylindrical aggregates. Furthermore, this slope is quite
constant over a large q range, changing to a −4 value only at
high q, as expected in the Porod region.29 Nevertheless, DLS
measurements revealed the presence of two aggregates quite
different in size (see Figure 2b,c).
This different experimental evidence can be matched either
by admitting that both aggregates shown by DLS have a
cylindrical shape or that the long cylindrical objects detected by
SANS dominate in number and in size over the smaller ones.
The former interpretation seems unrealistic on the basis of the
very low value of the mean hydrodynamic radius (7÷10 nm) of
the smaller aggregates. Thus, it appears more reasonable to
admit that in the studied system there is coexistence of
spherical or almost-spherical objects and long cylinders. In
order to clarify this point, we simulated the scattering profile
due to the simultaneous contribution to the form factor of
spheres and cylinders. The results are shown in Figure S2 of the
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vis spectra over a time of 6 days (150 h) for the 15:85 sample is
depicted in Figure 5. The absence of significant changes with
typical of bilayer structures that in the present case reflect the
presence of liposomes.
It is worth noting that in both profiles the slope α in the
intermediate q range is constantly ≥2, indicating the absence of
coexisting pure ToThyCholRu cylindrical micelles, for which
q−1 dependence should occur. It is thus possible to conclude
that ToThyCholRu is quantitatively inserted in the liposome
bilayer.
Since the α exponent is larger than 2, the aggregates were
modeled as multilamellar systems (see eq 6). However, α value
is only slightly larger than 2, and this is consistent with a small
number of lamellae. It is likely that both single lamellar and
some multilamellar vesicles are present.
The thickness of the layer changes from ∼4.0 nm for pure
POPC liposomes to ∼4.5 nm for ToThyCholRu-POPC (5:95)
and to ∼5.0 nm for ToThyCholRu-POPC (15:85) (see Table
1), indicating that the housing of the ToThyCholRu in the
bilayer produces a slight increase of its thickness.
Electron Paramagnetic Resonance (EPR). Spin-label EPR
spectroscopy has been proven to give substantial information
on the acyl chains structuring in the lipid bilayers.31−34 In this
study, different spin-labels were used. They presented a
nitroxide group, positioned at the levels of the 5, 7, 10, and
14 carbon atom of a phosphocholine backbone of a sn-2 chain,
from here on called n-PCSL. 5-PCSL bears the radical label
close to the molecule headgroup and consequently allows
monitoring of the behavior of the region of the membrane
inner core closer to the polar external layers. In contrast, 14-
PCSL bears the radical label close to the terminal methyl group
of the acyl chain, thus giving information on the more internal
region of the membrane hydrophobic core. The goal has been
to investigate how the membrane microstructure is influenced
by the presence of ToThyCholRu at different ToThyCholRu/
POPC ratios.
EPR spectra of n-PCSL in ToThyCholRu-POPC liposomes
at 15:85 molar ratio are shown in Figure 6. In the same figure,
the EPR spectra of the spin-labels in pure POPC vesicles are
also presented.
For pure POPC, the 5-, 7-, and 10-PCSL spectra present a
clearly defined axial anisotropy (see Figure 6, solid lines). In
contrast, 14-PCSL spectrum shows an almost isotropic three-
line signal, indicative of a rather free motion of the radical label.
The higher isotropy of the 14-PCSL spectrum indicates a
Figure 5. UV−vis time evolution of the ToThyCholRu-POPC 15:85
sample at 0.3 mmol kg−1 total concentration in pseudophysiological
conditions over a period of 150 h. The arrows indicate the sense of the
spectra evolution with time.
time in the spectra is a good indication of the formulation
stability; no precipitation was observed as well. Furthermore,
neither DLS nor EPR detected substantial changes with time in
the aggregate dimensions and bilayer fluidity, as discussed
below.
DLS measurements performed on the same 15:85 for-
mulation, both in water and in pseudophysiological conditions,
revealed for both systems the presence of aggregates with
monomodal distribution having a mean hydrodynamic radius of
60 and 90 nm, respectively (see Figure 2d). Their size was
invariant with time up to 5−6 days.
From the data obtained through SANS experiments, it was
possible to identify the aggregates as liposomes. In fact, as
shown in Figure 4b,c, the scattering profile of ToThyChol-
POPC vs q shows slopes with a power law decay, q−α, with α >
2 both in water and in pseudophysiological conditions. This is
a
Table 1. SANS, UV−vis, and DLS Results on Different ToThyCholRu/POPC Systems
SANS
shape
UV−vis
DLS
SANS
layer thickness/nm
solvent
molar ratio
100:0
degradation time/h
RH/nm
lamellae number
ToThyCholRu
pseudophys. buffer
cylinder
−
∼150 15
3.1 0.5 (cylinder radius)
ToThyCholRu:POPC
72
water
100:0
100:0
sphere
7.4 0.5
120 30
12 (*)
cylinder
sphere
pseudophys. conditions
4
cylinder
vesicles
85 (*)
pseudophys. conditions
water
0:100
15:85
5:95
stable
stable
stable
stable
75
64
78
80
7
5
7
6
1−2
4.0 0.4
pseudophys. conditions
pseudophys. conditions
vesicles
vesicles
1−2
1−2
4.5 0.7
5.0 0.5
15:85
a
Measurements marked with * have been only evaluated at 90°.
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Figure 6. EPR spectra of n-PCSL in POPC (continuous line) and in ToThyCholRu/POPC bilayers at the ratio of 15:85 mol/mol (dashed line)
freshly prepared (a) and ToThyCholRu/POPC bilayers at the molar ratio of 15:85 after 30 days (b).
flexibility increase in the segmental chain mobility going from
the polar head groups to the inner hydrophobic core, which is a
characteristic hallmark of the liquid-crystalline state of fluid
phospholipid bilayers.35
Spectra of the spin-labels at 15:85 molar ratio in Figure 6,
together with the 2Amax corresponding values in Table 2, show
bilayers, as indicated by the 2Amax values reported in Table 2
(Chol-POPC column). This effect could probably be due to the
headgroup of ToThyCholRu, which causes a more intense
change in the lipid packing density than that caused by
cholesterol, such as to affect the acyl chain mobility.
EPR measurements were repeated several times over a period
of 30 days obtaining substantial reproducibility of the results.
No degradation was observed for all the samples up to 15:85
molar ratio. An example of the invariance of the EPR spectra
with time is reported in Figure 6b.
Table 2. Outer Hyperfine Splitting Parameter 2Amax of n-
PCSL in POPC and ToThyCholRu/POPC Liposomes at 25
a
°C
In Vitro Bioactivity Study. We first analyzed the effect on
the cell viability of cholesterol-based nucleolipidic nano-
aggregates by culturing a panel of human tumor cell lines
(HeLa cervical cancer cells, CaCo-2 and WiDr epithelial
colorectal adenocarcinoma cells, MCF-7 breast adenocarcino-
ma cells, SH-SY5Y neuroblastoma cells). As shown in panels A
and C of Figure 7 with respect to MCF-7 and WiDr cell lines
after 72 h of incubation, ToThyChol did not exhibit a
significant cytotoxic effect. The cell viability was also
determined in the presence of conventional POPC liposomes,
currently used in a broad range of biotechnological
applications.36 As expected, POPC liposomes did not induce
cell growth inhibition at all the concentrations and time
intervals tested. Substantially, ToThyChol showed little if any
divergence compared to POPC liposomes. In this way, as
demonstrated by parallel biochemical and toxicological
investigations, we can assume that the cholesterol-containing
nucleolipidic technological platform ToThyChol is a safe
precursor potentially useful for drug delivery applications.
Consequently, we further investigated the conversion of
ToThyChol aggregates into Ru(III) complexes (ToThyChol-
Ru), as such or lodged in the biomimetic membrane formed by
the lipid POPC (ToThyCholRu/POPC). To gain an insight
into the antiproliferative activity of this novel cholesterol-based
nucleolipid−ruthenium complex, its cytotoxicity profile toward
human cancer cells was compared to that of a NAMI-A-like
ruthenium complex, named AziRu and prepared here for a
direct evaluation of their antiproliferative activity. Cell lines
were exposed to AziRu for 72 h in a growth inhibition assay,
and the cytotoxicity was determined in terms of IC50 values.
The detected cytotoxicities were rather moderate, and the IC50
values for AziRu were 900, 690, 515, 405, and 1500 μM in
HeLa, CaCo-2, WiDr, MCF-7, and SH-SY5Y cells, respectively.
ToThyCholRu-POPC
Chol-POPC
Chol/POPC molar
ratio
Ru/POPC molar ratio
n-PCSL 0:100 5:95 10:95 15:85 20:80
2Amax/G
20:80
5-PCSL
7-PCSL
10-PCSL
14-PCSL
52.4
50.6
47.2
33.1
52.7
50.7
46.7
33.4
53.1
51.2
46.1
33.6
53.2
51.4
45.5
33.4
54.0
51.9
45.9
33.5
53.4
51.7
48.5
34.9
a
The error on 2Amax value is 0.2 G.
that the presence of ToThyCholRu complex induces a
stiffening effect on the acyl carbon atoms closer to the
hydrophilic region of the complex as shown by the increased
value of 2Amax of 5-and 7-PCSL. An opposite effect, connected
with an increased fluidity of the membrane, is observed for 10-
PCSL. In the case of 14-PCSL, the fluidity of the more
hydrophobic region of the bilayer explored by this spin-label is
almost invariant. This complex behavior can be rationalized
admitting that the presence of a cholesterol moiety at C5 and
C7 vicinity creates more room at the level of the inner carbon
atom C10 that is not directly interested in the aliphatic chain-
cholesterol interaction, thus increasing the local segmental
mobility of the acyl chains, while for C14, the presence of the
Ru complex is ineffective.
Thus, the experimental results are a clear indication of the
perturbation caused by the presence of ToThyCholRu in the
phospholipid bilayers, which induces an increase in lipid
packing density of the more external acyl chain segments (n ≤
7) and enhanced fluidity of the inner chains region (n ≥ 10). It
is interesting to note that pure cholesterol reduces dynamics
and increases order of the whole lipid acyl chains in POPC
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Figure 7. Growth inhibition of MCF-7 (panel A) and WiDr cells (panel C) treated for 72 h with different formulations and with AziRu as indicated
in the legend, evaluated by MTT assay and cell counting. In panels B and D, for MCF-7 and WiDr cells, respectively, are reported the corresponding
concentration−effect curves obtained by normalizing for the actual amount of ruthenium contained within the ToThyCholRu/POPC liposome.
Data are expressed as percentage of untreated cells and are reported as mean of three independent experiments SEM.
First, under the same incubation conditions these results
indicate a significant difference in ruthenium cytotoxicity
exclusively depending on the cell line, suggestive of a selective
cell-dependent mechanism of action.37 Moreover, according to
many published studies on ruthenium complexes, our results
also showed that AziRu cytotoxicity was much lower than that
of a well-known reference drug based on transition metals as
the antitumor Cisplatin.38 However, concerning NAMI-A, it
should be considered that the concentration needed to reach
cytotoxicity in vitro is much higher than the NAMI-A
concentration capable of inhibiting the formation of metastases,
suggesting that the recognized anti-metastatic activity of NAMI-
A is probably not due exclusively to a reduction in cell
viability.39
Then, focusing on the more sensitive cells (MCF-7 and
WiDr lines) to ruthenium action in our in vitro experimental
model, we assessed the potential cytotoxic effect of ruthenium-
containing liposomes, and interestingly, we found that
ToThyCholRu/POPC liposome, even if containing only 15
mol % of ruthenium, was able to induce cell growth inhibition
in a similar manner to that induced by the same concentrations
of AziRu. In other words, the same cytotoxicity shown by
AziRu is reached at a ruthenium concentration ∼6 times
smaller if the metal is lodged in POPC liposomes. In particular,
in MCF-7 cells ToThyCholRu/POPC the concentration−
response curve was virtually identical to that of AziRu (Figure 7
panel A). Moreover, as reported in panels B and D of Figure 7,
by normalizing concentration−effect curves for the actual
amount of ruthenium contained within the liposomes, we
found that ToThyCholRu/POPC was more active than AziRu
in both cell lines. The latter is a noteworthy finding in the
design and biological evaluation of innovative supramolecular
systems for vectorization of the anticancer drugs. The
calculated IC50 values reported in Table 3 for the real amount
of ruthenium delivered to cells after 72 h of incubation in cell
culture media also suggested that the ToThyCholRu/POPC
complex was more effective than AziRu in inducing growth
inhibition in MCF-7 and WiDr cell lines. In addition, evaluation
of the potentiating factor (PF) values demonstrated that
ruthenium vectorization by this liposome potentiated the
growth inhibition induced by AziRu, reaching values higher
than 3 in cancer cells of different histogenesis (see Table 3).
Similar in vitro results emerged from bioassays on other
human cancer cell lines (data reported in the Supporting
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absence of POPC within these novel liposomal formulations
always reduces the effectiveness of the vehiculated anticancer
drug. In addition, stable liposome formulations can probably
more easily merge with target cell membranes, where the
presence of sterols and of other typical membrane constituents
might together play a crucial role in both liposome stability and
its affinity for cell membranes.
Table 3. IC50 Values of AziRu (a NAMI-A-like Ru Complex)
and of ToThyCholRu/POPC Liposome in the Indicated Cell
Lines Following 72 h of Incubation, And Potentiating
Factors (P.F.) of ToThyCholRu/POPC
a
b
IC50 /μM
P.F.
cell lines
AziRu
ToThyCholRu/POPC ToThyCholRu/POPC
MCF-7
WiDr
405 10
515 15
70 12
5.7
3.1
165 10
CONCLUSIONS
a
b
■
IC50 values are reported as mean
SEM. Potentiating factor
Herein, we report on a novel cholesterol-based nucleolipidic
ruthenium complex stabilized by lipid-based aggregates for
antineoplastic therapy. This work was aimed toward the
optimization of long-lived nanoaggregates containing high
amount of ruthenium complex.
calculated as the ratio of IC50 values of ToThyCholRu/POPC complex
to the IC50 of AziRu complex.
Information), albeit generally less sensitive to the action of the
antiproliferative ruthenium.
Hence, consistent with our recent report,40 different
aggregate compositions differentially impacts cell viability. As
demonstrated by a cytotoxic effect higher than that of AziRu,
the cholesterol containing nucleolipidic liposome stabilized
with POPC seems to deliver the ruthenium within the cells in a
very effective way. Within this context, it is believed that free
ruthenium complexes such as NAMI-A enter cells via a passive
or facilitated passive transport molecular mechanism, albeit
apparently with more difficulty than other antitumor drugs
based on transition metal such as Cisplatin.41 Together with
other considerations, this outcome may account for the lower
cytotoxicity of emerging ruthenium-based molecules assayed
for antineoplastic activity, thus emphasizing the critical role of a
proper vehiculation. The importance of liposome stabilization
by the presence of the naturally occurring POPC is also
supported by the fact that ToThyCholRu liposome per se
resulted in a significantly lower cytotoxic effect. In fact, the
This paper presents the synthesis and assembly properties of
the molecules settled, in the “bottom-up” engineering process
of the proposed nanovectors. Using thymidine as the central,
polyfunctional scaffold, a pyridine-methyl arm was introduced
on the nucleobase as a functional ligand able to form an
octaedric complex with Ru(III) ion. Insertion of one oligo-
(ethylene glycol) chain and one cholesterol residue on the
ribosidic moiety confers the desired amphiphilic behavior to
this nucleolipid in aqueous solution. The role of the cholesterol
moiety, inserted at the level of the sugar ring, is to favor the
inclusion of the polyfunctional molecule inside the POPC
liposome bilayer thus protecting the Ru complex from
degradation. A qualitative picture of the structure of the
ruthenium-cholesterol lipid vectors is depicted in Figure 8.
Moreover, the presence of the cholesterol residue within the
thymidine-based nucleolipidic ruthenium complex gives rise to
a safe precursor that results in an attractive platform with useful
Figure 8. Qualitative graphical representation of ToThyCholRu lodged into a bilayer with indication of some structural parameters.
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biotechnological features such as molecular carrier abilities for
potential applications in drug delivery.
A detailed study of the aggregation behavior has shown that
the synthesized compound forms a wide variety of supra-
molecular aggregates, spanning from spheres and cylinders to
oligolamellar vesicles. Aggregates formed only by the Ru-based
complex show a degradation kinetics that, depending on pH, is
almost complete from a few hours to days, as reported for
NAMI-A. However, when the ruthenium complex is lodged in
POPC liposomes up to 15 mol %, the degradation is markedly
reduced and the formulation is stable for at least several weeks.
According to the experimental data, the lipophilic cholesterol
residue of ToThyCholRu is lodged well inside the bilayer in the
typical position observed for pure cholesterol; meanwhile, the
ruthenium is hidden among the phospholipid heads.
It is worth mentioning that in vitro bioscreenings based on
the evaluation of concentration−effect curves reveal that
ToThyCholRu/POPC liposomes are more effective in
inhibiting the growth of human cancer cells of different
histogenesis with respect to the reference ruthenium-complex
NAMI-A-like AziRu. Therefore, this study introduces new
perspectives in the synthesis and formulation of highly
biocompatible transition-metal-based aggregates having poten-
tial application as antineoplastic agents.
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Ruthenium-based complex nanocarriers for Cancer Therapy. Bio-
materials 33, 3770−3782.
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Van Veghel, R., Tivel, K. L., Biessen, E. A. L., Van Berkel, T. J. C., and
Manoharan, M. (2000) Modulation of plasma protein binding and in
vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by
cholesterol conjugation. Nucleic Acids Res. 28, 2717−2725.
(10) Krieg, A. M., Tonkinson, J., Matson, S., Zhao, Q., Saxon, M.,
Zhang, L. M., Bhanja, U., Yakubov, L., and Stein, C. A. (1993)
Modification of antisense phosphodiester oligodeoxynucleotides by a
5′ cholesteryl moiety increases cellular association and improves
efficacy. Proc. Natl. Acad. Sci. U.S.A. 90, 1048−52.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Molecular characterization of ToThyColRu and its synthesis
intermediates, along with supplementary DLS and antiprolifer-
ative activity plots,is provided in a separate file. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: luigi.paduano@unina.it. Tel. +39 081 674250. Fax.
+39 081 674090. E-mail: daniela.montesarchio@unina.it. Tel.
+39 081 674126.
(12) Sava, G., Alessio, E., Bergamo, A., and Mestroni, G. (1999)
Sulfoxide ruthenium complexes: non-toxic tools for the selective
treatment of solid tumor metastases. Topics Biol. Inorg. Chem. 1, 143−
169.
Notes
The authors declare no competing financial interest.
(13) Bacac, M., Hotze, A. C. G., van der Schilden, K., Haasnoot, J. G.,
Pacor, S., Alessio, E., Sava, G., and Reedijk, J. (2004) The hydrolysis of
the anti-cancer ruthenium complex NAMI-A affects its DNA binding
and antimetastatic activity: an NMR evaluation. J. Inorg. Biochem. 98,
402−412.
ACKNOWLEDGMENTS
■
Project has been funded by MIUR (PRIN 2008 - prot.
20087K9A2J). We thank Forschungszentrum Julich for
provision of beam time. The SANS experiments were
supported by the European Commission, NMI3 contract
RII3-CT-2003-505925.
̈
(14) Berne, B. J., and Pecora, R. (1975) Dynamic Light Scattering:
with Applications to Chemistry, Biology, and Physics
(15) Brehm, G. A., and Bloomfield, V. A. (1975) Analysis of
polydispersity in polymer solutions by inelastic laser light scattering.
Macromolecules 8, 663−5.
(16) Paduano, L., Sartorio, R., and Vitagliano, V. (1998) Diffusion
Coefficients of the Ternary System α-Cyclodextrin-Sodium Benzene-
sulfonate-Water at 25 °C: The Effect of Chemical Equilibrium and
Complex Formation on the Diffusion Coefficients of a Ternary
System. J. Phys. Chem. B 102, 5023−5028.
(17) Vaccaro, M., Accardo, A., Tesauro, D., Mangiapia, G., Loef, D.,
Schillen, K., Soederman, O., Morelli, G., and Paduano, L. (2006)
Supramolecular Aggregates of Amphiphilic Gadolinium Complexes as
Blood Pool MRI/MRA Contrast Agents: Physicochemical Character-
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ABBREVIATIONS
■
AcOEt, ethyl acetate; Chol, cholesteryl residue; DCC, N,N-
dicyclohexylcarbodiimide; DCM, dichloromethane; DMAP, 4-
(N,N-dimethylamino)-pyridine; DMF, N,N-dimethylforma-
mide; DMT, 4,4′-dimethoxytriphenylmethyl; Py, pyridine; r.t.,
room temperature; t-Bu, tert-butyl; TEA, triethylamine; TEG,
triethylene glycol; THF, tetrahydrofuran; Thy, thymidine
residue; SANS, small angle neutron scattering; DLS, dynamic
light scattering; EPR, electron paramagnetic spectroscopy
(18) Vergara, A., Paduano, L., and Sartorio, R. (2001) Multi-
component Diffusion in Systems Containing Molecules of Different
Size. 4. Mutual Diffusion in the Ternary System Tetra(ethylene
glycol)-Di(ethylene glycol)-Water. J. Phys. Chem. B 105, 328−334.
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