Anticancer Cationic Ruthenium Nanovectors: From Rational Molecular

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Anticancer Cationic Ruthenium Nanovectors: From Rational Molecular Design to Cellular Uptake and Bioactivity Gaetano Mangiapia,†,‡ Giuseppe Vitiello,†,‡ Carlo Irace,§ Rita Santamaria,§ Alfredo Colonna,§,# Ruggero Angelico,∥,‡ Aurel Radulescu,⊥ Gerardino D’Errico,†,‡ Daniela Montesarchio,*,† and Luigi Paduano*,†,‡ †

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli “Federico II”, Complesso Universitario di M. S. Angelo, Via Cinthia, 80126 Naples, Italy ‡ CSGI − Consorzio interuniversitario per lo sviluppo di Sistemi a Grande Interfase, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy § Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, Via D. Montesano 49, 80131 Naples, Italy ∥ DISTAAM, Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso, Italy ⊥ Jülich Centre for Neutron Science, Garching Forschungszentrum, Lichtenbergstrasse 1, 85748 Garching bei München, Germany S Supporting Information *

ABSTRACT: An efficient drug delivery strategy is presented for novel anticancer amphiphilic ruthenium anionic complexes, based on the formation of stable nanoparticles with the cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP). This strategy is aimed at ensuring high ruthenium content within the formulation, long half-life in physiological media, and enhanced cell uptake. An in-depth microstructural characterization of the aggregates obtained mixing the ruthenium complex and the phospholipid carrier at 50/50 molar ratio is realized by combining a variety of techniques, including dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR), electron paramagnetic resonance (EPR), and zeta potential measurements. The in vitro bioactivity profile of the Ru-loaded nanoparticles is investigated on human and non-human cancer cell lines, showing IC50 values in the low μM range against MCF-7 and WiDr cells, that is, proving to be 10−20-fold more active than AziRu, a previously synthesized NAMI-A analog, used for control. Fluorescence microscopy studies demonstrate that the amphiphilic Ru-complex/DOTAP formulations, added with rhodamine-B, are efficiently and rapidly incorporated in human MCF-7 breast adenocarcinoma cells. The intracellular fate of the amphiphilic Ru-complexes was investigated in the same in vitro model by means of an ad hoc designed fluorescently tagged analog, which exhibited a marked tendency to accumulate within or in proximity of the nuclei.

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INTRODUCTION Since their first discovery in the mid 60s, the anticancer properties of platinum derivatives have been largely exploited in chemotherapy, with an estimated 70% of tumor-affected patients receiving platinum-based drugs as part of their treatment. The first effective antitumor platinum complex, cisplatin,1 entered in phase I clinical trials in 1971 and was approved by FDA in 1978. Other cisplatin-related analogues, such as carboplatin and oxaliplatin,2 have been successively approved. Despite their large application in medical therapy, platinum-based drugs show several disadvantages: typically, they are not effective against many common types of cancers, can develop drug resistance,3 and, above all, produce severe side effects.4 Some of these limitations can be overcome by ruthenium complexes that show a remarkable antitumoral and antimetastatic activity associated with a lower toxicity.5,6 Thus, they © XXXX American Chemical Society

represent a new and promising route toward a safer, more effective cancer therapy. Two ruthenium compounds are currently undergoing advanced clinical evaluation as anticancer drugs: NAMI-A and KP1019.7,8 Nevertheless, despite the encouraging preliminary results, there are still open questions regarding the effectiveness of ruthenium complexes, mainly due to their poor stability in physiological conditions. Indeed, these compounds show a short half-life in aqueous solution, because of degradation reactions involving exchange of chlorido ligands with hydroxyl ions, and leading to the formation of insoluble poly-oxo species.9 To develop more effective ruthenium-based treatments, we recently undertook the design of molecular vectors capable of Received: January 22, 2013 Revised: May 22, 2013

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dx.doi.org/10.1021/bm400104b | Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Molecular structures of the Ru-complexes: ToThyRu (A), HoThyRu (B), DoHuRu (C), and AziRu (D), along with the two anticancer drugs, NAMI-A (E) and KP1019 (F).

ruthenium, thus sensibly limiting the metal amount transported in these lipid aggregates. In this report we present a novel drug delivery strategy for the set of amphiphilic ruthenium complexes recently synthesized,10,13 aimed at enhancing their antineoplastic activity by increasing the ruthenium content within the aggregates and favoring their cell uptake. This goal has been achieved through the coaggregation of the amphiphilic ruthenium complexes (bearing a negative charge) with the cationic lipid 1,2-dioleyl-3trimethylammoniumpropane chloride (DOTAP). The resulting nanoaggregates are specifically designed to present high stability in aqueous environment even at high Ru-complex content. The aggregation behavior of the prepared nanoaggregates, as well as their stability as a function of time, has been investigated through an experimental strategy proved to be extremely informative.14 It combines dynamic light scattering (DLS) to estimate aggregate dimensions, small angle neutron scattering (SANS) to analyze the aggregate morphology and to determine their geometrical characteristics, neutron reflectivity and zeta potential to gain structural information on the bilayer, and electron paramagnetic resonance (EPR) to get information on

both transporting ruthenium in cells and protecting it against environmental degradation over long circulation times.10 With these goals in mind, we synthesized a novel ruthenium complex (AziRu), inspired to NAMI-A, to be used as a core molecular scaffold of a mini-library of amphiphilic ruthenium-containing molecules. Such a complex was in fact attached to the nucleobase of a nucleoside, which was further decorated with one or two long aliphatic chains able to promote the assembly into ordered nanosized aggregates in aqueous solutions and with one oligoethylene glycol chain of variable length, acting as a protective “stealth” agent for the metal complex nanoaggregates.11,12 To obtain highly biocompatible systems, stable under physiological conditions, these amphiphilic ruthenium complexes were coaggregated with 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC). Although these liposomes exhibited high stability and IC50 values much lower (∼6 times) than AziRu and other known organometallic complexes, on the other hand, they required quite long times (>6 h) to produce relevant cytotoxic effects, probably as a consequence of their slow cell uptake. Furthermore, Ru complex/POPC formulations completely stable against the complex degradation could be obtained only up to a maximum of 15% in mol of B


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Following the design previously exploited for ToThy, HoThy, DoHu, and other nucleolipids we have recently prepared, the lipophilic (oleic acid) and hydrophilic (hexa-ethylene glycol) appendages have been attached through ester linkages, chemically stable but in principle easily reversible in vivo by esterase-mediated degradation.10,21,22 The synthesis of HoThyDansRu has been accomplished in nine straightforward steps starting from uridine. Detailed synthetic procedures and schemes for the preparation of HoThyDansRu, as well as the 1H, 13C NMR, and ESI-MS characterization for all the synthesized compounds, are described in the Supporting Information. Preparation of the Samples and General Procedures. The procedure used for preparing the samples studied by means of physicochemical investigations (DLS, SANS, EPR, zeta potential, NR), along with their technical details are reported in the Supporting Information. Experimental details of fluorescence microscopy, cellular uptake of liposomes, light microscopy, anticancer activity measurements, and statistical analysis procedures are reported in the Supporting Information as well.

the dynamics of lipid hydrophobic tails in the bilayer. These investigations give detailed information on micro- and mesostructural characteristics of the liposomes, leading to results that constitute a reliable basis to cast light upon the mechanisms determining cellular uptake and antiproliferative activity. Experimental evidence on the in vitro antiproliferative activity of the aggregates on human cell lines, and their uptake kinetics are presented. Finally, the intracellular trafficking of the amphiphilic Ru complexes up to the cellular nucleus has been highlighted by means of an ad hoc designed fluorescently tagged Ru complex.

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MATERIALS AND METHODS

Synthesis of the Ruthenium Complexes ToThyRu, HoThyRu, and DoHuRu. The ruthenium complexes investigated, namely, ToThyRu, HoThyRu, and DoHuRu, depicted in Figure 1, along with AziRu, were prepared by reacting in stoichiometric amounts the starting nucleolipids named ToThy, HoThy, or DoHu10,15 with the Ru complex [trans-RuCl4(DMSO)2]−Na+ following a previously described procedure.10,15 In all cases the desired salt was obtained in a pure form, as confirmed by TLC and ESI-MS analysis, and almost quantitative yields. Synthesis of Fluorescently Labeled Ru(III) Complex HoThyDansRu. For a detailed investigation of the cellular mechanism of action of the synthesized Ru(III) complexes, particularly aiming at a deeper comprehension of their cell internalization process and metabolic fate, a fluorescently labeled nucleolipid has been designed using HoThyRu as the model compound. The dansyl group has been here selected to provide the fluorescent tag because it offers several advantages, for example, high chemical stability, limited steric hindrance compared to other commonly used fluorescent dyes, and simple installation protocols. In addition, dansyl derivatives are very sensitive to the solvent polarity conditions, thus providing relevant information on the local environment in which they are found.16−20 The dansyl-labeled nucleolipid Ru(III) complex (here indicated as HoThyDansRu) and depicted in Figure 2 thus contains the following moieties: • one pyridine-methyl arm at the N-3 position, as the privileged ligand for the ruthenium complexation; • a dansyl group attached via a sulfate bridge at the 2′ position; • one oleic acid residue at the 3′ position; • one hexa(ethylene glycol) chain, capped with a benzyl group, in 5′ position.

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RESULTS AND DISCUSSION Characterization of Amphiphilic Ruthenium Complexes/DOTAP Nanoaggregates. Structural characterization

Figure 3. Hydrodynamic radius distribution functions obtained through DLS measurements for DOTAP and amphiphilic ruthenium complexes/DOTAP nanoaggregates, as indicated in the legend.

of the mixed amphiphilic ruthenium complexes/DOTAP nanoaggregates was carried out through dynamic light scattering, small-angle neutron scattering, electron paramagnetic resonance, and zeta potential measurements in pseudophysiological conditions (i.e., 0.200 M phosphatebased buffer solution at pH 7.4). In Figure 3, an example of the hydrodynamic radius distribution functions at 90° for the different mixed aggregates formed by the amphiphilic ruthenium complexes is reported. For all the investigated systems, a single aggregate population was detected. Based on the molecular structures of DOTAP and Ru-based complexes, these aggregates are reasonably −1 vesicles. The hydrodynamic radii ⟨R−1 for all the systems, H ⟩ reported in Table 1, are located in the range 70−100 nm, which is the typical range of large vesicles.23,24 Furthermore, a moderate polydispersity is observed for all the aggregates, being ⟨iD⟩ ranged between 1.08 and 1.13.

Figure 2. Fluorescently labeled ruthenium(III) complex HoThyDansRu (Bn = benzyl). C


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Table 1. Structural Parameters Obtained by Means of DLS and SANS Investigations on Vesicle-Based Systemsa ⟨R0⟩ (nm)

system DOTAP ToThyRu/DOTAP 50/50 HoThyRu/DOTAP 50/50 DoHuRu/DOTAP 50/50

80.6 61.4 58.0 55.6

± ± ± ±

1.0 0.6 0.8 1.2

d (Å) 43.4 39.5 41.4 42.1

± ± ± ±

iR0 = ((Z + 1)(Z + 3))/(Z + 2)2

108⟨D⟩ (cm2 s−1)

± ± ± ±

2.76 ± 0.06 3.58 ± 0.11 3.7 ± 0.2 3.63 ± 0.16

0.9 0.7 0.8 0.8

1.13 1.12 1.13 1.12

0.02 0.02 0.03 0.01

−1 ⟨R−1 (nm) H ⟩

iD 1.08 1.08 1.11 1.13

± ± ± ±

0.02 0.02 0.02 0.02

88 68 65 67

± ± ± ±

2 2 4 3

The table reports the average inner radius ⟨R0⟩, the bilayer thickness d, the R0 polydisperity index iR0, the average diffusion coefficient ⟨D⟩, the −1 diffusive polydispersity iD, and the hydrodynamic radius ⟨R−1 H ⟩ .

a

parameter. The reported data indicate that the detected aggregates are slightly polydisperse, having a polydispersity index close to the unity for all the systems, according to the vesicle preparation method. Similarities are also observed for bilayer thickness, located around ∼4 nm for all the systems. It is noteworthy to compare the RH radii obtained by means of DLS and dimensions extracted from SANS data. Ideally, considering the spherical shape of vesicles, RH should agree to R0 + d quantity. Analysis of data shows that this is not the case; indeed a small discrepancy (∼3 nm) is observed. This difference arises from different contributions influencing the estimated parameters. First, it has to be highlighted that DLS provides the −1 inverse of the inversed average, namely, ⟨R−1 that, in H ⟩ dependence of the polydispersity level, is slightly different from the true average ⟨RH⟩. Furthermore, ⟨RH⟩ values are sensitive to solvation effects that contribute to overestimate the hydrodynamic radii:31,32 this effect is even enhanced in the presence of ionic headgroups, as well as of the longer oligo-ethylene glycol (OEG) chains that are highly hydrated.33,34 A third effect arises from polydispersity that is differently taken into account by DLS and SANS techniques. Finally, it has to be noted that the Guinier regime is only partially present in the q range spanned by SANS measurements, and this affects the accuracy of ⟨R0⟩. Zeta Potential. The zeta potential ζ is a convenient quantity for characterizing the electrostatic properties of aqueous dispersions of colloidal particles such as micelles and vesicles, since ζ is a measure of the surface charge at slipping plane of the aggregates.35 Concerning the vesicle systems composed by anionic nucleolipid-Ru complexes and cationic DOTAP, the measured ζ values confirm the formation of catanionic nanoaggregates. Indeed, the positive surface charge carried by bare DOTAP vesicles (+41.7 mV) is partially neutralized upon the addition of negatively charged nucleolipidbased Ru complexes (see Table 2). Analogous effect was detected in another catanionic system made by nucleolipids and CTAB.36 It is worthy to note that, for cationic systems, the zero-point potential is not reached when the molar ratio between the cations and the anions composing the aggregate is exactly unitary but, usually, when it is slightly different. Furthermore, only in a very narrow range it is possible to observe the inversion of ζ. The latter is less sensitive to the cationic/anionic molar ratio outside this range and more or less close to the values of pure cationic or anionic liposomes.37 The found values indicate that the liposome composition still results in a positive charge, even if this charge has been partially reduced, contributing to stabilize the Ru complex from degradation phenomena. Again, all the Ru complexes-based liposomes investigated exhibit a similar behavior if compared each other. Neutron Reflectivity (NR). Lipid bilayers of pure DOTAP and ruthenium complexes/DOTAP were characterized using

Figure 4. Scattering cross sections obtained through SANS measurements for DOTAP and amphiphilic ruthenium complexes/DOTAP nanoaggregates, as displayed in the legend. To allow for a better visualization, data have been multiplied for a scale factor, as indicated. Curves have been obtained from fitting the polydisperse unilamellar vesicle model to the experimental data.

Table 2. Zeta Potentials (ζ) for DOTAP-Based Liposomes at Different Lipid/Ruthenium Complex Ratios ratio 70/30 50/50

DOTAP (mV)

ToThyRu/ DOTAP (mV)

HoThyRu/ DOTAP (mV)

DoHuRu/ DOTAP (mV)

41.7 ± 1.5

36.5 ± 0.8 29.7 ± 0.3

38.8 ± 1.5 35.4 ± 1.0

37.2 ± 1.4 33.7 ± 0.3

To get more information about the morphology and structural characteristics of the aggregates revealed by DLS analysis, SANS measurements have been performed. Scattering cross sections, dΣ/dΩ, displayed in Figure 4, show the typical trends of unilamellar vesicles.25−28 The region where power law dΣ/dΩ ∝ q−2 occurs is due to the single layer scattering, whereas the Guinier regime is only glimpsed due to the size of the aggregates, as confirmed by DLS investigations. In order to extract quantitative information, scattering cross sections have been modeled as arising from collections of unilamellar spherical vesicles. It has to be noted that the extrusion process rarely produces rigorously monodisperse vesicles, even though in most cases the polydispersity is low.23 On these bases, the inner aqueous core of vesicles has been allowed to be sizepolydispersed, assuming a Schulz-Zimm distribution function of the core radius. For the described model, a close form for dΣ/ dΩ is obtainable.29,30 Microstructural parameters of the vesicles under investigation are reported in Table 1, namely, the average inner radius ⟨R0⟩, the bilayer thickness d, and the polydispersity index iR0 of R0 D


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Figure 5. Comparison of liposome bilayers composed by DOTAP and amphiphilic ruthenium complex/DOTAP nanoaggregates according to EPR and NR investigations. Liposome morphology is also represented according to DLS investigations. Finally, scattering length density profiles for the three studied systems are reported on the bottom.

different isotopic contrast solvents. Scattering length density profiles for the bilayers investigated in D2O are shown in Figure 5, whereas the parameters used to fit the curves simultaneously from all the contrasts are given in Table 3. In addition, the experimental curves are shown in Figure SI2. For all the lipid systems, a five layer model was found to best fit the data. The first two layers correspond to the silicon block and to the thin solvent layer interposed between the silicon surface and the adsorbed bilayer. The other three layers describe the bilayer, which is subdivided in the inner headgroups, the hydrophobic chains, and the outer headgroups layers. For all the considered bilayers, a model without the water layer between the substrate and the bilayer gave a worse fit to the data. The theoretical ρ values of the used lipids have been calculated and are reported in Table 4. Thus, the parameters obtained from the best fit procedure are the thickness and the roughness of each layer

plus the solvent content expressed as volume percent (see Table 3). The presence of DoHuRu and ToThyRu in DOTAP bilayers influences the bilayers microstructure. First, the variation in the scattering length density ρ values corresponding to all the bilayer regions clearly indicates that DoHuRu and ToThyRu molecules are stably lodged within DOTAP bilayers. In detail, this insertion causes an increase of the hydrophilic region thickness of 4 ± 1 Å, while the hydrophobic region is similar to that obtained for pure DOTAP. In addition, a slight increase in roughness values is observed for DoHuRu/DOTAP system, due to the presence of a longer OEG chains. Electron Paramagnetic Resonance (EPR). Spin-label EPR spectroscopy has been proven to give substantial information on the acyl chains structuring in the lipid bilayers.14,32,38−41 In this study, two amphiphilic spin probes E


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Table 5. Values of the Order Parameter S and Nitrogen Hyperfine Coupling Constant a′N of 5-PCSL and 14-PCSL in Bilayers of DOTAP and Ruthenium Complexes/DOTAP at 50/50 Molar Ratio

Table 3. Parameters Derived from Model Fitting of the Reflectivity Profiles for Pure DOTAP Lipid Bilayers and in the Presence of the Here Studied Amphiphilic Ruthenium Complexes interfacial layer

thickness (Å)

% solvent content

S

roughness (Å)

DOTAP water inner headgroups chains region outer headgroups

6±1 10 ± 1 25 ± 2 9±1

100 21 ± 10 8 ± 10 47 ± 10

5 4 7 4

± ± ± ±

1 1 2 1

water inner headgroups chains region outer headgroups

5±1 9±1 24 ± 2 13 ± 1

100 22 ± 10 4 ± 10 37 ± 10

5 4 6 3

± ± ± ±

1 1 2 1

water inner headgroups chains region outer headgroups

4±1 10 ± 1 24 ± 2 14 ± 1

100 20 ± 10 5 ± 10 40 ± 10

3 7 5 8

± ± ± ±

1 1 2 1

ToThyRu/ DOTAP50/50

Table 4. Molecular Properties of DOTAP Lipid and Amphiphlic Ruthenium Complexesa DOTAP

DoHuRu

ToThyRu

550 Å3 −0.46 Å−2 220 Å3 0.76 Å−2

550 Å3 −0.23 Å−2 440 Å3 1.04 Å−2 380 Å3 0.13 Å−2

280 Å3 −0.32 Å−2 440 Å3 1.04 Å−2 240 Å3 0.65 Å−2

DOTAP

5-PCSL 14-PCSL

0.58 ± 0.01 0.15 ± 0.02

5-PCSL 14-PCSL

15.3 ± 0.1 14.1 ± 0.2

HoThyRu/ DOTAP

0.67 ± 0.01 0.62 0.14 ± 0.01 0.14 (aN′ )/G 14.8 ± 0.1 15.6 13.3 ± 0.2 13.9

DoHuRu/ DOTAP

± 0.02 ± 0.02

0.63 ± 0.01 0.23 ± 0.01

± 0.1 ± 0.2

15.3 ± 0.1 14.1 ± 0.2

were alternatively inserted in the systems: 5-PCSL and 14PCSL. The former bears the radical nitroxide group close to the hydrophilic moiety of the molecule, while in the latter one the reporter group is located at the end of the hydrophobic tail. Consequently, 5-PCSL provides information on the aggregate microdomain just below the external surface, while 14-PCSL gives information on the inner core. The goal has been to investigate how the DOTAP membrane fluidity is influenced by the presence of HoThyRu, ToThyRu, and DoHuRu at 50/50 molar ratio. The spectra, depicted in Figure 6, show an anisotropic line shape for 5-PCSL and an almost isotropic line shape in the case of 14-PCSL. This is a characteristic hallmark of lipid bilayers in the liquid-crystalline fluid phase. A quantitative analysis of the spectra has been performed by determining the order parameter, S, and the coupling hyperfine constant, a′N, that is an index of the micropolarity experienced by the spin probe.42 These two quantities are reported in Table 5. Inspection of the Table shows that insertion of DoHuRu, HoThyRu, or ToThyRu in DOTAP causes an increase of the order parameter, S, for 5-PCSL indicating a stiffening effect on the carbon atoms closer to the hydrophilic region of the complex. From the comparison of the S values, it can be noted that all the ruthenium complexes have a stiffening effect on the outermost layer of the membranes of DOTAP. In detail,

DoHuRu/ DOTAP 50/50

Vchains 106·ρchains Vheadgroup 106·ρheadgroup VOEG 106·ρOEG

n-PCSL

ToThyRu/ DOTAP

a

Data for molecular volumes of complex headgroups have been evaluated through the Volume keyword available in Gaussian 09 package. The molecular volume of the hydrophobic chains has been evaluated according to Tanford.50 Finally, data for the OEG lateral chains have been acquired elsewhere.51.

Figure 6. EPR spectra of 5-PCSL (a) and 14-PCSL (b) for DOTAP, HoThyRu/DOTAP, ToThyRu/DOTAP, and DoHuRu/DOTAP bilayers at 50/50 ratio from freshly prepared solutions (continuous lines). For systems analyzed in the presence of 5-PCSL, data have also been collected 30 days after the preparation of the liposomes (dotted lines). F


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Figure 7. Fluorescent microphotographs of monolayers showing the cellular uptake of liposomes by human MCF-7 breast adenocarcinoma cells. MCF-7 were incubated with 100 μM of the rhodamine-added DoHuRu/DOTAP liposome solution for 30 min, 1 h, and 3 h. DAPI is used as a nuclear stain (shown in blue). Rhodamine-dependent fluorescence (Rhod) of DoHuRu/DOTAP liposomes is shown in green. In merged images (Merge), the two fluorescent emissions are overlapped. The images shown are representative of three independent experiments. Inset: higher magnifications of merged images showing rhodamine-dependent cytoplasmic fluorescence emission.

Figure 8. Fluorescent microphotographs of monolayers showing the cellular location of dansylated- HoThyDansRu complex into human MCF-7 breast adenocarcinoma cells subsequent to nanocarriers application. MCF-7 were incubated with 100 μM of the intrinsically fluorescent HoThyDansRu/DOTAP liposome solution for 30 min, 1 h, and 3 h. DAPI is used as a nuclear stain (shown in blue). Dansyldependent fluorescence (Dans) of HoThyDansRu/DOTAP liposomes is shown in red. In merged images (Merge), the two fluorescent emissions are overlapped. The images shown are representative of three independent experiments. Inset: higher magnifications of merged images showing cellular location of fluorescent dansylated ruthenium complexes.

ToThyRu, carrying a shorter OEG chain than HoThyRu and DoHuRu, has the greatest effect, indicating that OEG groups mitigate the impact of the complex on the bilayer structure. In addition, S values of 14-PCSL show that the bitailed molecule leads to a more compact structure of the inner regions of the bilayer, if compared to the monotailed derivatives. Concerning aN′ , it appears that this value is only marginally affected by the presence of the amhiphilic ruthenium complexes. In particular, ToThyRu causes a decrease in the local polarity, indicating a reduction of the water content in the external headgroup region, which is a consequence of the increased ordering and compactness induced by the presence of the ruthenium complexes. Finally, EPR spectra, performed on the same samples after three months, showed no variation of the signals, confirming the stability with time of the bilayers formed by DOTAP nanoaggregates hosting HoThyRu, ToThyRu, or DoHuRu. Examples of the invariance of the EPR spectra with time are also reported in Figure 6. Cellular Uptake of Ru-Containing Nanoaggregates. Because transition metal-based anticancer drugs target DNA in the nuclear cage, the cellular uptake characteristics and the nuclear entry ability have emerged as central factors influencing their antiproliferative efficacy. For both ruthenium and platinum complexes, the cytotoxicity profiles are significantly

correlated with the rate of cellular uptake, which in turn is positively coupled to their lipophilicities.43 In agreement with literature reports concerning low molecular weight ruthenium complexes, we have recently shown that AziRu is more cytotoxic than the well-known NAMI-A complex, presumably because of a higher lipophilic character leading to enhanced cellular uptake efficiency.10 In this study, fluorescent probes matched to 50/50 DoHuRu/DOTAP liposomes have been used to evaluate their uptake and localization into human MCF-7 cancer cells. First, by adding a rhodamine B derivative, we analyzed the interaction between cells and fluorescent liposomes. In the microphotographs reported in Figure 7, the blue areas indicate the cells nuclei stained by DAPI, whereas the green areas represent the rhodamine localization within cells. More in details, the rhodamine-dependent green fluorescence emission is produced from a rhodamine B lipid derivative, 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt, used as a fluorescent probe by 2% mol addition to nanocarriers, as described in the Supporting Information. As shown by time-course experiments starting after 30 min of incubation, DoHuRu/DOTAP liposomes show a high propensity to cross cell membranes, and the cell entry appears to be a very fast process. In fact, short G


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Figure 9. Cell survival index values, evaluated by MTT assay and total cell count, in MCF-7 (panel A), WiDr (panel C), and C6 (panel E) cell lines incubated for 48 h with DOTAP, with different Ru-containing formulations and with AziRu, as indicated in the legend. In panels B, D, and F, for MCF-7, WiDr, and C6, respectively, the corresponding concentration-effect curves are reported, as obtained by normalizing for the actual amount of ruthenium contained within DoHuRu/DOTAP, HoThyRu/DOTAP, and ToThyRu/DOTAP liposomes. Data are expressed as percentage of untreated control cells and are reported as mean of four independent experiments ± SEM.

cellular uptake of Ru-containing nanoaggregates (microphotographs are shown in Figure SI6). To further investigate cellular uptake, additional in vitro fluorescence experiments were performed on MCF-7 cells treated with the dansyl-labeled ruthenium complex HoThyDansRu coaggregated with DOTAP under the same conditions used for HoThyRu, ToThyRu, and DoHuRu. In this way, the fate of the active ruthenium complex can be directly assessed, thereby examining its location after nanocarriers application to the cells. Consistent with our previous results, fluorescently labeled HoThyDansRu localizes rapidly within the cells, and microphotographs in Figure 8 clearly show a time course accumulation as the duration of incubations increased. The analysis of fluorescent emission also suggests that the complexes lodged in DOTAP liposomes enter the cytoplasm before being predominantly delivered to discrete foci in the

incubations with liposomes result in a massive cell uptake, as highlighted by the rhodamine-dependent fluorescence emission (Rhod) localized widespread in the cytoplasm. Merged images (Merge), where fluorophore emission is overlapped at the same location, suggest also a nuclear localization of the aggregates. By means of their lipid properties, it is feasible that the here investigated Ru complexes/DOTAP nanoaggregates are taken up by cells directly via membrane fusion and by endocytosis. These processes apparently occur by nonspecific patterns involving multiple molecular mechanisms, probably starting from charge attraction and ultimately leading to close contact with the target membrane.44,45 As reported in the Supporting Information, control experiments have been also performed by exposing the cells to the rhodamine B adduct alone under the same in vitro experimental conditions used to evaluate the H


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anticancer activity with respect to low molecular weight ruthenium complexes is the result of an enhanced cellular uptake efficiency due to nanovectorization, as well as of the capacity of Ru(III) nucleolipidic aggregates themselves in inhibiting cell growth and proliferation. Consistent with our previous investigations,10 Ru(III) derivatives are more active against MCF-7 cells than against other cancer cells. However, the antiproliferative effects observed toward rat C6 glioma cells, a current model system useful for the evaluation of new therapies for the treatment of malignant glial neoplasm,46 are also worthy of note. Time-Dependent Degradation of Ruthenium Complexes Impairs Their Biological Activity. One of the major drawbacks of low molecular weight Ru complexes is their rather limited stability in aqueous solutions. Despite the good outcome throughout advanced clinical evaluation as anticancer drugs showed by NAMI-A and KP1019, this is a central issue that claims a reconsideration of the ruthenium complexes therapeutic effectiveness. Under physiological conditions, the degradation process is imputable to the replacement of chloride ions, as well as of the DMSO ligand, with water molecules and hydroxide ions, followed by the formation of poly-oxo species in relatively few hours.9 As we have recently reported, this process also occurs for Azi-Ru and for the derivative nucleolipidic complexes ToThyRu, HoThyRu, and DoHuRu, resulting in a visible change of the solution color and in the precipitation of brown particles.10,47 Because currently it is not clear if the formation of poly-oxo species interferes with the low molecular weight Ru complexes anticancer activity,48 and it is reported that the premature aquation and hydrolysis of Rubased anticancer drugs can deactivate or activate too early most of the administered complex,49 we have investigated the bioactivity time course by comparing freshly prepared and aged solutions of Azi-Ru and of HoThyRu, one of the Azi-Rurelated amphiphilic ruthenium complexes here investigated. The aging of the Azi-Ru and the HoThyRu solutions occurs spontaneously and the Figure 10 is obtained with Azi-Ru and HoThyRu taken for variable periods (48, 120, and 240 h) in pseudophysiological solutions. Interestingly, within a context of rather similar antiproliferative effects, in all the tested cell lines the trend over time of Azi-Ru and HoThyRu activity showed a slight but significant decrease, directly correlated with the aging of solutions. Hence, in accordance with our previous findings,10 in vitro data highlight that the effectiveness of low molecular weight Ru complexes, as well as of amphiphilic ruthenium complexes, is impaired over time, probably as a consequence of the formation of insoluble poly-oxo species under physiological conditions. Therefore, the discovery of innovative, stable Ru(III) formulations, such as those presented in this work, is a mandatory goal in the design of effective Ru-based antineoplastic agents.

Table 6. IC50 Values (μM) Relative to Azi-Ru and to DoHuRu/DOTAP, HoThyRu/DOTAP, and ToThyRu/ DOTAP Liposomes in the Indicated Cell Lines Following 48 h of Incubationa cell line

Azi-Ru

MCF-7

305 ± 16

WiDr

441 ± 20

C6

318 ± 12

ToThyRu/ DOTAP 50/50

HoThyRu/ DOTAP 50/50

DoHuRu/ DOTAP 50/50

19 ± 8 16 50 ± 11 9 54 ± 8 6

15 ± 7 20 65 ± 8 7 43 ± 11 7

13 ± 5 23 41 ± 10 11 34 ± 9 9

a IC50 values are reported as mean ± SEM. In bold, the Potentiating Factors (PF) of the nucleolipidic ruthenium complexes in DOTAP liposomes with respect to Azi-Ru are shown, calculated as the ratio of IC50 values of DoHuRu/DOTAP, HoThyRu/DOTAP, and ToThyRu/DOTAP complexes to the IC50 of AziRu complex.

perinuclear compartment; moreover, although attenuated by DAPI nuclear staining, dansyl-dependent fluorescence emission is also detectable in the nuclei area. Overall, in addition to demonstrating an effective process of cellular uptake, the fluorescent patterns seem to suggest an intracellular liposome degradation coupled with the release of the pharmacologically active agent within the cytoplasm. This would explain the widespread rhodamine-associated fluorescence and the concomitant generation of discrete dansylassociated spots following cellular uptake and disaggregation of nanocarriers. In Vitro Cytotoxicity of Ruthenium Complexes. The in vitro cytotoxicities of ruthenium-based nanocarriers against two human cancer cell lines (MCF-7 breast adenocarcinoma cells and WiDr epithelial colorectal adenocarcinoma cells) and one rat cancer line (C6 glioma cells) have been determined by the estimation of a “cell survival index” arising from the combination of cell viability evaluation with cell counting. The low molecular weight ruthenium complex AziRu, analog of NAMI-A and precursor of the here investigated nucleolipidic nanocarriers, as well as DOTAP, are included for comparison and tested under the same experimental conditions (Figure 9). As expected, the liposomes composed exclusively of DOTAP showed no significant interference with the cell viability and proliferation. Interestingly, based on the IC50 values (Table 6), the three mixed amphiphilic ruthenium complexes/DOTAP nanoaggregates are much more active than the precursor AziRu. In fact, by analyzing the results from in vitro bioscreening, it is to be considered that the nucleolipidic ruthenium complexes incorporated within DOTAP cationic liposomes enclose only 50% of ruthenium in moles. Consequently, results normalization according to the actual ruthenium content within the nanosystems largely modifies the concentration/effect curves, showing for these compounds higher antiproliferative activity than Azi-Ru (Figure 9B, D, and F for MCF-7, WiDr, and C6 cells, respectively). Overall, our results show that all the nanoaggregates of HoThyRu, DoHuRu, and ToThyRu mixed with DOTAP are notably effective. Primarily, as highlighted by the corresponding potentiating factors (PF, Table 6), these ruthenium complexes reach IC50 values around 15 μM on MCF-7, thus showing to be about 20 times more potent than Azi-Ru (305 μM) examined under the same conditions. This is a central finding of our project demonstrating the drug efficacy of nucleolipidic Ru(III) complexes toward cancer cells when lodged in cationic DOTAP liposomes. Presumably, their higher

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CONCLUSIONS Recently, we synthesized a mini-library of ruthenium complexes coordinating differently decorated nucleolipids, showing a marked propensity for aggregation in aqueous solutions and high in vitro antiproliferative activity. With the aim of developing suitable formulations which allow the transport of higher metal content within the aggregates and improve their cell uptake, still ensuring the complete stability for the active ruthenium complexes in physiological solutions, substantial progress has been achieved with the here presented studies, in which the amphiphilic ruthenium complexes, carrying a I


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Figure 10. Antiproliferative effect, evaluated by a cell survival index, of freshly prepared and aged solutions (taken at room temperature for 48, 120, and 240 h in a pseudophysiological solution) of Azi-Ru and of HoThyRu in MCF-7 (A and B, respectively), WiDr (C and D, respectively), and C6 (E and F, respectively) cell lines, as indicated in the legends. Data are expressed as percentage of untreated control cells and are reported as the mean of four independent experiments ± SEM; *p < 0.05; and ** p < 0.01 compared with cells incubated with freshly prepared solutions of Azi-Ru and of HoThyRu.

the positive net superficial charge present on the aggregates. The fate of the Ru-complex, once within the cells, was investigated by means of a fluorescently tagged amphiphilic Rucontaining derivative, showing a marked tendency to accumulate in proximity of the nuclei. In our opinion, these results provide convincing and definitive evidence that the investigated amphiphilic Rucomplexes, in the here described formulations, are a good option as improved candidate drugs, alternative to known Rucontaining complexes as NAMI-A and KP1019. Further studies are currently in progress to study their in vivo activity, particularly aiming at the definition of the metabolic targets and the physiological implications of these antiproliferative agents.

negative charge, are mixed up to 50/50 molar ratio with the cationic lipid DOTAP. These nanoaggregates form catanionic vesicles which are stable at least for three months under physiological conditions, showing no degradation phenomena. The physicochemical characteristics exhibited by the three Ru-based aggregates prepared were roughly similar. Remarkably, the Ru-complex/DOTAP nanoaggregates, tested in vitro on a panel of human and non-human cancer cell lines, exhibited IC50 values in the low μM range, proving to be 10−20-fold more cytotoxic against MCF-7 and WiDr cells than AziRu, a non-nucleolipidic Ru-complex, here used for control, built as a NAMI-A analog and showing a bioactivity pattern sensibly decreasing with time. Fluorescence microscopy studies, carried out with the amphiphilic Ru-complex/DOTAP formulations added with rhodamine-B, allowed following the efficient and rapid cell incorporation of the nanoaggregates, probably due to J


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ASSOCIATED CONTENT

S Supporting Information *

Detailed synthetic procedures and schemes for the preparation of HoThyDansRu, as well as the 1H, 13C NMR, and ESI-MS characterization for all the synthesized compounds, are described. The procedure used for preparing samples investigated, as well as the details of physicochemical techniques (DLS, SANS, EPR, Zeta Potential, NR) used. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; daniela.montesarchio@unina. it. Phone: +390 81 674229. Fax: +390 81 674090. Notes

The authors declare no competing financial interest. # Deceased.

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ACKNOWLEDGMENTS This work was supported by MIUR (PRIN 2010-prot. 2010BJ23MN_007), Università di Napoli Federico II-Compagnia di S.Paolo (Progetto FARO-III tornata) and by Strain Excellence Network − Innovative Therapeutic Strategies (POR Campania FSE 2007/2013, Asse IV - Ob. Specifico 1, Asse V − Ob. Operativo m3, A.G.C 06 D.D. n. 414 del 13.11.2009). One of the authors (G.V.) wishes to thank the Institute Laue Langevin for the provision of NR beam time performed during his stage there. Some of the authors (G.M., A.R., and L.P.) thank the Jülich Centre for Neutron Science for provision of beam time. SANS experiments were supported by the European Commission, NMI3.

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DEDICATION The authors wish to dedicate the present manuscript to Prof. A. Colonna that recently passed away for his valuable guide in their research.

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REFERENCES

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Anticancer Cationic Ruthenium Nanovectors: From Rational Molecular

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