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A new design for nucleolipid-based Ru(iii) complexes as anticancer agents

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A new design for nucleolipid-based Ru(iii) complexes as anticancer agents
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  DaltonTransactions PAPER  Cite this: DOI: 10.1039/c3dt52320a Received 24th August 2013,Accepted 24th September 2013DOI: 10.1039/c3dt52320a www.rsc.org/dalton A new design for nucleolipid-based Ru( III ) complexes asanticancer agents † Daniela Montesarchio,* a Gaetano Mangiapia, a,b Giuseppe Vitiello, a,b Domenica Musumeci, a Carlo Irace, c Rita Santamaria, c Gerardino D ’ Errico a,b andLuigi Paduano* a,b In continuation with our studies concerning the synthesis, characterization and biological evaluation ofnucleolipidic Ru( III ) complexes, a novel design for this family of potential anticancer agents is presentedhere. As a model compound, a new uridine-based nucleolipid has been prepared, named HoUrRu,following a simple and versatile synthetic procedure, and converted into a Ru( III ) salt. Stable formulationsof this highly functionalized Ru( III ) complex have been obtained by co-aggregation with either thezwitterionic lipid POPC or the cationic DOTAP, which have been subjected to an in-depth microstructuralcharacterization, including DLS, SANS and EPR measurements. The  in vitro  bioactivity pro fi le of HoUrRu,as a pure compound or in formulation with POPC or DOTAP, reveals high antiproliferative activity againstMCF-7 and WiDr human cancer cell lines. Introduction Even if its discovery as an antiproliferative agent dates back to1965 1 and its approval by FDA to 1978, cisplatin is still one of the anticancer drugs most largely adopted in the clinic, along  with its second-generation derivatives carboplatin and oxali-platin. 2 Despite the overall e ffi cacy of platinum-based drugs incancer therapy, their widespread use raises severe concernsdue to their low selectivity, ine ff  ectiveness towards most metastasized cancer cells, general drug resistance and toxi-city. 3 In the search for e ff  ective anticancer agents withreduced side e ff  ects, a variety of transition metal derivativesare currently investigated as a promising alternative toplatinum. 4 Great attention has been recently focused on rutheniumcomplexes, showing a remarkable antitumor and antimeta-static activity associated with lower toxicity, as is the case of two compounds currently undergoing advanced clinical trials:NAMI-A and KP1019 (Fig. 1). 5 Interestingly, these compoundshave displayed encouraging preliminary results, even if theirgeneral pharmacokinetic profile is not optimal, mainly due totheir poor stability in physiological environment. In fact, inaqueous solutions rapid exchange of the chlorido ligands withhydroxyl ions can occur, favoring the formation of insolublepoly-oxo species. 6  A dramatic consequence of these degra-dation processes is that probably only a limited amount of theadministered drug is e ff  ectively internalized into the cancercells.In an e ff  ort to improve the stability of the ruthenium-baseddrugs in the extracellular environment and also enhance theircellular uptake, we have recently proposed an innovativestrategy for their transport   in vivo . Our basic idea consisted of incorporating the cytotoxic ruthenium complex into a highly functionalized molecular structure, ensuring both an e ffi cient protection against degradation over long circulation times anda high cellular internalization of the metal. 7 More in detail, wehave designed and synthesized a novel NAMI-A analog, named AziRu (Fig. 2), showing higher  in vitro  cytotoxicity than Fig. 1  Chemical structures of NAMI-A and KP1019. † Electronic supplementary information (ESI) available: General syntheticmethods, synthesis and characterization data for compounds  1 ,  3 – 8 . See DOI:10.1039/c3dt52320a a  Department of Chemical Sciences, University of Napoli   “   Federico II  ”   , ComplessoUniversitario di Monte Sant  ’   Angelo, via Cintia 21, I-80126, Napoli, Italy. E-mail: daniela.montesarchio@unina.it  b CSGI  – Consorzio interuniversitario per lo sviluppo dei Sistemi a Grande Interfase, Italy. E-mail: luigi.paduano@unina.it; Fax: +39-081-674090; Tel: +39-081-674126  c  Department of Pharmacy, University of Napoli   “   Federico II  ”   , via D. Montesano 49, I-80131, Napoli, Italy This journal is © The Royal Society of Chemistry 2013  Dalton Trans.    P  u   b   l   i  s   h  e   d  o  n   2   4   S  e  p   t  e  m   b  e  r   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   1   2   /   1   1   /   2   0   1   3   2   1  :   4   7  :   5   0 . View Article Online View Journal  NAMI-A itself; AziRu has been then inserted into a nucleolipi-dic sca ff  old, as the core structural motif attached to thenucleobase of uridine or thymidine, which were further deco-rated with one or two lipid residues and with one oligoethyl-ene glycol chain of variable length. 8  Aiming at biocompatible systems, highly stable under physio-logical conditions, these amphiphilic ruthenium-complexes,named ToThyRu, HoThyRu and DoHuRu (Fig. 2), were co-aggregated with the zwitterionic lipid 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) 9 or the cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane chloride (DOTAP). 10 E ff  ective retardation of the ligand exchange processes inpseudo-physiological solutions, resulting in formulationsstable for months, has been obtained with the Ru( III ) complex present up to a maximum ratio of 15% in moles in mixture with POPC, 9 and up to 50% in mixture with DOTAP. 10 Remark-ably,  in vitro  bioscreenings on a panel of human and non-human cell lines showed that the nucleolipid-based ruthe-nium complexes stabilized in POPC are from 4 to 40 timesand, in the case of the DOTAP formulations, from 6 to 20times more e ff  ective than AziRu, which in turn was foundto have IC 50  values lower than other known Ru( III )complexes. 9 – 11 In order to expand the repertoire of available nucleolipidRu( III ) complexes and thus have a wider picture of the struc-ture – activity relationships for this family of potential anti-cancer drugs, we have decided to investigate novel Ru( III )-containing analogs based on di ff  erent decorations of thenucleolipid skeleton. In the first series of compounds we havedeveloped (ToThyRu, HoThyRu, and DoHuRu), the pyridineresidue  –  chosen as the privileged ligand for ruthenium  –  wasanchored to the N-3 position of the pyrimidine moiety. Wethen reasoned that, in order to take advantage of therecognition abilities of the nucleobases and their potentialinteractions with nucleic acids  via  Watson – Crick hydrogenbonds or stacking contacts, these should not be blocked by hindered groups. Therefore, on the basis of the promising results obtained for the first generation of ruthenium( III ) com-plexes, we have designed a novel nucleolipid where the pyri-dine ligand is not attached at the N-3 but at the C-3 ′  positionon the sugar, identified as a model compound for a secondgeneration of metal-complexed nucleolipids.Here described is the synthesis of a new amphiphilic Ru( III )complex   1  (which, in analogy with previously introduced acro-nyms, we have named HoUrRu, Fig. 3), as well as the micro-structural characterization and biological evaluation of itsformulations in POPC and DOTAP, discussed also in the light of our previous data. Fig. 2  Molecular structures of the Ru( III ) complexes ToThyRu, HoThyRu, DoHuRu and AziRu, synthesized in our group. 9,11 Fig. 3  Nucleolipidic ruthenium( III ) complex  1  (Bn = benzyl). Paper Dalton Transactions Dalton Trans.  This journal is © The Royal Society of Chemistry 2013    P  u   b   l   i  s   h  e   d  o  n   2   4   S  e  p   t  e  m   b  e  r   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   1   2   /   1   1   /   2   0   1   3   2   1  :   4   7  :   5   0 . View Article Online  Experimental section The general synthetic procedures, along with the synthesis andcharacterization data for compounds  3 – 8  and  1  are describedin the ESI. † Lipid-based aggregates preparation Samples of HoUrRu were prepared by dissolving a suitableamount of the complex in pure chloroform, in order to get aconcentration of   ∼ 1 mg ml − 1 . For pseudo-ternary systems con-taining POPC or DOTAP, an appropriate amount of this phos-pholipid was added to the Ru-complex solution, in order tohave the prefixed molar ratio (15% and 50% in moles of HoUrRu, respectively) and a final total solute concentration of 1 mM. The dissolution was favored by a slight warming (40 °C)and a very short sonication treatment ( ∼ 5 min). Subsequently,an appropriate amount of this solution was transferred intoround-bottom glass tubes. A thin film was obtained throughevaporation of the solvent and vacuum desiccation. Thesamples were then hydrated with di ff  erent media, namely,pure H 2 O (or D 2 O), a 0.9 wt% NaCl solution and, finally, apH 7.4 bu ff  er for miming physiological conditions. This bu ff  er was prepared by dissolving sodium dihydrogenphosphate(NaH 2 PO 4 ) and disodium hydrogenphosphate (Na 2 HPO 4 ) inD 2 O or H 2 O at concentrations equal to 0.0773 mol dm − 3 and0.123 mol dm − 3 , respectively. The pH was checked to be within0.1 pH unit using a Radiometer pHM220 pH-meter, equipped with a AgCl/Ag electrode and a glass electrode previously cali-brated with IUPAC standard bu ff  er solutions. All the solutions were vortexed and, for the DLS and SANS measurements, thesuspensions were then sonicated and repeatedly extrudedthrough polycarbonate membranes of 100 nm pore size, for at least 11 times.Samples prepared for EPR experiments also included 1%(w/w) of spin-labeled phosphatidylcholine (1-palmitoyl-2-[ n -(4,4-dimethyloxazolidine-  N  -oxyl)]stearoyl- sn -glycero-3-phospho-choline,  n -PCSL,  n  = 5 and 14), purchased from Avanti PolarLipids and stored at 20 °C in ethanol solution. Dynamic light scattering (DLS) Dynamic light scattering investigations were performed with asetup composed of a Photocor compact goniometer, a SMD6000 Laser Quantum 50 mW light source operating at 5325 Å and a PMT-120-OP/B and a correlator (Flex02-01D) purchasedfrom  Correlator.com . All the measurements were performed at 25.00 ± 0.05 °C with the temperature controlled through theuse of a thermostat bath. In dynamic light scattering, theintensity autocorrelation function  g  (2) ( t  ) is measured andrelated to the electric field autocorrelation function  g  (1) ( t  ) by the Siegert relation. 12  g  ð 2 Þ ð t  Þ ¼  1  þ  β  j  g  ð 1 Þ ð t  Þj 2 ð 1 Þ  where  β   is the coherence factor, which accounts for the devi-ation from ideal correlation and depends on the experimentalgeometry. The function  g  (1) ( t  ) can be written as the Laplacetransform of the distribution of the relaxation rates,  Г  , used tocalculate the translational di ff  usion coe ffi cient,  D :  g   1 ð Þ t  ð Þ ¼ ð  þ 1  1 τ   A  τ  ð Þ exp    t  τ    dln τ   ð 2 Þ  where  τ   = 1/  Г  . Laplace transforms were performed using a vari-ation of CONTIN algorithm incorporated in the PrecisionDeconvolve software. From the relaxation rates, the  z  -averageof the di ff  usion coe ffi cient   D  may be obtained as: 12  D  ¼  Γ  q 2  ð 3 Þ  where  q  = 4 π n 0 /  λ sin( θ  /2) is the modulus of the scattering  vector,  n 0  is the refractive index of the solution,  λ  is the inci-dent wavelength and  θ   represents the scattering angle. Pro- vided that the solutions are quite dilute, the Stokes – Einsteinequation, which rigorously holds at infinite dilution for non-interacting spherical species 13 di ff  using in a continuousmedium, may legitimately be used to evaluate the hydro-dynamic radius  R H  of the aggregates: 14  R H  ¼  kT  6 π η  D  ð 4 Þ  where  k   is the Boltzmann constant,  T   is the absolute tempera-ture and  η  is the medium viscosity. In the case of non-spherical particles,  R H  in eqn (4) represents the radius of equi- valent spherical aggregates with the same di ff  usion coe ffi cient. Small angle neutron scattering (SANS) SANS measurements were performed at 25 °C with the KWS2instrument located at the Heinz Meier Leibtnitz Source, Garch-ing Forschungszentrum (Germany). Monochromatic neutrons with a wavelength  λ  = 5 Å and a spread  Δ  λ /  λ  ≤  0.2 were used. A two-dimensional array detector at three collimations (C)/sample-to-detector (D) distance combinations (C 8m D 2m ,C 8m D 8m  and C 20m D 20m ), measured neutrons scattered from thesamples. These configurations allowed collecting data in arange of the scattering vector modulus  q  = 4 π /  λ sin( θ  /2)between 0.00240 Å  − 1 and 0.179 Å  − 1 , where  θ   is the scattering angle. The investigated systems were contained in a closedquartz cell, in order to prevent the solvent evaporation andkept under measurements for a period su ffi cient to have ∼ 1.5 million counts. The raw data were then corrected forbackground and empty cell scattering. Detector e ffi ciency cor-rection, radial average and transformation to absolute scatter-ing cross sections d  Σ  /d  Ω  were made with a secondary plexiglass standard. 15 Scattering cross sections obtained for the investigatedsystems have been modeled as arising from collections of uni-lamellar spherical vesicles with polydisperse inner aqueouscore  R 0  and monodisperse bilayer thickness  d  . A Schulz – Zimmdistribution function was assumed for  R 0  F R 0 ; Z  ;  R 0 ð Þ ¼  Z   þ  1  R 0   Z  þ 1  R Z  0 Γ   Z   þ  1 ð Þ exp   Z   þ  1  R 0  R 0    ð 5 Þ Dalton Transactions Paper  This journal is © The Royal Society of Chemistry 2013  Dalton Trans.    P  u   b   l   i  s   h  e   d  o  n   2   4   S  e  p   t  e  m   b  e  r   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   1   2   /   1   1   /   2   0   1   3   2   1  :   4   7  :   5   0 . View Article Online   where  R ˉ 0  represents the average inner radius,  Z   is the Zimmparameter and  Г  ( x ) is the Euler Gamma function Γ   x ð Þ ¼ ð  þ 1 0 exp   t  ð Þ t  x  1 d t  The theoretical expression for the scattering cross sections(d  Σ  /d  Ω ) of unilamellar polydisperse spherical vesicles isgiven by d  Σ  d  Ω  q ð Þ ¼  n p  P q ð Þ þ  d  Σ  d  Ω   incoh ð 6 Þ  where  n p  is the number density of the scattering objects ( i.e. the number of scattering objects per unit of volume), (d  Σ  /d  Ω ) incoh  takes into account the incoherent scattering contri-bution, mainly due to the presence of hydrogen atoms, and  P  ( q ) is the form factor of the objects that for unilamellar vesicles with a polydispersed core is given by   P q ð Þ ¼ ð  þ 1 0  f q ;  R 0 ; d  ð Þj j 2  F R 0 ; Z  ;  R 0 ð Þ d  R 0  ð 7 Þ  where  f q ;  R 0 ; d  ð Þ ¼  43 π  R 03  ρ 0    ρ v ð Þ 3   j  1  qR 0 ð Þ qR 0   43 π  R 0  þ  d  ð Þ 3  ρ 0    ρ v ð Þ 3   j  1  q R 0  þ  d  ð Þð Þ q R 0  þ  d  ð Þ ð 8 Þ  with  ρ 0  and  ρ  v   the scattering length densities of the solvent and the bilayer, respectively, whereas  j  1 ( x ) is the first orderspherical Bessel function   j  1  x ð Þ ¼  sin  x ð Þ   x cos  x ð Þ x  ð 9 Þ By inserting eqn (9), (8), (5) and (7) into eqn (6), it is poss-ible to obtain a theoretical expression for cross sections (d  Σ  /d  Ω ). Actually, for a Schulz – Zimm distribution function and fora core – shell spherical geometry, integral (7) can be expressedin a closed form. 16 Therefore, the resulting final expression tobe fitted to the experimental data is a function of   R ˉ 0 ,  Z   and  d  . Actually, as fitting parameters,  R 0 ,  d   and the  R 0  polydispersity index   i   R 0  were reported, where i   R 0  ¼ Р þ 1 0  F R 0 ; Z  ;  R 0 ð Þ  R 02 d  R 0 Р þ 1 0  F R 0 ; Z  ;  R 0 ð Þ  R 0  d  R 0   2  ð 10 Þ This parameter is related to the  Z   Zimm parameter by  i   R 0  ¼  Z   þ  1 ð Þ  Z   þ  3 ð Þ Z   þ  2 ð Þ 2  ð 11 Þ Electron paramagnetic resonance (EPR) EPR spectra were recorded using a 9 GHz Bruker Elexys E-500spectrometer (Bruker, Rheinstetten, Germany). Capillaries con-taining the samples were placed in a standard 4 mm quartzsample tube containing light silicone oil for thermal stability.The temperature of the sample was regulated at 25 °C andmaintained constant during the measurement by blowing thermostated nitrogen gas through a quartz Dewar. The instru-mental settings were as follows: sweep width, 120 G; resolu-tion, 1024 points; modulation frequency, 100 kHz; modulationamplitude, 1.0 G; time constant, 20.5 ms; incident power,5.0 mW. Several scans, typically 16, were performed to improvethe signal-to-noise ratio. Values of the outer hyperfine split-ting, 2  A max  , were determined by measuring, through a home-made MATLAB-based routine, the di ff  erence between the low-field maximum and the high-field minimum. Thisparameter is a useful empirical measure of the lipid chaindynamics and order in both gel and fluid phases of lipid bilayers. 17 Reproducibility of 2  A max   determination wasestimated by obtaining its value for selected independently prepared samples with the same nominal composition. Theuncertainty a ff  ecting the 2  A max   parameter was 0.2 G. Cell cultures and  in vitro  bioscreenings Human WiDr epithelial colorectal adenocarcinoma cells andMCF-7 breast adenocarcinoma cells were purchased from ATCC® (American Type Culture Collection, Manassas, Virginia,USA). MCF-7 and WiDr were grown in RPMI 1640 medium(Invitrogen, Paisley, UK). Media were supplemented with 10%fetal bovine serum (FBS, Cambrex, Verviers, Belgium),  L -gluta-mine (2 mM, Sigma, Milan, Italy), penicillin (100 units ml − 1 ,Sigma) and streptomycin (100  μ g ml − 1 , Sigma), according to ATCC recommendations. All the cells were cultured in ahumidified 5% carbon dioxide atmosphere at 37 °C.The anticancer activity of ruthenium-containing nucleolipi-dic nanoparticles and of Azi-Ru was investigated by the esti-mation of a  “ cell survival index  ” , arising from the combinationof cell viability evaluation with cell counting. MCF-7 and WiDrcells were washed with PBS bu ff  er solution (Sigma), collectedby trypsine (Sigma) and then inoculated in 96-microwellculture plates at a density of 10 4 cells per well. Cells wereallowed to grow for 24 h, then the medium was replaced withfresh medium and cells were treated for a further 48 h with arange of concentrations (10  →  100  μ M) of AziRu, and withHoUrRu alone or lodged in POPC and DOTAP liposomes(HoUrRu/POPC and HoUrRu/DOTAP, respectively). Cell viabi-lity was evaluated with the MTT assay procedure, whichmeasures the level of mitochondrial dehydrogenase activity using the yellow 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2  H  -tetrazolium bromide (MTT, Sigma) as a substrate. The assay  was based on the redox ability of living mitochondria toconvert dissolved MTT into insoluble purple formazan. Briefly,after the treatments the medium was removed and the cells were incubated with 20  μ l per well of an MTT solution (5 mg ml − 1 ) for 1 h in a humidified 5% CO 2  incubator at 37 °C. Theincubation was stopped by removing the MTT solution and by adding 100  μ l per well of DMSO to solubilize the purple forma-zan. Finally, the absorbance was monitored at 530 nm by using a multiwell plate-reader in a Perkin-Elmer LS 55 Lumi-nescence Spectrometer (Perkin-Elmer Ltd, Beaconsfield, UK). 18 Cell number and proliferation was determined using a TC10 Paper Dalton Transactions Dalton Trans.  This journal is © The Royal Society of Chemistry 2013    P  u   b   l   i  s   h  e   d  o  n   2   4   S  e  p   t  e  m   b  e  r   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   1   2   /   1   1   /   2   0   1   3   2   1  :   4   7  :   5   0 . View Article Online  automated cell counter (Bio-Rad, Milan, Italy), providing anaccurate and reproducible total count of cells and a live/deadratio in one step. The calculation of the concentration requiredto inhibit the net increase in the cell number and viability by 50% (IC 50 ) is based on plots of data carried out in triplicatesand repeated three times. IC 50  values were obtained by meansof a dose response curve by nonlinear regression using a curvefitting program, GraphPad Prism 5.0, and are expressed asmean ± SEM. Statistical analysis  All the data were presented as mean ± SEM. The statistical ana-lysis was performed using GraphPad Prism (GraphPad softwareInc., San Diego, CA) and ANOVA test for multiple comparisons was performed followed by Bonferroni ’ s test. Results and discussion Synthesis of amphiphilic Ru( III ) complex 1 (HoUrRu) Schematically, target compound  1  is composed of a uridinecore sca ff  old decorated with the following structural motifs:1. one carboxy-methyl pyridine at 3 ′  position, as the ligandfor the Ru complexation;2. one oleic acid residue at 2 ′  position, able to promote theformation of stable nano-aggregates in aqueous solutions;3. one hepta(ethylene glycol) unit, acting as a  “ stealth ” agent for the amphiphilic Ru complex nano-aggregates, deriva-tized with a COOH group at one extremity to allow its conden-sation at 5 ′  position.Derivative  1  was designed with the idea of maintaining thesame decoration set present in HoThyRu (Fig. 2), providing thenecessary functionalities for an appropriate balance betweenthe hydrophilic and lipophilic moieties, as well as the pyridineligand for the metal coordination. These appendages ulti-mately proved to guarantee in HoThyRu a rapid self-assembly in aqueous solution and a high antiproliferative activity. InHoUrRu all the three groups are fixed on the sugar, so that theuracil moiety is not hampered in hydrogen bonds formation orin stacking interactions with potential  in vivo  targets. The syn-thesis of   1  has been realized exploiting a modular and versatilestrategy using a uridine analog as the starting material.Remarkably, this design allows to introduce the di ff  erent functionalities on the uridine sca ff  old  via  selectively cleavablelinkages. In fact, the pyridine moiety is linked to the sugarthrough an amide bond, while the lipid and the polyetherchains are attached through ester linkages; therefore, if thepyridine ligand is strongly anchored to the nucleoside under in vivo  conditions, the lipid and polyether chains  –  having themain role to ensure a prolonged extracellular life and an easy cell uptake  –  in principle may be lost with time once the drug is internalized into cells, due to aspecific esterase activity.The key intermediate in the synthesis of compound  1  is themodified nucleoside 3 ′ -azido-3 ′ -deoxy-1- β - D -xylofuranosyluracil 2  (Scheme 1). This is a highly versatile building block, having three, easily di ff  erentiable functional groups on the sugarmoiety: two hydroxyls, of which one is primary and one sec-ondary, and an azido group, which can be easily reduced to aprimary amine on demand. By exploiting a rational scheme of protecting groups, it is therefore possible to insert on each of them a specific tag in a very selective and e ffi cient manner.Nucleoside  2 , known in the literature since the late 70s, 19 hasbeen recently prepared by some of us following a new, opti-mized protocol, requiring only straightforward and high yield-ing steps, 20 based on the use of Boc as a transient protecting group on the N-3 position. 21 Following our synthetic scheme,modified nucleoside  2  was obtained in 6 steps with 50%overall yield starting from uridine.Nucleoside  2  has been here easily converted into thedesired complex   1  through simple and high yielding manipu-lations, depicted in Scheme 1. In the first step, TBDMS hasbeen introduced to protect the 5 ′  position, leading to  3 , whichhas been then reacted with oleic acid, in the presence of DCCas the condensing agent, giving nucleolipid  4  (Scheme 1).Then the selective reduction of the azido group in  4  –  obtainedby catalytic hydrogenation under 1 atm. H 2  pressure in thepresence of Pd/C  –  a ff  orded amine  5  in almost quantitative yields and in a pure form simply filtering o ff   and washing thecatalyst. Then,  5  was reacted with 2-(pyridin-4-yl)acetic acidhydrochloride to give derivative  6 . This compound was succes-sively desilylated by reaction with Et  3 N × 3HF a ff  ording nucleolipid  7 , and then 5 ′ -functionalized with a residue of benzyl-capped hexa(ethylene glycol) acetic acid, synthesized aspreviously described, 8 thus providing the desired amphiphilicnucleolipid  8 .The reaction of   8  with an equimolar amount of [RuCl 4 (DMSO) 2 ] − Na + –  prepared as previously described 22 –  for4 h at 40 °C in CH 2 Cl 2  led to target complex   1  in quantitative yields (Scheme 2). This was obtained as a unique compound,as checked by TLC control on alumina, and its identity hasbeen proved by ESI-MS analysis. The presence of Ru( III ) in theprepared salt has been confirmed by its  1 H NMR spectra: asexpected, due to the paramagnetic nucleus, all the signalsshowed a general broadening and a diagnostic, dramaticupfield shift was observed for the signals relative to the pyri-dine residue (band centered at   δ   =  − 1.98 ppm) and the methylgroups (band centered at   δ   =  − 9.59 ppm) of the DMSO ligand,more directly involved in the ruthenium complexation. Lipid-based aggregates characterization The water stability of HoUrRu is limited. In fact, analogously to NAMI-A  6 and the Ru-based complexes ToThyRu, HoThyRuand DoHuRu, 9,10 marked changes of HoUrRu properties in itsaqueous solutions have been detected. In particular, uponsolubilization, a color variation of the HoUrRu solution from yellow to green was observed, followed by the formation of small dark particles, showing a pH-dependent kinetics: thehigher is the pH of the solution, the faster the rate of thedescribed process. It has been shown that this phenomenon isconnected to a degradation of the Ru complex, ascribable tothe replacement of chlorido ligands, as well as of DMSO, with Dalton Transactions Paper  This journal is © The Royal Society of Chemistry 2013  Dalton Trans.    P  u   b   l   i  s   h  e   d  o  n   2   4   S  e  p   t  e  m   b  e  r   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   1   2   /   1   1   /   2   0   1   3   2   1  :   4   7  :   5   0 . View Article Online
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