Modification of multiwalled carbon nanotubes with a ruthenium drug candidate-indazolium[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 ).

Dalton Transactions PAPER fi Modi cation of multiwalled carbon nanotubes — with a ruthenium drug candidate indazolium Citethis:DOI:10.1039/d0dt03528a H [tetrachlorobis(1 -indazole)ruthenate( )] III † (KP1019 ) MonikaRichert, *aGrzegorzTrykowski, bMariuszWalczyk, b MarcinJ.Cieślak,cJuliaKaźmierczak-Barańska,cKarolinaKrólewska-Golińska, c JanuszW.Sobczak dandStanisławBiniak b Functionalized carbon nanotubes are interesting, promising and unique delivery systems for anticancer drugs,whicharenowinthespotlightofnanomedicine.Connectingnanotubeswithanticancerdrugsor new compoundswithanticancerpropertiesaimsat improving theirstability, efficiencyand reducesthe toxic side effects of cancer treatment. In our research, we are interested in connecting functionalized MWCNTs-NH with [InH][trans-RuCl (In) ], (KP1019) which is one of the most promising anticancer 2 4 2 ruthenium(III)drugcandidates,knownmainlyasacytotoxicagentforthetreatmentofplatinum-resistant colorectalcancers.AsaresultoftheamidationofMWCNTs(1),MWCNTs-NH (2)wereobtained.Then, 2 they were modified with [InH][RuCl (In) ] (4) and the nanosystem [MWCNT-NH +][RuCl (In) −] (3) was 4 2 3 4 2 Received11thOctober2020, obtained. The characterization of the resulting products was performed using IR, Raman spectroscopy, Accepted21stOctober2020 thermalgravimetric,XRD,STEM-EDX,ESI-MS,ICP-MS,andXPSanalyses.Thecytotoxicactivityhasbeen DOI:10.1039/d0dt03528a testedonhumanlungcarcinoma(A549),chronicmyelogenousleukemia(K562)andhumancervixcarci- rsc.li/dalton noma(HeLa)cellswhichshowedthehighertoxicityofthenanosystemthantherutheniumcomplex. Introduction of the most important achievements isthe creation of a CNT- basedsystemtocarryanddeliverdrugsincancertherapy.The From among the unique and interesting properties of CNTs, main purpose of these activities was to solve the problems the most studied are those which may find biomedical appli- associated with the use of chemotherapeutics which involves cations, especially in drug delivery systems. They are exten- numerous side effects, low efficacy, or selectivity. The applied sivelyresearchedforthepurposesofcancertherapy,infectious CNTs successfully conjugated with chemotherapeutics, e.g. diseasesandneurodegenerativedisorders.1,2Inthecaseofbio- paclitaxel, taxoid, doxorubicin and cisplatin, and showed medical applications, a suitable functionalization of carbon sufficient stability under physiological conditions and higher nanotubes (which provide biocompatibility and stability in a tumor suppression efficacy compared to conventionally used biological environment) enables creating hybrids with drugs.1 different drugs and biomolecules as well as developing thera- Considering all the chemotherapeutics, platinum drugs, peuticanddiagnostictools,whichisanimportantissue.2One especially cisplatin, playan important role in treating cancers ofvarioustypes,e.g.headandneck,colorectal,andnon-small- cell lung cancers.3,4 Although the platinum drugs are charac- aFacultyofPharmacy,CollegiumMedicuminBydgoszcz,NicolausCopernicus terized by their great potency regarding different kinds of UniversityinToruń,Jurasza2,85-094Bydgoszcz,Poland. cancer, they also show a lot of side effects and drug resis- E-mail:monika.richert@cm.umk.pl;Fax:+48-52-585-38-04;Tel:+48-52-585-38-03 tance.5Oneofthestrategiestoovercometheseproblemsisthe bFacultyofChemistry,NicolausCopernicusUniversityinToruń,Gagarina7,87-100 Toruń,Poland use of an innovative drug delivery system with nanocarriers cCentreofMolecularandMacromolecularStudies,PolishAcademyofSciences, such as nanotubes. This method aims at increasing drug Sienkiewicza112,90-363Lódź,Poland efficacy and minimizing the side effects. Pristine nanotubes dInstituteofPhysicalChemistry,PolishAcademyofSciences,LaboratoryforSurface are insoluble in most solvents and toxic. However, upon suit- Analysis,Kasprzaka44-52,01-224Warsaw,Poland able functionalization, they become nontoxic, more hydro- †Electronic supplementary information (ESI) available: ESI-MS spectra of [RuCl4(In)2]− (Fig. S1 standard X-ray diffraction powder pattern of ruthenium philic and show enhanced biocompatibility.6,7 Interestingly, metal).SeeDOI:10.1039/D0DT03528A the use of the carbon nanotube delivery system of platinum Thisjournalis©TheRoyalSocietyofChemistry2020 DaltonTrans. .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online View Journal Paper DaltonTransactions drugsledtohighreactivitytowardscancercells.Cisplatinwas MWCNTsarecharacterizedbyhightoxicity;therefore,thefirst encapsulated into functionalized single-walled carbon nano- step was to purify and functionalize MWCNTs (1) with –NH 2 tubes (SWCNTs) and this system showed more cytotoxicity groups (by rendering them more biocompatible and lesstoxic than free cisplatin towards prostate cancer cells (PC3).8 The in comparison to pristine CNTs22–24) and then to modify in vivo activity of cisplatin in capped multi-walled carbon MWCNTs-NH (2) with [InH][trans-RuCl (In) ] (4). The charac- 2 4 2 nanotubes (MWCNTs) with functionalized 1-octadecanethiol terization of the resulting products was carried out with the gold nanoparticles (f-GNP) towards MCF-7 breast cancer cells use of IR, Raman spectroscopy, thermal gravimetric, XRD was enhanced compared to uncapped cisplatin–MWCNTs. STEM-EDX, ESI-MS, ICP-MS, and XPS analyses. The goals of This suggested good properties of f-GNP–MWCNTs as drug thisstudywerethefollowing:(i)toprepareacarbonnanotube- depots.9 Similar effects, i.e. increased cytotoxicity and higher basednanofunctionalcompoundsystem,(ii)tocharacterizeit, efficacyof nanosystems were observed forcarboplatin,10 oxali- and (iii) to show its biological activity by simple cytotoxic platin,11 and some Pt(IV) complexes.12 However, the fact that studies, which could result in further specific research of this cisplatin has no effect on metastasis, shows low selectivity, system. and demonstrates resistance towards many kinds of cancer13 promptedustosearchfornewsolutions.Thesolutiontothese problems may be the choice of non-platinum complexes of Materials differentstructuresandbiological activitiesandtakingadvan- tages of the new opportunities that they offer. Ruthenium(III) Hydrated ruthenium(III) chloride, indazole, imidazole and sol- and(II)complexesrepresentnewmaterialsthatmaybeapplied vents were purchased from Sigma-Aldrich and used as sup- as drugs and seem to be the most attractive and promising plied.[InH][trans-RuCl 4 (In) 2 ](KP1019)waspreparedaccording prodrugs developed within the past few years. Some ruthe- to procedures found in the literature.25 The complex was nium(III) complexes have undergone initial clinical tests, i.e. characterized by elemental analysis, IR spectroscopy and [IndH][trans-RuCl (Ind) ], (Ind = Indazole) (KP1019) and its thermogravimetric analysis. MWNTs with purity >95% and 4 2 sodiumsaltNa[trans-RuCl (Ind) ](NKP-1339)and(ImH)[trans- typical lengths of 5–15 µm (outside diameter: 10–30 nm and 4 2 RuCl (dmso)(Im)], (Im = imidazole) (NAMI-A).14 Despite their corediameter:5–10nm)werepurchasedfromNanostructured 4 remarkable antitumor and antimetastatic activities, together & Amorphous Materials, Inc. (NanoAmor), Los Alamos, New withtheirlowtoxicitytowardnormalcells,ruthenium(III)com- Mexico,USA. plexes show a lack of stability in aqueous solutions.15 PurificationandfunctionalizationofMWCNTs Therefore, the therapy based on the delivery of a ruthenium anticancer material with the use of inert carriers to cancer The purification26 andoxidationofMWCNTs,the acylationof cells would be a solution to this problem as it can result in OX-MWCNTs and the amidation of MWCNT acyl chloride27,28 reducing side effects, providing stabilityand improving thera- werecarriedoutaccordingtoproceduresintheliterature. peutic efficacy.16,17 The use of carbon nanotubes to improve PurificationofMWCNTs drug delivery seems reasonable when considering a new material,e.g.ruthenium(III)and(II)complexes. The pristine MWCNTs (650 mg) were sonicated at 323 K for TheresultsoftheresearchonthesystemofRu(II)polypyri- 1hinanultra-sonicationbath(250W,38kHz)with150mLof dyl complexes with carbon nanotubes are very promising and a mixture (70:30) of hydrochloric acid (36%) and hydrogen encouraging with regard to studying targeted drug delivery peroxide (30%). Then the sample was refluxed for 5 h at systems. The functionalized nanosystem RuPOP@MWCNTs boiling temperature. After this treatment, the mixture was fil- (RuPOP; [Ru(phen) p-MOPIP](PF ) ·2H O, where phen = 1,10- tered using a 0.8 μm PTFE membrane and washed with de- 2 62 2 phenanthroline, p-MOPIP = 2-(4-methoxyphenyl)-imidazo ionizedwaterseveraltimesuntilthepHvalueofabout7.0was [4,5f])18showedconsiderablyhigheranticancerefficacyagainst reached.ThenMWCNTs(1)weredriedundervacuumat423K R-HepG2cells(livercancercells)andreducedtoxicsideeffects for2h. in relation to free RuPOP in cancer radiotherapy.19 Synthesis of MWCNTs-NH (2). In a 250 ml flask, the puri- 2 Ru@SWCNTs as a bimodal photothermal and two-photon fied MWCNTs (1) (600 mg) were suspended in a H SO (98%, 2 4 photodynamic therapy (PTT-TPPDT) system gives new possibi- 50 ml) and H O (30%, 50 ml) solution under vigorous stir- 2 2 lities forcancer treatment. The results of biological studies of ring. The suspension was sonicated for 30 min. The mixture Ru(II) polypyridyl complex functionalized single-walled carbon was stirred andheated at 353 Kfor 5 h. Aftercoolingto room nanotubes (Ru@SWCNTs) against HeLa cells showed higher temperature, the solution was slowly diluted with deionized phototherapy effects than those of free Ru(II) complexes and water and filtered using a 0.8 μm PTFE membrane. SWCNTs.20 OX-MWCNTs (1) were washed several times until the yellow In our work, we focused on the preparation of a new colouredsolutiondisappearedandapHvalueofabout7.0was [MWCNTs-NH +][RuCl (In) − ] (3) hybrid functioningas a drug reached. Then, the OX-MWCNTs were dried at 313 K. In the 3 4 2 deliverysystem for [InH][trans-RuCl (In) ] (KP1019) (4), one of acylation reaction, the OX-MWCNT sample (500 mg) was 4 2 themostpromisingrutheniumcomplexespresentingexcellent refluxed in 100 ml of SOCl and 10 ml of DMF at 353 K for 2 anticancer activity.21 It is commonly known that pristine 50 h. The black precipitate was washed with anhydrous ethyl DaltonTrans. Thisjournalis©TheRoyalSocietyofChemistry2020 .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online DaltonTransactions Paper alcohol and filtered using a 0.8 μm PTFE membrane. 300 mg sive X-rayspectroscopy (EDS) (EDAX,RTEM model SN9577, 134 ofMWCNT-COCland600mgofNaN wereaddedto250mlof eV) was performed and the spectra were recorded in the desig- 3 DMF in a 500 ml flask. The reaction was carried out at 295 K nated areas of elements. The measurements were made in the for 30 h under vigorous stirring. Afterwards, the temperature TEM mode (bright-field) and STEM mode (HAADF and EDX was increased up to 373 K and the suspension was mixed for detectors). The samples were prepared as follows: A few milli- 20 h. The precipitate was filtered with the use of a 0.8 μm grams of the sample was dispersed in deionized water under PTFE membrane and mixed with HCl (30%) in a 1:1 ratio. ultrasoundfor5s.Then,adrop(5mL)wasplacedonacarbon- The suspensionwas sonicated for 1 h.MWCNTs-NH (2) were coatedcoppermeshwithholes(LaceytypeCu400mesh,Plano) 2 filtered using a 0.8 μm PTFE membrane, washed with de- andthenthesolventwasevaporatedatroomtemperature. ionizedwater,anddriedat313K. XPSanalysis Synthesis of MWCNTs-Ru (3) ([MWCNTs- NH +][RuCl (In) − ]). 30 mg of MWCNTs-NH (2) were soni- The XPS spectra were recorded with a PHI 5000 VersaProbe™ 3 4 2 2 cated in a 25 ml solution of HCl (pH ∼ 3) for 30 min. The scanning ESCA Microprobe using monochromatic Al-Kα radi- mixture was stirred in a flask at 295 K for about 24 h. After ation (hν = 1486.6 eV) from an X-ray source operating at that, the solution was diluted until the pH value reached ca. 100 µm spot size, 25 W powerand 15 kVacceleration voltage. 5.0. [InH][trans-RuCl (In) ] (4) (60 mg) was added to the sus- The high-resolution XPS spectra were collected with a hemi- 4 2 pension and sonicated for 30 min. The mixturewas stirred in spherical analyzer at a pass energy of 23.5 eV, an energy step a flask at 295 K for about 24 h. Next, the suspension was fil- size of 0.1 eV and a photoelectron take-off angle of 45° with teredusinga0.8μmPTFEmembrane,washedwithdeionized respecttothesurface.Themeasuredareawasdefinedasa250 waterandthenvacuumdried. ×250μm.TheShirleybackgroundsubtractionandpeakfitting Synthesis of [InH][trans-RuCl (In) ] (4). The purification of with Gaussian–Lorentzian-shaped profiles were performed for 4 2 RuCl × H O (method II) and the synthesis of KP1019 thehigh-resolutionXPSspectraanalysis. 3 2 [InH][trans-RuCl (In) ](whereInH=indazole)werecarriedout 4 2 accordingtoapreviouslyreportedprocedure.25 ICP-MS Analysis for C H N Cl Ru. Calculated (%): C, 42.16; H, Thequantificationofrutheniumionsinaqueoussolutionwas 21 19 6 4 3.20; N, 14.05. Found (%):C, 42.05; H, 3.15; N, 14.21; IR KBr performed on an inductively coupled plasma mass spectro- pellet, cm −1 and FIR (CsI pellet, cm −1): 3350s, 2925w, 1627s, meter with a quadrupole analyzer, model 7500 CX Agilent 1509m, 1439w, 1367m, 1354vs, 1290w, 1280w, 1154w, 1124w Technologies. Sample preparation29 of [MWCNTs- 1243s, 1080s, 1001m, 960m, 862w, 838w, 782vw, 750m, 734vs, NH +][RuCl (In) − ](3)andquantitativedeterminationofruthe- 3 4 2 660m,429m,348s,331sh,279m,189wand153wcm −1. nium using ICP-MS20,30 were carried out based on the litera- ture data. A sample of [MWCNTs-NH +][RuCl (In) − ] (3) was 3 4 2 digested in aqua regia at boiling temperature. After digestion, Experimental methods thesamplewascooledtoroomtemperatureandfilteredusing a0.8 μmPTFE membrane.The liquid fractionwasevaporated IRspectroscopy to 1/3 of the sample volume and diluted to 7% HCl and ana- IR spectra were recorded in transmission on a Vertex 70 V, lyzed.Quantificationwascarriedoutbyexternalfive-pointcali- Bruker Optik. The spectra were recorded from 4000 to bration. Ruthenium ICP standard RuCl in 7% HCl (1000 mg 600 cm −1 (MIR; KBr pellet) and 700–30 cm −1 (FIR; CsI pellet) l −1RuCertipur®,Merck,Germany)was 3 used. atascanrateof0.2cms −1,thenumberofscansis50,witha resolutionof4cm −1. ESI-MS ESI-MS spectra were obtained using a Shimadzu LC-MS 8030 Ramanspectroscopy fittedwithanelectrospraysourceandatandemmassanalyzerof The Raman spectra were measured using a Raman Senterra, quadrupole configuration. The capillary voltage was at 3.5 eV. Bruker Optik microspectrophotometer equipped with a Theelectrospraysolventwas95:5methanol:formicacid(0.1%). 532 nm laser at room temperature. The solid samples were Theotheroperatingparameterswereoptimizedasfollows:nebu- mounted on a glass slide. The maximum laser power on the lizergasflow,2.0Lmin −1;dryinggasflow,15Lmin −1;desolva- sampleswasabout5mWwithanexposuretimeof4×20s. tionline(DL)temperature,523K;heatblocktemperature,673K in ESI source. The spectra were acquired in negative ion mode Elementalanalysis over the range m/z 100–1000. The sample of the [MWCNTs- The elemental analysis of the obtained complex was per- NH +][RuCl (In) − ](3)(10mg)wassuspendedinmethanolwith 3 4 2 formedonaVarioELofElementarAnalysensystemeGmbH. 1%ofNaClandwassonicatedfor1hat323Kandfilteredonto 0.8μmPTFEmembrane.Thefiltratewasanalyzed. TEMandSTEM-EDX Thermogravimetricanalysis(TGA) The morphological characterization was performed using a transmissionelectronmicroscope(TEM)(FEI,G2F20X-Twin200 Thermal studies (TGA/DTA) were performed on an SDT 2960 kV,FEG).Toidentifythechemicalelementsused,energy-disper- TAanalyzerfor1–6(N ;heatingrate–10°min −1,under10ml 2 Thisjournalis©TheRoyalSocietyofChemistry2020 DaltonTrans. .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online Paper DaltonTransactions min −1nitrogenflowrate,heatingrangeupto1273Kwithpow- deredsamplesinacorundumcrucible;samplemass4–9mg. PXRDanalysis Powder X-ray diffraction data were collected with a Philips X′ Pert Pro diffractometer in 30–80° 2Θ ranges using Cu Kα irradiationandsamplespinning,stepsize0.1°,programmable receivingslits0.5°, measuringtime11sperpoint. Cellsandcytotoxicityassay HeLa (human cervix carcinoma), K562 (chronic myelogenous leukemia) and A549 (human lung carcinoma) cells were cul- tured in RPMI 1640 medium supplemented with antibiotics and 10% fetal calf serum (HeLa, K562) in a 5% CO –95% air 2 atmosphere.7×103cellswereseededoneachwellofa96-well plate(Nunc).24hlaterthecellswereexposedtothetestcom- poundsforadditional48hours.Thestocksolutionsofthetest Fig.1 IR spectra of MWCNTs (1), MWCNTs-NH (2), MWCNTs-Ru (3), 2 compounds were freshly prepared in DMSO (dimethyl- and[InH][RuCl (In) ](4). 4 2 sulfoxide). The final concentrations of compounds (3) and (4) testedinthecellcultureswere2×10 −1mM,1×10 −1mM,5× 10 −2mM,1×10 −2mM,1×10 −3mM,1×10 −4mMandcalcu- latedfor[RuCl 4 (Ind) 2 ] − .TheconcentrationofDMSOinthecell thefunctionalizationofthe–NH 2 groupsand[RuCl 4 (In) 2 − ]on culture medium was 1%. A cis-Pt stock solution of 5 mM was thenanotubes’surface. prepared in 0.9% NaCl. The final concentrations of cis-Pt in The bands at 3420 and 1637 cm −1 of (1) showed the pres- the cell culture were 5 × 10 −2 mM, 1 × 10 −2 mM, 2.5 × 10 −3 ence of –OH groups on the nanotube surface. It suggeststhat mM,1×10 −3mM,2.5×10 −4mMand1×10 −4mM.Thecyto- the nanotube surface was modified by OH groups in a purifi- toxicity study was carried out for MWCNTs (1) and MWCNTs- cation process with HCl/H O .26 In the region at 2 2 NH 2 (2)at10mgml −1.ThevaluesofIC 50 (theconcentrationof 3780–3270cm −1,inadditiontothebroadbandsofO–H,N–H thetestcompoundrequiredtoreducethecellsurvivalfraction bond vibrations can appear as well. Therefore, in the case of to50%ofthecontrol)werecalculatedfromthedose–response (2), the broad band at 3450 cm −1 was assigned to the N–H curves and used as a measure of cellular sensitivity to a given vibrations. This band is more intense and shifted to the left treatment. side of the spectrum with regard to (1). Moreover, the broad The cytotoxicity of all compounds was determined by the band with two maximums at 1611 and 1572 cm −1 and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bandat676cm −1of(2)wereassignedtoδ(N–H).28Thestretch- bromide; Sigma, St. Louis, MO] assay, as described.31 Briefly, ingvibrationscorrespondingtoC–Nbondswereexhibitedasa after48hofincubationwithdrugs,thecellsweretreatedwith series of bands in the region 950–1200 cm −1 for (2). After the MTT reagent and the incubation was continued for 2 h. MWNTs-NH (2)surfaceprotonationatthepH∼3,itsmodifi- 2 MTT–formazan crystals were dissolved in 20% SDS and 50% cation with [InH][trans-RuCl (In) ], KP1019 (4) at pH ∼ 5 was 4 2 DMFatpH4.7andabsorbancewasreadat570and650nmon conducted.Theprotonatedsurfaceof(2)wasmeanttoenable an ELISA plate reader (FLUOstar Omega). As a control (100% ionicbondformationbetweentherutheniumcomplexandthe viability), we used the cells grown in the presence of only nanotubesurfaceandtocreatea[MWCNTs-NH +][RuCl (In) − ] 3 4 2 vehicle(1%DMSO). systemasaconsequence.Thecharacteristicpeaksoftheruthe- niumcomplex(4)andMWCNTs-NH (2)wereobservedonthe 2 IRspectrumofMWCNTs-Ru(3)(Fig.1).Thevibrationpeaksat Results and discussion 1627 (ν(CvN)), 1354 and 1367 (ν(C–N)), 1243 (ν(N–H)) cm −1 wereinthesameplacefor(4)and(3)andconfirmedthepres- According to the literature,32 pristine MWCNTs are character- ence of coordinated indazole groups with the ruthenium ion. izedbyhightoxicity,sothefirststepwastopurifythemwitha However, the IR spectrum of MWCNTs-Ru (3) was obviously mixture(70:30)ofhydrochloricacid(36%)andhydrogenper- different from that of MWCNTs-NH (2) and [InH][trans- 2 oxide (30%) in order to remove impurities such as metal par- RuCl (In) ] (4). The band of ν(N–H) with the maximum at 4 2 ticlesandresidualcatalysts.26Inthenextstep,thesynthesisof 3470 cm −1 appeared in the range of 3500–3400 cm −1 for (3). MWNTs-NH (2) was carried out on the basis of the Curtius However, it was significantly smaller than the broad band of 2 rearrangementofcarboxylic acidazide.28 Acomparison ofthe (2) and was shifted to the left side of the spectrum. The infraredspectraofpurifiedMWCNTs(1),MWNTs–NH (2)and stretchingbandofν(N–H)ofcomplex(4)at3350cm −1didnot 2 MWCNTs-Ru(3)(Fig.1)revealeddifferences,whichconfirmed appear on the MWCNTs-Ru (3) spectrum; however, the broad DaltonTrans. Thisjournalis©TheRoyalSocietyofChemistry2020 .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online DaltonTransactions Paper band occurred at 3250 cm −1, which can be ascribed to the –NH +vibrationsaswell.Moreover,thedeformationvibrations 3 of –NH + exist in the regions of 1560–1625 cm −1 (m, asym- 3 metric)and1500–1550cm −1(m, symmetric).33 Inaddition,in these regions, the peaks occurring in the spectrum of (3) are differentfromthoseof(2)and(4).Thepeaksarecharacterized by lower intensity and bigger width than the peaks of the (4) spectrum; several peaks of very low intensityappeared as new intheregionof1560–1510cm −1aswell.Similarchangeswere observedintheregionof1510–1400cm −1,wheretheC–Hand C–N vibration bands of the pyrazole ring are present. It Fig.2 RamanspectraofMWCNTs(1),MWCNTs-NH (2),MWCNTs-Ru 2 suggests that both the nanotube surface and interactions in (3),and[InH][RuCl (In) ](4). 4 2 the ruthenium complex were changed. Probably the observed changesaretheeffectofprotonatingMWCNTs-NH andcreat- 2 ing ionic connections of [MWCNTs-NH +] with the ruthenium sensitivity to vibrations from functional groups that occur in 3 − complex as an anion [RuCl (In) ]. In addition to the for- carbonstructures.Forthisreason,itisnotusedtoidentifythe 4 2 mation of the N–H stretching vibrations band (a relative binding of the complexes to the MWCNTs.4,5 The spectra of increase in the band in the 3100–3600 cm −1 region), the rela- samples MWCNTs, MWCNTs-NH and MWCNTs-Ru (3) 2 tive increase in the absorption band near 1440 cm −1 can (Fig. 2) show three dominant bands at 1335, 1567 and confirmthetertiaryammoniacationpresenceaftertheadsorp- 2675cm −1,characteristicofcarbonmaterials.36 tion of the studied ruthenium complex on the carbon nano- [InH][trans-RuCl (In) ] spectrum shows the characteristic 4 2 tubesurface.Accordingtotheprotonatedindazoleresearch,34 bands (1072, 1146, 1187, 1349, 1378, 1430, 1469, 1590, the observation of the ν(N–N) peak at about 1080 cm −1 is 3066cm −1)forthecomplex[InH][trans-RuCl (In) ].37However, 4 2 important for the determination of protonation. In this case, there are no bands corresponding to the presence of the theintensityofthepeakat1080cm −1decreasedandtheCvN complexintheMWCNTs-Ru(3)sample.Onlyonemaximumis stretching band of the indazole disappeared at 1611 cm −1 on visible at 1605 cm −1 (band D′) but it results from structural the (3) spectrum according to (4). It may suggest the removal defects called Stone–Wales defects.38 Probably, the lackof the of InH+ from [InH][trans-RuCl (In) ] and the formation of apparent characteristic bands of the complex is caused by the 4 2 [MWCNTs-NH +][RuCl (In) − ]. It additionally confirmed low content of the complex concerning the amounts of nano- 3 4 2 changesintheregionlowerthan750cm −1wheretheC–H,N– tubesandbythefactthattheyoverlapeachother,especiallyin H and Ru–N bond vibrations are visible. In the [MWCNTs- the1300to1600cm −1region. NH +][RuCl (In) − ] (3) spectrum, the broadened band at ThelocationofthebandsandtherelativeintensityofI /I 3 4 2 G D 660 cm −1 (Ru–N) for the ruthenium(III) complex (4), where andI 2D /I G (Table1)clearlyindicatethatthebasisofthetested several sharp peaks (659, 630, and 618 cm −1) appeared, was carbonmaterialsismultiwallcarbonnanotubes. observed.However, inthe [MWCNTs-NH ]spectrum,onlyone They are characterized by a relatively small number of 2 broadpeakat676cm −1waspresent. defects and amorphous carbon, as indicated by the I /I G D Raman spectroscopy is a useful technique for identifying values above one. Among the tested materials, the MWCNTs- carbonmaterials andprovidesinformationaboutitsstructure NH sample has the most ordered structure, i.e. the highest 2 anddefects.Withitshelp,youcanidentifythetypeofcarbon value of the I /I parameter. The increase in the I /I para- G D G D nanotubes, determine the number of walls and estimate the meter is due to the thermal treatment that promotes the numberofdefectsandthecontentofamorphousstructures.35 removaloftheamorphouscarbonandtheprogressive,athigh CarbonnanotubeshavethreemainmodesintheRamanspec- temperatures, ordering of layers in MWCNTs. Moreover, the trum. The following bands are distinguished: D with a increaseintheD-bandintensityfor(3)couldbeinterpretedas maximum at about 1350 cm −1, G at 1580 cm −1 and 2D at a decrease in the structural order in MWCNTs. Probably, this 2700 cm −1. Peak G is associated with the ordered graphite disorder stems from the inhomogeneous decoration of − structure, and peaks D and 2D are associated with defects. In the nanotube surface by [RuCl (In) ] anions. However, the 4 2 addition,the2Dbandprovidesinformationonthenumberof determined I /I parameters indicate that the applied nano- 2D G wallsin MWCNTs.The orderofcarbon materialsismeasured tube modifications did not affect the number of walls in bytheintensityoftheGpeakrelativetotheDpeak.Itisdeter- MWCNTs.Theaveragevalueforallthesamplesisintherange minedbytheI /I parameter,whichistheratiooftheGtoD of0.53±0.03. G D band intensity. Another parameter is I /I which informs The presence of complex ruthenium on the surface of 2D G about the relative number of walls in MWCNTs. The greater MWCNTs-NH (2) was confirmed by the TEM and STEM-EDX 2 the intensity of this parameter, the more walls in the nano- analysis. In Fig. 3, MWCNTs-NH before and after the modifi- 2 tubes. Raman spectroscopy is one of the most sensitive tech- cation with complex Ru(III) (4) were presented. The lack of niques for characterizing the structure and defects in the defects on the surface of (1) confirmed the results of Raman layers of carbon nanotubes, while it does not have adequate spectroscopy. Moreover, it can be observed that the sample of Thisjournalis©TheRoyalSocietyofChemistry2020 DaltonTrans. .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online Paper DaltonTransactions Table1 ParametersdeterminedfromRamanspectra G D 2D Sample Position Intensity Position Intensity Position Intensity I /I I /I G D 2D G (1) 1567 12940 1337 11304 2672 7097 1.14 0.55 (2) 1568 12941 1338 9819 2674 6805 1.32 0.53 (3) 1572 12939 1340 11827 2680 6494 1.09 0.50 Fig.3 TEMimagesofMWCNTs-NH (2)andMWCNTs-Ru(3)withEDXspectra. 2 theRu(III)complexislocatedontheoutsidesurfaceofthemul- ligand. The characteristic peaks at 198.16 eV for Cl 2p 3/2 indi- tiwalled carbon nanotubes with a spherical shape as the dark cated the existence of Cl elements, which are assigned to Ru– areas where ruthenium dominates. The results of the Cl. Unfortunately, the C 1s lines can interfere with the Ru 3d STEM-EDX analysis are an additional verification of the nano- lines, and therefore, quantification is performed using the systemcreationcomposedofthe[MWCNTs-NH +][RuCl (In) − ]. high-resolution spectra and peak models to differentiate 3 4 2 An XPS analysis of [MWCNTs-NH +][RuCl (In) − ] (3) pro- between both chemical and elemental states. After deconvolu- 3 4 2 vided information about the surface functionalization of tion, the Ru 3d peaks are resolved as the doublet 3d and 5/2 MWCNTs and confirmed the presence of the ruthenium 3d at282.01and286.18eV,respectively,withadoubletsep- 3/2 complex. The XPS characteristic peaks of ruthenium(III) 3p 1/2 arationvalueof4.17eV.TheRu3d 5/2 signalcanbeassignedto and 3p 3/2 appeared at the binding energies of 485.27 eV and Ru in [RuCl 4 (In) 2−]. The C 1s signals appeared at 284.61 and 462.97eV,respectively(Fig.4and5).Thevalueof462.97eVis 285.38 eV, which are ascribed to the sp2- and sp3-hybridized an intermediate characteristic value of Ru(III) and Ru(IV).39 carbons attributed to the graphitic structure.40 Moreover, the Probably, it is caused by the presence of the ligands in the signalC1sat286.08eVcanbeassignedto–C–NH aswellas 2 coordinationsphereofruthenium(III)andtheionicinteraction –C–OH.Thepresenceofcomplexrutheniumonthesurfaceof − between [RuCl (In) ] and the protonated surface of MWCNTs-NH (2) was confirmed with TEM and STEM-EDX 4 2 2 [MWCNTs-NH +].Thebindingenergyofnitrogenwasfoundto analyses.41 3 be 399.55 eV and 400.66 eV, which is a characteristic of the In addition, the presence of ruthenium ion on the nano- –NH group and the pyrazole nitrogen ring of the indazol tubesurfacewasconfirmedbytheresultofelectrosprayioniza- 2 DaltonTrans. Thisjournalis©TheRoyalSocietyofChemistry2020 .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online DaltonTransactions Paper Fig.6 Thermogravimetric analysis of MWCNTs (1), MWCNTs-NH (2), 2 MWCNTs-Ru(3),and[InH][RuCl (In) ](4). 4 2 The thermal decomposition of KP1019 involved five endothermicsteps.Thecomplexstabilityuntil444Kindicates thelackofmoistureandthesolventusedinthesynthesis,and it confirms the complex purity. In the two first steps (443–563 K), the decomposition process of the KP1019 with 20%weightlosswasconnectedwiththeindazolegroupdegra- dation. The next steps were concerned with the loss of Cl 2 Fig.4 XPSspectraofC1s,N1s,andRu3pforMWCNTs-Ru(3). molecules (11%). The last two steps involved the degradation of indazole and two chloride ligands (30%). The residue decomposition process involving ruthenium metal and amor- phous carbon resulted from the indazole molecule degra- dation.Moreover,itwasconfirmedbytheXRDanalysis(Fig.7) ofpeaksat38,42,44,58,69,78,82,84,and 92°whichcorres- pondwiththestandardpeaksofrutheniummetal(ESI†). MWCNTs (1) and NH -MWCNTs (2) were characterized by 2 goodthermalstability.Afteraninitialweightloss(thepyrolytic evolutionofhydroxyland/orwater),26asignificantweightloss of (1) (6.4%) started at 928 K, proving the compact structure and good thermal stability. The thermal decomposition of (2) startedat443Kandwasconnectedwiththedecompositionof the amino groups.42 The thermal stability of MWCNTs-Ru (3) is shown as lower than that of NH -MWCNTs (2) and 2 [InH][trans-RuCl (In) ] (4). After the loss of moisture, the 4 2 decomposition process followed in three steps: the first step involving weight loss of approximately 3% was related to the Fig.5 Wide-scanXPSspectraofMWCNTs-Ru(3). degradation of NH + groups from the nanotube surface 3 (393–423 K); the second (423–723 K) and third (723–1223 K) stages of degradation, with 21% and 22.6% weight loss, tionmassspectrometry(ESI-MS).ESI-MSanalysisdetectedthe respectively,wereattributedtothedecompositionoftheruthe- [RuCl (In) ] − ion(m/z479.85,Fig.S1intheESI†). nium indazole and chloride ligands in both the steps. The 4 2 Moreover, inductively coupled plasma mass spectrometry remaining residual mass contains mainly MWCNTs–NH and 2 (ICP-MS)analysiswasusedtoquantitativelydeterminetheRu ruthenium metal, which was confirmed in the XRD analysis (III) ions in MWCNTs-Ru (3). The amount of ruthenium ion (Fig. 7) by the presence of the ruthenium peaks and the [RuCl In − ]onMWCNTs-Ru(3)was123.10µgml −1(18.9%). characteristic features of MWCNT carbon materials in the 2Θ 4 2 Thermogravimetric analyses were conducted on the range20–30,40–45,52–55,and78–80°. samples of MWCNTs (1) purified, MWCNTs-NH (2), Cytotoxicityanalyseswereperformedtoevaluatethepoten- 2 MWCNTs-Ru(3),and[InH][RuCl (In) ](4)(Fig.6). tialtherapeuticapplicationofthedrugdeliverysystems.Inthe 4 2 Thisjournalis©TheRoyalSocietyofChemistry2020 DaltonTrans. .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online Paper DaltonTransactions Fig.8 ThesurvivalrateofHeLacellsafter48hoursofincubationwith (3),(4)andcis-Pt. Fig.7 PXRD diffractograms of MWCNTs (1), MWCNTs-NH (2), 2 MWCNTs-Ru(3),[InH][RuCl (In) ](4),andrutheniumpatternlines(black 4 2 lines)withinterplanarspacingd(hkl). screening studies, MWCNTs (1), MWCNTs-NH (2), MWCNTs- 2 Ru (3), and[InH][RuCl (In) ](4) were testedfor theircytotoxic 4 2 properties in K562, A549, HeLa cells. Cells treated with 1% DMSO (vehicle) served as the control (100% viability in the MTT assay). Based on the viability of cells measured at different concentrations of test compounds (3) and (4), the IC valueswerecalculatedandarepresentedinTable2. 50 TheviabilitiesofK562,A549,andHeLacellswere72.0,71.6 and 98.8%, respectively, for MWCNTs (1) and 90.5, 99.4, and 100.1% for MWCNTs-NH (2) at 10 mg ml −1. The cytotoxicity 2 Fig.9 ThesurvivalrateofK562cellsafter48hoursofincubationwith studywasnotcarried outfor MWCNTs(1)and MWCNTs-NH 2 (3),(4)andcis-Pt. (2) in the full concentration range because these compounds did not show significant toxicity at high concentrations. However, there is a noticeable increase in cell viability for MWCNTs-NH (2) with reference to MWCNTs (1). This result 2 suggested no toxicity of the multiwalled carbon nanotubes with the –NH functional groups. The survival rates of HeLa, 2 A549, and K562 cells after 48 hours of incubation with com- pound(3)and(4)areshowninFig.8–10.Thesecelllineswere chosenarbitrarilyandincludedcancer(A549,K562andHeLa). Cancercells originated from different tissues,i.e. lung (A549), Table2 TheIC (mean±SD)valuescalculatedafter48hincubation 50 ofcellswithtestcompounds.Themeans±SDareshown IC [μM] 50 Compound HeLa K562 A549 MWCNTs-Ru(3) 14±4.6 15±4.9 25±9.4 [InH][RuCl (In) ](4) 40±8.2 44±6.7 70±8.6 Fig.10 ThesurvivalrateofA549cellsafter48hoursofincubationwith 4 2 cis-Pt 20±6 40±7 30±6.5 (3),(4)andcis-Pt. DaltonTrans. Thisjournalis©TheRoyalSocietyofChemistry2020 .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online DaltonTransactions Paper cervix (HeLa) and blood (K562) and represent solid and blood and 400.66 for N 1s, at 198.16 for Cl 2p , and 284.61 and 3/2 tumors. In addition, these cells display different growth 285.38 eV for C 1s, indicating the existence of the elements characteristics, i.e. adherent (A549, HeLa) and suspension Ru,N,ClandCinMWCNTs-Ru. (K562)cells. Cytotoxicityanalyseswereperformedtoevaluatethepoten- The creation of the hybrid of MWCNTs-NH (3) with tial therapeutic application of the drug delivery systems and 2 [InH][RuCl (In) ] (4) was aimed at increasing the stabilityand theyshowedthehighertoxicityofthenanosystem(thanthatof 4 2 efficacy of [InH][RuCl (In) ] (4) activity. Therefore, we have therutheniumcomplex)towardsthetestedcancercells. 4 2 compared the effects of MWCNTs-Ru (3) towards a series of cancer cells using the MTT assay with the free [InH][RuCl (In) ] (4). MWCNTs-Ru (3) were effective against Conflicts of interest 4 2 cancer cells in a dose-dependent manner (Fig. 8–10). Therearenoconflictstodeclare. Moreover, thetoxicityof(3)againstallthethree celllineswas higher than that of (4) and cis-Pt (Table2). The results of this study suggested that the functionalized MWCNT nanosystem Acknowledgements effectivelyenhancesthe anticanceractivityof MWCNTs-Ru (3) comparedtothefreerutheniumcomplex[InH][RuCl (In) ](4). 4 2 The authors wish to acknowledge the Polish National Science Centreforfinancialsupportgrantno.2011/03/D/NZ7/02283. Conclusions References Inthisstudy,MWCNTs-NH havebeensuccessfullymodifiedby 2 the complex [InH][RuCl (In) ] KP1019 (4) and the conjugation 1 M. I. Sajid, U. Jamshaid, T. Jamshaid, N. Zafar, H. Fessi 4 2 of [MWCNTs-NH +][InRuCl − ] (3) has been created. The result andA.Elaissari,Int.J.Pharm.,2016,501,278–299. 4 4 of IR analysis showed the presence of the NH + groups on the 2 V.S.Thakare,D.A.Prendergast,G.PastorinandS.Jain,in 4 surfaceofMWCNTs-Ru(3)bytheoccurrenceofthebroadband Targeted Drug Delivery: Concepts and Design, ed. at 3250 cm −1 (ν(N–H)) and the relative increase of the absorp- P. V. Devarajan and S. Jain, Springer International tionbandat1440cm −1.Moreover,thedecreasedintensityν(N– Publishing, Cham, 2015, pp. 615–645. DOI: 10.1007/978-3- N)ofthepeakat1086cm −1andthedisappearanceoftheCvN 319-11355-5_19.. stretchingbandoftheindazoleat1611cm −1inthespectrumof 3 L.Kelland,Nat.Rev.Cancer,2007,7,573–584. (3)withregard to(4)suggestthelackofInH+from[InH][trans- 4 S.P.Fricker,DaltonTrans.,2007,4903–4917. RuCl (In) ] on the surface of (3). The broad band at 660 cm −1 5 R. Agarwal and S. B. Kaye, Nat. Rev. Cancer, 2003, 3, 502– 4 2 (Ru–N)of(3)confirmedthechange inthe coordinationsphere 516. ofruthenium(III)complex(4). 6 M. Bottini, N. Rosato and N. Bottini, Biomacromolecules, TheRamanspectroscopyshowedthatthefunctionalization 2011,12,3381–3393. of MWCNTs did not cause damage to the skeleton of 7 A. Bianco, K. Kostarelos and M. Prato, Chem. Commun., MWCNTs, which was confirmed by the results of the STEM 2011,47,10182–10188. analysis. The modification of the MWCNT sample led to an 8 C. Tripisciano, K. Kraemer, A. Taylor and E. Borowiak- increase in the order of the structure of the MWCNTs-NH Palen,Chem.Phys.Lett.,2009,478,200–205. 2 sample,asitwascausedbytheremovalofamorphouscarbon. 9 J. Li, S. Q. Yap, S. L. Yoong, T. R. Nayak, G. W. Chandra, These changes led to an increase in the I /I parameter. W. H. Ang, T. Panczyk, S. Ramaprabhu, S. K. Vashist, G D Probably, the increase in the D-band intensity for [MWCNTs- F.S.Sheu,A.TanandG.Pastorin,Carbon,2019,50,1625– NH +][InRuCl − ](3)isconnectedwiththeinhomogeneousdec- 1634. 4 4 − orationofthenanotubesurfaceby[RuCl (In) ]anions. 10 S. Hampel, D. Kunze, D. Haase, K. Krämer, 4 2 Moreover, the results of the STEM-EDX analysis provided M. Rauschenbach, M. Ritschel, A. Leonhardt, J. Thomas, strong evidence of the successful functionalization of multi- S. Oswald, V. Hoffmann and B. Büchner, Nanomedicine, walled carbon nanotubes with complex Ru by the presence of 2008,3,175–182. a dark area aggregation on the surface of MWCNTs and Ru 11 L. Wu, C. Man, H. Wang, X. Lu, Q. Ma, Y. Cai and W. Ma, signals in this area. The existence of ruthenium was alsoveri- Pharm.Res.,2013,30,412–423. fiedintheresidueofthermaldecompositionduringthePXRD 12 R. P. Feazell, N. Nakayama-Ratchford, H. Dai and study. Thermal analysis showed the high thermal stability of S.J.Lippard,J.Am.Chem.Soc.,2007,129,8438–8439. the nanotubes, which is an important feature for a drug 13 N. J. Wheate, S. Walker, G. E. Craig and R. Oun, Dalton carrier.Thepresenceof[RuCl (In) ] − onthenanotubesurface Trans.,2010,39,8113–8127. 4 2 wasconfirmedbytheresultofESI-MSanalysis. 14 E.AlessioandL.Messori,Molecules,2019,24,1995–2015. The XPS characteristic peaks of ruthenium(III) 3p 1/2 and 15 C. G. Hartinger, S. Zorbas-Seifried, M. A. Jakupec, 3p appearedatthebindingenergiesof485.27eVand462.97 B. Kynast, H. Zorbas and B. K. Keppler, J. Inorg. Biochem., 3/2 eV, respectively. The characteristic peaks appeared at 395.55 2006,100,891–904. Thisjournalis©TheRoyalSocietyofChemistry2020 DaltonTrans. .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online Paper DaltonTransactions 16 K.H.Son,J.H.HongandJ.W.Lee,Int.J.Nanomed.,2016, 30 T. Hopp, M. Fischer-Gödde and T. Kleine, J. Anal. At. 11,5163–5185. Spectrom.,2016,31,1515–1526. 17 N. K. Mehra, K. Jain and N. K. Jain, Drug Discovery Today, 31 M. Maszewska, J. Leclaire, M. Cieslak, B. Nawrot, 2015,20,750–759. A. Okruszek, A. M. Caminade and J. P. Majoral, 18 T. Chen, Y. Liu, W.-J. Zheng, J. Liu and Y.-S. Wong, Inorg. Oligonucleotides,2003,13,193–205. Chem.,2010,49,6366–6368. 32 S. Vardharajula, S. Z. Ali, P. M. Tiwari, E. Eroğlu, K. Vig, 19 N.Wang, Y.Feng,L. Zeng,Z. ZhaoandT. Chen,ACS Appl. V. A. Dennis and S. R. Singh, Int. J. Nanomed., 2012, 7, Mater.Interfaces,2015,7,14933–14945. 5361–5374. 20 P. Zhang, H. Huang, J. Huang, H. Chen, J. Wang, K. Qiu, 33 Biointerface Characterization by Advanced IR Spectroscopy, D. Zhao, L. Ji and H. Chao, ACS Appl. Mater. Interfaces, ed. C. M. Pradier and Y. J. Chabal, Elsevier, Amsterdam, 2015,7,23278–23290. 2011,ch.iv,pp.83–113. 21 R. Trondl, P. Heffeter, C. R. Kowol, M. A. Jakupec, 34 J. Oomens, G. Meijer and G. von Helden, Int. J. Mass W. Berger and B. K. Keppler, Chem. Sci., 2014, 5, 2925– Spectrom.,2006,249–250,199–205. 2932. 35 M. S. Dresselhaus, G. Dresselhaus, R. Saito and A. Jorio, 22 M.FoldvariandM.Bagonluri,Nanomedicine,2008,4,183– Phys.Rep.,2005,409,47–99. 200. 36 L. Stobinski, B. Lesiak, L. Kövér, J. Tóth, S. Biniak, 23 A.Figarol,J.Pourchez,D.Boudard,V.Forest,J.-M.Tulliani, G. Trykowski and J. Judek, J. Alloys Compd., 2010, 501, 77– J.-P. Lecompte, M. Cottier, D. Bernache-Assollant and 84. P.Grosseau,J.Nanopart.Res.,2014,16,2507. 37 A. Vergara, G. D’Errico, D. Montesarchio, G. Mangiapia, 24 S. Jain, V. S. Thakare, M. Das, C. Godugu, A. K. Jain, L. Paduano and A. Merlino, Inorg. Chem., 2013, 52, 4157– R. Mathur, K. Chuttani and A. K. Mishra, Chem. Res. 4159. Toxicol.,2011,24,2028–2039. 38 S. A. Curran, J. A. Talla, D. Zhang and D. L. Carroll, 25 K. G. Lipponer, E. Vogel and B. K. Keppler, Met.-Based J.Mater.Res.,2005,20,3368–3373. Drugs,1996,3,243–260. 39 D.J.Morgan,Surf.InterfaceAnal.,2015,47,1072–1079. 26 R.Das, M. E. Ali, S. Bee Abd Hamid, M. S. M. Annuarand 40 V.Datsyuk,M.Kalyva,K.Papagelis,J.Parthenios,D.Tasis, S.Ramakrishna,J.Nanomater.,2014,2014,945172. A. Siokou, I. Kallitsis and C. Galiotis, Carbon, 2008, 46, 27 N. Karousis, N. Tagmatarchis and D. Tasis, Chem. Rev., 833–840. 2010,110,5366–5397. 41 F. G. Pacheco, A. A. C. Cotta, H. F. Gorgulho, A. P. Santos, 28 A. Gromov, S. Dittmer, J. Svensson, O. A. Nerushev, W. A. A. Macedo and C. A. Furtado, Appl. Surf. Sci., 2015, S. A. Perez-García, L. Licea-Jiménez, R. Rychwalski and 357,1015–1023. E.E.B.Campbell,J.Mater.Chem.,2005,15,3334–3339. 42 P. Hou, C. Liu, Y. Tong, S. Xu, M. Liu and H. Cheng, 29 M.Balcerzak,Crit.Rev.Anal.Chem.,2002,32,181–226. J.Mater.Res.,2011,16,2526–2529. DaltonTrans. Thisjournalis©TheRoyalSocietyofChemistry2020 .MP 93:53:4 0202/21/11 no ytisrevinU etatS ocsicnarF naS yb dedaolnwoD .0202 rebotcO 22 no dehsilbuP View Article Online