The synthesis and photophysical and biological

investigation of Ru(II)-polypyridyl stabilized watersoluble,

luminescent gold nanoparticles (AuNPs) are described.

These structures bind to DNA and undergo rapid

cellular uptake, being localized within the cell cytoplasm and

nucleus within 4 h

Bell A. T.

Boger D. L.

Bonnet C. S.

Brust M.

Bunz U. H. F.

Chithrani B. D.

Cosgrave L.

Coskun A.

D. Clive Williams

Daniel M.-C.

David A. G.

Elias B.

Elmes R. B. P.

Erkkila K. E.

Fernandez-Moreira V.

Ghosh P.

Gill M. R.

Herman L.

Huang T.

Kelly J. M.

Kim C.

Kim N. Orange

Labande A.

Lecomte J.-P.

Massue J.

Matson M.

Mayer C. R.

Metcalfe C.

Moloney M. P.

Montalti M.

Moucheron C.

Nonat A. M.

Ozbay E.

Puckett C. A.

Rau S.

Robert B. P. Elmes

Rosi N. L.

Ryan G. J.

Saha K.

Suzanne M. Cloonan

Tan C. P.

Thorfinnur Gunnlaugsson

Tian X.

Veale E. B.

Vos J. G.

Zeglis B. M.

Zhao Q.

Zhu M.

English

Crossref

Journal of the American Chemical Society

Luminescent Ruthenium(II) Polypyridyl Functionalized Gold Nanoparticles; Their DNA Binding Abilities and Application As Cellular Imaging Agents

Elmes, Robert B.P.

Orange, Kim N.

Cloonan, Suzanne M.

Williams, D. Clive

Gunnlaugsson, Thorfinnur

NUI Maynooth Eprint Archive

Luminescent Ruthenium(II) Polypyridyl Functionalized Gold

Nanoparticles; Their DNA Binding Abilities and Application

As Cellular Imaging Agents

Maynooth University ePrints and eTheses Archive

Maynooth EPrints and ETheses Archive

MURAL - Maynooth University Research Archive Library

Published: September 16, 2011r 2011 American Chemical Society 15862 dx.doi.org/10.1021/ja2061159 | J. Am. Chem. Soc. 2011, 133, 15862–15865COMMUNICATIONpubs.acs.org/JACSLuminescent Ruthenium(II) Polypyridyl Functionalized GoldNanoparticles; Their DNA Binding Abilities and ApplicationAs Cellular Imaging AgentsRobert B. P. Elmes,†,§ Kim N. Orange,‡,§ Suzanne M. Cloonan,‡,§ D. Clive Williams,*,‡,§ andThorfinnur Gunnlaugsson*,†,§† School of Chemistry, Centre for Synthesis and Chemical Biology, Trinity College, Dublin, Dublin 2, Ireland‡School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland§Trinity College Biomedical Sciences Institute, Dublin 2, IrelandbS Supporting InformationABSTRACT: The synthesis and photophysical and biolog-ical investigation of Ru(II)-polypyridyl stabilized water-soluble, luminescent gold nanoparticles (AuNPs) are de-scribed. These structures bind to DNA and undergo rapidcellular uptake, being localized within the cell cytoplasm andnucleus within 4 h.The development of functional supramolecular nanostruc-tures for applications in photonics,1 sensing,2 catalysis3 andmedicine,4 etc. is a fast emerging interdisciplinary research field.The design and synthesis of nanoparticles5 and, in particular,functionalized gold nanoparticles (AuNPs) has been at the fore-front of this effort in recent times, with many examples beingdeveloped for use in biological and medical applications,6 due totheir biocompatibility, unique size- and shape-dependence, andoptoelectronic properties. Similarly, Ru(II)-polypyridyl complexeshave been intensively studied due to their photophysicalproperties,7 where they have been employed for example inluminescent recognition and sensing,8 as sensitive and structure-specific DNA probes.9 Luminescent d6 transition metal ioncomplexes have often been proposed as useful fluorophores forcellular imaging,10,11 but until very recently their use in actualapplications has remained scarce. With our interest in the devel-opment of luminescent novel cellular targeting (therapeutic/imaging) agents7a,b,12 and surfacemodifiedAuNPs,13 we envisagedthat the combination of Ru(II)-polypyridyl complexes, spatiallyseparated from the surface ofAuNPs, by a covalent spacer, could beemployed as luminescent probes/imaging agents for various bio-logical applications.15 Herein we describe the synthesis of theRu(II) complexes 13, Figure 1, all of which possess a terminalalkyl thiol group which facilitates their adsorption onto AuNPs,leading to the formation of the three water-soluble systemsAuNP-1, AuNP-2 and AuNP-3 (Figure 1). We demonstrate that theseluminescent AuNPs offer attractive photophysical properties, idealfor application in cellular imaging, which we demonstrate usingHeLa cells. These are, to the best of our knowledge, the firstexamples of such Ru(II)-polypyridyl functionalized AuNPs to beemployed for such cellular applications.The syntheses of 1, 2, and 3 are shown in Scheme S1 (see alsofull details in Supporting Information) and were achieved in afew steps. The common ligand for all of these complexes is 4,the synthesis of which was achieved by employing peptidic(carbodiimide) coupling of 11-mercaptoundecanoic acid14a with5-amino,-1,10 phenanthroline in CH2Cl2, yielding 4 as an off-white solid in 78% yield. The microwave irradiation of 4 in thepresence of the Ru(II) bispolypyridyl dichlorides Ru(bpy)2Cl2,Ru(phen)2Cl2, and Ru(TAP)2Cl2 gave 1, 2, and 3, respectively,after 40 min.14b These were isolated, by precipitation from water,using excess NH4PF6, followed by purification using automaticcolumn chromatography [flash silica; 40:4:1 CH3CN/H2O/NaNO3 (sat)], yielding 13 in 75%, 65%, and 54%, respectively.These isolated NO3 salts were reconverted to their correspond-ing PF6 salts and were fully characterized, which included theuse of 1D and 2D NMR. The characteristic MLCT basedabsorbance spectra of 13, together with the excitation andemission spectra, are shown in the Supporting Information. All ofthe complexes were found to be fully water-soluble as their Clsalts (see Supporting Information), and their photophysicalproperties were investigated in 10 mM phosphate-bufferedaqueous solutions at pH 7.4. The absorption spectrum of 1showed characteristic ππ* intraligand transitions centered atFigure 1. Structure of the complexes 1, 2, and 3 (Ru = Ru(II)), ligand 4,and cartoon representation of their corresponding AuNPs-13 systems.Received: July 1, 201115863 dx.doi.org/10.1021/ja2061159 |J. Am. Chem. Soc. 2011, 133, 15862–15865Journal of the American Chemical Society COMMUNICATION285 nm (ε = 49 500 cm1 M1) assigned to the ancillary ligands,while the characteristic MLCT transitions of the Ru(II) centerwere observed at 450 nm (ε = 12 400 cm1 M1). Similartransitions were found for 2 and 3. Excitation into the MLCTbands of 13 gave rise to, on all occasions, MLCT basedemission occurring at λmax = 610 nm (ΦF = 0.054), 605 nm(ΦF = 0.056), and 635 nm (ΦF = 0.028), for 13, respectively.For all of these, the excitation spectra structurally matched thoseof the absorption spectra.The syntheses of AuNP-13 were achieved using the two-phase Brust method which gave tetraoctylammoniumbromide(TOAB) stabilized AuNPs.15a Exchange of TOAB on the surfaceof AuNPs with 13 was achieved upon stirring TOAB-AuNPwith 13, respectively, for 12 h at room temperature, followedby centrifugation and the removal of any unbound complex byemploying a method developed by Mayer et al.15b involvingdouble anion exchange. The AuNPs were characterized usingUVvisible spectroscopy, where the surface plasmon resonance(SPR) band was found at 520 nm (Supporting Information)slightly overlapping with the MLCT absorption bands of 13,Figure 2A. Using UVvis spectroscopy, AuNP-13 were foundto be stable in aqueous-buffered solution at pH 7.4 at roomtemperature for many months. Excitation into the MLCTabsorption bands of AuNP-13 gave rise to, on all occasions,MLCT centered emission with λmax at 610, 605, and 635 nm, forAuNP-13 respectively, with ϕF of ∼0.002, demonstrating thatthese were significantly less emissive after adsorption to theAuNP surface, as has previously by seen for [Ru(bpy)3]2+ in thepresence of AuNPs coated with thiolate monolayers.15c AuNP-13 were further characterized using TEM, which showedspherical nanoparticles with an average diameter of 4.0 ( 1.3,4.3 (1.3, and 3.2 (1.1 nm for AuNP-1, AuNP-2, and AuNP-3respectively, with no evidence of aggregation, Figure 2A, andusing DLS which gave hydrodynamic radii of 37 nm(Supporting Information).Ru(II)-polypyridyl complexes are known to bind to DNA andcause photosensitized DNA damage, but the binding constantand the photocleavage efficiency are highly structurally depen-dent.7,16 Consequently, the DNA binding affinities of 13, andAuNP-13, were evaluated in 10 mM phosphate buffer atpH 7.4 using salmon testes DNA. In the case of 13, DNAbinding gave rise to hypochromic effects in the MLCT band inthe UVvis absorption spectra, which in the case of 1 was ca.30% (Supporting Information). From these, a binding constantKb = 5.5 105 M1 was determined, while, for 3, Kb = 6.4 105M1 was calculated. Concomitantly, the MLCT centered emis-sion was also modulated, being most noticeable for 3, with ca.75% quenching being observed (see Supporting Information).Similarly, the binding of AuNP-13 also gave rise to changes inthe photophysical properties of the surface modified Ru(II)complexes. In the case of AuNP-1, an initial red shift in theUVvis absorption spectra, followed by a ca. 26% hypochroism,was observed upon binding to DNA. Similar changes were seenfor AuNP-2 and AuNP-3. Significant changes were also seen inthe emission spectra of AuNP-13, as demonstrated in Figure 3,upon binding of AuNP-3 to DNA, giving rise to 71% quenchingin the MLCT based emission (see Supporting Information forAuNP-12). This is in agreement with the behavior of otherπ-deficient Ru(II) complexes of the general formula [Ru(TAP)2(L)]2+ (L = phen or bpy),16 as those complexes contain-ing two TAP ligands have been shown to photo-oxidize guanine-containing nucleotides through a proton coupled photoinducedelectron transfer (PCET) process.17 The affinities of AuNP-13for DNA were further confirmed by carrying out ethidiumbromide (EtBr) displacement assays (Supporting Information),18which demonstrated that AuNP-13 displaced EtBr with anapparent binding constant (Kapp) of ∼107 M1. From theseresults, it is clear that AuNP-13 all interact with DNA in astrong manner. To further confirm this, we investigated theability of these nanostructures to bind to DNA using confocalfluorescence imaging, by treating a solution of DNA (4 mM)with a solution of AuNP-13. This led to immediate forma-tion of a precipitate and the formation of fibrous networks andsmaller aggregates, which are currently under investigation inour laboratory.Having demonstrated high DNA binding affinities of AuNP-13, we next carried out cellular uptake and localization studiesof these systems using confocal fluorescence microscopy. Weanticipated that AuNP-13 would be taken up by cells as hasbeen seen with many AuNP conjugates.19 This may occurthrough receptor mediated transport (e.g., endocytosis), or byplasma membrane driven transport due to their cationic nature.The results are shown in Figure 4. In Figure 4A, a brighfieldimage shows the presence of the AuNP (as aggregates) withinlive HeLa (cervical cancer) cells as dense ‘dark spots’, afterincubation with AuNP-1 (∼16 nM) 4 h prior to imaging.Fluorescence confocal laser scanning microscopy confirmed thisuptake and localization, as demonstrated in Figure 4B, whichshows an image of these cells upon excitation at 450 nm, wherethese dense areas of Figure 4A are seen to overlap perfectly withFigure 2. (A) MLCT absorption band of 1, andAuNP-1 in buffered H2Oat pH 7.4. (B) Transmission electron microscopy image of AuNP-1.Figure 3. Changes in the emission spectrum of AuNP-3 (λex 450 nm)with increasing concentration of DNA (077 μM). The blue and thered spectra demonstrated the non-DNA and the DNA bound AuNP-3.Inset: Plot of the change in integrated MLCT intensity as a function ofP/D ratio.15864 dx.doi.org/10.1021/ja2061159 |J. Am. Chem. Soc. 2011, 133, 15862–15865Journal of the American Chemical Society COMMUNICATIONthe red fluorescence emanating from within the cell interior. Theimage clearly confirms that AuNP-1 is luminescent within thecells; with apparent localization in the cytoplasm and the nucleus,where these can be seen as discrete ‘packets’ or aggregates offluorescence. In addition, Z-stack experiments clearly show thatthe observed luminescence is spherical in three dimensions;corresponding to the AuNPs being located inside the cells andnot bound to their exterior, Figure 5, for AuNP-2 (See largerimage in Supporting Information as well as for AuNP-1 andAuNP-3).Costaining of the nucleus, using DAPI, further confirmed thenuclear localization of AuNP-1, as demonstrated in Figure 4C,where the costained image is overlapped with the bright fieldimage (Figure 4A) and the luminescent image (Figure 4B).Similar behavior was observed for AuNP-2, Figure 5, and AuNP-3with the luminescence intensity being slightly diminished in thecase of AuNP-3 due to the PCET process discussed above(Supporting Information). The uptake of AuNP-1 was alsoconfirmed by TEM imaging within the HeLa cells, confirmingtheir localization within both the cytosol, Figure 4D, and thenucleus. We also carried out time dependent fluorescenceconfocal imaging studies, where the cellular uptake of AuNP-1was imaged at 2 and 6 h in addition to the 4 h discussed above.The results (see Supporting Information) clearly demonstratedthat only minimal uptake had occurred after 2 h. After 6 h, resultssimilar to those seen at 4 h were observed, though with slightlyenhanced cellular contrast.To assess the antiproliferative effects of these AuNPs weattempted to determine the EC50 value of AuNP-1 by carryingout an Alamar blue cytotoxicity assay with a large range ofconcentrations of AuNP-1 in the mesothelioma cell lineCRL5195. In short, and as is evident from Figures 4 and 5 usingHeLa cells, no significant apoptosis was observed for AuNP-1,and in fact, an IC50 value could not be determined for AuNP-1from the concentration range used as % viability did not dropbelow 60% (see Supporting Information).In summary, we have developed, to the best of our knowledge,the first examples of novel supramolecular DNA targeting motifsbased on Ru(II) stabilized AuNPs. We have shown that AuNP-13 all bind to DNA with high affinity, while at the same timebeing nontoxic, and that we can monitor the binding of these toDNA using various spectroscopic techniques. Moreover, by usingfluorescence confocal microscopy and TEM imaging studies, wehave demonstrated their cellular uptake and localization within thecytoplasm and nucleus. These studies confirm the potential use ofsuch Ru(II) polypyridyl functionalized AuNPs as highly sensitivecellular imaging agents. We are currently investigating the mecha-nism of their uptake, and their fate, in greater detail.’ASSOCIATED CONTENTbS Supporting Information. Synthesis and characterizationof 1, 2, and 3; Figures S1S30. This material is available free ofcharge via the Internet at http://pubs.acs.org.Figure 4. Live cell confocal laser scanning microscopy images of AuNP-1 (∼16 nM gold concentration20) with HeLa cells: (A) the bright field image;(B) emission arising from 600 to 700 nm; (C) overlay of the luminescence from AuNP-1 (red), nuclear costain DAPI (blue), and the bright field image;(D) TEM image of AuNP-1 following incubation with HeLa cells.Figure 5. Confocal fluorescence Z-section live cell images of AuNP-2 (20 μM) with Hela cells showing luminescence in three dimensions. Shown isthe image obtained with cells costained with nuclear stain DAPI (blue) and AuNP-2 (red). Scanning from left to right.15865 dx.doi.org/10.1021/ja2061159 |J. Am. Chem. Soc. 2011, 133, 15862–15865Journal of the American Chemical Society COMMUNICATION’AUTHOR INFORMATIONCorresponding Authorclive.williams@tcd.ie; gunnlaut@tcd.ie’ACKNOWLEDGMENTWe thank the Science Foundation Ireland (SFI RFP 2009),HEA PRTLI Cycle 4, The Irish Research Council for Science,Engineering and Technology (IRCSET Postgraduate Student-ship to R.B.P.E.), and TCD for financial support’REFERENCES(1) Ozbay, E. Science 2006, 311, 189.(2) (a) Bunz, U. H. F.; Rotello, V. M. Angew. Chem., Int. Ed. 2010,49, 3268. (b)Moloney,M. P.; Gun’ko, Y. K.; Kelly, J. M.Chem. Commun.2007, 3900. (c) Labande, A.; Ruiz, J.; Astruc, D. J. Am. Chem. Soc. 2002,124, 1782.(3) Bell, A. T. Science 2003, 299, 1688.(4) (a) Kim, C.; Agasti, S. S.; Zhu, Z.; Isaacs, L.; Rotello, V. M. Nat.Chem. 2010, 2, 962. (b) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005,105, 1547.(5) (a) David, A. G.; Dwight, S. S.; Weston, L. D.; Matthew, D. M.;Pinal, C. P.; Chad, A. M. Angew. Chem., Int. Ed. 2010, 49, 3280. 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Luminescent Ruthenium(II) Polypyridyl Functionalized Gold Nanoparticles; Their DNA Binding Abilities and Application As Cellular Imaging Agents

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