Journal of Colloid and Interface Science 439 (2015) 28–33

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Journal of Colloid and Interface Science

www.elsevier .com/locate / jc is

Gold nanoparticles obtained by aqueous digestive ripening: Theirapplication as X-ray contrast agents

http://dx.doi.org/10.1016/j.jcis.2014.10.0250021-9797/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding authors at: Institute of Molecular Science and Technologies(ISTM-CNR), Via G. Fantoli 16/15, 20138 Milan, Italy (L. Polito). Fax: +39 0250313927.

E-mail addresses: laura.polito@istm.cnr.it (L. Polito), claudio.evangelisti@istm.cnr.it (C. Evangelisti).

Alessandro Silvestri a,b, Laura Polito a,⇑, Giacomo Bellani c, Vanessa Zambelli c, Ravindra P. Jumde a,Rinaldo Psaro a, Claudio Evangelisti a,⇑a CNR – ISTM, Nanotechnology Lab., Via G. Fantoli 16/15, 20138 Milan, Italyb Department of Chemistry, University of Milan, Via C. Golgi 19, 20133 Milan, Italyc Department of Health Science, University of Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy

a r t i c l e i n f o

Article history:Received 26 September 2014Accepted 16 October 2014Available online 23 October 2014

Keywords:Au nanoparticlesMetal vapor synthesisDigestive ripeningX-ray contrast agents

a b s t r a c t

A preparative protocol to synthesize large quantities of size-controlled gold nanoparticles (Au NPs), sta-bilized by CH3O-PEG5000-SH (PEG-SH) in aqueous medium, is reported. The combination of metal vaporsynthesis (MVS) technique with digestive ripening process allowed to obtain PEGylated Au NPs withmean core particle size of 3.8 nm and hydrodynamic diameters centered at 8.0 nm which were effectivelyused as computed tomography (CT) contrast agents for in vivo experiments on mice. The surface function-alization together with the small hydrodynamic diameters of the engineered Au nanoparticles permittedtheir efficient renal clearance, still retaining a prolonged blood circulation and a stealth capability.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

In the last decades medical imaging has undergone enormousimprovements due to the development of nanomaterial-based con-trast agents which are quickly becoming valuable and potentiallytransformative tools to enhance medical diagnostics for a widerange of in vitro and in vivo imaging modalities [1–3]. To date, var-ious gold nanostructures including spheres, rods, shells, and cageshave been widely studied for biodiagnostic assays, photothermaltherapy, surface enhanced Raman scattering (SERS), photoacousticimaging (PAT), and controlled drug release [4–6]. Stabilized goldnanoparticles (Au NPs), in particular, show interesting behavioras computed tomography (CT) contrast agents owing to their highX-ray absorption coefficient, low toxicity and high biocompatibil-ity [4,7–11]. The performance of these materials, in terms of pro-longed circulation time, enhanced renal clearance and littleaccumulation in reticuloendothelial system (RES) organs, stronglydepends on their particle size, shapes and interactions betweensurface atoms and stabilizing ligands [12–15]. Among the largenumber of methodologies to synthesize Au NPs (including alcoholsand water–oil emulsions or the synthesis in a nonpolar solvent

with a simultaneous use of a stabilizing agent) [5,16] the mostcommon and simplest one is the chemical reduction in water med-ium [17–19]. According to this method, positively charged goldcomplex ions are reduced to the zero valence state, forming a col-loidal solution. However, in many cases colloidal particles areunstable in time and the addition of a proper stabilizer has to beperformed to prevent gold clustering processes. Even if the reduc-tion protocol is extremely common and reliable, every batch ofreaction affords only few milligrams of stabilized Au NPs, depictinga drawback for their widespread development and applications. In2002, Klabunde et al. demonstrated that metal vapor synthesistechnique (MVS) [20–22] combined with digestive ripening, allowsto prepare monodisperse Au colloids soluble in organic phase inlarge quantity and reproducible quality, avoiding the presence ofby-products of metal salts in solution [23,24]. Following thisapproach, polydisperse Au solvated metal atoms (SMA), obtainedby co-condensation of Au and acetone vapors, can be convertedinto a nearly monodisperse one by refluxing the colloidalsuspension at the boiling point of non-polar organic solvents (e.g.toluene) in the presence of an excess of long chain aliphatic thiolsor other capping ligands (e.g. amines, phosphines, silanes) [25,26].Recently, many studies have been performed to exploit this syn-thetic route for the large scale preparation of mono- and bimetallicNPs (Au, Ag, Cu, etc.) soluble in organic solvents, whereas only fewexamples were devoted to the synthesis of water-soluble Au NPsstabilized by short chain thiols, exploiting this approach [27].

A. Silvestri et al. / Journal of Colloid and Interface Science 439 (2015) 28–33 29

Here, we report the synthesis of Au NPs by combining MVStechnique with aqueous digestive ripening process in the presenceof CH3O-PEG5000-SH ligand (PEG-SH). A regime of preparative con-ditions to synthesize large quantities of size-controlled PEGylatedAu NPs is highlighted. The morphological and structural featuresof Au NPs were evaluated by transmission electron microscopy(TEM), Inductively Coupled Plasma-Optical Emission Spectrome-ters (ICP-OES), UV–Vis spectroscopy, dynamic light scattering(DLS) analysis and 1H NMR diffusion ordered spectroscopy (DOSY)experiments. In vivo experiments, involving micro-CT imaging,were conducted to evaluate PEG-capped Au NPs properties asX-ray contrast agents, the time course of their systemic distribu-tion and their biocompatibility.

2. Materials and methods

2.1. Materials

All operations involving the MVS products were performedunder a dry argon atmosphere. Gold shots, 2–8 mm, 99.999% tracemetals basis, were purchased from Strem Chemicals. Acetone(Aldrich product) were dried over molecular sieves and storedunder dry argon atmosphere. CH3O-PEG5000-SH ligand, purchasedfrom Rapp Polymer GmbH, was used as received and stored underdry argon atmosphere at �20 �C.

2.2. Synthesis of Au/acetone SMA

The synthesis of Au/acetone SMA was carried out in a staticMVS reactor, similar to those previously described [28–30]. In atypical experiment, gold vapors, generated by resistive heating ofan alumina crucible filled with ca. 500 mg of gold shots wereco-condensed at liquid nitrogen temperature with acetone(100 mL) in the glass reactor chamber of the MVS apparatus inca. 2 h (P = 5 � 10�4 mBar). The reactor chamber was warmed tothe melting point of the solid matrix (ca. �80 �C) and the resultingdeep purple solution was siphoned at low temperature in a Schlenktube and kept in a refrigerator at �20 �C. The content of the metalin SMA was 4.65 mg Au/mL, as determined by Inductively CoupledPlasma-Optical Emission Spectrometers (ICP-OES; iCAP 6200 Duoupgrade, Thermofisher). For ICP-OES, a sample (1 mL) of SMA solu-tions was heated over a heating plate in a porcelain crucible in thepresence of aqua regia (2 mL) for four times, dissolving the solidresidue in 0.5 M aqueous HCl. The limit of detection (lod) calcu-lated for gold was 0.01 ppm.

2.3. Synthesis of PEGylated Au NPs

In a typical experiment, a portion of the resulting Au/acetoneSMA (0.46 mL, 0.011 mmol of Au) was added to a solution of15 mg of CH3O-PEG5000-SH (0.003 mmol) in 15 mL of MilliQ water,cooled in an ice-bath. The mixture was stirring 1 h at 0 �C and 1 hat room temperature. Then the mixture was refluxing for a prede-termined time: 1, 3, 6 or 18 h. CH3O-PEG5000-SH-capped Au NPswere recovered by centrifuge using Amicon� Ultra centrifugal fil-ters with a 30 KDa cut-off. The PEGylated Au NPs were stored asstable colloidal solution in MilliQ water for months.

For a larger scale synthesis: a portion of the resulting Au/ace-tone SMA (5 mL, 0.12 mmol of Au) was added to a solution of150 mg of CH3O-PEG5000-SH (0.03 mmol) in 150 mL of MilliQwater, cooled in an ice-bath. The mixture was stirring 1 h at 0 �Cand 1 h at room temperature. Then the mixture was refluxing for3 h and PEG-SH capped Au NPs were recovered by centrifuge usingAmicon� Ultra centrifugal filters with a 30 KDa cut-off.

2.4. Au NPs characterization

The main particle diameter, particle size distribution, and mor-phology of Au NPs were evaluated using transmission electronmicroscopy (Zeiss LIBRA 200FE, equipped with: 200 kV FEG, in col-umn second-generation omega filter for energy selective spectros-copy (EELS) and imaging (ESI), HAADF STEM facility, EDS probe forchemical analysis, integrated tomographic HW and SW system).TEM specimens were prepared by dropping an aqueous solutioncontaining PEGylated Au NPs onto on a carbon-coated copper grid(300 mesh) and evaporating the solvent. Histograms of the particlesize distribution were obtained by counting onto the micrographsat least 500 particles; the mean particle diameter (dm) was calcu-lated by using the formula dm = Rdini/Rni where ni was the numberof particles of diameter di. UV/Vis spectra were recorded on an Agi-lent 8453 instrument. DLS and f-potential measurements werecarried out on a Zetasizer Nano ZS instrument (Malvern Instru-ments Corp., Malvern, Worcestershire, UK) at a wavelength of633 nm with a solid state He–Ne laser at a scattering angle of173�, at 298 K on diluted samples. Each hydrodynamic diameterwas averaged from at least three measurements. NMR experimentswere carried out on a Bruker BioSpin FT-NMR Avance 500equipped with a 11.7 T superconducting ultrashield magnet. DOSY(diffusion-ordered spectroscopy) NMR spectrum was acquiredwith a pulsed gradient unit capable of producing magnetic fieldpulse gradients of 53.5 G cm�1 in the z-direction. Bipolar gradientpulses were used for diffusion using two spoil gradients. The dura-tion of the magnetic field pulse gradients (d) was optimized foreach diffusion time (D) to obtain a 1–5% residual signal with themaximum gradient strength. In each PFG-NMR (pulsed-field gradi-ent NMR) experiment, a series of 16–32 spectra on 16 K data pointswere collected and the eddy current delay (Te) was set to 5 ms. Thevalue of d was set between 1 and 2.5 ms, while the value of D wasset between 50 and 120 ms; the pulse gradients (g) were incre-mented from 2% to 95% of the maximum gradient strength in a lin-ear ramp. After Fourier transformation and baseline correction, thediffusion dimension was processed with the Bruker TopSpin NMRsoftware package (version 2.1).

2.5. In vivo CT analysis

CD1 mice (20–25 g) were obtained from Harlan Laboratories(Udine, Italy) and maintained under standard laboratory conditionin University of Milano-Bicocca in Monza (Italy). Proceduresinvolving animals and their care were conducted in conformitywith the institutional guidelines complying with national andinternational laws and policies. The study was approved by theethical committee of our institution. Anesthetized (Ketamine andXilazine) mice received PEG-capped Au NPs (7.01 mg Au/mL,200 ll) injection via tail vein (i.v.) and underwent CT scans (Sky-scan 1176, Bruker, Belgium) at different time points (immediatelybefore [baseline], 5, 60 and 240 min after injection). CT scans wereperformed with the following parameters: exposure time 60 ms,voltage 58 kV, current 431 lA, resolution 35 lm and rotation step0.70� (acquisition time about 5 min). The projection data were cor-rected for distortion and reconstructed by adjusting smoothing,correction of ring artifact and beam hardening. Reconstructed iso-tropic voxel size was 35 lm3. Images were reconstructed and ana-lyzed with the scanner softwares (NRecon v.1.6.9 and CTAn v.1.13,Skyscan, Bruker, Belgium). For image analysis, prior to measureHounsfield Unit (HU), as index of X-ray attenuation, a HU calibra-tion was done by scanning a phantom containing air and water andby ascribing the values 0 HU to water and �1000 HU to air (stan-dard unit of X-ray CT density). Then HU measurement was per-formed by constructing Regions of Interest (ROI) into the bladder,heart and liver and by analyzing each ROI at each time point.

30 A. Silvestri et al. / Journal of Colloid and Interface Science 439 (2015) 28–33

3. Results and discussion

3.1. Synthesis and characterization of PEGylated Au NPs

The synthetic procedure to obtain PEG-capped Au NPs is sche-matically illustrated in Fig. 1. Acetone was used as preliminary sta-bilizing solvent for the preparation of highly concentrated Au SMA(>4 mg Au/mL) [21–23]. The further addition of a portion of Au/ace-tone SMA to a water solution of an excess of CH3O-PEG5000-SHligand allowed to obtain a deep purple solution of Au aggregatesstabilized by the strong soft–soft interaction between the sulfuratom of the ligand and Au atoms on the nanoparticle surface [31].These aggregates appeared predominantly comprised of highlydefective particles, well separated from each other with a broadparticle size distribution (2.5–21.5 nm) and a mean particle diame-ter of 7.5 nm (Fig. 2A). Higher magnifications showed the presenceof both individual crystallites and particles held together to formlarger aggregates (Fig. 2A inset). In order to evaluate the role ofwarming-up on the gold particle size distribution, different sampleswere refluxed for 1, 3, 6, 18 h, respectively, and a sample of each ofthem was analyzed by TEM microscopy (SI, Fig. S1). The Au NPsobtained after 1 h of refluxing in water were observed undergoneto a remarkable effect of the digestive ripening which affected themorphologies and size distribution of Au NPs. However, a loweramount of large Au aggregates was still detected indicating thatthe process was not completed in this time frame. Samplesobtained, after 3 and 6 h (SI, Fig. S1) of refluxing showed very sim-ilar particle size distributions. The procedure allows to turn quanti-tatively Au aggregates into nearly monodisperse spherical Au NPs(Fig. 2B), with a mean core size of 3.8 nm (particle size ranging:2.5–7.5 nm). This phenomenon can be described as an inter-particlediffusion of Au atoms from large particles to smaller ones resultingin a narrowing of particle size distribution [32]. The ligand plays acrucial role in this process acting as both a strong stabilizing agentand an etchant of metal atoms, transferring materials among thenanoparticle surfaces.

On the other hand, after refluxing for long time (18 h) (SI,Fig. S1), an increase in polydispersion and in mean particle sizeof Au NPs was observed, indicating the onset of the unfavorableOstwald ripening process [32].

On the basis of the above reported results, we selected, as pref-erential targets, PEG-capped Au NPs obtained after a digestive rip-ening occurring during 3 h of refluxing in water. A careful analysisof micrographs of a sample of this system (Fig. 2B), obtained byevaporation of the aqueous solution onto lacey carbon film ofTEM grid, revealed a coherence in the packing order of Au NPs with

Fig. 1. Synthesis of PEGylated Au NPs by combined MVS

a regular inter-particle edge–edge spacing of ca. 15 nm. Thisdistance is consistent with the end-to-end chain length of CH3O-PEG5000-SH [33] of ca. 7.5 nm, suggesting the presence of PEGchains surrounding each Au NPs core. UV–Vis absorption spectraof PEGylated Au NPs in water solutions exhibited surface plasmonresonance (SPR) peaks at 519 nm (Fig. 3), which is in agreementwith the Au particle size observed by TEM analysis [34]. A broadplasmon absorption with a red shift in the SPR peak was observedfor Au aggregates before digestive ripening treatment, confirmingthe low homogeneity in Au particle sizes and shape of the startingsystem.

In order to get a deeper insight into the structural properties ofPEG-capped Au NPs and to verify their feasibility as in vivo contrastagents, we needed to evaluate their dimension in aqueous solution.Indeed, the measurement of the hydrodynamic diameter gives afundamental picture of the NPs core and their coating behaviorwhen dispersed in a medium. We evaluated the hydrodynamicradius distribution by means of 1H NMR diffusion ordered spec-troscopy (DOSY) (SI, Figs. S2 and S3) and dynamic light scattering(DLS) experiments (SI, Fig. S4). 1H NMR DOSY experiments[35,36], carried out on a sample of Au NPs dissolved in D2O showedtranslational diffusion coefficients ranging 2.82 � 10�11 to5.62 � 10�11 m2/s centered at 4.22 � 10�11 m2/s. On the basis ofthe Stokes–Einstein relationship (Eq. (1)), a mean hydrodynamicdiameter of ca 8.0 nm in solution can be estimated for PEG-cappedAu NPs after aqueous digestive ripening process (3 h).

Stokes–Einstein equation

D ¼ KT6pgRH

ð1Þ

k is the Boltzmann constant, T the absolute temperature, and gis the solution viscosity.

1H NMR DOSY data are in accordance with hydrodynamicradius obtained by DLS experiments (SI, Fig. S4), which resultedto be ca 3.7 nm. A mean radius increase of 2.1 nm was observed,which can be attributed to the PEG shell on the metallic surface.It is known that PEG chains can be displayed as ‘‘mushrooms’’ or‘‘brush’’ conformations when anchored onto surfaces [37,38]. Thelatter conformation is characterized by an high package densityand extended polymer chains, event that can increase the meanradius of nanostructures up to ca 8 nm (for PEG-SH, MW 5 KDa).The ‘‘mushrooms’’ conformation, instead, is characterized by a ran-dom oriented chains displacement and a low packing density. Inthis case the increase in mean radius is smaller and is calculatedon the basis of Flory radius (F) theory [39] (F, ca 5.9 nm [40],considering a PEG with MW of 5 kDa). The noted value in our

technique and aqueous digestive ripening process.

Fig. 2. TEM micrographs of PEGylated Au NPs: before (A) and after (B) aqueous digestive ripening process (3 h).

Fig. 3. UV–Vis absorption spectra of PEGylated Au NPs before (—) and after (--)aqueous digestive ripening process (3 h).

A. Silvestri et al. / Journal of Colloid and Interface Science 439 (2015) 28–33 31

experiments is smaller than the expected one, suggesting that, dur-ing coating reaction, PEG chains were able to assume a sort ofshrunk ‘‘mushroom’’ conformation on the gold nanoparticle sur-face. This event highlights an interesting surface behavior of goldsolvated metal atoms respect to the most common citrate-cappedgold nanoparticles.

Fig. 4. Micro-CT grayscale (upper panel) and colored (lower panel) images of water(A) and PEGylated Au NPs (B).

3.2. CT analysis

Once having in our hands a reliable and reproducible platformto produce high quantity of size-controlled and water stable PEGy-lated Au NPs, we tested them as suitable CT contrast agents.

Fig. 5. In vivo micro-CT images at different time points (baseline, 5, 60 and 240 min after PEGylated Au NPs injection). Reconstruction of images under three projections(coronal, axial and sagittal, from top to bottom) allows to better visualize the liver, the heart and the bladder.

32 A. Silvestri et al. / Journal of Colloid and Interface Science 439 (2015) 28–33

PEG-capped Au NPs, obtained after digestive ripening processoccurring during 3 h of refluxing in water, underwent an in vitromicro-CT scan to evaluate the CT attenuation intensity prior injec-tion in the live mice. Fig. 4 shows the micro-CT images of the AuNPs versus water. The results were promising as the attenuationin tube containing PEGylated Au NPs (608 ± 18 HU) were 8 timeshigher than the normal in vivo tissue (about 80 HU).

Then, to evaluate the biodistribution of PEG-capped Au NPs,in vivo micro-CT imaging experiments were performed (Fig. 5). Abaseline scan was performed before injection to take into accountany background CT signal (such as some food minerals in the stom-ach or intestine). PEG-capped Au NPs i.v. administration was welltolerated by mice, considering that until 24 h from the injectionthey did not show any sign of toxicity. The organ distribution ofAu NPs in function of the time, is reported in Fig. 6.

Moreover, it was possible to monitor in real time the renalclearance of PEG-capped Au NPs, observing the increasing X-rayattenuation in bladder. It is worth to underline that the particleswere able to circulate in blood stream up to 240 min of observa-tion, as indicated by the relative change in the attenuation withinthe heart (100% increase). No apparent relevant accumulation was

Fig. 6. X-ray attenuation change versus baseline in function of the time and organaccumulation of PEGylated Au NPs.

observed in liver, in which the X-ray attenuation increased mini-mally (probably due to the intraparenchymal blood pool) andremained stable over time. Noticeably, the X-ray attenuation inthe bladder rapidly increased from ca 50 to 1900 HU, just a fewminutes after Au NPs injection. After 240 min from injection, theurine was collected and their behavior under X-ray was evaluated(Fig. 7), showing a value of ca 1955 HU.

This result points out that PEGylated Au NPs were effectivelycleared from the blood through the kidneys and the bladder, yield-ing to a profound increase of the concentration of gold in urines, as

Fig. 7. Micro-CT grayscale (upper panel) and colored (lower panel) images of urineat the baseline (A) and 240 min after PEGylated Au NPs injection (B).

A. Silvestri et al. / Journal of Colloid and Interface Science 439 (2015) 28–33 33

compared with the original solution. Moreover, TEM analysis per-formed on a sample of the urine showed the presence of Au NPswithout significant change in particle size respect to the freshlyprepared sample before the injection (SI, Fig. S5).

4. Conclusions

In this paper we developed an efficient protocol to deliver largequantity of monodispersed water soluble PEG-SH capped Au NPs.The regime of preparation exploited a combination of high quan-tity synthesis of Au NPs by MVS technique and digestive ripeningprocess in aqueous media. The proposed regime, offers an efficientand reliable system to synthesize PEGylated Au NPs stable formonths at room temperature which acted as effective CT contrastagents in mice models. Moreover, the combination of MVS tech-nique and surface functionalization permitted to obtain an unex-pected shrinkage of hydrodynamic radius, which resulted innano-contrast agents characterized by an increased renal clear-ance. This result is noteworthy, considering that improvementsin the development of nanosystems for biomedicine applicationsare often related to the assemblies of stealthy nanoparticles withprolonged circulation time, enhanced renal clearance and littleaccumulation in RES organs. Moreover, the reliability and effi-ciency of the reported synthetic protocol suggests that it couldbe conveniently extended to the synthesis of size-selected AuNPs protected by a wide range of biocompatible hydrophilicligands or drugs.

Acknowledgments

The authors thank Dr. Daniela Maggioni (University of Milan) forhelp in DLS measurement and Dr. Enrico Caneva (CIGA, Universityof Milan) for 1H NMR and DOSY spectra. A.S. and L.P. thank ‘‘RSPP-TECH-Convenzione Operativa inserita nella Raccolta Convenzioni eContratti della Regione Lombardia (Grant number 18095/RU)’’, andMIUR-Italy (PRIN 2010-2011: contract 2010JMAZML_003). C.E. andR.P.J. thank MIUR-Italy (FIRB 2010: contract RBFR10BF5V).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jcis.2014.10.025.

References

[1] R. Bardhan, S. Lal, A. Joshi, N.H. Halas, Cancer Acc. Chem. Res. 44 (2011) 936–946.

[2] M.E. Davis, Z.G. Chen, D.M. Shin, Nat. Rev. Drug Discov. 7 (2008) 771–782.[3] D.P. Cormode, P.A. Jarzyna, W.J.M. Mulder, Z.A. Fayad, Adv. Drug Deliv. Rev. 62

(2010) 329–338.

[4] D. Xi, S. Dong, X. Meng, Q. Lu, L. Meng, J. Ye, RSC Adv. 2 (2012) 12515–12524.[5] C.J. Murphy, A.M. Gole, J.W. Stone, P.N. Sisco, A.M. Alkilany, E.C. Goldsmith, S.C.

Baxter, Acc. Chem. Res. 41 (2008) 1721–1730.[6] M.-C. Daniel, D. Astruc, Chem. Rev. 104 (2004) 293–346.[7] E.C. Dreaden, A.M. Alkilany, X. Huang, C.J. Murphy, M.A. El-Sayed, Chem. Soc.

Rev. 41 (2012) 2740–2779.[8] D. Kim, S. Park, J.H. Lee, Y.Y. Jeong, S. Jon, J. Am. Chem. Soc. 129 (2007) 7661–

7665.[9] V.W.K. Ng, R. Berti, F. Lesage, A. Kakkar, J. Mater. Chem. B 1 (2013) 9–25.

[10] C. Peng, L. Zheng, Q. Chen, M. Shen, R. Guo, H. Wang, X. Cao, G. Zhang, X. Shi,Biomaterials 33 (2012) 1107–1119.

[11] C. Peng, J. Qin, B. Zhou, Q. Chen, M. Shen, M. Zhu, X. Lu, X. Shi, Polym. Chem. 4(2013) 4412–4424.

[12] C. Zhou, M. Long, Y. Qin, X. Sun, J. Zheng, Angew. Chem. Int. Ed. 50 (2011)3168–3172.

[13] J. Liu, M. Yu, X. Ning, C. Zhou, S. Yang, J. Zheng, Angew. Chem. Int. Ed. 52 (2013)12572–12576.

[14] Z. Wang, L. Wu, W. Cai, Chem. Eur. J. 16 (2010) 1459–1463.[15] C. Xu, G.A. Tung, S.H. Sun, Chem. Mater. 20 (2008) 4167–4169.[16] E. Boisselier, D. Astruc, Chem. Soc. Rev. 38 (2009) 1759–1782.[17] F.H. Fry, G.A. Hamiltox, J. Turkevich, Inorg. Chem. 5 (1966) 1943–1946.[18] T. Yonezawa, T. Kunitake, Colloids Surf. A 149 (1999) 193–199.[19] N.R. Jana, L. Gearheart, C.J. Murphy, Langmuir 17 (2001) 6782–6786.[20] K.J. Klabunde, Free Atoms, Clusters and Nanoscale Particles, Academic Press,

New York, 1994.[21] M. Franklin, K.J. Klabunde, High-Energy Processes in Organometallic

Chemistry, ACS Symposium Series, American Chemical Society, Washington,DC, 1987. pp. 246–259.

[22] S.-T. Lin, M.T. Franklin, K.J. Klabunde, Langmuir 2 (1986) 259–260.[23] S. Stoeva, K.J. Klabunde, C.M. Sorensen, I. Dragieva, J. Am. Chem. Soc. 124

(2002) 2305–2311.[24] D. Jose, J.E. Matthiesen, C. Parsons, C.M. Sorensen, K.J. Klabunde, J. Phys. Chem.

Lett. 3 (2012) 885–890.[25] B.L.V. Prasad, S.I. Stoeva, C.M. Sorensen, K.J. Klabunde, Langmuir 18 (2002)

7515–7520.[26] B.L.V. Prasad, S.I. Stoeva, C.M. Sorensen, K.J. Klabunde, Chem. Mater. 15 (2003)

935–942.[27] S.I. Stoeva, A.B. Smetana, C.M. Sorensen, K.J. Klabunde, J. Colloid Interf. Sci. 309

(2007) 94–98.[28] J.S. Bradley, in: G. Schmid (Ed.), Clusters and Colloids. From Theory to

Applications, VCH, Weinheim, 1994, pp. 459–537.[29] K.J. Klabunde, G. Cardenas-Trivino, in: A. Fürstner (Ed.), Active Metals, VCH,

Weinheim, 2007, pp. 237–278 (Chapter 6).[30] G. Vitulli, C. Evangelisti, A.M. Caporusso, P. Pertici, N. Panziera, S. Bertozzi, P.

Salvadori, in: B. Corain, G. Schmid, N. Toshima (Eds.), Metal Nanoclusters inCatalysis and Materials Science: The Issue of Size-Control, Elsevier,Amsterdam, 2008, pp. 437–452 (Chapter 32).

[31] J.E. Marrhiensen, D. Jose, C.M. Sorensen, K.J. Klabunde, J. Am. Chem. Soc. 134(2012) 9376–9379.

[32] D.S. Sidhaye, B.L. Prasad, New J. Chem. 35 (2011) 755–763.[33] W.P. Wuelfing, S.M. Gross, D.T. Miles, R.W. Murray, J. Am. Chem. Soc. 120

(1998) 12696–12697.[34] K.L. Kelly, E. Coronado, L.L. Zhao, G.C. Schatz, J. Phys. Chem. B 107 (2003) 668–

677.[35] C.S. Johnson Jr., Prog. NMR Spectrosc. 34 (1999) 203–256.[36] D. Zuccaccia, A. Macchioni, Organometallics 24 (2005) 3476–3486.[37] C.S. Levin, S.W. Bishnoi, N.K. Grady, N.J. Halas, Anal. Chem. 78 (2006) 3277–

3281.[38] K. Larson-Smith, D.C. Pozzo, Langmuir 28 (2012) 13157–13165.[39] P.G. de Gennes, Macromolecules 13 (1980) 1069–1075.[40] J.V. Jokerst, T. Lobovkina, R.N. Zare, S.S. Gambhir, Nanomedicine 6 (2011) 715–

728.