DOI: 10.1021/la102758f 14799Langmuir 2010, 26(18), 14799–14805 Published on Web 08/26/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Phagocytosis of Biocompatible Gold Nanoparticles

�Zeljka Krpeti�c,† Francesca Porta,*,† Enrico Caneva,‡ Vladimiro Dal Santo,§ and Giorgio Scarı̀^

†Dipartimento di Chimica Inorganica Metallorganica e Analitica “Lamberto Malatesta”, University of Milan,Via Venezian 21, 20133Milan, Italy, ‡Centro Interdipartimentale Grandi Apparecchiature (C.I.G.A.), Via Golgi 19,20133 Milan, Italy, §CNR-Istituto di Scienze e Tecnologie Molecolari, Via Golgi, 19, Milano I-20133, Italy, and

^Dipartimento di Biologia 7b, University of Milan, Via Celoria 26, 20133 Milan, Italy

Received November 16, 2009

We report the evidence for the cellular uptake of gold nanoparticles via the phagocytosis mechanism inmurine macrophage cells strongly supported by TEM and optical microscopy. Nanoparticles were prepared usingseveral biocompatible molecules of choice (5-aminovaleric acid, L-DOPA, melatonin, and serotonin hydrochloride)as stabilizers for gold colloids. Their surface chemistry was fully characterized by UV-vis, ATR-FTIR, 1H NMR,and HR-MAS 1H NMR spectroscopies, and size distribution was determined by CPS disc centrifuge and TEM.Differences in coatings were evaluated against cellular uptake, and a preferential movement of macrophagestoward 5-aminovaleric acid-modified gold nanoparticles was shown, leading to the fast accumulation of nanoparticlesin the cytosol.

Introduction

Macrophages are remarkably versatile cells that have origi-nated over time through the evolution ofmulticellular organisms,and their specialized recognition receptor and their phagocyticcapabilities are undisputed1 and well understood.2-4 What ren-ders macrophages very intriguing cell line is their ability torecognize, migrate, and phagocytise supposed “non-self” compo-nents with sharp discrimination between what is appetizingfor them. Deriving from a monocyte/macrophage cell lineage,5

it is of interest to understand which molecules are appetizingto these cells.

There are still unsolved issues related to macrophage contribu-tion to tumor immunity, such as role of tumor-associated macro-phages (TAMs) during the tumor infiltration processes.6 Tumorsinduced via chemical carcinogens, i.e., transplantable tumors, canpossess up to 55% macrophages.7,8 While some macrophages aretumor destroyers, some others may promote tumor cell growth byangiogenic factor secretion.9

Here we report the study of novel biocompatible gold conjugatesand their cellular uptake into macrophage cells, reporting phagocy-tosis as the uptake mechanism of gold nanoparticles stronglysupported by TEM and optical microscopy. This report aims tocontribute to targeting and delivery studies involving gold nanopar-ticles (GNPs) in order to address these nanomaterials to possiblehyperthermia treatments.

Experimental Section

Spectroscopy andMicroscopy Techniques Used for Nano-

particles Characterization. UV-vis spectra were recorded on aJascoV-530UV/vis spectrophotometer.ATR-FTIR spectrawererecorded on a BioradFTS-40 spectrophotometer equippedwith aSpecac Golden Gate ATR platform with diamond crystal.

1HNMR spectra were recorded on a 400MHz Bruker Avanceinstrument in D2O. For analysis colloidal gold solutions wereconcentrated by centrifugation and redispersed inD2O. Chemicalshifts were referenced to 3-(trimethylsilyl)propionic-2,2,3,3-d4acid sodium salt (δMe= 0).

1H-13C experiments were performed with the HRMAS (highresolution magic angle spinning) facility, installed on a FT-NMRspectrometer Avance 400 (1H frequency of 400 MHz) of BrukerBioSpin S.r.l. group, equipped with a superconducting ultrashieldmagnet of 9.4 T spinning the samples at the rotation of 4 kHz andoperating at 303K.The followingparameters for the 1Hexperiment(standard zg sequence) were set up: sw= 4006.4 Hz, d1= 3 s, andnt = 24. For the 13C experiment (zgig inverse gated 1H all-decoupled sequence) parameters were sw = 22058.8 Hz, d1 = 3s,and nt = 8000. For the analysis of GNPs, a sample was concen-trated by centrifugation and redispersed in D2O (50 μL) andsubsequently placed into a 12 mm high-precision ceramic rotor.

Transmission electron microscopy (TEM) micrographs of thecolloidal dispersions were obtained using anEFTEMLEO912abinstrument operated at an accelerating voltage of 120 kV. Speci-mens for TEM imagingwere preparedby evaporating a droplet ofgold colloidal solutions onto carbon-coated copper grids. Ahistogram of the particle size distribution and average particlediameter was obtained by measuring about 50-100 particles.

The particle size and distribution were measured using the CPSdisc centrifuge DC24000 (CPS Instruments Inc.). For all thesamples, 8-24 wt % sucrose solution in Milli-Q water was usedand filled successively in nine steps into the disc, starting with thedilution of highest density. For analysis the disc rotation speedwasset to 24 000 rpm. Calibration was performed using 0.377 μm sizedPVC particles as calibration standard (Analytik Ltd.), and particlesamples were sonicated before injection in the disc centrifuge.

A Leica TCS-NTmicroscope equipped with an argon ion laser(emission at 405-640 nm) operating at 200 mW was used forconfocal microscopy observations. Nanoparticles scattering inthe cytosol was evaluated using a 405-640 nm bandpass filter for

*Corresponding author: Phþ39 0250314361, Fax þ39 0250314550, e-mailFrancesca.porta@unimi.it.(1) Rees, R. C.; Parry, H. In TheMacrophage; Lewis, C. E., McGee, J. O'D., Eds.;

Oxford University Press: New York, 1992; pp 330-357.(2) Luck�e, B.; Strumia, M.; Mudd, S.; McCutcheon, M.; Mudd, E. B. H.

J. Immunol. 1933, 24, 455–491.(3) Schlesinger, R. B.; Fine, J. M.; Chen, L. C. Toxicol. Sci. 1992, 19, 584–589.(4) Dushkin, V. A.; Emel’yanenko, I. N.Bull. Exp. Biol.Med. 1975, 79, 290–291.(5) Gerbod-Giannone, M.-C.; Li, Y.; Holleboom, A.; Han, S.; Hsu, L.-C.;

Tabas, I.; Tall, A. R. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3112–3117.(6) McBride, W. H. Biochim. Biophys. Acta 1986, 865, 27–41.(7) Norman, S. J.; Sorkin, E. J. Natl. Cancer Inst. 1976, 57, 135–140.(8) Loeffler, D. A.; Keng, P. S.; Baggs, R. B.; Lord, E.M. Int. J. Cancer 1990, 45,

462–467.(9) Kuwano, H.; Sonoda, K.; Yasuda, M.; Sumiyoshi, K.; Nozoe, T.; Sugimachi,

K. J. Surg. Oncol. 1998, 65, 188–193.

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emission and dichroic or neutral filter for 3D scanning in trans-mission mode with a 40� oil immersion objective. Data capturewas performed with Leica Confocal Software (LCS).

To monitor the movements of macrophage cells assisting thechemotaxis step of the phagocytosis mechanism, a Nikon La-bomed TCM400 phase trinocular optical microscope was used,photographing a small number of macrophages every 30 min for6 h at magnification of 320� using a D5000 Nikon camera.

Cell Extraction and Preparation of the Samples for Con-

focal Microscopy. For all the experiments, macrophages wereextracted by an intraperitoneal wash of a CD1mouse. CD1 strainmice were bred in our Animal Care Facility. A male mousebetween 5 and 7 weeks of age was used. 5 mL of DulbeccoD-MEM cell culture medium was injected into the mouse andsubsequently removed. Extracted cells (ca. 1 � 106) were pro-cessed in solution, dividing the volume in aliquots of 1 mL.Peritoneal-elicited macrophages were incubated with gold nano-particles (300 μL of 10 nM solution of GNPs were added to 1 mLof extracted cells in D-MEM) for 30 min at 37 �C in a humidifiedatmosphere of 5% CO2 and subsequently centrifuged at 9g for3min.The supernatantwas removed, and10μLof the centrifugedcell suspensionwas placedonto amicroscope cover glass slide andsprayed with CryoSpray in order to fix the cells. Subsequently, acover glass slide containing fixed cells was placed onto a micro-scope glass slide containing 10 μL of Gel Mount aqueous-basedmounting medium. Prepared glass slides were glued and kept in afreezer/dark room before microscopy observations. In the de-scribed protocol, use of 4%paraformaldehyde fixative solution isavoided in order to remove any false signals during confocalmicroscopy observations.

Preparation of Animal Cells for TEM. Peritoneal-elicitedmacrophageswere incubatedwith gold nanoparticles at 37 �C in a5% CO2 atmosphere for 30 min. Subsequently, the pellets werespun at 13500 rpm for 2 min, then the supernatant was removed,and previously warmed 2% agarose solution added to the cellsand allowed to solidify. Subsequently, solidified gel was removed,and cells were fixed with 4%paraformaldehyde/2.5% glutaralde-hyde in phosphate buffer for 1 h. After fixation, the samples werewashed with PBS and postfixed using 1% OsO4 for 1 h (caution:extremely toxic!) and stained using 0.5% uranyl acetate. Cellswere then gradually dehydrated using a series of ethanol solutions(30, 60, 70, 80, and 100%), embedded in Epon-Araldite resin, andleft to polymerize for 48 h at 60 �C. Ultrathin sections (100-150 nm) were cut using a diamond knife on a Leica ultrome IIImicrotome and mounted on Formvar-coated copper grids. Thesectionswere poststained using 5%uranyl acetate in 50%ethanoland 2% lead citrate solution and imaged with EF TEMLEO912ab at 120 kV using AnalySIS software (Soft ImagingSystems).

Evidencing the Chemotaxis Step of Phagocytosis Me-

chanism by Optical Microscopy. For chemotaxis experiments,50 μL of GNP-embedded agarose gel (1.5% at 37 �C) was placedin the center of a 5 cm wide dish and allowed to solidify. As acontrol, the same amount of pure agarose was used. Subse-quently, cell culture media (D-MEM) containing 10% fetalbovine serum, 2% glutamine, and 1% penicillin-streptomycinwas added to the dishes and 2mL ofmacrophage cells seeded at adistance of 1 cm from solidified agarose gel. Confining areabetween the gel and cell culture media was photographed every30 min for 6 h.

Preparation of Gold Nanoparticles. Au-AminovalericAcid (35 nm) by Citric Acid Reduction Method. Aqueoussolution of NaAuCl4 (12.5 μmol; 85 mM) was added to 40 mL ofMilli-Q water at room temperature. Under stirring, an aqueoussolution of 5-aminovaleric acid (12.5 μmol; 35 mM) was added.Subsequently, aqueous solution of citric acid was added (250μmol; 1 M), and after 1 h formation of a raspberry red sol wasobserved. Colloidal solution was left under stirring for 24 h.Particles were purified by centrifugation (3500 rpm for 30 min).

Au-L-DOPA and Au-Melatonin (35-45 nm) by CitricAcid Reduction Method. To a 40 mL ofMilli-Q water, aqueoussolution of NaAuCl4 (12.5 μmol; 85 mM) was added at roomtemperature. Under stirring, aqueous solution of L-DOPA ormelatonin (12.5 μmol; 8.5 mM L-DOPA solution or 0.34 mMmelatonin solution) was added. Subsequently, aqueous solutionof citric acid was added (250 μmol; 1 M) under continuousstirring, and a pink-red sol started to form after 1 h. Obtainedcolloids were left under stirring for 24 h. The particles werepurified by centrifugation (3500 rpm for 30 min).

Au-Serotonin. Aqueous solution of NaAuCl4 (25 μmol;122 mM) was added to 40 mL of Milli-Q water at roomtemperature. Under stirring aqueous solution of serotonin hydro-chloride (12.5 μmol; 10 mM) was added, and after 30 minformation of brownish-red sol observed. Colloidal solution wasleft under stirring for 96 h. The particles were purified bycentrifugation (1500 rpm for 15 min).

Results and Discussion

In recent years many papers have appeared in the literaturereporting studies of the uptake of gold nanoparticles by differentcell lines.10-14 Moreover, eradication of tumor tissues using goldparticles capable of causing local hyperthermia was studied.15,16

We have contributed to these studies by investigating the selectivecellular uptake of gold nanoparticles byK562 cancer cells.17 Herewe report the study of novel gold conjugates and their cellularuptake into macrophage cells reporting phagocytosis as thecellular uptake mechanism.

For this study we have utilized biologically active compounds5-aminovaleric acid (AVA), (S)-2-amino-3-(3,4-dihydroxy-phenyl)propanoic acid (L-DOPA), N-acetyl-5-methoxytryptamine(melatonin), and 5-hydroxytryptamine hydrochloride (serotonin)as stabilizers in the preparation of biocompatible gold nano-particles (Figure S1). By reduction of NaAuCl4, used as goldprecursor, with citric acid in the presence of biocompatiblestabilizers of choice, we have obtained coated Au(0) colloidalnanoparticles with different surface chemistry. In the particularcase of serotonin-coated GNPs, serotonin was used as bothstabilizer and reducing agent. By carefully setting up componentsmolar ratio (AuCl4

-/reducing agent/stabilizer= 1:1:0.01-1) andoperating at room temperature for 24-96 h, we obtained goldparticles having dimensions in the 30-50 nm size range. InFigure1a TEM micrograph of 35 nm sized Au-AVA nanoparticles isshown and in Figure 1b its relative size distribution. Consideringrelatively slow reduction and growth of these nanoparticles, it isexpected that particle size distribution will be fairly polydispersein our experimental conditions. To show this, we have used a CPSdisc centrifuge to measure particle size and corresponding sizedistributions. By this technique, particles are separated by sizeusing centrifugal sedimentation in a liquidmedium. The sedimen-tation is stabilized by a slight density gradient within the liquid.In the case of Au-AVA weight distribution profile (Figure 2a)of the original nanoparticles solution, three separate peaksare shown demonstrating that the sample consists of 43.4, 34.2,

(10) Nativo, P.; Prior., I. A.; Brust, M. ACS Nano 2008, 2, 1639–1644.(11) Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J. E.; Rosi, N. L.;

Mirkin, C. A. Nano Lett. 2007, 7, 3818–3821.(12) Khan, J. A.; Pillai, B.; Das, T. K.; Sigh, Y.;Maiti, S.ChemBioChem 2007, 8,

1237–1240.(13) Chitrani, B. D.; Chan, W. C. W. Nano Lett. 2007, 7, 1542–1550.(14) Chitrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Nano Lett. 2006, 6, 662–

668.(15) Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M.; Smilowitz, H. M. Br. J.

Radiol. 2006, 79, 248–253.(16) Sperling, R. A.; Gil, P. R.; Zhang, F.; Zanella, M.; Parak, W. J. Chem. Soc.

Rev. 2008, 37, 1896–1908.(17) Krpeti�c, �Z.; Porta, F.; Scari, G. Gold Bull. 2006, 39, 66–68.

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and 22.6 nm sized nanoparticles. Considering the number dis-tribution (Figure 2b), where peak intensities are relative tothe particle number, the same sample consists mostly of 43.4and 34.2 nm sized particles, while the peak with lower inten-sity (22.6 nm) indicates the fraction of initial “seed” particles thatstill remain in the sample. The formation and growth of goldcolloids were followed by changes in the surface plasmon reso-nance (SPR) peaks using UV-vis spectroscopy. Typically, forgold colloids, λmax of the SPR peaks was found in the range from515 to 550 nm.

In Figure 1c,dUV-vis spectra of Au-L-DOPA (λmax=531 nm)and Au-serotonin (λmax=544 nm) nanoparticles are reported.Figure 1c, in particular, represents slow reduction and growth ofspherical nanoparticles, while Figure 1d represents gold nanotrian-gles formation within 24 h of reaction. In this particular case, usingserotonin hydrochloride as both stabilizer and reducing agent in thepreparation of gold particles, a significant number of gold nano-triangles are formed (Figure 1e,f and Figure S2), and their growthcan be appreciated by UV-vis monitoring the region from 750 to1100nm.The peakmaximum (not shown) is reached over 1100 nm,rendering these nanoparticles particularly suitable for hyperthermiatreatments.

In order to confirm the presence of the organic compounds ofchoice on gold nanoparticle surface and analyze the ligand shellcomposition, samples were analyzed by 1H NMR.

In Figure 3 the 1H NMR spectrum of Au-AVA in D2O isreported in comparison with the spectra of free 5-aminovalericacid and citric acid. The spectrum of free 5-aminovaleric acid insolution shows a multiplet at 1.58 ppm due to the two internalmethylene groups, a triplet at 2.14 ppm due to-CH2COOH, anda triplet at 2.92 ppm assigned to -CH2NH2. Proton NMRspectra of citric acid show a doublet of doublets centered at2.88 ppm. Spectra of Au-AVAnanoparticles show amultiplet at1.65 ppm, practically unchanged after binding with gold, and atriplet centered at 2.40 ppm assigned to protons of the R-methy-lene group bound to COOH, while the signal of CH2NH2 over-laps with citric acid signals. Thus, the ligand shell of thesenanoparticles consists of citric acid mixed with 5-aminovalericacid on gold surface. The interaction of 5-aminovaleric acid withgold is shownwith a remarkable shift ofmethylene protons boundto -COOH (Δ = 0.26 ppm), indicating the interaction of thecarboxylate group ofAVAwith gold through a polar electrostaticbond.

1H-13C experiments of 5-aminovaleric acid have been per-formed using HRMAS NMR (Figures S5-S8). To assign thesignals, a double quantum gradient COSY experiment wascarried out, obtaining clear and intense proton cross-peak corre-lations. For attribution of CH2-CO vicinal protons heteronuc-lear 1H-13C long-range 2D sequence was acquired obtaining aheteronuclear multiple bond coupling (HMBC) map showing across-peak between CdO carbon (182.77 ppm) and CH2 protons(2.10 ppm). This long-range C-H correlation allows the unam-biguous assignment of the CH2-CdO signal.

Magnetic resonance spectra of free L-DOPAandAu-L-DOPAin D2O are reported in Figure S4, showing a small change inchemical shift of CHR-COOH methylene group as well as achange in the peak shape observed between L-DOPA and Au-L-DOPA, while signals due to aromatic protons disappear com-pletely. These evidences show polarized binding of L-DOPA ongold particle surface trough the phenyl OH groups.

ATR-FTIR analysis of Au-AVA nanoparticles showed veryintense signals due to the citric acid (Figure S9). To be able toobserve IR signals due to 5-aminovaleric acid as well, the samplewas furthermore washed by centrifugation.

The ATR-FTIR spectrum of Au-AVA shows two convolutedbroad groups: one located between 3300 and 2600 cm-1 wherebands located at 3200, 3043, 2958, 2920, and 2851 cm-1 arepresent; and another group between 1300 and 850 cm-1 wherebands at 1225 (f), 1131 (m, sh), 1074 (m, sh) 1032 (m), 943 (m),and 874 cm-1 (f) are present; finally, a sharp band located at 768cm-1 (s, sh) is evident. With respect to the pure AVA (solidsample) spectrum, the differences are clearly evident: a largenumber of bands present in the pure compound spectrum arealmost absent in the Au-AVA spectrum, and there are evident

Figure 1. (a) TEM micrograph of 35 nm sized Au-AVA nano-particles (scalebar100nm); (b) correspondingparticle sizedistributionhistogram; (c)UV-vis spectraofAu-L-DOPAobtainedbycitricacidreduction; (d) UV-vis spectra of Au-serotonin; (e, f) TEM ofAu-serotoninnanotriangles (scale bars 100and200nm, respectively).

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shifts of the residual bands. These evidences reveal strong inter-action of the AVAmolecules with gold surface. Vibrations can beforbidden by the surface selection rule (i.e., vibrationalmodes notperpendicular to the gold surface can have a very low intensity), orother phenomena can be operative. Ooka et al.18 reported thecondensation polymerization of 5-aminopentanoic acid on goldsurface, and Castro et al.19 reported anomalous spectra of4-aminobutyric acid on colloidal silver and attributed it to aClaisen condensation-like reaction. However, spectra of oursample donot showbands attributable to amide or to the presenceof CdC double bonds.

The Au-serotonin ATR spectrum shows weak bands locatedat 1262, 1235, 1158, 876, and 917 cm-1 a broad convolutedabsorption centered at 1050 cm-1 and one intense band at 780cm-1; such bands fall in the range of CH (ring vibrations) andCH2 (twists, waggings) absorptions, but it is hard to make sureattributions on the basis of the literature.20 At higher wavenum-bers three very weak bands are evident at 2963, 2928, and 2855cm-1 attributable to ν(CH2) stretchings.

20

Both ATR spectra of Au-AVA and Au-serotonin, shown insection 7 of the Supporting Information, are very different withrespect to the ones of pure compounds (not shown), indicating astrong interactionwith gold surface even if its exact nature cannotbe easily derived from IR data.

For biological experiments, macrophage cells were extractedby an intraperitoneal wash of a CD1 mouse, having the CD1strain being bred in our Animal Care Facility. Intraperitonealmacrophageswere incubatedwith gold particles (10mM) at 37 �Cin a 5% CO2 for 30 min, and the cellular uptake of gold particleswas investigated using TEM, confocal, and optical microscopy.

Although the cellular uptake of gold nanoparticles into differ-ent cancer cell lines has been widely investigated, the uptake intomacrophage cells has been reported only within few examples in

the literature.21-26 The use of gold nanorods has been reportedfor murine macrophage cell targeting,27 and the GNP toxicity tomacrophages and the pathway of the particle uptake has beendetermined.28

For this study, we have chosen mature macrophage cellsnormally present in the peritoneal cavity of CD1 mice. Whilemonocyte/macrophage systems that are widely present in thevertebrate bloodstream do not transform themselves into TAMsable to migrate to the tumor stroma,29 macrophages are able torecognize and infiltrate tumor cells and also promote generationof new capillaries through angiogenesis. It was recently shownthat bone marrow-derived macrophages can be activated withpeptide-coated GNPs.30 Therefore, we believe that nanoparticlescoated with biologically active compounds that are capable ofsuccessfully penetrating macrophage cells can be used as vehiclesfor drug delivery applications. This can be achieved introducingthe additional drugmolecules of choice. In suchwaymacrophageswould acquire a potential use in cancer diagnostics as metastatictracers, drug delivery, and therapeutics.10,31

Phagocytosis of Gold Nanoparticles. It was demonstratedby Chitrani and co-workers that gold particles are taken up bycells via the endocytosis mechanism.13 Using macrophages as acellmodel of choice, we propose that gold particles are takenup bythese cells via the phagocytosis mechanism. In our experiments,each step of the phagocytosis mechanism was evidenced by TEMmicroscopy and represented as a cartoon in Figure 4. The initialstep of thephagocytosis, i.e., chemotaxis, is shown inFigure 4a.Atthis stage macrophage cell is approaching nanoparticles whilegiving forth its pseudopodia. The second step of phagocytosis isshown inFigure 4b, i.e., the formationof phagosomes. In the thirdstep phagosome vesicles merge with lysosomes and peroxisomevesicles, forming phagolysosomes. Lysosomes usually containlytic enzymes while peroxisomes contain strong oxidants such asH2O2, both deputed to digest phagocytised food. As shown inFigure 4c, lysosome and peroxisome vesicles are ready to merge

Figure 2. Distribution of Au-AVA nanoparticles as measured by analytical disk centrifugation: (a) weight distribution; (b) numberdistribution.

(18) Ooka, A. A.; Kuhar, K. A.; Cho, N.; Garrell, R. L.Biospectroscopy 1999, 5,9–17.(19) (a) Castro, J. L.; Sanchez-Cortes, S.; Ramos, J. V. G.; Otero, J. C.; Marcos,

J. I. Biospectroscopy 1997, 3, 449–455. (b) Castro, J. L.; Otero, J. C.; Marcos, J. I.J. Raman Spectrosc. 1997, 28, 765–769.(20) Bayarıa, S.; Saglamb, S.; Ustundag, H. F. J. Mol. Struct. (THEOCHEM)

2005, 726, 225–232.(21) Liu, C. J.;Wang, C. H.; Chien, C. C.; Yang, T. Y.; Chen, S. T.; Leng,W.H.;

Lee, C. F.; Lee, K. H.; Hwu, Y.; Lee, Y. C.; Cheng, C. L.; Yang, C. S.; Chen, Y. I.;Je, J. H.; Margaritondo, G. Nanotechnology 2008, 19, 295104/1–295104/5.(22) Kong, T.; Zeng, J.; Wang, X.; Yang, X.; Yang, J.; McQuarrie, S.; McEwan,

A.; Roa, W.; Chen, J.; Xing, J. Z. Small 2008, 9, 1537–1543.(23) Oo, M. K.; Yang, X.; Du, H.; Wang, H. Nanomedicine 2008, 3, 777–786.(24) Patra, H. K.; Banerjee, S.; Chaudhuri, U.; Lahiri, P.; Dasgupta, A. K.

Nanomedicine 2007, 3, 111–119.

(25) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Cancer Lett. 2006, 239, 129–135.

(26) Bast�us, N. G.; S�anchez-Till�o, E.; Pujals, S.; Farrera, C.; L�opez, C.; Giralt,E.; Celada, A.; Lloberas, J.; Puntes, V. ACS Nano 2009, 3, 1335–1344.

(27) Pissiwan,D.; Valenzuela, S.M.;Killinhsworth,M.C.; Xu,X.; Cortie,M. B.J. Nanopart. Res. 2007, 9, 1109–1124.

(28) Shukla, R.; Bansal, V.; Chaudhary,M.; Basu, A.; Bhonde, R. R.; Sastry,M.Langmuir 2005, 21, 10644–10654.

(29) Evans, R. Transplantation 1972, 14, 468–473.(30) Bast�us, N. G.; S�anchez-Till�o, E.; Pujals, S.; Farrera, C.; Kogan, M. J.;

Giralt, E.; Celada, A.; Lloberas, J.; Puntes, V. Mol. Immunol. 2009, 46, 743–748.(31) Gil, P. R.; Parak, W. J. ACS Nano 2008, 2, 2200–2205.

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with phagosomes, which contain gold particles and form phago-lysosomes. We have confirmed the presence of gold colloids in thecytosol of macrophage cells also by confocal microscopy (FiguresS12 and S13).

The evaluation between different coatings can be appreciated indifferent behavior of AVA, melatonin, serotonin, and L-DOPA-coated gold nanoparticles regarding phagocytosis (Figure 4 andFigure S13). Melatonin-coated GNPs were found in phagosomevesicles close to the nucleusmembrane and enclosed in the cell podo-somes (Figure 4d and Figure S13d). As expected, serotonin-coated

gold particles were found in the macrophage cytoplasm (Figure 4eandFigure S13e) in a large amount, being serotonin ubiquitous to allcellular systems. On the contrary, we have found that L-DOPA-coated gold colloids are not taken up by macrophages under thesame experimental conditions, having nanoparticles present outsidethe macrophage cell membrane even after 6 h of incubation,rendering these particles remotely appetizing for macrophage cells(Figure 4f and Figure S13f).

These experiments showed that chemical nature of the stabiliz-ing ligand molecule on the surface of gold particles affects theamount of the phagocytised gold. For L-DOPA-coated goldparticles we observed no phagocytosis event, as expected, sincethis molecule is a highly specific mediator for cells of the centralnervous system, and thus it is not recognized by macrophages.Large quantities of serotonin-coated gold particles were found inthe macrophage cell cytoplasm as serotonin molecule is normallypresent in a large amount in the circulatory system playing animportant role in wound healing performed by serotonin-filledplatelets.31 With regard to this phenomenon, macrophages areimportant in a secondary step of wounds cleaning and assisting incapillary restoration processes. Moreover, serotonin itself iscrucial in the regulation of angiogenesis, affecting angiogenicfactor expression in colon, lung, and breast cancer TAMs.32 Thecolloidal gold-serotonin system shows very high level of bio-compatibility, being a positive control. However, it is interestingto note that sections of the cells containing Au-serotoninobserved by TEM did not contain any gold nanotriangles butonly nanospheres also contained in the colloidal solution, indicat-ing that gold spheres represent the preferential shape for theuptake in the described experimental conditions.14

A very modest quantity of melatonin-coated gold particles hasbeen taken up intomacrophage cells. It is well-known thatmacro-phages show specific receptors for melatonin.33,34 Melatonin isalso able to stimulate secretion of cytokines, such as tumornecrosis factor (TNFR) and interleukin 1β,35 and can interferewith inflammatory processes. In the case of melatonin-coatedgold particles, melatonin itself could be released once inside thecells, lowering the angiogenic activity.36

With particular attention, we have investigated phagocytic activ-ity of 35 nm sized 5-aminovaleric acid-coated gold particles, havingobserved in a previous work the selective entrance of 7 nm sizedparticles into cancer cells.17 AVA-coated gold particles proved to bea robust system showing very low toxicity both in vivo (data notshown) and in vitro.Moreover,macrophage cells have taken up verylarge quantities ofAu-AVA ina short period of time, i.e., after only5 min of incubation (Figures S12 and S13a-c). Being an δ aminoacid, this molecule shows similarities with the GABA molecule (γ-aminobutyric acid) that is able to inhibit both inflammation andimmune response in T-cells37 and macrophages.38,39 In accordancewith a literature article describing analogous 5-aminolevullinic acid-stabilized gold particles,23 we highlight here the importance of

Figure 3. 1H NMR spectra of (a) Au-AVA, (b) AVA, and (c)citric acid in D2O (400 MHz).

(32) Nocito, A.; Dahm, F.; Jochum, W.; Jang, J. H.; Georgiev, P.; Bader, M.;Graf, R.; Clavien, P. A. Cancer Res. 2008, 68, 5152–5158.

(33) Guerrero, J. M.; Reiter, R. J. Curr. Top. Med. Chem. 2002, 2, 167–179.(34) Garcia-Perganeda, A.; Guerrero, J. M.; Rafii-El-Idrissi, R.; Paz, R. M.;

Pozo, D.; Calvo, J. R. J. Neuroimmunol. 1999, 95, 85–94.(35) Morrey, K.M.; McLachlan, J. A.; Serkin, C. D.; Bakouche, O. J. Immunol.

1994, 153, 2671–2680.(36) Mei, Q.; Yu, J. P.; Xu, J. M.; Wei, W.; Xiang, L.; Yue, L. Acta Pharmacol.

Sin. 2002, 23, 882–886.(37) Peng, L.; Yu-Long, Y.; Defa, L.; Sung, W. K.; Guoyao, W. Br. J. Nutr.

2007, 98, 237–252.(38) Stuckey, D. J.; Anthony, D. C.; Lowe, J. P.; Miller, J.; Palm, W.M.; Styles,

P.; Perry, V. H.; Blamire, A. M.; Sibson, N. R. J. Leukocyte Biol. 2005, 78, 393–400.

(39) Tian, J. D.; Lu, Y. X.; Zhang, H. W.; Chau, C. H.; Dang, H. N.; Kaufman,D. L. J. Immunol. 2004, 173, 5298–5304.

14804 DOI: 10.1021/la102758f Langmuir 2010, 26(18), 14799–14805

Article Krpeti�c et al.

aminovaleric acid in the construction of biocompatible nanogoldsystems able to deliver bioactive principles to macrophages.

Furthermore, to verify and assist the chemotaxis step of thephagocytosis mechanism, we have performed an experiment thatclearly showed the appetite of macrophage cells for AVA-coatedgold particles with respect to the control (Figure 5). For thisexperiment, a droplet of agarose gel, premixed with colloidalsolutions of Au-AVA nanoparticles (20 nM), was placed on acell culture dish, and subsequently macrophages in cell culturemedia were placed on a distance of 1 cm far from the gel. Themovement of macrophages toward nanoparticles embedded inagar gel was followed for 6 h taking the images every 30 min.Figure 5a shows macrophage cells that have penetrated agar geldroplet containing nanoparticles (after 1 h), while in the control

experiment even after 5 h of observation macrophages remainedat a borderline agar/cell culture media (Figure 5b).

Furthermore, we have observed a particular appetite ofmacro-phages for AVA-coated nanoparticles with respect to othermolecules we have chosen for this study, while confirming thechemotaxis step of the phagocytosis mechanism as well as diges-tion. A gallery of images (Figures S17-S21) representing thisexperiment and a short movie are available in the SupportingInformation. Because of the large amount of gold accumulated inthe cells, AVA-coated nanoparticles possess the requisites for theuse of these materials in hyperthermia treatments that couldefficiently and selectively destroy both tumor cells and TAMs.Further studies are in progress to test these particles in tumorprogression in vivo.

Figure 4. Cartoon representing three stages of the phagocytosis mechanism represented by TEM images of Au-AVA nanoparticles: (a)chemotaxis stage: macrophage is forming a vacuole with its pseudopodia (scale bar 1000 nm); (b) phagocytosis: formation of aphagolysosome (PL) containing GNPs upon subsequent fusion with lysosomes (L) (scale bar 200 nm); (c) digestion stage: peroxisomes(Pe) and lysosomes (L) ready to fuse with the phagosome (Ph) forming a phagolysosome (PL) (scale bar 500 nm); (d) Au-melatonin in themacrophage podosomes after 30 min incubation; (e) Au-serotonin in macrophage cell cytoplasm after 30min incubation; (f) Au-L-DOPAoutside the macrophage cell membrane after 6 h incubation.

Figure 5. Optical microscopy images of macrophage cells movement toward nanoparticles embedded in agar gel recognized as “food” asdemonstration of chemotaxis and phagocytosis. (a) Macrophage cells (arrows) penetrate the agar gel droplet containing Au-AVAnanoparticles after 1 h. Numerous red blood cells present in the sample remain on the borderline agar/cell culture media. (b) Macrophagecells approach the agar droplet and remain outside the gel in the absence of gold nanoparticles after 5 h.

DOI: 10.1021/la102758f 14805Langmuir 2010, 26(18), 14799–14805

Krpeti�c et al. Article

Conclusions

Summarizing, we have prepared gold nanoparticles functio-nalized by different bioactive molecules of choice, fully charac-terized them, and studied their interaction with murine mac-rophage cells. We have demonstrated the uptake of gold nano-particles in macrophages via the phagocytosis mechanism byTEM and assisted the chemotaxis step of this mechanism ina simple experiment followed by optical microscopy, registeringmovements ofmacrophage cells toward nanoparticles.Webelievethat accumulation of gold nanoparticles in TAMs could providean important path toward tumor eradication in its early andmetastatic stage.

Acknowledgment. We thank the Fondazione CARIPLO forfinancial support and Prof Franco Fraschini (Dept. of Pharma-cology, Chemotherapy and Toxicology of Milan University) for

helpful discussions. We thank Umberto Fascio for microscopysuggestions and Pasquale Illiano for NMR.

Supporting Information Available: (1) Chemicals; (2)ligands for gold particle stabilization; (3) TEM imagesof Au-serotonin nanotriangles; (4) CPS distribution ofAu-serotonin nanoparticles; (5) 1H NMR of Au-L-DOPAinD2O; (6) NMRof 5-aminovaleric acid onHRMAS probe;(7) ATR-FTIR analysis; (8) Au-AVA nanoparticles inmacrophage cells cytosol shown by confocal microscopy;(9) additional confocal and TEM microscopy images ofmacrophage cells incubated with GNPs of different surfacecoatings; (10) additional UV-vis spectra of gold colloids;(11) phagocytosis mechanism evidenced by optical micro-scopy; a short movie showing movements of macrophagestoward agar embedded GNPs. This material is available freeof charge via the Internet at http://pubs.acs.org.