Nanoscale

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aNanobiophotonics and Laser Microscopy

Bio-Nano-Sciences, and Faculty of Phys

Laurian Street 42, 400271 Cluj-Napoca, R

ubbcluj.ro; monica.potara@phys.ubbcluj.robMolecular Biology Center, Interdisciplinary

Babes-Bolyai University, T Laurian 42, 4002cInstitute of Biology, Romanian Academy, Sp

RomaniadWiTec GmbH, D-89081 Ulm, Germany

† Electronic supplementary informatioexperimental details of chitosan-coatedDOI: 10.1039/c3nr00005b

Cite this: Nanoscale, 2013, 5, 6013

Received 14th March 2013Accepted 16th April 2013

DOI: 10.1039/c3nr00005b

www.rsc.org/nanoscale

This journal is ª The Royal Society of

Chitosan-coated triangular silver nanoparticles as anovel class of biocompatible, highly sensitive plasmonicplatforms for intracellular SERS sensing and imaging†

Monica Potara,*a Sanda Boca,a Emilia Licarete,b Annette Damert,b

Marius-Costel Alupei,b Mircea T. Chiriac,b Octavian Popescu,bc Ute Schmidtd

and Simion Astilean*a

There is a need for new strategies for noninvasive imaging of pathological conditions within the human

body. The approach of combining the unique physical properties of noble-metal nanoparticles with

their chemical specificity and an easy way of conjugation open up new routes toward building bio-

nano-objects for biomedical tracking and imaging. This work reports the design and assessment of a

novel class of biocompatible, highly sensitive SERS nanotags based on chitosan-coated silver

nanotriangles (Chit-AgNTs) labeled with para-aminothiophenol (p-ATP). The triangular nanoparticles are

used as Raman scattering enhancers and have proved to yield a reproducible and strong SERS signal.

When tested inside lung cancer cells (A549) this class of SERS nanotags presents low in vitro toxicity,

without interfering with cell proliferation. Easily internalized by the cells, as demonstrated by imaging

using both reflected bright-light optical microscopy and SERS spectroscopy, the particles are proved to

be detectable inside cells under a wide window of excitation wavelengths, ranging from visible to near

infrared (NIR). Their high sensitivity and NIR availability make this class of SERS nanotags a promising

candidate for noninvasive imaging of cancer cells.

Introduction

The emergence of nanotechnology at the interface betweenbiology, chemistry, physics and engineering has spawned newopportunities for a wide range of applications such as targeteddrug delivery and controlled release,1 protein and DNAsensing,2,3 tissue engineering,4 and sensing-based early diag-nostic and biomedical imaging.5,6 Nanoparticles fabricated tofulll several precise requirements in terms of size, composition,stability and biocompatibility are becoming the most appealingnanotechnology tools for implementing the above applications.Indeed, the nanometric size not only creates new materialproperties but becomes essential to facilitate nanoparticleincorporation and information extraction from specic

Center, Interdisciplinary Research in

ics, Babes-Bolyai University, Treboniu

omania. E-mail: simion.astilean@phys.

Research Institute in Bio-Nano-Sciences,

71 Cluj-Napoca, Romania

l. Independentei 296, 060031 Bucharest,

n (ESI) available: Fig. S1–S4, thespherical nanoparticles synthesis. See

Chemistry 2013

microscopic locations in biological systems.7 On the other handthe composition and surface properties of nanoparticles allowconjugation with specic biomolecules in order to endowstability, biocompatibility and receptiveness toward the targetedorgans or pathologic sites.8 In particular, beyond their biocom-patibility and versatile surface chemical properties noble-metal(gold, silver) nanoparticles stand out as versatile tools in nano-biophotonics due to their strong interaction with light. Indeed,when incoming light couples with the oscillation frequency ofthe conduction electrons in noble metal nanoparticles, a so-called localized surface plasmon resonance (LSPR) arises, which ismanifested as a strong UV-visible-NIR extinction band.9 A maineffect of plasmon excitation is the electromagnetic eldenhanced at the surface of the nanoparticles under such reso-nant excitation. The effect is currently exploited via a number ofsurface-enhanced spectroscopies in sensing and biodetection.Surface-enhancedRaman scattering (SERS) can be used to detectand identify the molecular species and provide structural infor-mation based on their unique vibrational Ramanngerprint.10,11

In SERS, the so-called hot-spots created in the gaps betweennanoparticles or at the edges and tips of anisotropic nano-particles provide sufficiently intense electromagnetic elds topromote a tremendous increase in the Raman intensity ofmolecules located in these regions,making it possible to detect aSERS signal even from single molecules.10,11 SERS facilitates thedetection, characterization and precise identication of trace

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amounts of the substance, thus being a powerful tool for diag-nosis and monitoring disease progression through tracking ofspecic biomarkers.12 Beyond the ultrasensitive analyticaldetection, an increasing interest is given nowadays to the designof SERS nanotags used for imaging of living cells. Typically, SERSnanotags are obtained by anchoring a strong Raman activemolecule (the reporter molecule) onto the surface of metalnanoparticles (usually gold or silver nanoparticles). The result-ing reporter-nanoparticles are characterized by a specic SERSsignal. Subsequently, they are coated with a biocompatible,protective layer of dielectric or a polymer. Thereby a core–shellstructure is formed with the outer shell potentially conjugatedwith antibodies for specic targeting. There are several examplesin the literature where SERS-encoded nanoparticles weresuccessfully explored for probing and imaging of cancer cells.Doering and Nie suggested the application of silica embeddedgold nanoparticles as SERS nanotags for cell detection andspectroscopy.13 A few years ago, Qian et al. reported that PEGy-lated gold nanoparticles can be implemented as SERS probes forin vivo tumor targeting and spectroscopic detection.14 Morerecently, Maiti et al. developed bovine serum albumin encapsu-lated gold nanoparticles as SERS nanotags for in vivo multiplextargeted imaging.15 However, most of the fabrication methodsrely on the exploitation of spherical nanoparticles as Ramansignal enhancers. As individual spherical nanoparticles show arather small Raman enhancement and limited in vivo applica-bility, controlled aggregation was considered as a rst choice topromote the formation of hot-spots between nanoparticles.16,17

Although aggregated nanoparticles have strong SERS enhance-ment, the control over the size and stability of aggregates isextremely difficult in biological environments. Alternatively,anisotropic nanoparticles with sharp geometries can give rise tohot-spots localized at their corners and edges and, enveloped in aprotective shell, can operate individually as a highly sensitive andreproducible SERS substrate.18–21 In addition, the particles ofanisotropic shape exhibit multiple plasmonic resonances andlarge tunability whichmake them versatile SERS substrates for awide range of laser excitation from UV to NIR. This feature is ofparticular interest because most biological molecules absorb inthe UV region of the spectrum. Therefore, the use of SERSnanotags that are active in a spectral region close to theabsorption maximum of biomolecules is benecial for proof ofconcept in vitro experiments. Based on these considerations theuse of multi-wavelength responsive SERS nanotags could enablea better in vitro localization of nanoparticles inside cells. Inaddition, simultaneous sensing and imaging in vivo will makediagnostics and therapy more effective. More importantly,anisotropic nanoparticles with a plasmonic response close to theNIR region can simultaneously perform optical imaging andfunction as effective photothermal transducers for cancertherapy.22,23 Our group has recently reported that chitosan-coated triangular silver nanoparticles can be used as effectivephototherapeutic agents against cancer cells.24 In another studywe demonstrated that chitosan-coated anisotropic silver nano-particles are sensitive enough to be implemented as effectiveplasmonic substrates for single-molecule detection by SERS.25

Considering the interesting optical and biological properties

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already demonstrated, here we introduce a novel class of highlysensitive SERS-nanotags basedon triangular silver nanoparticlesenveloped in chitosan (Chit-AgNTs) for performing noninvasiveintracellular SERS imaging under multiple wavelength excita-tions. A thiolmarker, para-aminothiophenol (p-ATP) is anchoredonto the nanoparticle surface through silver–sulfur interactionsto generate a stable, robust and consistent Raman signal indifferent liquid environments. The chitosan biopolymer playsmultiple roles, from preventing the aggregation of nanoparticlesto conferring biocompatibility and functionality to them. Whentested on lung carcinoma cells (A549) this class of SERS nanotagspresents low in vitro toxicity and does not interfere with cellproliferation. Easily internalized by the cells, the particles areproven to be spectroscopically detectable inside cells under threelaser lines excitation (532, 633 and 785 nm). Their biocompati-bility combined with multi-wavelength SERS responsivenessendows this class of SERS nanotags with the potential to befurther developed as effective medical imaging tools.

ExperimentalMaterials

Chitosan akes (high molecular weight, >75% deacetylated),silver nitrate (AgNO3), and p-aminothiophenol (p-ATP) werepurchased from Aldrich. Glacial acetic acid (99.8%) wassupplied by Sigma-Aldrich and diluted to a 1% aqueous solu-tion before use. Ascorbic acid, sodium borohydride (NaBH4)and trisodium citrate (C6H5Na3O7$2H2O) were obtained fromMerck. All chemicals were used without further purication.Solutions were prepared using ultrapure water with a resistivityof at least 18 MU cm. All other reagents used in solution prep-aration were of analytical grade. All glassware used was cleanedwith aqua regia solution (HCl : HNO3 3 : 1) and then rinsedthoroughly with ultrapure water.

Nanoparticles preparation

Chitosan-coated silver nanoplates were prepared via seed-mediated growth according to our method described previ-ously.26 Briey, in the rst step a stock aqueous solution of silverparticles called ‘seeds’ was prepared through reduction reactionof silver nitrate with sodium borohydride at 0 �C. In the secondstep, aqueous solutions of seeds (200 ml), trisodium citrate(25 mM, 200 ml), ascorbic acid (0.1 M, 50 ml) and chitosan (2 mgml�1, 10 ml) were pre-combined and brought to 35 � 2 �C. Tothis mixture, AgNO3 (0.01 M, 300 ml) was added dropwise undercontinuous stirring. The growth of silver nanoplates wascomplete aer 3 min. under magnetic stirring. Chitosan-coatedspherical nanoparticles were synthesized using a seed-mediatedgrowth method reported previously27 (more details in the ESI†).For SERS nanotag preparation, 990 ml colloidal solution ofchitosan-coated silver nanoparticles (spherical or triangular)were incubated at room temperature with 10 ml aqueous solu-tion of p-ATP (10�3 M). The mixture was kept for 24 h until thesystem reached equilibrium, aer which the nanoparticles werecentrifuged and re-suspended in ultrapure water to remove anyunused reactants.

This journal is ª The Royal Society of Chemistry 2013

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Cell culture and nanoparticle incubation

Human lung carcinoma cells (A549, Cell Line Service, Germany)were cultured in a Dulbecco's modied Eagle's medium (Lonza)supplemented with 2 mM L-glutamine, penicillin/streptomycin100 U ml�1, 10% fetal calf serum and incubated in a humidiedincubator (37 �C, 5% CO2). For SERS measurements, about 3 �104 A549 cells were cultured on 24� 50mm cover slips (0.17mmthickness) and placed in cell culture dishes overnight. Next day,the culturemediumwas replacedwith amediumcontaining 6mgml�1 nanoparticles. Aer 2 hours, themediumwas removed andthe cells were washed 3 times with phosphate buffered saline(PBS). The glass coverslip was then inverted over a glass slidewith �0.2 mm spacers and PBS solution was inserted betweenthe two glasses. The cells sandwiched between the cover slip andthe glass slide (with PBS) were sealed and mounted for micro-scopic inspection and spectroscopic measurements.

Cytotoxicity assay

Cytotoxic effects of silver nanoparticles were assayed using aWST-1 dye (Millipore) assay based on the enzymatic cleavage ofthe tetrazolium salt WST-1 to formazan by mitochondrialdehydrogenases active in the living cells. 5 � 103 cells per wellwere cultured in a 96-well plate, in duplicate. Aer 24 h, theculture medium was removed and a fresh medium containingbetween 4 and 10 mg ml�1 nanoparticles was added to the testwells. Subsequently, the cells were placed in the incubator foradditional 24 h. Cells without nanoparticles were used as apositive control. At the end of the incubation period, themedium was removed and 100 ml of the fresh medium con-taining 10% WST-1 solution were added to each well. Emptywells with amedium containing theWST-1 reagent were used asblank. Aer 30 min of incubation, the absorbance wasmeasured at 450 nm, using a microplate reader (BMG Labtech)with a reference wavelength of 650 nm.

Cell proliferation assay

To determine the effects of silver nanoparticles on cell prolif-eration, 1 � 103 A549 cells per well were seeded in a 96-wellplate for 24 h. Cell proliferation was assessed using an ELISABrdU-colorimetric immunoassay (Roche Applied Science)according to the manufacturer's instructions. This method isbased on the incorporation of the pyrimidine analogue – bro-modeoxyuridine (BrdU) – instead of thymidine into the DNA ofproliferating cells. A549 cells were incubated with BrdU solutionfor 24 h and the culture media were completely removed fromeach well. Following this step, the cells were xed and the DNAwas denatured by addition of 200 ml of FixDenat solution to eachwell. A monoclonal peroxidase-conjugated anti-BrdU antibodywas added to each well in order to detect the incorporated BrdUin the newly synthesized cellular DNA. The antibody wasremoved aer 1 h of incubation and the cells were washed threetimes with PBS. A peroxidase substrate (tetramethyl-benzidine)was added and the immune complexes were detected bymeasuring the absorbance of the reaction product at 450 nmwith a reference wavelength of 655 nm. The effects of

This journal is ª The Royal Society of Chemistry 2013

nanoparticles were tested at different concentrations rangingfrom 4 to 10 mg ml�1. All concentrations were tested in dupli-cate, and untreated cells were used as controls.

Nanoparticle characterization

UV-visible-NIR extinction spectra were measured in a 2 mmquartz cell using a Jasco V-670 spectrometer with 1 nm spectralresolution. The morphology of the silver nanoparticles wasexamined using a JEOL JEM 1010 transmission electronmicroscope (TEM). For TEM measurements the samples wereprepared by placing a drop of colloidal dispersion onto carbon-coated copper grids. The samples were le to dry at roomtemperature. The average size and distribution of silver nano-particles in TEM images were determined using ImageJ so-ware.28 The concentrations of silver colloids were measured byatomic absorption spectroscopy (Avanta PM, GBC-Australia).The zeta potentials of Chit-AgNTs and p-ATP labeled Chit-AgNTswere determined by a laser Doppler micro-electrophoresistechnique using a Malvern Zetasizer Nano ZS-90. The Nano ZScontains a He–Ne laser operating at a wavelength of 633 nm andan avalanche photodiode detector. The zeta potentialmeasurements were performed at a temperature of 25 �C.

The Raman and SERS spectra as well as the spectroscopic cellimages presented in this work were recorded using a ConfocalRaman Microscope (alpha 300R from WITec GmbH, Ulm, Ger-many) equipped with three laser lines (532 nm from Nd-YAGlaser, 633nmfromHe–Ne laser and785nmfromdiode laser) andtwo ultrahigh throughput spectrometers (UHTS 300) for visibleand near-infrared (NIR) Raman light analysis, respectively.

The cells sandwiched between the two glasses were mountedon a piezolelectric scanning stage. A 100� oil immersionobjective (Nikon Fluor), with a numerical aperture (NA) of 1.25and a working distance (WD) of 0.23 mm was used to performall spectroscopic measurements from live cells. The Ramanbackscattered light collected through the objective was passedthrough a holographic edge lter, before being focused onto amultimode optical ber of 100 mm diameter which provides theoptical pinhole for confocal measurement. The light emergingfrom the output optical ber was analyzed by ultrahighthroughput spectrometers equipped with a back-illuminateddeep-depletion 1024 � 128 pixel CCD camera operating at �60�C, either in visible (DV401-BV, Andor) or NIR (DU401-BR-DD,Andor). The individual Raman and SERS spectra collected fromspecic spots in living cells were acquired with all three laserlines (532 nm, 633 nm and 785 nm) at a power ranging from0.77 to 10mW and an integration time per spectrum of 10 s. TheSERS spectra at different z-depth positions from the cell surfacemembrane were recorded with a 532 nm laser excitation, at apower incident on the sample of 3 mW, and an integration timeof 0.5 s. The experiment was performed by focusing the laserbeam on the cell surface selected at a position z ¼ 0 mm andrecording several spectra at different z-depth positions insidethe cell. The Raman and SERS cell maps were acquired by usinga piezo-electrically driven scan stage. The cells cultured on thebottom side of the cover slip were scanned through the laserfocus in a raster pattern. The spectroscopic cell imaging was

Nanoscale, 2013, 5, 6013–6022 | 6015

Fig. 1 UV-Vis-NIR extinction spectra of colloidal Chit-AgNTs: (a) as prepared and(b) p-ATP labeled nanoparticles. The arrows indicate the laser excitation lines. Theinset shows a representative TEM image of Chit-AgNTs.

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performed over a 40 � 40 mm2 area (75 � 75 pixels) by choosingan integration time of 0.5 s for each spectrum to prevent sampledegradation at the focal point. The cells were excited using the532 nm laser line. With the above parameters about yminutes were necessary for a complete image acquisition. Thereference SERS spectra of p-ATP labeled Chit-AgNTs wererecorded directly from the colloidal solution through a 20�objective (NA ¼ 0.4), employing for excitation the 532 nmwavelength from a Nd-YAG laser (�5 mW power on sample).The integration time was set at 10 s per spectrum. The WITecProject Plus soware was used for spectral analysis and imageprocessing. The chemical images were computed from the two-dimensional collection of Raman/SERS spectra by integratingthe intensity of a specic band over a dened wavenumberrange aer baseline subtraction. For instance, we used the bandat 2800–3100 cm�1 assigned to C–H stretching vibration of themembrane lipid bilayer to image the cell morphology and theband at 1596 cm�1 assigned to C–C stretching vibration of the p-ATP molecule to image the distribution of nanoparticles insidecells. Besides the univariate image generation described above,multivariate image generation was used to further distinguishbetween components in a cell. Specically the K-mean clusteranalysis algorithm allows an independent clustering of therecorded spectra. A detailed description of this method can befound in ref. 29. Reected-light bright-eld optical images ofliving cells were captured with a color video camera attached tothe eyepiece output of the same microscope using for illumi-nation a super-bright white LED source.

Results and discussionCharacterization of p-ATP labeled Chit-AgNTs

The optical extinction spectrum of as prepared Chit-AgNTs inFig. 1a exhibits characteristic LSPR bands corresponding tonanoparticles of triangular shape, specically two dipolar reso-nances at 605 nm (in-plane) and 410 nm (out-of-plane) and twoquadrupolar resonances at 445 nm (in-plane) and 338 nm (out-of-plane).30

The overall optical extinction around 410–445 nm is broaderthan expected for pure triangular nanoplates, and a certaincontribution originating from the plasmon resonances of othershapes can be assumed. However, the occurrence of the plas-mon quadrupolar mode indicates a sufficiently high concen-tration of nanoprisms with relatively narrow particle size andshape distributions as previously reported.31 Fig. 1b illustratesthe spectrum of Chit-AgNTs aer incubation with p-ATP mole-cules. The decrease in the intensity of the in-plane dipolarplasmonic band with a concomitant red shi (12 nm) can beexplained by a local change in the refractive index aer theadsorption of reporter molecules. As previously reported, thep-ATP Raman marker is strongly adsorbed on silver nano-particles through its thiol group, while the chitosan shellprovides colloidal stability for encoded nanoparticles.26 Conse-quently, the resulting SERS nanotags are very stable in differentliquid environments. Size and shape of the silver nanoparticleswere investigated using TEM measurements. The inset in Fig. 1shows a representative TEM image which reveals individual

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silver nanoparticles mainly of triangular and truncated-trian-gular shapes with an average edge length of 65 � 10 nm. Thesuccessful use of p-ATP labeled Chit-AgNTs for imaging livingcells proves their robustness under simulated biologicalconditions. Unlike spherical nanoparticles which possess thelowest surface energy, thus being the most stable, anisotropicnanoparticles exhibit crystallographic facets with differentsurface energies. The energy differences between crystallo-graphic planes can cause nanoparticle etching and a conse-quent change in particle shape. This in turn modies theplasmonic resonances of nanoparticles and promotes therelease of silver ions which can contribute to the overall toxicityof the colloidal suspension.32,33 Therefore, the size and shapestability of anisotropic nanoparticles should be examined underdifferent physical and chemical conditions. We have alreadydemonstrated the very good stability of colloidal Chit-AgNTsduring storage, under conditions of different ionic strengthsand aer transfer to various aqueous media.26 Here we investi-gated the colloidal stability around the physiological tempera-ture of 37 �C. We found that the original extinction spectrum isfully recovered at temperatures between 19 �C and 58 �C andaer cooling down back to 19 �C. However, a reversible spectralshi (2 nm) in the LSPR band positions is observed which couldbe explained by the variation of the refractive index of water as afunction of temperature (see Fig. S1 in the ESI†).

Previous studies have demonstrated the relevance of nano-particle surface charge in their internalization and cell prolif-eration.34 In our case we found that the potential of Chit-AgNTsincreases from +36 mV to +47 mV aer the adsorption of p-ATP.As uptake efficiency is correlated with the positive surfacecharge, p-ATP labeling results in a more efficient uptake.

Cell viability assays

A key issue for a contrast agent to be suitable for spectralimaging of living cells is that it must be compatible with bio-logical environments. Therefore, we rst performed in vitro cell

This journal is ª The Royal Society of Chemistry 2013

Fig. 2 (A) Cytotoxicity assay of A549 cells incubated for 24 h with increasingconcentrations of Chit-AgNTs and p-ATP labeled Chit-AgNTs. (B) Proliferationassay of A549 cells incubated for 24 h with various concentrations of Chit-AgNTsand p-ATP labeled Chit-AgNTs. Values obtained for cells incubated in the absenceof nanoparticles were set as 100%.

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viability assays to evaluate the cytotoxic effects of Chit-AgNTson A549 cells. Cells were incubated for 24 h with increasingconcentrations of either Chit-AgNTs or p-ATP labeled Chit-AgNTs.

As shown in Fig. 2A, no cytotoxic effect could be observed.This nding is in accordance with numerous studies reportingthat chitosan provides a highly biocompatible coating to metalnanoparticles.35 As comparable results were obtained with andwithout p-ATP labeling we conclude that the Raman reportermolecules were chosen below any potentially harmfulconcentration. To investigate the effects of Chit-AgNTs on cellproliferation, A549 cells were incubated with increasingconcentrations of Chit-AgNTs ranging from 4 to 10 mg ml�1 for

Fig. 3 Top: bright field images of individual A549 cells incubated for 2 h in the abBottom: (D) Raman spectra collected from a single A549 cell without nanoparticles, rthe laser power was 10 mW. (E) SERS spectra collected from a single A549 cell incubaexcitation line was 532 nm and the laser power was 0.77 mW. (F) SERS spectra collecthe positions marked in image (C). The excitation line was 532 nm and the laser powecolloidal solution is presented in Fig. 3F (the spectrum marked by *). The excitationsolution was recorded through a 20� objective. The different collection efficiency madifficult since they were recorded through a 100� oil immersion objective.

This journal is ª The Royal Society of Chemistry 2013

24 h. No anti-proliferative effect of Chit-AgNTs (with or withoutp-ATP) was observed, at any of the concentrations tested(Fig. 2B). Moreover, the results showed that in comparison tocontrol cells, the presence of Chit-AgNTs stimulated the growthand proliferation of A549 cells. A previous report in the litera-ture suggested that the chitosan biopolymer enhances cellproliferation.36 In addition, a great number of publicationsdemonstrated the high medical potential of chitosan. Thesestudies showed that chitosan is a non-toxic, biodegradable,biocompatible material that can be used in various biomedicalapplications such as drug delivery, wound dressing, tissueengineering, skin substitutes, nerve regeneration, hemostaticaction, implants, antibacterial coating, etc.37,38 Thus, the use ofchitosan for nanoparticles stabilization offers not only theadvantage of biocompatibility, but also the possibility to inte-grate multiple functions onto the same plasmonic platform,such as combined therapeutic and diagnostic abilities. There-fore, not only a fundamental understanding of the interactionbetween this class of nanoparticles and cells but also thenanoparticles' localization inside cells is crucial for such futureapplications and represents the main objective of this study.

In vitro characterization of p-ATP labeled Chit-AgNTs

For in vitro characterization of p-ATP labeled Chit-AgNTs, thecells were incubated at different time intervals (2, 6 and 24hours) with nanoparticles (6 mg ml�1). Control cells were

sence (A), in the presence of Chit-AgNTs (B) and of p-ATP labeled Chit-AgNTs (C).ecorded at the positions marked in image (A). The excitation line was 532 nm andted with unlabeled Chit-AgNTs, recorded at the positions marked in image (B). Theted from a single A549 cell incubated with p-ATP labeled Chit-AgNTs, recorded atr was 0.77 mW. For comparison, the SERS spectrum of p-ATP labeled Chit-AgNTs inline was 532 nm and the laser power was 5 mW. The spectrum (*) in colloidal

kes comparison of this spectrumwith the other spectra in the figure quantitatively

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Table 1 Assignments of the major Raman and SERS bands of A549 cells

Raman(cm�1)

SERS(cm�1) Tentative assignment40,41

756 763 Ring breathing DNA/RNA bases856 — Tyrosine (deformation CCH aliphatic), tyrosine

(ring), proteins928 933 Proline (ring stretching CC)1008 — Phenylalanine (ring breathing)1076 1082 Phosphate (stretching PO2, stretching CC,

stretching COC) glycosidic link1134 1134 Proline— 1161 Stretching (CC, CN), rocking CH3

1265 1270 Amide III, deformation CH2, CH3, CH lipids1312 1312 Adenine, proteins (stretching ring), twisting CH2,

CH3

1453 1450 Lipids, proteins (deformation CH2, CH3)— 1530 Guanine1589 1605 Phenylalanine (deformation CC), proteins1664 1664 Amide I (stretching CO coupled with deformation

NH)1750 1750 Lipids (stretching CO)2728 — Lipids (symmetric stretching CH2, CH3)2856 — Lipids and proteins (asymmetric stretching CH2)2890 2875 Lipids and proteins (asymmetric stretching CH2)2939 2920 Stretching CH3014 — Lipids (stretching HCC)

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cultured under identical conditions in the absence of nano-particles. Fig. 3 shows three bright-eld optical images repre-senting the reference cells cultured in the absence ofnanoparticles (A) and the cells incubated for 2 hours with Chit-AgNTs (B) and p-ATP labeled Chit-AgNTs (C), respectively.

The bright spots in images can be related to metallic nano-particles known as strong light scatterers relative to backgroundlight scattered by subcellular structures as nucleus and mito-chondria. It is noteworthy that the bright spots appear mainlygrouped in a clustered pattern located outside of subcellulararea assigned to nucleus, in accordance with other reports fromthe literature.39 There is, however, a drastic difference betweenthe images in Fig. 3B and C. Specically, in Fig. 3B an importantnumber of bright spots are also localized outside the cell whilein Fig. 3C almost all spots are inside the cell. To check thereproducibility of this result the experiment was repeated threetimes by comparing 6 different samples identically prepared (3samples incubated with Chit-AgNTs and 3 with p-ATP labeledChit-AgNTs). More than 90 individual cells (�15 cells in eachsample) were analyzed and bright eld microscopy imagesrevealed similar features (see Fig. S2 in the ESI†). Thisdemonstrates a more efficient uptake of p-ATP labeled Chit-AgNTs, which is not a surprising result considering their higherpositive surface charge as conrmed by zeta potentialmeasurements. Similar distribution of nanoparticles in cellswas found aer longer incubation interval (6 and 24 hours, datanot shown). As the uptake of Chit-AgNTs by the cells was clearlyproven by the bright eld images, we examined if enhancedRaman signals could be collected and the nanoparticles used asreliable SERS contrast agents. Fig. 3D shows some examples ofordinary (unenhanced) Raman spectra collected from differentlocations inside a single A549 cell free of nanoparticles (seeFig. 3A). The tentative assignment of vibration Ramanngerprints of the A549 cell is given in Table 1 which is ingood agreement with other similar data published in theliterature.40,41

To test the reproducibility, several single A549 cells wereanalyzed and there was no signicant change in the spectralpositions of tabulated Raman bands. The major vibrationalbands are consistent with themain chemical components of theA549 cell. For instance the prominent bands located at 2856 and2939 cm�1 originate from C–H symmetric and asymmetricvibrations of lipids in the cellular membrane.40 The band at1664 cm�1 can be attributed to the protein vibration mode(amide I, C]O stretching vibration).40,41 However, the intensityprole of recorded bands slightly differs from point to pointwithin the cell, depending on the prevalence of intracellularcomponents. For instance the spectra 1 and 2 in Fig. 3D showmore intense bands associated with nucleus and proteins whilethe spectrum 3 shows more intense bands associated withlipids in the membrane. When we proceeded to record Ramanspectra from the cell incubated with non-labeled Chit-AgNTs werst reduced the laser intensity by 10–15 times to the level whereno signal could be detected, from regions free of nanoparticles(see spectrum 1 in Fig. 3E corresponding to spot 1 in Fig. 3B).However, under similar conditions but with the laser moved toa bright spot, a typical Raman spectrum was recorded (see

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spectrum 2 in Fig. 3E corresponding to spot 2 in Fig. 3B) withseveral bands assignable to cellular constituents (see Table 1).This is a key result indicating that the Raman signal generatedfrom the intracellularly localized Chit-AgNTs is surfaceenhanced, namely a true SERS spectrum. It is obvious that somevibrational bands appear to be spectrally shied with theirintensity ratios modied in comparison with the ordinary(unenhanced) Raman bands (Fig. 3D) due to orientation ofchemical groups relative to the metal surface. The SERS activityof nanoparticle edges and tips as well as cluster–cluster aggre-gations inside the cell is believed to be the most efficient “hotsites” for Raman enhancements. We assume that the packingdensity of chitosan at the surface of particles hinders the directinteraction between cellular components and the metal core,but allows chemical groups in proteins or lipids to come in closevicinity with the metal surface. Thus, unlabeled Chit-AgNTs canoperate as SERS nanoprobes to deliver spatially localizedchemical information from the cellular environment at a verylow laser power which is good for imaging purpose. In addition,the ability of chitosan to provide a separation between the silversurface and cellular components makes Chit-AgNTs a highlybiocompatible class of spectral contrast agents. A similar resultwas recently reported by Sivanesan et al.42 They found that thechitosan biopolymer allows the partial diffusion of proteins intoits layer, but not the direct interaction with the silver core.

Fig. 3F illustrates three representative SERS spectra recordedfrom distinct points in a single A549 cell incubated with p-ATPlabeled Chit-AgNTs at an identical laser power as used in theprevious experiments (see the corresponding spots in Fig. 3C).By comparison with the reference spectrum of p-ATP recordedin colloidal solution in Fig. 3F, the main intense bands at 1093

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cm�1, 1596 cm�1 and 1171 cm�1 are assigned to the vibrationmodes of p-ATP, according to the literature.43 The strong anddistinct SERS spectrum of p-ATP attached to silver nanoparticlesmakes p-ATP labeled Chit-AgNTs suitable as contrast agents.However, the SERS enhancement is not limited to the p-ATPmolecules but is also experienced by the molecular groups inthe vicinity of the nanoparticles. Thus, the composite SERSspectra present vibrational information from the cell compo-nents in addition to the SERS signature of p-ATP. As for examplethe bands located at 2890 cm�1 and 2939 cm�1 originate fromthe lipid components of the cell.40 It is noteworthy that the lipidcontribution from cell components relative to the p-ATPcontribution in the composite SERS spectra of pATP-AgNTsinside the cell is lower than the contribution of the lipid bandsobserved in the case of non-labeled Chit-AgNTs relative to thecellular bands in the 900–1700 cm�1 spectral region anddepends on the degree of co-adsorption of both kinds ofmolecules to the hot-spots.

Further evidence of nanoparticle internalization wasprovided by recording the SERS spectra of labeled nanoparticlesat different z-depth positions from the cell surface membrane,at a specic local point inside the same cell (Fig. 4).

In order to obtain in depth information about the localiza-tion of the nanoparticle inside cells we adjusted the laser powersuch as to be able to obtain both the Raman response from thecellular constituents (lipids bands at 2890 cm�1 and 2939 cm�1)and the SERS response from the particles. We selected the lipidbands because these bands are most prominent and are presentthroughout the cell (Fig. 3D). The occurrence of the bandscharacteristic of the lipid components solely at the position z ¼0 mm (Fig. 4) corroborated with the microscopic le inset imagein Fig. 4, suggest that the particles are not located on the cellmembrane surface. Discrete but obvious variations of the SERS

Fig. 4 Selected SERS spectra of p-ATP labeled Chit-AgNTs in a single living A549cell at different z-depth positions (0, �3, �5 and �8 mm). The spectra werecollected using a 532 nm excitation at the location marked in the microscopicimage. The laser power was 3 mW and the integration time was 0.5 s. The insetsshow bright field microscopic images of the A549 cell at the relative position of:left: 0 mm (cell surface) and right: 5 mm inside from the cell surface.

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nanotags signal from one spectrum to another could beobserved at different z-depths. This proves that the Raman tagsare located not on a xed position on the cell surface but theycan be accurately detected at a 3D level inside cells thereforebeing able to operate as SERS contrast agents for spectralimaging inside living cells.

SERS imaging of living A549 cells with p-ATP labeled Chit-AgNTs

By combining vibrational spectroscopy with sample scanningthrough the laser focus in a raster pattern, confocal Ramanmicroscopy facilitates the investigation of molecular composi-tion and structures without requiring any external labels.XY-plane slices of a structure at different z-depths can beimaged at a submicron lateral resolution which is comparablewith confocal uorescence microscopy and exceeds the resolu-tion of various histological techniques.40 Moreover, confocalRaman microscopy has the advantage of high specicity,sensibility and optical stability of the used contrast agents. Oneof the main aims of our study was to demonstrate the ability ofp-ATP labeled Chit-AgNTs to function as contrast agents forSERS imaging of living cancer cells. Besides their intrinsicRaman signature, the capability of the particles to generateamplied Raman and SERS signals from native biologicalmolecules gives us the possibility to obtain precise informationabout the localization of the nanoparticle inside cells. This canbe achieved by combining simultaneous SERS and Ramanimaging of the same living A459 cell at one single z-plane whichminimizes the exposure time. A laser excitation line in thespectral region close to the absorption of cell componentsseems to be the best choice for such in vitro experiments. We,therefore, selected the 532 nm laser excitation line and adjustedthe power at 6 mW, such as to be able to obtain both the Ramanresponse from the cellular constituents and the SERS responsefrom the particles. The Raman maps of one selected cell incu-bated in the presence of p-ATP-labeled nanoparticles are pre-sented in Fig. 5 (images B, C and D).

Image (B) in Fig. 5 was constructed from the C–H stretchingvibration of lipids (2800–3100 cm�1) and resembles closely themicroscopic image of the cell shown in (A). By plotting theintensities of the most prominent band at 1536 cm�1, which ischaracteristic for the labeled nanoparticles, sufficient contrastwas obtained to facilitate visualization of the nanoparticledistribution inside the cell (Fig. 5C).

The majority of the particles appear to be localized in theperinuclear region of the cell, most probably in the endo-plasmatic reticulum and inside late endosomes. No particleswere detected inside the nucleus. This is consistent with theinternalization results described above.

As stated above, we were also interested in discriminatingbetween the various subcellular components at the z-planewhere particles are localized. Several multivariate methods arerecently used for the extraction of such reliable spectral infor-mation.29 By applying K-means cluster analysis we were ableto visualize internalized nanoparticles, to determine topologicaldistribution of nanoparticles inside the cell and more

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Fig. 5 (A) Bright-field optical image of a single A549 cell incubated with p-ATPlabeled Chit-AgNTs. (B and C) Raman maps of the cell illustrated in image (A). Themaps were generated by plotting the intensity of a characteristic Raman peakover the scanned area. The basic cellular structure (B) was visualized using thepeak at 2800–3100 cm�1 (C–H stretching vibrations of lipids). The nanoparticlelocalization (C) was obtained using the peak at 1536 cm�1 (C–C stretchingvibration of p-ATP molecule). (D) Map obtained by multivariate data analysisalgorithms (K-means cluster analysis). This algorithm allows the discriminationbetween the cell body (dark yellow), nucleoli (yellow), mitochondria (blue) and p-ATP labeled Chit-AgNTs (individual and small clusters – red; big cluster – pink). (E)Corresponding extracted spectra from distinctly colored areas in image (D). Theexcitation line was 532 nm and the laser power was 6 mW.

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importantly, identify multiple subcellular components. Thecolor-coded image constructed by using 6 clusters and thecorresponding extracted spectra are presented in Fig. 5D and E.Few nanoparticle-arrangements, hardly visible at opticalmicroscopy resolution are visualized in the color coded Ramanimage in red while bigger ones are coded in pink (image (D)).The two types of nanoparticle patterns also evidenced somespectral differences: composite bands of reporter moleculesand of cellular components were obtained for small nano-particle-clusters while only p-ATP bands were obtained for thebigger ones. Further analysis of the cellular ngerprint regionfrom 750 to 1800 cm�1 revealed discrete spectral features thatcould be assigned to specic subcellular components. Nucleusand nucleoli (yellow) or heterogeneously distributed mito-chondria (blue) could be visualized at a resolution comparableto that of uorescence microscopy. Taken together, the mapgenerated through K-means cluster analysis provides additionalproof of efficient nanoparticle internalization by cells andfacilitates the establishment of their localization at the intra-cytoplasmic level.

Fig. 6 SERS spectra of p-ATP labeled Chit-AgNTs in a single A549 cell collectedwith different laser excitation wavelengths: (a) 532 nm; (b) 633 nm and (c) 785nm. The spectra were collected at the location indicated in themicroscopic image.The inset shows a bright field microscopic image of a single A549 cancer cell.

SERS active nanotags from visible to NIR excitation

A real advantage of nanoparticle-tags would be their SERSactivity extended to multiple laser excitation lines from visibleto NIR biological optical window. Taking into consideration theversatile optical properties of Chit-AgNTs, e.g.multiple plasmonresonances and enhanced local elds at tips and edges we

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assume that labeled Chit-AgNTs can function as SERS activenanotags inside cells under multiple wavelengths laser excita-tion.26 Indeed we successfully recorded SERS spectra of inter-nalized p-ATP-labeled Chit-AgNTs at three different laserexcitation: from visible (532 and 633 nm) to NIR (785 nm)(Fig. 6). The analysis of the spectra reveals amostly reproducibleresponse at each wavelength excitation. The specic p-ATP SERSngerprint is persistent in each spectrum, providing strongsupport that nanoparticles can be identied at any laser exci-tation inside living A549 cancer cells. We mention that at 785nm excitation the collection efficiency of our setup decreasesstrongly at wavenumbers beyond 1400 cm�1, this being thereason why the p-ATP band at 1536 cm�1 is barely visible. Asusually nanoparticles gather in endosomal compartmentsinside cells, we cannot exclude the formation of small aggre-gates. In this case the superiority of triangular nanoparticlestoward randomly shaped particles could be questionable, sinceaggregated particles are also a source of high SERS intensitiesand the wavelength of the exciting light becomes tunable. Toverify this, a mirror experiment was performed by investigatingthe response of p-ATP labeled spherical silver nanoparticleswith a mean diameter of 18–20 nm together with the triangularones.

As the NIR availability of any SERS contrast agent is imper-ative for in vivo applications, we focused our experiment on785 nm laser excitation. Multiple SERS spectra were collectedfrom randomly sized bright spots localized inside cells (seeFig. S4 in the ESI†), giving each time higher intensities in thecase of triangular nanoparticles than for spherical ones. More-over, the limited SERS sensibility of spherical nanoparticlesunder NIR excitation made impossible the collection of thespectral signal from smaller bright spots of small dimers orindividual nanoparticles. We believe that the study presentedhere is consistent with demonstration of practical superiority ofanisotropic nanoparticles as SERS contrast agents. However, adecisive comparison should take into consideration several

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unknown parameters which are difficult to investigate sepa-rately, as for instance the number of reporter molecules pernanoparticle, the fraction of nanoparticles internalized by thecells (which is a shape dependent property), nanoparticle sizeand the degree of their aggregation. Based on these experimentswe anticipate that p-ATP labeled Chit-AgNTs are reliablecontrast agents for intracellular applications.

Conclusions

Herein we proposed a new class of biocompatible SERS nano-tags based on p-ATP labeled Chit-AgNTs and demonstratedtheir applicability as multi-responsive contrast agents for SERSimaging of living cancer cells. Specically, we demonstratedthat this kind of nanoparticle provides a strong, distinct SERSsignal inside A549 lung cancer cells under a wide window ofexcitation wavelengths. Responding from visible to NIR, p-ATPlabeled Chit-AgNTs overcome the limited availability at the NIRregion of spherical nanoparticles. In addition to the signature ofthe reporter molecule, our nanotags enable us to acquiresensitive molecular information on cellular components thusgiving us the possibility to obtain multiple information, bothabout nanoparticle localization and specic interaction insidecells. The promising results presented here open perspectivesfor integrating a therapeutic agent onto these imaging probes,hence designing a theranostic agent.

Acknowledgements

This work was supported by CNCSIS-UEFISCSU, project numberPNII-ID_PCCE_129/2008. The authors are grateful to Dr LucianBarbu Tudoran for TEM measurements and to Dr Imre LucaciFlorica for atomic absorption measurements.

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