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Biomaterials 34 (2013) 8344e8351

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Nanocarriers with ultrahigh chromophore loading for fluorescencebio-imaging and photodynamic therapy

Julien R.G. Navarro a, Frédéric Lerouge a, Cristina Cepraga a,b,c, Guillaume Micouin a,Arnaud Favier b,c, Denis Chateau a, Marie-Thérèse Charreyre b,c, Pierre-Henri Lanoë a,Cyrille Monnereau a, Frédéric Chaput a, Sophie Marotte b,c,e, Yann Leverrier e,Jacqueline Marvel e, Kenji Kamada a,d, Chantal Andraud a, Patrice L. Baldeck a,f,Stephane Parola a,*

a Laboratoire de Chimie UMR 5182, Ecole Normale Supérieure de Lyon, CNRS, université Lyon 1, 46, allée d’Italie, Lyon cedex 07 F-69364, Franceb École Normale Supérieure de Lyon, Laboratoire Joliot-Curie, USR CNRS3010, Lyon F-69364, Francec INSA-Lyon, Laboratoire Ingénierie des Matériaux Polymères, UMR CNRS UMR5223, Villeurbanne F-69621, FrancedResearch Institute for Ubiquitous Energy Devices, National Institute of Advanced Industrial Science and Technology, AIST Kansai, 1-8-31 Midorigaoka,Ikeda, Osaka 563-8577, Japane INSERM, U851, Université Lyon1, 21, Avenue Tony Garnier, Lyon F-69007, FrancefUniversité Grenoble 1/CNRS, LIPhy UMR 5588, Grenoble F-38041, France

a r t i c l e i n f o

Article history:Received 13 June 2013Accepted 8 July 2013Available online 31 July 2013

Keywords:GoldNanocarriersBio-imagingFluorescencePDT

* Corresponding author. Tel.: þ33 (0) 4 72 44 81 67E-mail address: stephane.parola@univ-lyon1.fr (S.

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.07.032

a b s t r a c t

We describe the design of original nanocarriers that allows for ultrahigh chromophore loading whilemaintaining the photo-activity of each individual molecule. They consist in shells of charged biocom-patible polymers grafted on gold nanospheres. The self-organization of extended polymer chains resultsfrom repulsive charges and steric interactions that are optimized by tuning the surface curvature ofnanoparticles. This type of nano-scaffolds can be used as light-activated theranostic agents for fluores-cence imaging and photodynamic therapy. We demonstrate that, labeled with a fluorescent photosen-sitizer, it can localize therapeutic molecules before triggering the cell death of B16-F10 melanoma withan efficiency that is similar to the efficiency of the polymer conjugate alone, and with the advantage ofextremely high local loading of photosensitizers (object concentration in the picomolar range).

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Compared to small molecules, nanoparticles (NPs) presentnumerous advantages such as the possibility to increase drasticallythe amount of carried agents, to introduce multi-modality bycombining different agents, or to improve targeting using specificmoieties [1e4]. They also allow selective accumulation of nano-particles loaded with therapeutic molecules in tumor tissues bythe Enhanced Permeability and Retention (EPR) effect [5e8].Typical nanocarriers are polymeric micelles, liposomes, den-drimers, porous silica particles, metal oxide particles, metalnanoparticles [9]. The loading capacity is a crucial factor regardingimaging resolution and therapeutic efficiency. Depending on the

.Parola).

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size of the nanoparticles (NP), it varies from few hundreds tothousands of molecules, making NP attractive nano-scaffolds [10].In the specific case of photosensitizing drugs, interactions withoxygen and water in the bio-environment are primordial. Thus,localization of the photosensitizers at the surface of the nano-object and not inside should be preferred. This makes core-shellnanoparticles, with the drug located in the outer shell, inter-esting scaffolds in that regard.

Gold NP have attracted particular attention due to their ease ofsynthesis, biocompatibility, chemical stability and optical proper-ties [11e18] and surface functionalization that can be achievedwitha large library of organic molecules [19e22], including proteins[23e25] and DNA [26e28]. However, the interaction of a photo-active probe with a metal surface may strongly modifies its pho-tophysical properties [20,29]. The interaction depends on severalparameters such as the NP size and shape, the chromophore toparticle distance and orientation and the spectral overlap of the

J.R.G. Navarro et al. / Biomaterials 34 (2013) 8344e8351 8345

chromophore with the surface plasmon band [30e37]. In mostcases, small chromophore to surface distances (<10 nm) lead tofluorescence quenching [38], and reduced singlet-oxygen genera-tion. Previously, fluorescent hybrid gold nanoparticles have beenreported with spacer layers based on DNA [30,32,37], poly-electrolyte [31,39,40] or silica shell deposition [41,42].

In this paper we report on a novel nanoscaffold strategy basedon thiol-terminated charged block copolymers, labeled with photo-activemolecules, which are grafted on spherical gold NP presentinghigh curvature angles. The presence of the charges along thepolymer chains induces electrostatic repulsions, which, combinedwith steric hindrance, lead to an extension of the chains that favorsthe positioning of the chromophores away from the gold surface(Fig. 1), in a structure reminiscent of a “hedgehog”. As a firstapproach, the polymer was labeled with Lucifer Yellow (LY), afluorescent dye that is commonly used in cell biology [43], thatallowed to achieve ultrahigh chromophore loading without sig-nificant quenching. Then, we extended our study to a dibromo-benzene based chromophore (DBB), an efficient photosensitizerwith significant residual fluorescence allowing both in vitro fluo-rescence imaging and efficient photodynamic therapy (PDT) onB16-F10 melanoma.

2. Material and methods

2.1. Materials

Tetrachloroauric acid (HAuCl4, 3H2O, 99.9%), sodium citrate tribasic dehydrate,acetylacetone, potassium cyanide, ruthenium (IV) chloride and sodium per-iodatewere obtained from SigmaeAldrich and used as received. Dimethylformamide(DMF: Fischer Scientific Analytical Reagent Grade, 99.99%), diethyl ether (Et2O:ACROS,>99%) were used as received. LYCadaverine is a negatively chargedmoleculeand was purchased from Interchim (FluoProbe� dye) as a potassium salt. 4-(2-aminoethylmorpholine) was purchased from Aldrich (99%). All suspensions were

Fig. 1. Schematic representation of the luminescent polymer structure depending on the paaround the particle in green (For interpretation of the references to color in this figure leg

synthesized in milliQ water. Reactors were cleaned using aqua regia. Absorptionspectra were recorded using a PerkineElmer UV-Vis-NIR Lambda 750 spectrometer.Transmission electron microscopy (TEM) images for gold nanoparticles were ob-tained using a TOPCON EM-002B microscope (80 & 120 kV).

2.2. Synthesis of the luminescent polymer

The poly(NAMeco-NAS) copolymers was synthesized by the RAFT polymeriza-tion technique using the synthetic procedure described elsewhere [44]. The poly(-NAMeco-NAS) were then react with the Lucifer Yellow. In a round bottom flask of25 mL equipped with a magnetic stirrer were sequentially added: the copolymersolution in DMF (10mg/mL), LY chromophore solution inwater (water/DMF¼ 10/90,v/v), and triethylamine (TEA) (2 molar equivalents with respect to LY chromophore).The reaction mixture under vigorous stirring was heated at 50 �C in a thermostatedoil bath during 48e96 h, until reaching the maximum chromophore to polymercoupling yield (calculated by SEC/UV). Purification of LY-conjugates was carried outby precipitation in diethyl ether. LY-polymer conjugates, insoluble in diethyl ether,precipitated, leaving in solution a yellow supernatant (the color of the free chro-mophore). Several precipitation-centrifugation cycles were repeated until completediscoloration of recovered supernatant, thus complete removal of the free unreactedchromophore.

2.3. Synthesis of the sodium acetylacetonate

Sodium hydroxide (1.6 g) was dissolved in water (2 mL). The solution was thendiluted with 8 mL of EtOH. The solution was then added to the acetylacetone (4 g).The white precipitate were filtered and washed with acetone. Recrystallization inacetone: EtOH. The white powder was dried over vacuum for 5 h.

2.4. Synthesis of gold nanoparticles

Spherical citrate-stabilized gold nanoparticles (xCS-AuNP), with an averagediameter of 13 nme90 nm were obtained following a modified Turkevich citrate-reduction procedure.

Briefly, a solution of sodium acetylacetonate (327mM) and sodium citrate (8mM)were separately prepared. 360 mL of the sodium acetylacetonate solution wereadded to the solution of sodium citrate (30 mL). A solution containing chloroauricacid (HAuCl4, 0.3 mM, 95 mL) is boiled. The appropriate volume (30e200 mL) ofNa(Acac) is added under vigorous stirring to the boiling solution, immediately

rticle size and curvature angle. The gold core is in yellow, the plasmon influence areaend, the reader is referred to the web version of this article.).

Fig. 2. Structure of the luminescent water-soluble LY-labeled block copolymer.

J.R.G. Navarro et al. / Biomaterials 34 (2013) 8344e83518346

followed 10 s later by the fast addition of 5 mL of the prepared mixture of Na(Acac)and sodium citrate. The initial yellow solution rapidly turns red which indicate thetransformation from a dissolved salt to gold nanoparticles. The gold nanoparticlessizes can be tuned by simply adjusting the sodium acetylacetonate volume (30 mLe200 mL). The resulting suspension was characterized using UV/Vis spectroscopy,dynamic light scattering and TEM microscopy.

2.5. Functionalization of the gold nanoparticles

To suspension of gold nanoparticles (9 mL, 1 equivalent) was slowly added, dropby drop, the diluted polymer solution (1 mL, 5 equivalents/binding site) understirring. The solution was then allowed to warm, in the dark at 75 �C for 12 h. Thefunctionalized gold nanoparticles were then purified thought several centrifugationcycles (8000 RPM, 20 min). The supernatants, composed of unbound fluorescentdye, were carefully removed and replaced with fresh water. The addition of waterallowed a dispersion of the concentrated gold nanoparticles, localized in the bottomof the centrifugation tube. The purifications were continued until a clear superna-tant was observed. Moreover, each of the supernatants was analyzed with UV-Visible spectroscopy to ensure the disappearance of the chromophore absorptionband.

2.6. Dissolution of the gold nanoparticles core

The gold nanoparticles are dissolved by a cyanide solution (KCN, 100 mM).The cyanide oxidizes the nanoparticles, in presence of oxygen, to form a gold-

Table 1Summary of the polymer-LY and polymer-DBB conjugate characteristics.

Conjugate Mna PDI NAM units/chain A

Polymer-LY 32400 1.1 139 5Polymer-DBB 33800 1.2 173

a Mn is expressed in g mol�1.

Fig. 3. TEM of 13 nm (left), 50 nm (middle

cyanide complex. The gold core dissolution was followed with UV-Visiblespectroscopy.

2.7. Cell growth and incubation

B16-F10 melanoma cells were cultivated in cell culture medium at 37 �C with 6%CO2 as previously described. B16-F10 melanoma cells were then incubated for 24 hwith increasing concentrations of 90 nm AuNP-DBB. B16-F10 cells were seeded at6� 104 cells mL�1 into 24-well plates, grown over-night and incubated for 24 h withthe indicated concentrations of 90 nm AuNP-DBB.

3. Results and discussion

Gold NP were synthesized in the presence of sodium acetyla-cetonate [Na(acac)] allowing the complexation and reduction ofAuIII (HAuCl4) to AuI immediately followed by the reduction of AuI

to Au0 by the sodium citrate (See SI for details). TEM imagingreveals monodispersed spheres with size ranging of13 nm � 3 nm, 50 � 6 nm and 90 � 8 nm (statistics from 500objects), depending on the experimental conditions. The NP con-centration and the number of thiolate potential binding sites attheir surface were estimated from the UV-Visible absorbance us-ing previously described procedures [19,28,45,46]. Gold NP werefunctionalized with the luminescent polymer by mixing an excess(ca 5 eq./number of binding sites at the surface, see SI) ofmacromolecular ligand (polymer-LY) with the citrate-stabilizedgold NP to cover the entire particle surface. The structure of theluminescent LY-labeled water-soluble block copolymer is shown inFig. 2. The synthesis followed previously reported procedures[44,47]. Characteristics of the prepared polymer conjugates areshown in Table 1.

The resulting nano-objects were purified through severalcentrifugation cycles. The polymer shell thickness was estimatedusing TEM microscopy images (80 kV) using RuO4 as stainingagent [48] for a better contrast between the gold structure and theorganic part (Fig. 3). Polymer shell thicknesses of 2.5, 6.5 and12 nm were estimated for 13 nm, 50 nm and 90 nm gold NP,respectively. Since the TEM images, obtained on dried samples,induce shrinkage of the polymeric shell, the real shell thicknessesin water were estimated from cryoTEM measurements as 5, 13 and24 nm respectively. Interestingly, the increase of the polymer shellthickness with the gold core diameter indicates that the polymerchains adopt a more extended conformation when the curvature

EM units/chain COO� units/chain Chromophore units/chain

1.5 0 5.20 56 4.0

) and 90 nm (right) gold-polymer NP.

Fig.

4.Fluo

rescen

cesp

ectraof

AuN

P-po

lymer-LY(13nm

,50nm

,90nm

)an

dthepo

lymer-LYafterdissolutionof

thego

ldco

re.

J.R.G. Navarro et al. / Biomaterials 34 (2013) 8344e8351 8347

of the NP decreases (increase of NP diameter), going from a folded(13 nm NP) to a stretched-out (90 nm NP) conformation as shownon Fig. 1. This can be compared to the maximal theoretical lengthof the extended polymer which should be about 50 nm.

In order to study the influence of spherical gold NP on thephotophysical properties of the polymer-LY, we used a procedurebased on the dissolution of gold cores by cyanide salts (KCN, seeSI for the exact protocol and calculation details) to restore the LYinitial luminescence, i.e. the free polymer fluorescence in solu-tion. Thus, we can measure the LY concentration in the solution,that determine the number of polymer chain per NP, and we cancalculate the luminescence quenching that results from theinteraction with the metal surface, by comparison between theluminescence signal intensity before and after KCN treatmentthat does not affect the LY luminescence (Fig. 4). For the differentgold particles sizes, 13 nm, 50 nm and 90 nm, the relative fluo-rescent brightness was estimated to be 60%, 71% and 85%,respectively. This confirms that reducing the curvature of thenano-object, increases the polymer extension, and improves itsfluorescence quantum efficiency. Similarly, the particle coveragewas estimated to be 175, 3400, 13,900, polymer chains per NP for13 nm, 50 nm and 90 nm hybrids, respectively. This correspondsto 0.32; 0.43; 0.55 ligand unit per nm2 or else, 1.6; 2.15; 2.5equivalent chromophore per nm2. Thus, the chromophore loadingfor a 90 nm NP could be estimated to be 70,000 units of LY whichis about ten times higher to what can be loaded in a mesoporoussilica particle of same size [10]. Mesoporous silica NP being oftenconsidered as archetypical high-capacity cargoes for nanomedicalapplications, our finding confirms the potential of our newapproach towards multifunctional nano-objects that can be usedas scaffolds for the delivery of ultrahigh loading of chromophoresinto the biological environment. Table 2 summarizes the differentcharacteristics.

Individual nano-objects were characterized by optical micro-scopy. Quantitative images of dark-field scattering emission(Fig. 5a), and epifluorescence (Fig. 5b) with co-localization could beobtained for 50 nm and 90 nm AuNP-polymer-LY. The fluorescenceintensity of individual NP is plotted versus the dark-field scatteringintensity in Fig. 5c. Intensity variation corresponds to size variationof NP. The dark-field scattering intensity of spherical gold NP in-creases proportionally with their surface, thus it can be used as ananosize ruler when calibrated with TEM measurements. Fig. 6displays the fluorescence intensity of single NP as a function oftheir NP diameters, that have been calibrated with their averageTEM sizes 50� 6 nm and 90� 8 nm. The fluorescence intensity hasa general tendency to increase with the NP surface, as shown byquadratic curve fittings. The difference in fitting slopes S90/S50¼ 1.5 is in good correlationwith the expected ratio of 1.4 whichcan be calculated from the difference on their chromophore con-centration per NP surface (2.15 vs 2.5 chromophores per nm2), andon their fluorescence quantum yields (70% vs 85%), for S50 and S90respectively. Overall, this photophysical study with LYallowed us tocharacterize the loading capacity of hedgehog nanocarriers, and tocomprehend how this type of nanoscaffold geometry can be tunedto reduce the influence of the metal core surface on chromophoreproperties.

Our next objective was to adapt this strategy with a chromo-phore suited for light-activated theranostic based on fluorescenceimaging and photodynamic therapy (PDT). DBB is a dibromo-benzene derivative that has both an efficient photo-generation ofsinglet-oxygen (FO2 ¼ 0.45) for PDT, and a significant residualfluorescence (Ff ¼ 0.22) to visualize its localization in target cells[49]. Synthesis of this photosensitizer has been previously re-ported [50]. The procedure used for polymer coupling and NPfunctionalization was similar than for LY, except that the

Fig. 5. a)dark-field and b)fluorescence microscopy images of single gold NP with co-localization of the NP in both modes (yellow circles) c)Plot (logarithmic scale) of fluorescence vsscattering intensity for two batches of AuNP-polymer-LY with different averages sizes (red circle: 50 nm and green triangle: 90 nm) (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.).

Table 2Summary of the photophysic properties of luminescent NP.

AuNP size Quenching/% Relative brightness/% Polymer ligand/NP Eq. number of chromophore/nm2

Polymer-LY 0 100 na na13 nm 40 60 175 1.650 nm 29 71 3400 2.1590 nm 15 85 13,900 2.55

J.R.G. Navarro et al. / Biomaterials 34 (2013) 8344e83518348

remaining activated ester units (N-Acryloxysuccinimide) of thepolymer were hydrolyzed to provide negative charges along thechain to induce the electrostatic repulsion effect. This slightlydiffers from the polymer-LY for which negative charges are

Fig. 6. Emission intensity vs NP diameters, calculated from scattering data, of AuNP-polymer-LY with different average sizes (red circle: 50 nm and green triangle:90 nm). Fit of the data to quadratic functions proportional to NP surfaces (For inter-pretation of the references to color in this figure legend, the reader is referred to theweb version of this article.).

directly provided by the charged fluorophore. The chemicalstructure of the polymer-DBB conjugate and a TEM image of a90 nm AuNP-polymer-DBB functionalized NP with its polymercorona are shown in Fig. 7.

Finally, we explored the ability of the nanocarrier to be inter-nalized by cancer cells, and to induce their death afterphotoactivation. B16-F10 melanoma cells were incubated withAuNP-polymer-DBB and their internalization was visualized byconfocal fluorescence microscopy owing to the residual DBB fluo-rescence (Fig. 7 left). Cellular uptake of 90 nm AuNP-polymer-DBBwas quantified by flow cytometry and found to be dose-dependent(Fig. 7 middle) [51]. We next assessed whether the nano-objectscould induce the death of cancer cells upon one-photon irradia-tion. B16-F10 cells incubated with increasing concentrations of90 nm AuNP-polymer-DBB were irradiated (16 J cm�2 at 365 nm)and cell mortality was assessed 5 h later by flow cytometry. In theabsence of photoactivation, 90 nm AuNP-polymer-DBB were nottoxic for the cells (data not shown). Photoactivation did not inducethe death of untreated cells or cells incubated with control nano-particles that did not carry DBB (AuNP-REF). Importantly photo-activation triggered the death of cells incubated with AuNP-polymer-DBB and this was dose-dependent (Fig. 7 right). Cellmortality after photoactivation was observed with concentrationsof the nano-object as low as 2 � 10�12 mol of nano-object L�1. Thisis very similar in terms to results obtained with the polymer-DBBconjugate in the same sensitizer concentration but with theadvantage of providing EPR effect for targeting and extremely highlocal photosensitizer concentration (Fig. 8).

Fig. 7. Structure of the polymer-DBB conjugate and TEM image of the 90 nm AuNP-polymer-DBB functionalized NP.

Fig. 8. Fluorescence image of B16-F10 cells cultured for 24 h with AuNP-polymer-DBB (6.7 � 10�8 mol of DBB.L�1) (Left). B16-F10 cells were cultured for 24 h without or withincreasing concentrations of 90 nm AuNP-polymer-DBB (respectively 2 � 10�8, 6.7 � 10�8 and 2 � 10�7 mol of DBB L�1). As a control, cells were incubated with AuNP-REF(9 � 10�12 mol of object L�1). Mean fluorescence intensity (Middle) and cell mortality 5 h after photoactivation (16 J cm�2 at 365 nm) (Right) were recorded using flow cytom-etry. Results are expressed as � SD of 2e4 independent experiments.

J.R.G. Navarro et al. / Biomaterials 34 (2013) 8344e8351 8349

4. Conclusions

In conclusion, multi-charged AuNP-block copolymer-chromophores conjugates are efficient nanocarriers for the de-livery of ultrahigh loadings of light-activated theranostic agents forfluorescence imaging and photodynamic therapy within cancercells. The presence of negatively charged units along the polymerchain drives the organization of the polymer corona around the NP,resulting in what can be seen as a “hedgehog” structure. Thisbehavior is dependent on the size of the spherical NP, with thepolymers chains adopting an increasingly stretched-out confor-mation as the radius of the particles increase, as clearly evidenced

with LY-conjugates. Thus, this type of controlled nano-objects is anefficient scaffold geometry to adjust the distance between fluo-rophores and a metallic surface, and to prevent fluorescencequenching by the surface plasmon resonance. Efficiency of thesenano-objects in terms of increased local concentration of photo-active unit, high brightness, improved cell internalization, andfinally photoinduced cell death was evidenced with a AuNP-polymer-DBB conjugate, and it was shown that the resultingnano-object was of similar efficiency than the polymer-DBB con-jugate alone in that respect, with the advantage of carrying largeconcentration of sensitizers with possible targeting using EPR ef-fect. Further works are in progress to optimize those scaffolds by

J.R.G. Navarro et al. / Biomaterials 34 (2013) 8344e83518350

changing the surface topography of the NP and thus the self-organization of the polymers in order to obtain plasmon-enhancement of the optical response of the dyes.

Acknowledgments

This work was supported by grants from French NationalResearch Agency (ANR) P3N project nanoPDT # ANR-09-NANO-027-04 #, INSERM, the Association pour la Recherche contre leCancer, the Ligue du Rhône, the Région Rhône-Alpes, CNRS, Uni-versité Claude Bernard Lyon 1. SM, YL and JM acknowledge thecontribution of the cytometry platform of SFR Biosciences Gerland-Lyon Sud (UMS344/US8).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2013.07.032.

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