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Photodynamic therapy and two-photon bio-imaging applications ofhydrophobic chromophores through amphiphilic polymer delivery

Thibault Gallavardin,a Mathieu Maurin,b Sophie Marotte,c Timea Simon,b Ana-Maria Gabudean,b

Yann Bretonniere,a Mikael Lindgren,a,d Frederic Lerouge,a Patrick L. Baldeck,b Olivier Stephan,b

Yann Leverrier,c Jacqueline Marvel,c Stephane Parola,a Olivier Maury*a and Chantal Andraud*a

Received 16th December 2010, Accepted 21st February 2011DOI: 10.1039/c0pp00381f

The synthesis and photophysical properties of two lipophilic quadrupolar chromophores featuringanthracenyl (1) or dibromobenzene (2) were described. These two chromophores combined significanttwo-photon absorption cross-sections with high fluorescence quantum yield for 1 and improved singletoxygen generation efficiency for 2, in organic solvents. The use of Pluronic nanoparticles allowed asimple and straightforward introduction of these lipophilic chromophores into biological cell media.Their internal distribution in various cell lines was studied using fluorescence microscopy andflow-cytometry following a successful staining that was achieved upon 2 h of incubation. Finally,multiphoton excitation microscopy and photodynamic therapy capability of the chromophores weredemonstrated by cell exposure to a 820 nm fs laser and cell death upon one photon resonant irradiationat 436 ± 10 nm, respectively.

Introduction

Following pioneering works of Webb and co-workers,1 two-photonexcited luminescence2 and photo-dynamic therapy3 (PDT) arebecoming promising tools for biological applications like imaging,diagnostic or even therapy.4 In the last decade, such multiphotonexcitation-based techniques found tremendous development dueto the commercial availability of confocal microscopes andtuneable fs-pulsed laser sources. The two-photon excitation (orabsorption, TPA) presents several intrinsic advantages: (i) thetight focusing restricts the irradiation to an approximately 1 mm3

volume, limiting out-of-focus photo-damage of living tissues andphoto-bleaching of the probes. (ii) The small TPA excitationvolume makes the associated techniques ‘self-confocal’ allowing3D resolved imaging or PDT. (iii) The two-photon excitationwavelength is typically in the 700–1100 nm spectral range (cor-responding to the tuneable Ti-sapphire laser source), called “thebiological window”, where water, biomolecules and tissue areless absorbing and scattering, allowing a deeper penetration intothe specimen. (iv) TPA is of particular interest in the case of

aUniversite de Lyon, CNRS UMR 5182, Institut de Chimie de Lyon,Universite Lyon 1, Ecole Normale Superieure de Lyon, Site Monod, 46allee d’Italie, 69364, Lyon Cedex 07, France. E-mail: Chantal.Andraud@ens-lyon.fr; Fax: (+33)472728860bLaboratoire de Spectrometrie Physique Universite Joseph Fourier, CNRS –UMR 5588, BP 87 F-38402, Saint Martin d’Heres, FrancecINSERM, U851, Universite Lyon1, IFR128, 21 Avenue Tony Garnier, Lyon,F-69007, FrancedDepartment of Physics, Norwegian University of Science and Technology,7491, Trondheim, Norway

PDT, for which the generation of cytotoxic singlet oxygen isobtained by energy transfer from a stabilized state of a two-photon excited chromophore whose energy must be larger than94 kJ mol-1 (which corresponds to 1270 nm).4 In this context,numerous chromophores featuring optimized TPA cross-sectionhave been developed and reported in the literature.4 Some of thempresent additional excellent quantum yields of luminescence andothers exhibit enhanced efficiency for singlet oxygen generation,leading to potential bio-probes for imaging4 or PDT applications,5

respectively. Such two-photon absorbing chromophores generallycomprise an extended p-conjugated skeleton, which makes themextremely lipophilic and therefore difficult to apply in water-containing biosystems such as cells or the extracellular matrix.Therefore a great challenge is to ensure a good hydrosolubility andbio-compatibility, keeping the desired photophysical propertiesintact. To that end, many synthetic endeavours have been focusedto the functionalization of TPA chromophores with hydrosolu-bilizing moieties like polyethyleneglycols,6,7 poly-carbohydrates,8

cationic ammoniums, pyridinium,9 or anionic sulfonate or car-boxylate groups.10 The resulting chromophores are generally goodbioprobes, but the hydrosolubilization is achieved at a very highsynthetic cost, which limits further commercialisation and clinicaluse.

To overcome this major drawback, an elegant way is toheterogenize the lipophilic dyes in an aqueous medium using aphysico-chemical approach to transport photoactive and thera-peutic agent into the target cells without any time-consuming andcostly functionalization. For example, cyclodextrin can provide aninternal hydrophobic cavity acting as host for the dye, where at the

1216 | Photochem. Photobiol. Sci., 2011, 10, 1216–1225 This journal is © The Royal Society of Chemistry and Owner Societies 2011

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same time its hydrophilic exterior ensures the hydrosolubility ofthe host–guest supramolecular complex.11 In addition, lipophilicmicelles like vesicles or polymer nanoparticles are widely used fordrugs or cosmetics-release.12 Wilson and co-workers demonstratedthe photocytotoxicity increase of the hydrophobic protoporphyrinIX in a diblock copolymer.13 In this context, some of us recentlydescribed the use of such particles as alternative hydrophobic TPAdyes vehicules for in vivo imaging of mice brain vascularisation.14

In this study, we describe the use of Pluronic R© polymers as efficientcarriers for lipophilic chromophores 1 and 2 (Chart 1) into livingcells for two-photon excitation imaging or PDT applications.

Chart 1 Structure of the chromophores 1, 2 and of the Pluronic R©

polymers F68 (n = 76, m = 29) and F127 (n = 100, m = 65). PO: propyleneoxide; EO: ethylene oxide.

Pluronic R© polymers (or poloxamers) are non-ionic triblockcopolymers composed of a hydrophobic poly(propylene)oxidecore terminated by two hydrophilic poly(ethyleneoxide) chains onboth sides.15 These triblock copolymers can easily form micellesand self-assembled structures above some critical micellar con-centration due to their amphiphilic structures, and are frequentlyused for their surfactant properties in cosmetics and biomedicalapplications16 or for nanostructuring in materials science.17 Bothchromophores 1 and 2 present a classical quadrupolar struc-ture with strong electro-donating end-groups and extended p-conjugated skeleton. Similar chromophores for PDT featuringdiarylamino fragments were previously described by the groupsof Strehmel18 and Ogilby,7a respectively. These chromophores areknown to show significant TPA cross-sections combined with highfluorescence quantum yield for the anthracenyl derivative (1) andlarge singlet oxygen generation for the dibromobenzene one (2)via an intersystem crossing mechanism induced by the bromineatoms. Here, their dihexylamino counterparts are demonstratedto be appropriate models to study the introduction of lipophilicTPA dyes into living cells. Specifically, we report on the synthesisand photophysical spectroscopic properties of 1 and 2 along withtheir incorporation into living cell via Pluronic F68 or F127nanoparticles (Chart 1). The chromophores uptake kinetics andtheir cytotoxicity were estimated by fluorescence microscopy andflow-cytometry techniques. Finally, their successful introductioninto living cells allowed us to carry out and demonstrate two-photon excitation imaging microscopy and PDT experiments.

Results and discussion

Preparation of compounds

The two chromophores 1 and 2 involved in this study featurestrong electro-donating amino end-groups linked to a central1,4-dibromobenzyl or anthracenyl core. As already describedin the literature,7a,16,19 their synthesis is readily achieved by aSonogashira cross-coupling reaction starting from 1,4-dibromo-2,5-diiodo-benzene or 9,10-dibromoanthracene. It is important tonote that the synthesis of 2 is performed at lower temperature toavoid a loss of regioselectivity of the coupling reaction. The twochromophores are recovered after column chromatography andwere fully characterized using common chemical characterizationtechniques (see Experimental section). As their photophysicalproperties are essential for the applications discussed here theywill be further outlined in sections below. Due to their extendedp-conjugated structure and to the presence of four hexyl chainslinked to the amino fragments, 1 and 2 are very lipophilicmolecules. Consequently they are highly soluble in chlorinatedsolvents or in toluene but their solubility decreases in alcohol andbecomes zero in water where it is not possible to perform any highlydiluted photophysical measurements. Hence, it is not straight-forward to use 1 and 2 alone for any applications in biologicalmedia.

Spectroscopic properties and intersystem crossing

The photophysical properties of 1 and 2 were studied in organicsolvents and compared to those of the corresponding dopedPluronic nanoparticles dispersed in water. The determined photo-physical parameters are collected in Table 1 and spectral ap-pearances and key issues are further discussed as follows. Owingto the presence of strong dialkylamino donor groups, the twochromophores present broad, intense structureless absorption andemission bands localised in the visible part of the spectrum (exceptfor very hydrophobic solvents like toluene, vide infra). Thesetransitions can be assigned to charge transfer (CT) transitionsfrom the periphery of the molecule to the central core acting aselectron tank for following reasons: (i) the two chromophorespresent a weak positive solvatochromism in absorption thatbecomes more marked in emission (Fig. 1 and Table 1). (ii) Theprotonation of the amino moieties results in the suppression of thedonor part and consequently of the CT transition.20 As illustratedfor 1 in chloroform, protonation induces the disappearance of thebroad transition at 561 nm accompanied by the apparition of ablue-shifted structured transition at 479 nm assigned to a classicalp–p* transition from the local excited (1LE) state (Fig. 2). (iii) Sincecharge transfer is a thermally activated process,21 it is hindered atlow temperature. Indeed, in the case of 2, the broad CT transitiondisappears at 77 K in an organic glass (MeOH–EtOH, v/v 1/4)and the structured 1LE is recovered (Fig. 3). (iv) In toluene, bothcompounds exhibit a blue-shifted structured transition in markedcontrast with the broad transition observed in other more polarsolvents. It is well known that CT transitions are blue-shiftedwhen the solvent polarity decreases (positive solvatochromism). Insolvents presenting a very low polarity like toluene or cyclohexane,the CT state is not the lower energy excited state and the 1LEstate becomes the emitting state, which explains the modificationobserved in the shape of the emission bands.

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Table 1 Photophysical data for 1 and 2: absorption maximum (lmax), extinction coefficient (e), emission maximum (lem), fluorescence quantum efficiency(f) and two-photon absorption cross-section (s 2) for a designated wavelength (l(2))

lmax/nm e/L mol-1 cm-1 lem/nm f s 2/GMc (l(2)/nm)

Compound 1Toluene 486 545CHCl3 513 52000 561 1a 518 (810)MeTHF 495 568DMSO 519 634Pluronic suspension in H2O 494 591 <0.01a

Compound 2Toluene 412 441CHCl3 421 91500 458 0.25b 570 (730)MeTHF 417 467EtOH–MeOH 4 : 1 298 K 416 488DMSO 427 540Pluronic suspension in H2O 422 469 <0.01b

a Using fluorescein as reference (f = 0.92 in NaOH 1 M, lex = 470 nm). b Using coumarin 153 as reference (f = 0.45 in MeOH, lex = 417 nm). c 1 GM(Goeppert–Mayer) = 10-50 cm4 s photon-1

Fig. 1 Emission solvatochromism at 298 K of chromophores 1 (upperpanel) and 2 (lower panel): toluene (dashed dot), chloroform (dot), MTHF(dash), DMSO (solid), EtOH–MeOH (dash dot dot).

In all solvents, the absorption and emission bands are signif-icantly red-shifted for compound 1 compared to 2 (for instanceDlabs = 92 nm; Dlem = 105 nm in chloroform, Table 1). This iscertainly resulting from the additional delocalisation pathways al-lowed by the anthracenyl moieties compared to the dibromophenylones. However, the most striking difference between the twocompounds is observed in the fluorescence quantum yield (f, Table1). Whereas 1 is extremely luminescent with a quantum yield close

Fig. 2 Absorption (solid lines) and emission (dashed lines) spectra(CHCl3, 298 K) of 1 before (thin lines) and after (bold lines) protonation(H2SO4).

Fig. 3 Emission of dye 2 in MeOH–EtOH (1/4), lexc = 405 nm at 298 K(red solid), 77 K under argon (blue dash) and time-gated emission (delay50 ms) at 77 K under argon (black dash-dot).

to 1, 2 gives only a modest 0.25 efficiency. This difference is dueto the presence of the two bromine heavy atoms that favour theformation of a triplet excited state by intersystem crossing, toultimately provide an efficient energy transfer to solvated oxygento form singlet oxygen.

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Fig. 4 Left: singlet oxygen emission sensitized by irradiation of 2 (lexc = 410 nm, OD = 0.05) in chloroform at 298 K; right: determination of fD relativelyto phenalenone using the Stern–Volmer plot method. The molecule 2 (triangles) and reference (squares) were irradiated at 410 nm.

The formation of singlet oxygen is widely described in the liter-ature, and illustrated by the observation of the emission band at1275 nm in CHCl3, characteristic of the luminescence of the singletexcited state of oxygen upon excitation of the chromophore CTabsorption transition (Fig. 4, left). The phenomenon is of courseabsent in solvents that have been properly degassed, e.g. withargon. However, even in degassed conditions the phosphorescenceemission of the triplet state is too weak to be detected due toself-quenching when the solute is freely diffusing in the solvent.At low temperature (77 K) under fully degassed atmosphere,a weak phosphorescence emission transition is detected in thered tail of the fluorescence band (dotted curve in Fig. 3).To enhance the signal-over-noise ratio, a time-gated emissionspectrum was recorded with a delay of 50 ms between excitationand detection. Under such conditions, the short living fluorescenceemission is suppressed and long-lived phosphorescence signalscan more easily be detected. In the present system, 2 exhibitsa phosphorescence emission with a structured band centred at586 nm (black curve in Fig. 3). The decay time-constant of thephosphorescence was determined to 2.9 ms.

The quantum efficiency of singlet oxygen generation fD wasestimated to 0.53 ± 0.10 in chloroform using the Stern–Volmerintegration of the singlet oxygen luminescence signal relative tophenalenone in the same solvent (fD = 0.98 ± 0.05 in CHCl3)22

(Fig. 4, right). This result is in agreement with already publisheddata on related compound using alternative fD determinationtechniques in toluene.

Two-photon absorption properties

The two-photon cross-section of 1 and 2 were determined usingchloroform as solvent, employing the two-photon excited fluo-rescence technique. For calibration of the two-photon excitationspectra in the 700–900 nm spectral range using a fs-Ti:sapphirelaser source, coumarin-307 and fluorescein were used as referencematerials (see experimental section for more details). Compound1 has a distinct maximum at 800 nm whereas for compound 2, itis at around 730 nm (Fig. 5). In the spectral presentation of theTPA cross-section of Fig. 5, the one-photon absorption (OPA)(dashed lines) is superimposed with the suitable wavelength scaleto compare energies reached by one and two photon absorption.Thus, for both compounds it is observed that the two-photon

Fig. 5 Two-photon absorption spectra of 1 (squares) and 2 (circles) inCHCl3 (bottom abscissa). For comparison, the one-photon absorptionspectra of 1 (dash) and 2 (dot) were superimposed using a wavelengthdoubled scale (upper abscissa).

excitation spectra are significantly blue-shifted compared to thewavelength doubled one-photon absorption spectra. It can beconcluded that the lowest S0→S1 transition is not two-photonallowed in agreement with the centrosymmetric structure of thedyes and that only states of different parity and higher energy arereached by the two-photon excitation. The maximal two-photoncross-section of 1 was determined to 518 GM at 810 nm (Table1) and matches well data already published in the literature foranalogous compounds.18 The TPA cross-section of 2 is in the sameorder of magnitude (570 GM at 730 nm) but at a wavelengthsignificantly blue-shifted compared to 1, following the same trendsobserved in the one-photon absorption spectra. Finally, it is worthnoting that for both dyes, the two-photon efficiency is large enoughto envisage in cellulo experiments to be discussed more in detailbelow.

Pluronic nanoparticle suspensions

The aqueous dispersion of chromophore-Pluronic nanoparticleswas prepared by mixing a chloroform solution of dyes withan aqueous solution of amphiphilic polymer. After stirring and

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ultrasonicating, the organic solvent was removed by keeping themixture at 50 ◦C for 30 min. The colloidal suspension was filtered(100 nm pore size) to remove a small fraction of excessively largeparticles. The process is thus handy and allows dispersing anhydrophobic chromophore in water up to approximately 100 mgL-1 concentration. As a consequence, the spectroscopic propertiesof the chromophore-Pluronic nanoparticle dispersion could bedetermined in water (Fig. 6) and the sample could be subsequentlyadded to biological sample systems. The absorption and emissionspectra of chromophore 1 and 2 are similar to that obtained inorganic solvent and owing to the solvatochromism study (Table1 and Fig. 1), it could be concluded that the internal polarityof the particle appears similar to that of MeTHF. This resultis not surprising, keeping in mind that the hydrophobic blockof Pluronic is made of polyethylene oxide and this linear etherstructure is rather similar to the cyclic ether encountered inMeTHF. It seems that water is largely excluded from the micellecore. In addition it is worth pointing out that the emission isalmost completely quenched in the nanoparticles with very lowquantum yield efficiency (Table 1). This luminescence extinc-tion can be explained by intermolecular de-excitation processesfrequently occurring in locally high concentrated media likealso observed using silica particles.23 Finally, the two-photonexcitation spectra of 1 and 2 was recorded for the aqueousnanoparticle suspension, resulting in a very similar profile tothat obtained for the pure solute in chloroform with a maximumcentered at 740 and 850 nm, respectively (Fig. 6, inset). However,the two-photon cross-section of the nano-objects could notbe precisely determined due to the large uncertainty in theconcentration determination and the poor luminescence quantumyield.

Fig. 6 Absorption (solid) and emission (dash) spectra of 1 (bold line)and 2 (thin line) incorporated in a Pluronic F68 nanoparticles dispersedin water. Inset: Normalized two-photon excitation spectra of 1 (squares)and 2 (circles) in Pluronic F68 nanoparticles dispersed in water.

Bio-imaging and dye loading of cells

The chromophore-Pluronic nanoparticle suspension was incu-bated with various cell lines in culture medium (see Experimentalfor details) and the uptake was studied either by fluorescencemicroscopy or by flow cytometry. Qualitatively, an efficient celluptake was observed by the internalisation of dye 2 in glioma

(Fig. 7). Upon one-photon excitation, the fluorescence of thedye clearly indicated that the chromophore has efficiently stainedthe cells and was mainly localised in the cytoplasm. In Fig. 7,the staining efficiencies of Pluronic F68 and F127 nanoparticlessuspension are compared. After 24 h incubation with the PluronicF127 nanoparticles suspension, a smaller amount of dyes wasfound inside the cells as illustrated by the weaker fluorescencesignal compared to that of Pluronic F68 nanoparticles suspensionafter 4 h incubation. This result can be explained by the differentstructures of the two amphiphilic polymers (Chart 1): PluronicF68 presents a larger proportion of polyethylene oxide and istherefore more hydrophilic than Pluronic F127, emphasizing thekey-role of the hydrophilic/hydrophobic balance in the cell uptakeprocess. To assess the kinetics of this internalisation process, theincrease of the fluorescence signal of the cells with time wasmeasured in the case of glioma cells incubated with the dye2-Pluronic F68 nanoparticles suspension. The results indicateda rapid loading into the cells (Fig. 8); however, only a smallnumber of cells can be analysed using fluorescence microscopy.Therefore, we used flow cytometry, a powerful technique thatprovides fluorescence measurements of large numbers of cells, toquantify the uptake of 2 by approximately 10 000 live cells. Inthis experiment, two murine cell lines (the haematopoietic nontransformed pro-B Baf-3 cells and the melanoma B16-F10 cells)were first incubated for 3 h with two concentrations of 2 (2 or 10 mgmL-1) in DMSO or in Pluronic (Fig. 9A). The shift in fluorescenceintensity reflected the amount of uptake. The fluorescent profileswere narrow, consistent with a homogeneous uptake. The highestfluorescence intensity was observed when 2 was in Pluronic.Kinetic experiments showed that the fluorescence intensity of thecells increased with time in contact with 2 indicating a continuousuptake over the 24 h incubation period (Fig. 9B). Altogether, theseresults show that Pluronic greatly facilitates uptake. In conclusion,experiments clearly indicated that the lipophilic chromophore2 is able to stain both rapidly and efficiently various kinds ofcells types when incorporated into the Pluronic F68 nanoparticlessuspension.

Fig. 7 100¥ oil-immersion images of F98 glioma cells (left : transmission;middle: fluorescence; right: merge): first line, after 4 h incubation with2-Pluronic F68; second line, after 1 day incubation with 2-Pluronic F127probes.

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Fig. 8 Internalisation kinetics of 2-F68 Pluronic nanoparticles suspen-sion in glioma cells measured by epifluorescence microscopy.

TPA imaging and PDT demonstration

Finally, the two lipophilic chromophores 1 and 2 were chosen fortheir particular intrinsic properties namely, two-photon excitedfluorescence and singlet oxygen generation. In order to fullyvalidate the Pluronic-mediated delivery of lipophilic TPA dyes, wechecked that the in cellulo internalized chromophores conservedtheir properties. To that end, glioma cells were incubated with anaqueous suspension of dye 1 in F68 Pluronic nanoparticles usingthe above-described procedure and were successfully stained bythe lipophilic chromophore 1. The images presented in Fig. 10

Fig. 10 TPA imaging in Z-projection of 10 two-photon images (2 mmstep width) of F98 glioma cells after 4 h incubation with 1 in Pluronicnanoparticle. Images were acquired using an 820 nm excitation wavelength,with a 20 mW incident laser power. The acquisition time was 0.9 s perimage.

were recorded using a laser scanning microscope using a 100 fstwo-photon excitation at 820 nm. The chromophore fluorescencewas recorded above 710 nm (BG39 filter) and scanned in Z-stackswith a 2 mm step. The resulting image stack (Fig. 10) clearly showsthat the chromophore is localised in the perinuclear region of thecell and appears as brilliant spots in the endoplasmic reticulumarea. This localisation is similar to that observed with 2 using aclassical one-photon fluorescence microscopy technique (Fig. 7).In addition, preliminary PDT experiments were carried out usingchromophore 2. Glioma cells were incubated for 1 h in the presenceof 2 in Pluronic F68 nanoparticles. After washing, the stained cellswere irradiated at 436 ± 10 nm using a mercury lamp deliveringa 2 mW power on the sample for 5 min. 45 min after irradiation,the cells were incubated in presence of 1 mM propidium iodide for

Fig. 9 Uptake of chromophore 2 by Baf-3 and B16-F10 cells (A) Chromophore 2 incorporation by Baf-3 (left panel) and B16-F10 cells (right panel).Cells were incubated for 3 h with 10 mg mL-1 (solid line) or 2 mg mL-1 (dotted line) of 2 in Pluronic (dark) or in DMSO (grey), or left untreated (tintedhistogram). The uptake was quantified by flow cytometry. (B) Kinetic of chromophore 2 uptake by Baf-3 (left panel) and B16-F10 cells (right panel).Cells were incubated with Pluronic (dashed line), 2 in Pluronic (dark line) or 2 in DMSO (grey line). Uptake was evaluated by flow cytometry. Results areexpressed as the average of the mean fluorescence intensity (MFI) ± SD of two independent experiments.

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Fig. 11 Transmission (left) and fluorescence (right) images (¥10) of F98glioma cells stained with chromophore 2, 45 min after a 300 J cm-2

irradiation. Fluorescence is due to the propidium iodide labelling deadcells nucleus.

1 min to reveal cell death. The same experiment was carried outin the presence and the absence of chromophore 2. The imagesdepicted in Fig. 11 unambiguously show a significantly morecomplete cell mortality in the irradiated area in the presence ofchromophore 2. Furthermore, the evolution of the fluorescenceintensity of 2 was monitored and found to decrease steadily duringthe irradiation. The plot in Fig. 12 shows a steady decrease thefluorescence signal of 2 in the cells with only 10% of the initialfluorescence conservation after 120 s of irradiation. This decreasecould be due to photobleaching process, suggesting that duringirradiation singlet oxygen and other radical organic species aregenerated. Both could be responsible of both cell death andchromophore destruction. Similar phenomena and concomitantcell and chromophore degradation have been observed also inother PDT systems.24

Fig. 12 Evolution of the fluorescence of 2 in glioma cells duringphotodynamic therapy experiment.

Summary and conclusion

This report presents a photophysical study of two hydropho-bic chromophores functionalized in the view of multiphotonexcitation microscopy and photodynamic therapy capabilities,respectively. The model chromophores were chosen for theirparticular properties namely high TPA cross-section combinedwith high quantum yield for 1 and high singlet oxygen generationefficiency for 2. In order to distribute the chromophores in cellularsystems a Pluronic nanoparticle technique was employed andwas shown to deliver the photo-agents in a simple incubationprocedure. The bio-imaging and PDT properties were conservedupon cell internalisation since it was possible to image cells by two-photon scanning microscopy or to induce cell death upon resonantirradiation. Conclusively, the Pluronic nanoparticle was found tobe a useful method to test in cellulo the nonlinear chromophoreproperties without any time-consuming hydrosolubilizing func-tionalisation of active chromophores.

Experimental

General

Pluronic F68 (n = 76, m = 29) and Pluronic F127 (n = 100, m = 65)were purchased from Aldrich. The NMR spectra (1H, 13C) wererecorded at room temperature on a BRUKER 500 operating at500.105 and 135.32 MHz for 1H and 13C, respectively. NMR. Dataare listed in parts per million (ppm) and are reported relativeto tetramethylsilane, residual solvent peaks of the deuteratedsolvents were used as an internal standard. Infrared spectra wererecorded on a Mattson 3000 spectrometer using KBR pellets.High-resolution mass spectrometry measurements and elementalanalysis were performed at the Service Central d’Analyze duCNRS (Vernaison, France). 4-Ethynyl-N,N-dihexylaniline25 and1,4-dibromo-2,5-diiodobenzene26 were synthesised as previouslyreported

1. In a Schlenk flask, 9,10-dibromoanthracene (300 mg, 0.89mmol), 4-ethynyl-N,N-dihexylaniline (521 mg, 1.83 mmol) weredissolved in a triethylamine/THF mixture (v/v = 20/20 mL). Thesolution was degassed by bubbling argon for 20 min. Pd(PPh3)2Cl2

(12 mg, 2 mol%) and CuI (8 mg, 5 mol%) were added and thereaction was stirred overnight at 55 ◦C. After cooling at roomtemperature, dichloromethane was added and the mixture wasfiltered through a Celite plug, the organic layer was washedwith a solution of saturated ammonium chloride, water, andbrine. After drying on Na2SO4, the solvent was evaporatedunder reduced pressure. The crude material was purified by flashchromatography on silica gel (cyclohexane/ethyl acetate 98 : 2) toprovide a fluorescent red solid. (464 mg, 70%). mp: 116–117 ◦C.FTIR (KBr, cm-1):n 2923, 2854, 2183, 1602. 1H NMR (200 MHz,CDCl3, ppm): d 8.69 (q, J = 3.3 Hz, 4H), 7.60 (d, J = 9 Hz, 4H),7.59 (q, J = 3.3 Hz, 4H), 6.67 (d, J = 9 Hz, 4H), 3.33 (t, J = 7.2Hz, 8H), 1.62 (m, 8H), 1.35 (m, 24H), 0.92 (t, J = 6.5 Hz, 12H).13C NMR (125 MHz, CDCl3, ppm): d 148.2, 133.0, 131.8, 127.5,126.3, 118.5, 111.4, 109.1, 104.1, 84.8, 77.3, 77.1, 76.8, 51.1, 31.8,27.3, 26.9, 22.7, 14.1. HRMS calcd for [C54H68N2 + H]+ 745.5461,found 745.5443.

2. In a Schlenk flask, 1,4-dibromo-2,5-diiodobenzene (544 mg,1.11 mmol), 4-ethynyl-N,N-dihexylaniline (650 mg, 2.27 mmol)

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were dissolved in a triethylamine/THF mixture (v/v = 25/25mL). The solution was degassed by bubbling argon for 20 min.Pd(PPh3)2Cl2 (14 mg, 2 mol%) and CuI (9 mg, 5 mol%) were addedand the reaction was stirred overnight at 55 ◦C. After cooling atroom temperature, dichloromethane was added and the mixturewas filtered through a Celite plug and washed with a solution ofsaturated ammonium chloride, water, and brine. After drying onNa2SO4, the solvent was evaporated under reduced pressure. Thecrude material was purified by flash chromatography on silica gel(cyclohexane/ethyl acetate 95 : 5) to provide a yellow fluorescentsolid. (502 mg, 63%). mp: 68–69 ◦C. FTIR (KBr, cm-1):n 3085,3045, 2952, 2924, 2852, 2206, 1604. 1H NMR (200 MHz, CDCl3):d 7.67 (s, 2H), 7.37 (d, J = 8.4 Hz, 2H), 6.55 (d, J = 8.4 Hz, 2H),3.26 (t, J = 7 Hz, 8H), 1.56 (m, 8H), 1.30 (m, 24H), 0.88 (t, J =6.3 Hz, 12H). 13C NMR (500 MHz, CDCl3): d 148.6, 135.4, 133.4,126.4, 123.3, 98.7, 85.7, 51.2, 31.9, 27.4, 27.0, 22.9, 14.3. Elementalanaylsis calcd for C46H62N2Br2: C, 68.82; H, 7.78; N, 3.49%. Found:C, 68.56; H, 7.90; N, 3.65. HRMS calcd for [C46H63N2Br2 + H]+

801.3358, found 801.3333.

Synthesis of Pluronic nanoparticle aqueous dispersion

Chromophores 1 or 2 (2 mg) were dissolved in chloroform(100 mL) and added to a Pluronic F68 (2 wt.%) or Pluronic F127(1 wt.%) aqueous solution (5 mL). After 15 min stirring for pre-emulsification, the micro-emulsion was prepared by ultrasonicat-ing for 15 min (bath, 150 W). The mixture was then stirred at50◦ C until complete evaporation of the chloroform (30 min). Thecolloidal particle size was fixed by mechanical filtration (100 nm)of the solution. The process leads to a ca. 100 mg L-1 aqueouschromophore suspension.

Cell culture

F98 glioma cells (rat brain tumor models) were used for in vitroassays. Cells were cultured in complete culture medium (DMEMGlutaMAX-I) containing 10% FBS, 4.5 g L-1 glucose, sodiumpyruvate, 1% of an antibiotic mixture (10000 units Penicillin-Gper mL, 10 mg mL-1 streptomycin in normal saline), and nonessential amino acids (BioWhittaker) in a 37 ◦C thermostattedincubator with 5% CO2. Mouse Interleukin-3 dependent pro-B Baf-3 cell line was cultivated in DMEM medium (InvitrogenLife Technologies) supplemented with 6% heat inactivated FBS(Lonza, Belgium), 10mg ml-1 gentamycin (Invitrogen) and 5%Wehi-3B cell-conditioned medium as a source of IL-3. Mousemetastatic B16-F10 melanoma cell line was cultivated in DMEMmedium (Invitrogen Life Technologies) supplemented with 10%heat inactivated FBS and 10 mg ml-1 gentamycin. Cells were grownat 37 ◦C in a humid atmosphere with 7% CO2.

Uptake and biodistribution

Prior to the bio-distribution analysis, cells were plated on LabTekchambered cover glass (Nunc) and incubated with 40 mL Pluronicnanoparticle in 400 mL culture medium for 4 h. Then, transmissionand fluorescence images were acquired using an oil-immersionobjective (¥100). For uptake measurements, cells were platedon Petri box and incubated with ten time diluted Pluronicnanoparticles in culture medium (2 mL) for different time (10,20, 30, 50 and 80 min). Fluorescence images were acquired using

the ¥10 objective and the cytoplasmic fluorescence intensity wasestimated using Image J R© software.

Flow cytometry analysis

For uptake measurements, B16-F10 cells were plated on 24 wellculture plates three days before the experiment in order to reachhalf confluence. Baf-3 cells were used at a concentration of 2 ¥105 cells ml-1. Cells were incubated at 37 ◦C with 2 or 10 mgmL-1 of 2 for the indicated periods of time. Incorporation wasanalysed with a Canto II flow cytometer (BD Biosciences) usinga 405 nm laser and a 450 nm emission filter. At least 10 000 cellsper condition were recorded and mean fluorescence intensity wascalculated among live cells using an FSC/SSC gate to exclude deadcells. Results were analysed using FlowJo software (Tree star).

Optical characterisations

UV-Visible spectra were recorded with Jascow 670 UV-Visiblespectrophotometer. The luminescence spectra were measured us-ing a Horiba–Jobin Yvon Fluorolog-3 R© spectrofluorimeter. Thesteady-state luminescence was excited by unpolarized light from a450 W xenon CW lamp and detected at an angle of 90◦ for dilutedsolution measurements (10 mm quartz cuvette) by a red-sensitiveHamamatsu R928 photomultiplier tube. Spectra were referencecorrected for both the excitation source light intensity variation(lamp and grating) and the emission spectral response (detectorand grating). Uncorrected near infra-red spectra were recorderusing a liquid nitrogen cooled, solid indium/gallium/arsenicdetector (850–1600 nm). Fluorescence quantum yields Q weremeasured in diluted water solution with an optical density lowerthan 0.1 using the Stern–Volmer plot method based on followingequation:

Qx/Qr = [Ar(l)/Ax(l)][nx2/nr

2][Dx/Dr]

where A is the absorbance at the excitation wavelength (l), nthe refractive index and D the integrated luminescence intensity.“r” and “x” stand for reference and sample. Here, references arecoumarine 153 in MeOH (Qr = 0.45) at 417 nm for 2 and fluoresceinin NaOH 1 M (Qr = 0.92) at 470 nm for 1 and phenalenonefor singlet oxygen generation (fD = 0.98 in CHCl3). Excitationof reference and sample compounds was performed at the samewavelength.

Two-photon excited luminescence measurements

The TPA cross-section spectra were obtained by up-conversionfluorescence using a Ti:sapphire femtosecond laser in the range700–900 nm. The excitation beam (5 mm diameter) is focalizedwith a lens (focal length 10 cm) at the middle of the fluorescence cell(10 mm). The fluorescence, collected at 90◦ to the excitation beam,was focused into an optical fiber (diameter 600 mm) connected toan Ocean Optics S2000 spectrometer. The incident beam intensitywas adjusted to 50 mW in order to ensure an intensity-squareddependence of the fluorescence over the whole spectral range.The detector integration time was fixed to 1 s. Calibration ofthe spectra was performed by comparison with the published700–900 nm coumarin-307 and fluorescein two photon absorptionspectra.27 The measurements were done at room temperature indichloromethane and at a concentration of 10-4 or 10-5 M.

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Microscopy set-up

One photon microscopy studies were performed with an invertedmicroscope Zeiss Axiovert optimized for fluorescence. Excitationis made by a mercury lamp, and passes through an excitation filter(436 ± 10 nm). Then, light is reflected by a dichroic mirror anddirected toward the sample using oil-immersion objectives (¥10or ¥100). Emitted fluorescence collected by the objective passesthrough the dichroic and an emission filter (480 ± 20 nm). Tofinish, images were recorded by a numeric cam (1 megapixels).

Two-photon laser scanning microscopy was performed with aconfocal microscope consisting of a Biorad (MRC 1024) scanheadand an Olympus BX50WI microscope. A 820 nm excitation beamoriginating from a femtosecond Ti:sapphire laser (100 fs, 80 MHz,5 W pump; Spectra-Physics, Millenia V) was focused into thecortex using a 20¥ water-immersion objective (0.95 numericalaperture, Xlum Plan FI Olympus). The laser beam was thenscanned in the x–y plane to acquire a 512 ¥ 512 image (0.9 sper image). The z scan (variation of the observation depth) wasrealized by translation of the motorized objective. Fluorescencewas directly collected with an external photomultiplier (PMT)protected by a BG39 filter (Schott Glass–Jena, Germany). Planarscans of the fluorescent intensity were acquired at successivedepths with a 2 mm z step between scans. Image reconstructionand analysis were performed using Image J R© software.

Photodynamic therapy

F98 cells were incubated for 1 h in presence of dibromobenzenederivative (2) encapsulated in Pluronic F68 nanoparticle (10 mgmL-1 in culture medium). Irradiation was performed with aninverted microscope Zeiss Axiovert optimized for fluorescence.Excitation is made by a mercury lamp, and passes through anexcitation filter (436 ± 10 nm). Then, light is reflected by a dichroicmirror and directed toward the sample using a ¥10 objective.Excitation area of 0.2 mm2 was selected using an iris diaphragmconjugated to the focal plane. The cells mono-layer was irradiatedfor 5 min using 2 mW power excitation (total light dose of 300 Jcm-2). 45 min after irradiation, cells were incubated in presence of1 mM propidium iodide (IP) for 1 min to show the cellular mor-tality. For comparison, similar experiments have been performedwithout chromophore 2. The evolution of the fluorescence of thedye in cells is monitored during irradiation. Fluorescence imagesare acquired every 5 s, and the cytoplasmic fluorescence intensityis estimated using Image J R© software.

Acknowledgements

Authors thank the region Rhone-Alpes (MACODEV cluster andcluster 5) for a grant to TG and financial support, and theANR program (NanoPDT) for financial support. We also thankJean Bernard for technical support in two-photon absorptionmeasurements. We thank the staff from the Cytometry facility(IFR128 Lyon BioSciences).

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