Dr Aude Sourdon

Dr Magali Gary-Bobo

Dr Marie Maynadier

Dr Marcel Garcia

Jean Pierre Majoral

A.-M Caminade

Dr Olivier Mongin
email: olivier.mongin@univ-rennes1.fr

Dr Mireille Blanchard-Desce
email: mireille.blanchard-desce@u-bordeaux.fr

Jean-Pierre Majoral

Anne-Marie Caminade

Dr J.-P Majoral

Dr A.-M Caminade

Dr J.-P Majoral

Dr A.-M Caminade

Photophysical Properties, Innocuousness in Daylight and Cytotoxicity under Two-Photon Irradiation in the NIR

Keywords: dendrimers, photophysics, fluorescence, imaging, photodynamic therapy

come

Introduction

Photodynamic therapy (PDT) is a clinical treatment used in oncology for the treatment of certain types of tumors including esophagus, skin, bladder and non-small cell lung carcinomas, as well as in ophthalmology for the treatment of age-related macular degeneration (ARMD). [1] PDT is based on the activation by light of photosensitizers; non-toxic in the absence of light excitation, a photosensitizer is able upon excitation by a laser source, to generate singlet oxygen and/or other reactive oxygen species (ROS), which leads to the destruction of the targeted tissues. [2] The use of a two-photon excitation (2PE) offers several advantages for PDT, especially for the treatment of small areas, such as small solid tumors. [3] These include the intrinsic threedimensional resolution allowed by 2PE, offering the ability for highly spatially resolved excitation as well as increasing penetration depth in tissues thanks to the use of near infra-red (NIR) light. [4] Yet, in order to achieve efficient treatments, optimized 2P photosensitizers (2PP) -i.e., combining very large 2PA cross-sections in the biological spectral window (700-1000 nm) and high singlet oxygen (or ROS production) quantum yields -are required. In addition, if the 2PP remains fluorescent, in vitro monitoring is allowed, paving the way to theranostics applications. [3] Most of the current clinical or preclinical photosensitizers are porphyrin derivatives, such as porfimer sodium (Photofrin) and verteporfin (Visudyne). As model tetraphenylporphyrin (TPP, 12 GM), [5] these compounds exhibit high singlet oxygen quantum yields, significant fluorescence but low 2PA cross sections (10 GM and 50 GM, respectively) in the near infra-red (NIR). [6] Giant 2PA cross-sections have been obtained with expanded porphyrins, [7] conjugated porphyrin dimers, trimers and oligomers, [8] planarized fused or bridged porphyrins [9] and supramolecular assemblies, [8a, 10] however at the expense of a strong modification of the other photophysical properties. In particular, it often leads to decrease or suppression of fluorescence and onset of residual one-photon absorption (1PA) overlapping with the 2PA band located in the NIR region. The huge gain in 2PA response therefore results in the loss of some of the intrinsic advantages of selective 2PA, in particular of the 3D confinement of excitation and therefore spatial resolution in laser-assisted tumor resection.

To increase the 2PA cross-sections (while avoiding one-photon absorption at the same wavelength) and still retain the excellent photosensitization and luminescence properties of the porphyrins, different systems have been designed, such as symmetrically or dissymmetrically -extended phosphorescent metalloporphyrins, [11] as well as dendritic antenna systems based on resonant energy transfer (FRET) from peripheral 2P absorbers towards a single porphyrin core, [12] and "semidisconnected" assemblies with a weak conjugation between 2P absorbing dendrons and a porphyrin core, [13] or between a central 2PA unit and peripheral porphyrins. [14] FRET between 2P absorbers and porphyrinic photosensitizers could also be achieved without covalent architectures by coencapsulating them within silica nanoparticles. [15] Other types of nanoparticles based on energy transfer have been designed, including quantum dot-porphyrin [16] and quantum dot-phthalocyanine [17] conjugates and porphyrin-doped conjugated polymers. [18] Non-porphyrinic 2P absorbing photosensitizers have also been developed, [19] among which should be mentioned quadrupolar chromophores substituted with heavy atoms such as bromine or iodine, [20] which leads to efficient intersystem crossing and singlet oxygen generation quantum yields but concomitantly to vanishing fluorescence. We have previously developed alternative 2P fluorescent photosensitizers based on bananashaped quadrupolar systems, that combine large 2PA crosssections and significant singlet oxygen quantum yields, while maintaining fair fluorescence quantum yields. [21] These symmetric photosensitizers were covalently incorporated within mesoporous silica nanoparticles for efficient two-photon photodynamic therapy of solid tumors. [4e, 4f] As an alternative to these hard nanoparticles, our aim was to develop soft and monodisperse fully organic nanodots by gathering quadrupolar 2P photosensitizers in phosphorus-based dendrimer architectures, and to graft at their surface watersolubilizing groups. Among the major advantages of phosphorus-based dendrimers [22] are their low toxicity [23] and their accessibility for both inner and surface functionalization using covalent chemistry. This highly modular "organic nanodots" strategy was previously used successfully to obtain covalent assemblies of fluorophores, leading to luminescent nanoparticles that can outperform semiconductor quantum dots in terms of one-and two-photon brightness. [24] These fluorescent nanodots have been proved to be of major interest for in vivo imaging. [25] We report here the first two-photon photosensitizing nanodots and their use in photodynamic therapy.

Results and Discussion

Synthesis of the graftable two-photon photosensitizer

The photosensitizer needed for the synthesis of 2-G2 (Fig 1) should exhibit two different orthogonal grafting moieties, a phenol function to attach it to the dendrimeric scaffold, and an aldehyde function to continue the dendrimer synthesis and introduce water-solubilizing peripheral groups. The synthesis of this dissymmetric photosensitizer is described in Scheme 1. The Mitsunobu condensation of N-ethyl-N-hydroxyethylaniline (1) with hydroquinone afforded 2a. Phosphonium salts 2b and 6 were synthesized according to the procedures reported. [START_REF] Porrès | [END_REF] Wittig condensations of 5-iodo-2-thiophenecarboxaldehyde (3) [27] with phosphonium salts 2b and 6 led to mixtures of stereoisomers. Their isomerization with iodine afforded 4a and 7a, respectively, as pure trans isomers (Scheme 1).

Scheme 1. Reagents and conditions. a) hydroquinone, PPh3, DIAD, THF, RT, 16h (36%); b) PPh3, paraformaldehyde, toluene, NaI, H2O, acetic acid, 66h, 61°C, (83% for 6 and 84% for 2b); c) 3, tBuOK, anhyd CH2Cl2, RT, 16h (50% for 7a and 54% for 4a); d) acetyl chloride, DMAP, Et3N/CH2Cl2, RT, 16h (97%); e) Pd(PPh3)2Cl2, CuI, ethynyltrimethylsilane, Et3N/toluene, 40°C, 16h (87% for 8 and 94% for 5); f) 4 hydroxybenzaldehyde, PPh3, DIAD, THF, RT, 16h (81%); g) 8, 9, Pd(PPh3)2Cl2, CuI, TBAF/THF, 40°C, 16h (77%); h) 5, Pd(PPh3)2Cl2, CuI, TBAF/THF, 40°C, 16h (70%); i) NaOH, EtOH/THF, RT, 20 min (84%).

Accepted Manuscript

Scheme 2. Synthesis of spherical dendrimer 2-G1.

On one hand, the phenol function of 4a was esterified with acetyl chloride, giving 4b, which was reacted with ethynyltrimethylsilane in a Sonogashira coupling to afford 5, and on the other hand, Mitsunobu condensation of 7a with 4hydroxybenzaldehyde gave 7b, which was also coupled with ethynyltrimethylsilane, leading to 8. Finally, graftable chromophore 10 was obtained as a single E,E-stereoisomer, from 9,9-dibutyl-2,7-diiodo-9H-fluorene (9) [28] by means of two successive Sonogashira couplings with 8 and 5, followed by saponification of the acetate protective group (Scheme 1). The synthesis of the spherical first generation dendrimer 2-G1 was carried out by grafting 12 equivalents of the 2PA chromophore 10 in the presence of caesium carbonate, to the P(S)Cl2 end groups of the first generation phosphorous dendrimer 1-G1, [22a, 29] built from a cyclotriphosphazene core [30] (Scheme 2). The substitution of the twelve chlorine atoms of 1-G1 with such a bulky substituent was slow and needed 7 days to go to completion. This reaction was monitored by 31 P NMR which shows a total disappearance of the singlet at δ = 62.3 ppm corresponding to the phosphorus P1 in 1-G1 on behalf of a new singlet at δ = 64.3 ppm due to the phosphorus P1 in 2-G1. An intermediate signal observed at δ = 69.3 ppm and corresponding to the monosubstitution (i.e. P(S)ClOAr) totally disappears when the reaction is over. The second step consisted in the condensation of 12 equivalents of the phosphorhydrazide 11 with the aldehyde functions of 2-G1 in chloroform at room temperature, and afforded the second generation of the dendrimer 1-G2 (Scheme 3).

Scheme 3. Synthesis of spherical dendrimer 2-G2.

Accepted Manuscript

Cpd

λabs /nm εmax [a] /M -1 .cm -1 εmax/n [b] /M -1 .cm -1 λem /nm Stokes shift /10 3 cm -1  F [c]  /ns [d]  [e] 10 434 1.2 10 The reaction was monitored by 1 H NMR, its completion being characterized by the total disappearance of the aldehyde singlet. The 31 P NMR spectrum of 2-G1 consists of three singlets δ = 8.6 ppm for the core, δ = 63.3 ppm for P1, δ = 64.4 ppm for the newly grafted phosphorus P2. The next step involved the grafting of 24 equivalents of the monodisperse triethyleneglycol chain 12 on 1-G2 to improve solubility in water, affording dendrimer 2-G2 (Scheme 3). Here again, the reaction was monitored by 31 P NMR. Almost no change occurs for the signal corresponding to the core, but we observed the total disappearance of the singlet at δ = 63.4 ppm corresponding to the phosphorus P2 in 2-G1 on behalf of a new singlet, slightly deshielded. An overlap occurs for the signals of phosphorus P1 and P2 at δ = 64.6 ppm. To ascertain the full substitution or condensation, we used in all cases a slight excess of reagents (10%), which was relatively easy to eliminate by washings.

One photon absorption (1PA) and fluorescence properties

The compared photophysical properties of 10, 2-G1, and 2-G2 in THF are shown in Table 1 and their absorption and emission spectra are represented in Fig 2 . THF was chosen as a common solvent as all three compounds are soluble in this solvent. All compounds exhibit an intense absorption band from the near UV to the blue visible range and an emission band from the green visible range to the yellow visible range (Fig 2). Dendrimers also show strong one-photon absorption in the UV. The low-energy absorption band located at 434 nm can be ascribed to a periphery-to-core intramolecular charge transfer transition, characteristic of bis-donor quadrupolar dyes built from a fluorene core. [31] We note a broadening on the high energy side for organic nanodots 2-G1 and even more for 2-G2 as compared to the isolated dye 10 in solution (Fig. 2). This can be related to the residual absorption of the dendrimeric backbone.

In addition, a hypochromic effect is observed: the extinction coefficients of the chromophoric building blocks are reduced by about 17 % for 2-G1 and 30% for 2-G2 (Table 1). This suggests that different conformations or relative positioning of the dyes within the dendrimeric architecture might occur, in particular in the case of 2-G2, suggesting that partial interactions and more compact arrangement might occur in that case. Dye 10 exhibits a significant singlet oxygen quantum yield as well as a reasonable fluorescence quantum yield (Table 1), as was initially required for allowing both therapy (by photosensitized production of singlet oxygen) and monitoring (by fluorescence imaging).

The grafting of photosensitizers on the dendrimeric scaffold (from 10 to 2-G1) leads to a slight red shift and broadening of the emission (Fig 2 ), in relation with an increase of the polarity produced by polarizable environment produced by the dendrimeric scaffold and the closeness of adjacent quadrupolar dyes. [32] Interestingly, in spite of the confinement of the dyes chromophores in a reduced volume, 2-G1 maintains the same fluorescence quantum yield than the isolated dye 10. This indicates that no significant through-space interchromophoric interactions which would lead to fluorescence quenching occurs within 2-G1. At opposite, fluorescence quenching of fluorescence is often observed when widespread fluorescent dyes (such as Nile red, dioxaborine…) are grafted in close proximity onto a similar dendritic architecture. [33] In contrast, rigid extended dendrimers where quadrupolar subunits are directly connected by triphenylamine nodes were reported to show strong throughbond coupling leading to cooperative enhancement of 2PA and preservation of high fluorescence quantum yield. [34] In comparison, the design of dendrimers 2-G1 and 2-G2 does not allow through-bond coupling between dye subunits due to the

Accepted Manuscript

presence of alkyl linkers (CH2-CH2) between conjugated moieties. We also note that the fluorescence decay is biexponential most probably in relation with conformational flexibility of the dye. Such phenomenon was not observed in the case of ultra-bright organic nanodots confining more rigid and shorter quadrupolar dyes. [24a] A more pronounced red shift of the emission band is observed with 2-G2, indicating that the addition of a water-solubilizing layer increases the polarity of the environment of the buried chromophores. The most prominent feature is a significant decrease of the fluorescence quantum yield. This behavior indicates that competing deactivation process takes place and/or interaction between the dyes occurs within dendrimer 2-G2 compared to 2-G1. Most probably, the presence of the hydrophilic layer created by the TEG surface moieties produces a different external environment which may induce different organization of the dendritic backbone, as discussed earlier, and favor interchromophoric ground-state interactions that are deleterious to fluorescence. The singlet oxygen production () is also reduced in dendrimers 2-G1 (by 37 %) and 2-G2 (by 56%) as compared to isolated dye 10. We note that this progression is similar to the decrease in extinction coefficient and could be related to the different conformations and possible interactions of the dyes within the dendrimeric backbone. This may also originate from the hindered access of dissolved dioxygen molecules to the buried dyes subunits in dendrimer 2-G2, the TEG surface moieties creating a barrier to the diffusion of O2 molecules. . This is consistent with symmetry breaking occurring in the excited state prior to emission. [35] However, it appears clearly that the emission of nanodot 2-G2 in a low polarity solvent such as toluene is strongly red-shifted in comparison with the emission of 10 in the same solvent, which confirms that the dendrimeric scaffold and the triethyleneglycol chains do influence the polarity of the environment of the chromophores by creating a cybotactic region reminiscent of that of ethyl acetate.

Solvatochromism

Two-photon absorption

2PA measurements were conducted by investigating the twophoton excited fluorescence (2PEF) of dendrimers 2-G1 and 2-G2 in THF. The 2PA spectrum of monomeric dye 10 was also determined for comparison. The quadratic dependence of the 2PEF signal on the excitation intensity was checked for each data point, indicating that no photodegradation or saturation occurs.

As expected from its symmetric quadrupolar type structure, dye 10 shows an intense 2PA band located around 720 nm (with maximum 2PA cross-section of about 1900 GM) which can be ascribed to a higher energy excited state which is strongly twophoton allowed (see figure S2). Yet the lowest-energy excited state also leads to significant 2PA response at 870 nm (Table 2), indicating that this state is both one-photon and two-photon allowed. This effect can be attributed to a breaking of centrosymmetry induced by the dissymmetrical environment of the dye. Indeed, dye 10 bears an alkoxybenzaldehyde moiety only on one side. The presence of this dipolar unit (> 5 D) [36] generates a local electric field which in turns affects the polarization of the quadrupole thus breaking its centrosymmetry. This effect is clearly noticeable, in agreement with the reported strong effect of local electric field on the 2PA of dyes. [37] Along this line, we also note that the two-photon allowed excited state is also slightly onephoton allowed (see Figure S2). Hence the breaking of centrosymmetry results in multiple absorption bands (and thus broader response) as compared to purely symmetrical quadrupolar chromophores.

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Table 2. Two-photon absorption data of compounds 10, 2-G1, 2-G2 in THF.

Cpd

21PA max /nm 2 /GM [a] 2/n /GM 2PA max1 /nm [b] 2 max1 /GM [c] 2 max1 /n /GM 2PA max2 /nm [b] 2 max2 /GM [c] 2 max2 /n /GM 2F /GM [d]  2 /GM [e]  Dendrimers 2-G1 and 2-G2 also show the same intense 2PA bands located at 820 and 730 nm as clearly seen from Fig. 5, leading to very large 2PA responses at 730 nm (up to 19200 GM for 2-G1 and 13200 GM for 2-G2,). Yet, we note that the molecular response of each individual dye is affected by the confinement and the modification of the environment induced by the dendrimeric scaffold (see Figure S1). As seen from Table 2, a reduction of the 2PA cross-sections per chromophoric subunit of about 20-25 % (resp. 40-45 %) is observed at 870 nm, 820 nm and 730 nm for 2-G1 (resp. 2-G2). Addition of the water-solubilizing groups leads to a further decrease of about 30% of the 2PA crosssections for dendrimer 2-G2 as compared to 2-G1. This decrease is reminiscent of the decrease noted in one-photon absorption, although the trend is even stronger. This once again confirms that different positioning and possibly partial ground-state interactions between dye subunits occur in the dendrimeric architecture, leading to both decrease of 1PA and 2PA. Yet we note that dendrimers 2-G1 and 2-G2 retain very large 2PA cross-sections at 730 nm (19200 and 13200 GM respectively) and maintain high 2PA response over 5000 GM down to 900 nm. This effect can be ascribed to symmetry breaking (also noted in dendrimers 2-G1 and 2-G2, see Figure S3 and S4) promoted by the presence of dipolar moieties in close proximity. This results in a broadening of the 2PA spectrum, which is of interest for biological applications and use in two-photon bioimaging.

Investigation of cell internalization by fluorescence imaging

Based on its properties (1PA and 2PA responses, fluorescence properties and presence of water-solubilizing surface groups), dendrimer 2-G2 was tested in one-and two-photon excited fluorescence (2PEF) cell imaging. In order to analyze its penetration in cancer cells, dendrimer 2-G2 was incubated for 24 h with MCF-7 human breast cancer cells, whose nuclei were stained with Hoechst 33342 as described in the experimental section. As shown in Figure 6, confocal fluorescence microscopy showed that 2-G2 is efficiently internalized. Accepted Manuscript The cell distribution of dendrimer 2-G2 was also investigated by co-staining with a membrane marker. MCF-7 breast cancer cells were incubated for 3 h with 2-G2, then plasma membranes were stained with CellMask as described in the experimental section (Fig 7). Merged images of membranes and 2-G2 showed that NDs were efficiently internalized after 3 h of incubation. The semi-circular shape of NDs localization in cell suggests that 2-G2 molecules are located in lysosomes. Hence, 2-G2 is internalized by cancer cells. The amount of internalized dendrimers depends on the incubation time. We found that more dendrimers were present in cells after 24 h incubation (Fig 6) than after 3 h (Fig 7). In all cases, internalization occurs and 2-G2 was found to be nontoxic at 50 µg.mL -1 without irradiation (not shown). An incubation time of 20 h was thus selected for PDT experiments.

Two-photon excited photodynamic therapy (2PE-PDT) experiments

2PE-PDT experiments were performed on MCF-7 breast cancer cells incubated with or without 2-G2 (50 g. mL -1 ) for 20 hours. After incubation, cells were washed, maintained in fresh culture medium and then submitted or not to laser irradiation. Twophoton irradiation was performed on a confocal microscope equipped with a femtosecond pulsed Ti:sapphire laser. The laser beam was focused using a microscope objective lens (10X, NA 0.4). Cells were irradiated at 760 nm by 3 scans of 1.57 sec each at an average power of 80 mW. The surface of the scanned areas was 1.5 x 1.5 mm 2 (mean energy of 16.6 J cm -2 ). Two days after irradiation, the percentage of living cells was determined by MTS enzymatic assay. Results are reported in Figure 8, and 2-G2 was found to be non-toxic without irradiation.

It was also shown that irradiation alone did not damage the cells.

Hence, the photodynamic therapeutic potential of 2-G2 is demonstrated, with 78% cell death under two-photon irradiation of MCF-7 cells incubated with 2-G2. The in vitro efficiency of this compound is thus comparable to that of mesoporous silica nanoparticles (MSNs) incorporating a closely related chromophore towards the same MCF-7 cell line, these MSNs leading to 56% cell death after 4 h incubation time and 100% cell death after 24 h incubation time, followed by irradiation (3 x 1 s) at the same wavelength and power. [4e] As these MSNs had also been demonstrated to induce tumor regression (70% decrease in tumor size) on nude mice bearing tumor xenografts upon 9 min of irradiation at the same wavelength (i.e. 760nm) and power, a required irradiation time of 10-20 min can be anticipated with organic dendrimer 2-G2 for in vivo 2PE-PDT. It should be added that 2PE-PDT drugs bearing peptides specific of tumor vasculature targeting, already led to even higher tumor regressions for deep-tissue cancer treatment on mice, with comparable irradiation times. [4c, 4j] This indicates that further surface functionalization of dendrimer 2-G2 with similar targeting peptides would be of major interest for PDT.

Accepted Manuscript

The two-photon induced toxicity of 2-G2 can originate from different processes including singlet oxygen production. Indeed 2-G2 displays a two-photon induced singlet oxygen generation (2), which is 120 times larger than that of TPP (see Table 2). Yet formation of reactive oxygen species via different photoinduced processes (in particular electron transfer sensitized by quadrupolar subunits, which become strong electron donor in the excited state) may also contribute to the two-photon induced cell death. Interestingly, no cell death was observed when the cells were exposed to daylight for 4 hours, indicating that dendrimer 2-G2 is nontoxic under daylight illumination conditions (Figure 9). Dendrimer 2-G2 is thus nontoxic in the dark and even under daylight exposition. This represents an important progress in view of therapeutics as it allows circumventing side effects in current daylight. The daylight toxicity represents a common drawback of photodynamic therapy medical treatment as it requires patients to avoid direct light for a certain time. This effect can be related to the large number of UV absorbers present on the outskirt of the phosphorous dendritic backbone of 2-G2, which form an effective UV-screen, avoiding excitation of the inner quadrupolar photosensitizers by UV from sunlight. At opposite, these constitutive UV absorbers are very poor 2P absorbers in the 700-950 nm spectral range, thus permitting 2P excitation of the inner quadrupolar subunits. This leads to a twophoton versus one-photon excitation on-off process (i.e. the dendritic platform is transparent to two-photon excitation in the NIR region but absorbing upon UV excitation).

Conclusions

In summary we have described a new class of dendrimeric nanoparticles for theranostics, which combine unique properties for bioimaging and anticancer therapy. These fully organic, soft, yet monodisperse nanoparticles show: (i) high two-photon absorption in the biological spectral window, which allows highly confined and three-dimensional photoaddressing in tissues, thus limiting side damages, (ii) large two-photon brightness, which allows fluorescent tracking in cells and investigation of cell internalization, (iii) efficient two-photon photosensitizing properties in the NIR region leading to highly spatially controlled cell death.

In vitro experiments conducted on human breast cancer cells demonstrated these dendrimeric nanoparticles to be efficient nanomedicine tools as their distribution and penetration within cells can be efficiently monitored by fluorescence imaging, while cancer cell death can be induced efficiently and selectively upon two-photon excitation in the NIR. Moreover they were found to be non-toxic in the dark and, more importantly, under daylight exposition, which is a major advantage in comparison with other photodynamic therapy medical treatments. The grafting of targeting functions could further improve the therapeutic promise of these nano-objects by adding to the spatial selectivity of the two-photon activation a high affinity for cancer cells. We are currently exploring this way.

Experimental Section

5-Iodo-2-thiophenecarboxaldehyde (3).

A mixture of 2thiophenecarboxaldehyde (1.79 g, 15.92 mmol), iodine (2.00 g, 7.96 mmol) in carbon tetrachloride (4 mL), iodic acid (0.70 g, 3.98 mmol), distilled water (3 mL), acetic acid (8 mL) and concentrated sulfuric acid (0.12 mL) were stirred under reflux for 1h and at room temperature for another 12h. After washing with 2% NaHCO3 and Na2S2O3 the organic layer was dried over anhydrous MgSO4 and the solvent was removed in vacuum. The crude product was purified by column chromatography eluting with ethyl acetate/ heptane (1:9) to yield 2.95 g (78%) of 3 as yellowish crystals, mp 53 °C; 1 H NMR (CDCl3): 9.8 (s, 1H), 7.4 (s, 2H).

13

C NMR (CDCl3): 181.1, 149.6, 138.2, 137.0, 87.8.

2-(Ethyl-[4-[2-(5-iodothiophen-2-yl)vinyl]phenyl]amino)ethanol (7a).

Air was removed from a mixture of 3 (1.95 g, 8.19 mmol), phosphonium salt 6 (6.32 g, 12.29 mmol) and tBuOK (1. 84 g, 16.38 mmol) by bubbling with argon for 20 min. Fresh distilled CH2Cl2 (50 mL) was added. The solution was stirred at room temperature for 16 h. It was then filtered through a short pad of silica gel. The solvent was removed under reduced pressure, and the crude product was purified by column chromatography eluting with CH2Cl2/ AcOEt (98:2 to 92:8) to yield 2.16 g of a mixture of Z/E stereoisomers. To 1.34 g of this mixture of isomers dissolved in Et2O (50 mL) was added a solution of iodine in Et2O (1 g.L -1 , 3.4 mL). The mixture was then stirred 21h at room temperature under light. After washing with sat Na2S2O3, the organic layer was dried over anhydrous MgSO4 and the solvent was removed in vacuum to yield 1.02 g (50%) of 7a as yellow crystals. M.p: 93 °C. 1 H NMR (CDCl3): 7.3 (d, J= 8.8 Hz, 2H), 7.1 (d, J=3.7 Hz, 1H), 6.9 (d, J= 16 Hz, 1H), 6.78 (d, J=16 Hz, 1H), 6.74 (d, J=8.8 Hz, 2H), 6.6 (d,J=3.7 Hz, 1H), 3.8 (q, 2H), 3.5 (m, 4H), 1.6 (t, J= 5.8 Hz, 1H), 1.2 (t, J= 7 Hz, 3H). 13 C NMR (CDCl3): 149.7, 147.7, 137.2, 129.1, 127.5, 125.6, 124.7, 116.5, 112.3, 70.2, 60.0, 52.2, 45.4, 11.8.

4-[2-(Ethyl-[4-[2-(5-iodothiophen-2-yl)vinyl]phenyl]amino)ethoxy]benzaldehyde (7b).

Under argon, a mixture of 7a (0.5 g, 1.25 mmol), 4hydroxybenzaldehyde (0.31 g, 2.51 mmol) and triphenylphosphine (0.66 g, 2.51 mmol) was dissolved in THF (15 mL) and bubbled with argon for 20 min. A solution of DIAD (0.5 mL, 2.51 mmol) in THF (3 mL) was then added dropwise and the mixture was stirred overnight at room temperature. The solvent was removed in reduce pressure and the crude product was purified on column chromatography with CH2Cl2 to yield 0.51

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FULL PAPER g (81%) of yellow crystals. M.p: 108 °C. 1 H NMR (CDCl3) 9.9 (s, 1H), 7.8 (d, J= 8.8 Hz, 2H), 7.3 (d, J= 8.8 Hz, 2H), 7.1 (d, J= 3.7 Hz, 1H), 7.0 (d, J= 8.7, 2H), 6.9 (d, J= 15,5 Hz, 1H), 6.78 (d, J= 16 Hz, 1H), 6.72 (d, J= 8.9 Hz, 2H), 6.65 (d, J=3.7 Hz, 1H), 4.2 (t, J= 6 Hz, 2H), 3.8 (t, J= 5.9 Hz, 2H), 3.5 (q, 2H), 1.3 (t, J= 7 Hz, 3H). 13 C NMR (CDCl3): 190.7,163.6,147.2,145.6,133.4,131.9,130.19,130.16,129.5,127.9,124.3,120.2,117.1,114.7,111.9,99.4,98.2,65.8,49.5,45.8,12.3.

4-[2-(Ethyl-[4-[2-(5-trimethylsilanylethynylthiophen-2-yl)vinyl]

phenyl]amino)ethoxy]benzaldehyde (8): Air was removed from the solution of 7b (0.77 g, 1.54 mmol) dissolved in toluene (15 mL) and Et3N (3 mL) by bubbling with argon for 20 min. After heating at 40 °C, CuI (6 mg, 0.03 mmol), Pd(PPh3)2Cl2 (22 mg, 0.03 mmol) and ethynyltrimethylsilane (0.33 mL, 2.31 mmol) were added. The mixture was stirred at 40 °C overnight. Solvent was removed by reduce pressure and the crude product was purified by column chromatography with CH2Cl2/ Heptane (6:4 to 7:3) to yield 0.63 g (87%) of 8. 1 H NMR (CDCl3): 9.9 (s, 1H), 7.9 (d, J= 9 Hz, 2H), 7.4 (d, J= 9 Hz, 2H), 7.1 (d, J=3.7 Hz, 1H), 7.01 (d, J= 8.7 Hz, 2H), 6.97 (d, J= 16 Hz, 1H), 6.85 (d, J= 16 Hz, 1H), 6.82 (d, J= 3.8 Hz, 1H), 6.7 (d, J= 8.9 Hz, 2H), 4.2 (t, J= 6 Hz, 2H), 3.8 (t, J= 5.9 Hz, 2H), 3.5 (q, 2H), 1.3 (t, J= 7 Hz, 3H), 0.3 (s, 9H). 13 C NMR (CDCl3): 190.7,163.6,147.2,145.6,133.4,131.9,130.19,130.16,129.5,127.9,124.3,120.2,117.1,114.7,111.9,99.4,98.2,65.8,49.5,45.8,12.3.

4-[2-(Ethylphenyl-mino)ethoxy]phenol (2a):

2-[ethyl-(phenyl)amino]ethanol (1) (3.00 g, 18.07 mmol), hydroquinone (3.98 g, 36.14 mmol) and PPh3 (9.51 g, 36.14 mmol) were dissolved in THF (55 mL). Air was removed by bubbling with argon for 20 min. A solution of DIAD (7. 16 mL, 36.14 mmol) in THF (35 mL) was then added dropwise and the mixture was stirred overnight at room temperature. The solvent was removed in reduce pressure and the crude product was purified on column chromatography with CH2Cl2 to yield 1.68 g (36%). 1 H NMR (CDCl3): 7.38 (q, 2H), 6.87 (m, 7H), 6.58 (s, 1H), 4.17 (t, J= 6.3 Hz, 2H), 3.80 (t, J= 6.3 Hz, 2H), 3.59 (q, 2H), 1.31 (t, J= 6.9 Hz, 3H). 13 C NMR (CDCl3): 152. 6, 149.6, 147.4, 129.3, 116.1, 115.5, 111.9, 66.0, 49.6, 45.5, 12.1.

[[4-[Ethyl[2-(4-hydroxyphenoxy)ethyl]amino]phenyl]methyl]triphenylphosphonium iodide (2b):

Compound 7 (0.600 g, 2.34 mmol), PPh3 (0.552 g, 2.34 mmol) and paraformaldehyde (0.063 g, 0.70 mmol) were dissolved in toluene (8 mL). NaI (0.434 g, 2.34 mmol), water (0.4 mL) and acetic acid (1.2 mL) were then added. The solution was stirred for 66 h at 61 °C. Water (30 mL) was added and the mixture was stirred for 10 min. The aqueous layer was extracted with CH2Cl2 and the combined organic layers were washed with NaHCO3 (20 mL) and water (20 mL), dried and evaporated under reduced pressure. The crude product was washed with Et2O to afford 1.362 g of 2b (88%). 1 H NMR (CDCl3): 7.64 (m, 15H), 7.05 (d, J= 8.7 Hz, 2H), 6.76 (m, 4H), 6.44 (d, J=8.7 Hz, 2H), 4.88 (d, J= 12.9 Hz, 2H), 4.05 (t, J=6.3 Hz, 2H), 3.77 (s,1H), 3.71 (t, J=6.3 Hz, 2H), 3.48 (q, 2H), 1.22 (t, J=6.9 Hz, 3H). 13 C NMR (DMSO-d6): 151.2, 151.0, 147.2 (d, J = 2.7 Hz), 134.8 (d, J = 2.9 Hz), 133.9 (d, J = 9.7 Hz), 131.0 (d, J = 9.7 Hz), 129.9 (d, J = 12.2 Hz), 118.1 (d,J = 84.7 Hz),115.6,115.2,112.4 (d,J = 8.7 Hz),111.5,65.7,48.8,44.5,27.5 (d,J = 45.0 Hz),11.6. 31 P NMR (DMSO-d6): 21.1.

4-[2-(Ethyl-[4-[2-(5-iodothiophen-2-yl)vinyl]phenyl]amino)ethoxy]phenol (4a):

Air was removed from a mixture of 2b (2.09 g, 3.18 mmol), 3 (0.556 g, 2.34 mmol) and tBuOK (0.53 g, 4.67 mmol) by bubbling with argon for 20 min. Fresh distilled CH2Cl2 (25 mL) was added. The solution was stirred at room temperature overnight, then filtered through a short pad of mixed celite and silica gel. The solvent was removed under reduce pressure, and the crude product was purified on column chromatography with mixed solvent CH2Cl2 to yield 0.62 g of Z/E stereoisomers (45/55). The mixture of isomers was then mixed with I2 in Et2O under beam of light to obtain 4a (54%). M.p: 114 °C. 1 H NMR (CDCl3): 7.34 (d, J= 8.8 Hz, 2H), 7.11 (d, J= 3.8 Hz, 1H), 6.98 (d, J= 16 Hz, 1H), 6.78 (d, J=16 Hz, 1H), 6.77 (d, J= 2.5 Hz, 4H), 6.71 (d, J= 9 Hz, 2H), 6.64 (d, J= 3.8 Hz, 1H), 4.53 (s, 1H), 4.09 (t, J=6.3 Hz, 2H), 3.74 (t, J=6 Hz, 2H) 3.53 (q, 2H), 1.24 (t, J= 6.9 Hz, 3H). 13 C NMR (CDCl3): 152.7,149.9,149.7,147.4,137.4,129.4,127.8,125.8,124.4,116.5,116.1,115.5,111.8,70.3,66.0,49.6,45.6,12.3. (36 mL), DMAP (22 mg, 0.18 mmol) was added and stirred 5 min at 0°C. At 0°C, Et3N (0.39 mL) was then added and acetyl chloride (0.19 mL, 2.73 mmol) was added dropwise. The mixture was stirred overnight at room temperature. Ice was added in solution followed by NaHCO3sat (20 mL) and stirred 5 min. The organic layer was then extracted (3 × 15 mL), dried and concentrated under vacuum. The crude product was purified on column chromatography with CH2Cl2 to yield 0.61 g (97%) of 4b. 1 H NMR (CDCl3): 7.34 (d, J= 8.7 Hz, 2H), 7.11 (d, 3.7 Hz 1H), 7.01 (d, 9 Hz, 2H), 6.98 (d, J= 16 Hz, 1H), 6.89 (d, J= 9.3 Hz, 2H), 6.78 (d, J= 16 Hz, 1H), 6.71 (d, J= 9 Hz, 2H), 6.64 (d, J= 4 Hz, 1H). 13 C NMR (CDCl3): 169.8, 156.3, 150.0, 147.3, 144.4, 137.4, 129.5, 127.8, 125.8, 124.6, 122.4, 116.6, 115.0, 111.82, 111.8, 70.3, 65.8, 49.6, 45.7, 12.3.

Acetic

Acetic acid 4-[2-(ethyl-[4-[2-(5-trimethylsilanylethynylthiophen-2yl)vinyl]phenyl]amino)ethoxy]phenyl ester (5):

Air was removed from the solution of 4b (0.37 g, 0.70 mmol) dissolved in toluene (5 mL) and Et3N (1 mL) by bubbling with argon for 20 min. After heating at 40 °C, CuI (3 mg, 0.02 mmol), Pd(PPh3)2Cl2 (12 mg, 0.02 mmol) and ethynyltrimethylsilane (0.19 mL, 1.31 mmol) were added. The mixture was stirred at 40 °C overnight. Solvent was removed by reduce pressure and the crude product was purified by column chromatography with CH2Cl2/ Heptane (6:4 ) to yield 0.33 g (94%) of 5. 1 H NMR (CDCl3): 7.37 (d, J= 9 Hz, 2H), 7.12 (d, J= 3.9 Hz, 1H), 7.03 (d, J=9 Hz, 2H), 6.98 (d, J= 16 Hz, 1H), 6.90 (d, J= 9 Hz, 2H), 6.87 (d, J= 16 Hz, 1H), 6.83 (d, J= 3.9 Hz, 1H), 6.72 (d, J= 9 Hz, 2H), 4.13 (t, J= 6 Hz, 2H), 3.77 (t, J= 5.9 Hz, 2H), 3.54 (q, 2H), 2.29 (s, 3H), 1.26 (t, J= 6.9 Hz, 3H), 0.30 (s, 9 Hz). 13 C NMR (CDCl3): 169.7, 156.11, 156.09, 147.2, 145.5, 144.14, 144.11, 142.2, 133.2, 132.2, 130.6, 129.9, 129.5, 127.7, 127.2, 124.0, 122.17, 122.15, 119.9, 119.7, 116.7, 114.8, 111.6, 111.2, 99.1, 98.0, 65.6, 49.42, 49.39, 45.5, 45.4, 31.7, 31.28, 31.22, 29.97, 29.91, 29.48, 29.45, 29.41, 29.29, 29.15, 28.9, 28.7, 22.5, 20.8, 13.9, 12.1.

4-[2-[[4-[(1E)-2-[5-[(9,9-dibutyl-7-iodo-9H-fluoren-2-yl)ethynyl]2thienyl]ethenyl]phenyl]ethylamino]ethoxy]benzaldehyde (9a):

Air was removed from a mixture of 8 (0.63 g, 1.34 mmol), 9,9-dibutyl-2,7diiodo-9H-fluorene 9 (2.13 g, 4.01 mmol) and Et3N (2.4 mL) dissolved in toluene (12 mL) by bubbling with argon for 20 min. After heating at 40°C, CuI (5.7 mg, 0.03 mmol) and Pd(PPh3)2Cl2 (21 mg, 0.03 mmol) were added. Air was removed from the solution of TBAF in THF (1 M, 0.80 mmol) by blowing argon for 5 min and then added to the mixture. The solution was stirred overnight at 40 °C. Solvent was removed by reduce pressure and the crude product was purified by column chromatography with mixed solvent CH2Cl2/Heptane (6:4) to yield 0.83 g (77%) of orange crystals of compound 9a. M.p: 146 °C. 1 H NMR (CDCl3): 9.89 (s, 1H), 7.85 (d, J= 8.9 Hz, 2H), 7.68 (dd, J= 1.6 Hz, 1H), 7.66 (s, 1H), 7.63 (d, J= 8.6 Hz, 1H), 7.51 (dd, J= 1.4 Hz, 1H), 7.46 (s, 1H), 7.42 (d, J= 8.4 Hz, 1H), 7.39 (d, J= 9 Hz, 2H), 7.18 (d, J= 3.6 Hz, 1H), 7.01 (d, J= 8.7 Hz, 2H), 6.96 (d, J= 16 Hz, 1H), 6.90 (d, J= 3.3 Hz, 1H), 6.89 (d, J= 15.3 Hz, 1H), 6.73 (d, J= 9 Hz, 2H), 4.25 (t, J= 6.3 Hz, 2H), 3.83 (t, J= 5.7 Hz, 2H), 3.56 (q, 2H), 1.96 (m, 4H), 1.26 (t, J= 7.2 Hz, 3H), 1.14 (q, 4H), 0.72 (t, J= 7.2 Hz, 6H), 0.56 (m, 4H). 13 C NMR (CDCl3): 190.6,163.4,153.2,150.0,147.0,145.5,140.2,139.8,135.8,132.5,131.87,131.82,130.3,

Accepted Manuscript

FULL PAPER 129.9, 129.3, 127.8, 124.7, 124.6, 121.7, 121.5, 120.2, 119.6, 116.9, 114.5, 111.7, 94.6, 92.9, 83.7, 65.6, 55.1, 49.3, 45.6, 39.9, 29.5, 25.7, 22.8, 13.6, 12.2. HRMS (ESI) calcd for C46H46NO2INaS [(M+Na) + ] m/z 826.21862, found 826.2185.

4-[2-[[4-[(1E)-2-[5-[2-[9,9-dibutyl-7-[2-[5-[(1E)-2-[4-[ethyl-[2-[4-(formyl)phenoxy]ethyl]amino]phenyl

]ethenyl]-2-thienyl]ethynyl]-9Hfluoren-2-yl]ethynyl]-2-thienyl]ethenyl]phenyl]ethylamino]ethoxy]phenyl acetate (9b): Air was removed from 5 (0.33 g, 0.66 mmol), the previous compound 9a (0.48 g, 0.60 mmol) and Et3N (2.8 mL) dissolved in toluene (14 mL) by bubbling with argon for 20 min. After heating at 40°C, CuI (2.3 mg, 12 µmol) and Pd(PPh3)2Cl2 (8.4 mg, 12 µmol) were added. Air was removed from the solution of TBAF in THF (1 M, 0.42 mmol) by blowing argon for 5 min and then added to the mixture. The solution was stirred overnight at 40 °C. Solvent was removed by reduce pressure and the crude product was purified by column chromatography with mixed solvent CH2Cl2/Heptane (7:3) to yield 0.46 g (70%) of orange crystals of compound 9b. M.p: 160 °C. 1 H NMR (CDCl3): 9.89 (s, 1H), 7.85 (d, J= 9 Hz, 2H), 7.68 (d, J=8.6 Hz, 2H), 7.52 (m, 4H), 7.39 (dd, J=3 Hz, 4H), 7.19 (d, J=4 Hz, 2H), 7.02 (d, J= 16 Hz, 2H), 7.01 (d, J= 9 Hz, 2H), 6.90 (s, 4H), 6.89 (s, 2H), 6.85 (d, J=16 Hz, 2H), 6.74 (dd, J=3 Hz, 4H), 4.24 (t, J=6 Hz, 2H), 4.14 (t, J=6 Hz, 2H), 3.82 (m, 4H), 3.53 (qd, 4H), 2.29 (s, 3H), 2.03 (m, 4H), 1.28 (td, J= 3.9 Hz, 6H), 1.16 (m, 4H), 0.73 (t, J= 7.2 Hz, 6H), 0.63 (m, 4H). 13 C NMR (CDCl3): 190.9,170.0,163.8,156.4,151.2,145.8,144.5,132.8,132.2,130.6,130.3,129.8,128.1,125.83,125.76,125.0,124.0,124.81,124.77,122.5,120.6,120.1,117.3,115.2,114.9,111.9,65.94,65.92,55.3,49.8,49.62,49.60,45.96,45.86,26.0,23.2,21.1,13.9,12.5. HRMS (ESI) calcd for C72H70N2O5NaS2 [(M+Na) + ] m/z 1129.46184, found 1129.4620.

Photosensitizer 10: The compound 9b (0.46 g, 0.42 mmol) was dissolved in a mixture of EtOH (10 mL) and THF (28 mL). NaOH (4 mL) was then added and the solution was stirred 20 min at room temperature, and HCl (15 mL) was added. Aqueous layer was extracted with CH2Cl2 (10 mL), and organic layer was then washed with water (5 mL). The organic layer was dried and concentrated under vacuum. The crude product was purified on column chromatography with CH2Cl2 to yield 0.37 g (84%) of orange crystals of 10. M.p: 94 °C. 1 H NMR (CDCl3): 9.89 (s, 1H), 7.85 (d, J= 9 Hz, 2H), 7.68 (d, J=8.6 Hz, 2H), 7.52 (m, 4H), 7.39 (dd, J=3 Hz, 4H), 7.19 (d, J=4 Hz, 2H), 7.01 (d, J= 9 Hz, 2H), 6.99 (2×d, J= 16 Hz, 2H), 6.89 (m, 2H), 6.87 (d, J=16 Hz, 2H), 6.77 (s, 4H), 6.74 (dd, J=3 Hz, 4H), 5.19 (s, 1H), 4.24 (t, J=6 Hz, 2H), 4.10 (t, J=6 Hz, 2H), 3.82 (t, J= 5.7 Hz, 2H), 3.73 (t, J=5.7 Hz, 2H), 3.53 (m, 4H), 2.01 (m, 4H), 1.28 (2×t, J= 3.9 Hz, 6H), 1.16 (m, 4H), 0.73 (t, J= 7.2 Hz, 6H), 0.63 (m, 4H). 13 C NMR (CDCl3): 191.1,163.8,152.8,151.2,149.9,147.6,147.3,145.9,145.8,140.76,140.71,132.77,132.16,130.6,130.1,129.8,129.6,128.04,128.01,125.7,124.93,124.86,124.7,124.6,121.83,121.77,120.5,120.3,120.1,117.2,116.9,116.1,115.6,114.8,111.9,111.8,105.0,95.0,84.07,84.01,66.1,65.9,55.3,49.8,49.5,45.9,45.7,40.3,25.9,23.1,13.9,12.43,12.40. HRMS (ESI) calcd for C70H69N2O4S2 [(M+H) + ] m/z 1065.46933, found 1065.4691. ATR-FTIR (cm -1 ): 3363, 2926, 2189, 2037, 1685, 1596, 1507, 1446, 1397, 1349, 1256, 1221, 1182, 1158, 1136, 1074, 1020, 946, 885, 806, 751.

4-[2-[2-(2-Methoxyethoxy)ethoxy]ethoxy]phenol (12).

In a solution of triethylene glycol monomethyl ether (6.00 g, 36.58 mmol) in CH2Cl2 (10 mL) at 0°C, was added Et3N (5.83 mL). The mixture was cooled below 5°C. Stirring vigorously and maintaining the reaction mixture at that temperature, a solution of p-toluenesulfonyl chloride (3.25 mL; 42.07 mmol) in 3mL of CH2Cl2 was added dropwise. Stirring was continued overnight at room temperature. A white precipitate was filtered and the filtrate was washed with NaHCO3 (2 × 10mL). The aqueous layer was extracted with CH2Cl2. Organic layers were combined, dried over Na2SO4 then evaporated under reduce pressure to yield 8.46 g (95%) of yellow oil.

In a solution of this oil (8. 46 g, 34.97 mmol) in 25 mL of DMSO, was added hydroquinone (3.86 g, 34.97 mmol), and KOH (5.89 g, 104.91 mmol). The mixture was stirred overnight at room temperature (water bath) and 20 mL of water was then added. The solution was extracted (3 × 30 mL) with Et2O to remove both unreacted tosylate and disubstituted byproduct. The solution was then acidified with concentrated HCl and extracted with CHCl3 (3 × 20 mL). The combined organic layers were washed with water until neutrality, dried over Na2SO4, and concentrated in vacuo to yield 3.69 g (41%) of brown oil of 12. 1 H NMR (CDCl3): 6.82 (s, 1H), 6.73 (d, J = 9 Hz, 2H), 6.69 (d, J = 9 Hz, 2H), 3.98 (t, J = 4.8 Hz, 2H), 3.79 (t, J = 4.8 Hz, 2H), 3.70 (m, 6H), 3.55 (m, 2H), 3.34 (s, 3H). 13 C NMR (CDCl3): 152.3, 150.1, 115.9, 115.5, 71.7, 70.5, 70.4, 70.3, 69.7, 67.8, 58.8. HRMS (ESI) calcd for C13H20N2O5Na [(M+Na) + ] m/z 279.12029, found 279.1203. ATR-FTIR (cm -1 ): 3333.3, 2873.0, 1508.4, 1448.9, 1355.7, 1214.9, 1096.0, 1064.3, 824.4, 753.1. Dendrimer 2-G1. The reaction was shielded from the light. To a solution of photosensitizer 10 (152 mg, 142.72 mol) in distilled THF (0.75 mL) was added Cs2CO3 (93 mg, 285.43 mol). The solution was stirred 2h at 35°C. A solution of dendrimers bearing P(S)Cl2 1-G1 (20 mg, 10.95 mol) in 0.75 mL of distilled THF was then added and stirred 7days at 35°C. The reaction was monitored by 31 P using a C6D6 capillary. The resulting mixture was filtered and dendrimer was precipitated in pentane (200 mL). The orange precipitate was purified on column chromatography (pentane: THF: 1:1 until THF 100%) and washed several times with AcOEt, to eliminate the excess of 10, and dried under reduce pressure to yield 106 mg (69%) of orange powder. Mp: 178 °C. 1 H NMR (CD2Cl2) 9.86 (s, 12H), 7.82 (d, J = 8.7 Hz, 24H), 7.67 (dd, J = 7.8 Hz, 24H), 7.59 (m, 66H), 7.36 (d, J = 9 Hz, 24H), 7.30 (d, J = 8.4 Hz, 24H), 7.16 (dd, J = 3.9 Hz, 24H), 7.05 (m, 132 H), 6.73 (d, J = 8.7 Hz, 48H), 6.64 (d, J = 7.5 Hz, 24H), 4.24 (t, J = 6 Hz, 24H), 3.98 (t, J = 6 Hz, 24H), 3.80 (t, J = 6 Hz, 24H), 3.62 (s br, 24H), 3.54 (q, 24H), 3.40 (s br, 24H), 3.21 (d, J = 12 Hz, 18H), 2.00 (m, 48H), 1.21-1,02 (m, 120H), 0.77-0.48 (m, 120H). 13 C NMR (CD2Cl2) 191.42,164.63,157.13,152.18,148.45,146.77,145.22,141.67,133.73,132.72,131.31,131.16,130.59,128.82,126.64,125.77,123.22,122.68,122.25,121.19,121.00,117.70,115.97,115.65,112.84,95.75,84.76,84.67,66.95,61.16,56.14,55.08,54.72,54.36,50.39,46.68,41.03,34.00,31.01,26.92,23.99,14.93,14.57,13.01. 31 P (CDCl3),64.40,8.63. LC/MS: calcd avg mass for C888H852N39O54NaP9S30 [(M+Na) + ] m/z 14198.2 found: 14197.1.

Dendrimer 1-G2. The reaction was shielded from the light. 2-G1 (86.4 mg, 6.10 mol) was cooled at 0 °C and 11 (508.3 L, 91.5 mol) in solution in CHCl3 was added. The solution was stirred 5 days at room temperature (monitored by 1 H NMR). Dendrimer was precipitated in pentane (200 mL), to remove excess of 11, filtered under argon then dried under reduce pressure to yield 85 mg (86%) of orange powder. M.p: 184 °C. 1 H NMR (CDCl3) 7.68 (m, 120H), 7.38 (d, J = 8.4 Hz, 24H), 7.31 (d, J = 8.4 Hz, 24H), 7.16 (dd, J = 3.7 Hz, 24H), 7. 06-6.63 (m, 192 H), 4.19 (t, J = 4.5 Hz, 24H), 3.96 (s br, 24H), 3.80 (s br, 24H), 3.62 (s br, 24H), 3.53 (m, 84H), 3.19 (d, J = 9.6 Hz, 18H), 1.98 (m, 48H), 1.15 (m, 120H), 0.69 (m, 120H). 13 C NMR (CDCl3): 160. 31,156.04,151.50,151.11,147.34,145.72,144.23,141.71,140.65,135.75,132.68,130.52,129.56,128.95,128.23,127.95,127.19,125.59,125.50,124.72,124.58,122.29,121.73,121.37,120.41,120.33,120.02,117.08,116.95,115.03,114.75,111.84,94.92,83.94,65.75,65.56,55.17,49.58,45.76,45.63,40.18,34.21,33.07,31.75,30.31,25.90,23.05,21.18,13.85,12.34,12.31. 31 P (CDCl3) 64.41,63.30,8.66.

Dendrimer 2-G2. The reaction was shielded from the light. To a solution of 12 (12 mg, 46.50 mol) in distilled THF (2mL), was added Cs2CO3 (29 mg, 89.28 mol) and stirred overnight at room temperature. 1-G2 (30.1 mg, 1.86 mol) in 1.5 mL of distilled THF was then added to the mixture and stirred 5 days at room temperature. (monitored by 31 P NMR). The Accepted Manuscript PAPER solution was filtered, and dendrimer was precipitated in pentane (200 mL). The resulting powder was washed with Et2O to removed excess of 12 then solubilized in THF and centrifuged to remove cesium traces. Dendrimer was again precipitated in pentane, filtered under argon, and dried under reduce pressure to yield 30 mg (75%) of orange powder. M.p: 178 °C. 1 H NMR (CDCl3) 7.68-7.28 (m, 168H), 7.13-6.52 (m, 317H), 4. 16-3.16 (m, 558H), 1.97 (m, 48H), 1.11 (m, 120H), 0.66 (m, 120H). 31 P (CDCl3) 64.59, 8.61.

Photophysical studies

UV-Vis absorption and emission spectroscopy. All photophysical measurements have been performed with freshly-prepared solutions in air-equilibrated THF at room temperature (298 K). UV/Vis absorption spectra were recorded on a Jasco V-570 spectrophotometer. Steadystate and time-resolved fluorescence measurements were performed on dilute solutions (ca. 10 -6 M chromophore concentration, optical density < 0.1) contained in standard 1 cm quartz cuvettes using an Edinburgh Instruments (FLS920) spectrometer in photon-counting mode. Emission spectra were obtained, for each compound, under excitation at the wavelength of the absorption maximum. Fluorescence quantum yields were measured according to literature procedures using quinine bisulfate in 0.5 M H2SO4 as a standard (quantum yield  = 0.546). [38] The lifetime values were obtained from the reconvolution fit analysis (Edinburgh F900 analysis software) of decay profiles obtained using the FLS920 instrument under excitation with a nitrogen-filled nanosecond flash-lamp. The quality of the fits was evidenced by the reduced ² value (² <1.1).

Measurements of singlet oxygen quantum yield ().

Measurements were performed on a Fluorolog-3 (Horiba Jobin Yvon), using a 450 W Xenon lamp. The emission at 1272 nm was detected using a liquid nitrogen-cooled Ge-detector model (EO-817L, North Coast Scientific Co).

Singlet oxygen quantum yields  were determined in dichloromethane solutions, using tetraphenylporphyrin (TPP) in dichloromethane as reference solution ([TPP] = 0.60) and were estimated from 1 O2 luminescence at 1272 nm.

Two-photon absorption. Two-photon absorption cross sections (2) were obtained from the two-photon excited fluorescence (2PEF) cross sections (2) and the fluorescence emission quantum yield (). 2PEF cross sections in toluene (10 -4 M chromophore concentration) were determined using a Ti-sapphire laser delivering 150 fs excitation pulses, according to the experimental protocol established by Xu and Webb. [39] This experimental protocol allows avoiding contributions from excited-state absorption that are known to result in largely overestimated 2PA crosssections. Fluorescein in 0.01 M NaOH, whose 2PEF cross-sections are well-known, [39] served as the reference, taking into account the necessary corrections for the refractive index of the solvents. [40] The quadratic dependence of the fluorescence intensity on the excitation intensity was verified for each data point, indicating that the measurements were carried out in intensity regimes in which saturation or photodegradation do not occur. More details about the experimental setup have been previously published. [31] Cellular imaging. One-and two-photon imaging have been performed on Montpellier RIO Imaging (MRI) platform. For cell culture, MCF-7 human breast cancer cells (ATCC) were routinely cultured in Dulbecco's Modified Eagle Medium F-12 Nutrient Mixture (Ham) supplemented with glutamax, 10% foetal bovine serum, and 1/100 antibiotics (penicillin/ streptomycin). Cells were allowed to grow in humidified atmosphere at 37°C under 5% CO2. On the one hand, cells were incubated 24 h with dendrimer 2-G2 (50 µg.mL -1 ). Thirty minutes before the end of incubation, cells were loaded with 2 µg.mL -1 Hoechst 33342 for nuclear staining. After 30 min cells were washed and maintain in fresh culture medium without serum and phenol red. For confocal imaging, a Zeiss LSM 780 microscope equipped with DIC plan-apochromat 63x/1.4 NA oil immersion objective was used. Nuclear staining was excited at 405 nm and 2-G2 at 548 nm. On the other hand, cells were incubated 3 h with dendrimer 2-G2 (50 µg/ml). Thirty minutes before the end of incubation, cells were loaded with 1 mg.mL -1 CellMask for plasma membrane stains. For multi-photon imaging, a Zeiss LSM 780 microscope with tuneable Chameleon Ultra II laser (Coherent, mode-locked Ti: sapphire laser, 680-1080 nm) and equipped with DIC plan-apochromat 63x/1.4 NA oil immersion objective was used. 2-G2 and plasma membrane stains were excited at 750 nm. Two-photon excited photodynamic therapy. For experiments of 2PE-PDT, MCF-7 breast cancer cells were seeded in a 384 multi-well plate with 0.17 mm glass bottom and incubated with or without 2-G2 (50 g. mL -1 ) for 20 hours. After incubation, cells were washed, maintained in fresh culture medium and then submitted or not to laser irradiation. Twophoton irradiation was carried out on MRI platform. PDT was then performed on a multi-photon confocal microscope Zeiss LSM 780 equipped with tuneable Chameleon Ultra II laser (Coherent, 680-1080 nm) generating 140 fs wide pulses at 80 MHz rate. The laser beam was focused by a microscope objective lens (10x, NA 0.4). The wells were irradiated at 760 nm by 3 scans of 1.57 sec each at an average power of 80 mW. The surface of the scanned areas was 1.5 x 1.5 mm 2 (mean energy of 16.6 J cm -2 ). Two days after irradiation, the percentage of living cells was determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl) -2-(4sulfophenyl)-2H-tetrazolium) enzymatic assay. Briefly, cells were incubated in the presence of 0.5 mg. mL -1 MTS for 4 h to determine mitochondrial enzyme activity. Absorbance was read at 492 nm with a spectrophotometer Thermoscientific Multiskan FC.

Daylight study.

For experiments of daylight toxicity, MCF-7 cancer cells were seeded in a 96 multi-well plate and incubated with or without 2-G2 (20 g. mL -1 ) for 5 hours. 2-G2 was previously solubilized in THF at 5 mg/mL and sonicated. After incubation, cells were washed, maintained in fresh culture medium and then submitted or not to daylight for 4 hours. The percentage of living cells was determined by MTT (3-(4,5dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide) enzymatic assay. Briefly, cells were incubated in the presence of 0.5 mg. mL -1 MTT for 4 hours to determine mitochondrial enzyme activity. Then medium was removed and purple precipitate was dissolved in 150 l ethanol/DMSO (1:1) solution and stirred 30 min. Then absorbance was read at 540 nm.

Figure 1 .

 1 Figure 1. Molecular design.

  [a] Molar extinction coefficient. [b] Molar extinction coefficient per chromophoric subunit [c]F = fluorescence quantum yield, using as a standard quinine bisulfate (F= 0.546) in 0.5M aq H2SO4. [d] Fluorescence lifetime. [e] = singlet oxygen formation quantum yield determined relative to tetraphenylporphyrin in methylene chloride ([TPP] = 0.60 in methylene chloride).

Figure 2 .

 2 Figure 2. Normalized absorption and emission spectra of compounds 10, 2-G1 and 2-G2, in THF.

Fig 3 and 4

 4 Fig 3 and 4 illustrate the dependence of the absorption and emission properties of dye 10 and soft organic nanodot 2-G2 on solvent polarity. Increasing the solvent polarity induces a slight red shift of the absorption band and a marked bathochromic shift of the emission band as clearly illustrated in Fig 3 for 10 and Fig 4 for 2-G2. This is consistent with symmetry breaking occurring in the excited state prior to emission.[35]

Figure 3 .

 3 Figure 3. Normalized absorption and emission spectra of quadrupolar dye 10 in solvents of different polarities.

Figure 4 .

 4 Figure 4. Normalized absorption and emission of dendrimer 2-G2 in solvents of different polarities.

  [a] 2PA cross section at 21PA max .[b] Maximum 2PA wavelength.[c] 2PA cross section at 2PA max .[d] 2P-brightness at 2PA max2 .[e] Figure of merit of the 2Pinduced singlet oxygen generation at2PA max2 .1 GM = 10 -50 cm 4 .s.photon -1 .

Figure 5 .

 5 Figure 5. 2PA spectra of 10, 2-G1, 2-G2 in THF (with solid curves as guides for the eyes only).

Figure 6 .

 6 Figure 6. Nuclear marker (left, excited at 405nm), 2-G2 (middle, excited at 458 nm) and merge (right) confocal microscopy images of living MCF-7 human breast cancer cells, incubated for 24 h with 50 µg.mL -1 2-G2.

Figure 7 .

 7 Figure 7. Merged multiphoton confocal microscopy images of living MCF-7 breast cancer cells, incubated for 3 h at 37°C with 50 µg.mL -1 2-G2 excited at 750 nm (green) and membrane marker (yellow).

Figure 8 .

 8 Figure 8. 2PE-PDT efficiency on MCF-7 breast cancer cells incubated or not (control) for 20 h with 50 g. mL -1 of dendrimer 2-G2 and then irradiated or not (control) at 760 nm for 3×1.57 s. Data are mean ± SD of 3 experiments.

Figure 9 .

 9 Figure 9. Absence of toxicity on MCF-7 breast cancer cells of 2-G2 in the dark (right) or under daylight exposition (left).

  ]phenyl ester (4b): Under argon, to a mixture of 4a (0.62 g, 1.26 mmol) in CH2Cl2

Table 1 .

 1 Photophysical properties of compound 10, 2-G1 and 2-G2 in THF.

Acknowledgements

We acknowledge financial support from CNRS and Région Bretagne for a fellowship to AS (ARED, Biphobio grant). We wish to thank Vincent Hugues for his contribution to two-photon excited fluorescence experiments. We thank MRI (Montpellier RIO Imaging platform) for confocal facilities.

Entry for the Table Contents


Ngurah Indra
email: indra@unud.ac.id

Deep Kamal

Singh

Jean-Marie Bonnin
email: jean-marie.bonnin@irisa.fr

DC4LED: A Hierarchical VDTN Routing for Data Collection in Smart Cities

In smart cities, the mobility of vehicles can be used to collect data produced by connected objects and to deliver them to several applications which are delay tolerant. The Vehicular Delay Tolerant Networks (VDTN) can be utilized for such services. This paper introduces DC4LED (Data Collection for Low Energy Devices): a hierarchical VDTN routing which takes advantage of the specific mobility patterns of the various type of vehicles. It provides a low-cost delivery service for applications that need to gather data produced from the field. The idea is to propose a simple routing scheme where cars, taxis, and buses route data hierarchically in a storecarry-forward mechanism to any of the several available Internet Point-of-Presences in the city. This paper aims to explore the possibility of implementing a very simple mechanism for very simple services, as opposed to utilizing complex VDTN mechanisms proposed in the literature. Our simulations compare the performance of DC4LED routing with two legacy VDTN routing schemes which represent the extreme ends of VDTN routing spectrum: First-contact and Epidemic routing. It show that DC4LED has much lower network overhead in comparison with the two legacy routing schemes, which is advantageous for its implementation scalability. The DC4LED also maintains comparable data delivery probability and latency to Epidemic routing.

I. INTRODUCTION

Smart cities today, and in the future, will have to cope with huge amounts of data coming from various low energy devices, such as wireless sensors and connectedobjects, for multiple kinds of applications. Some of these applications will be delay-tolerant such as the collection of air pollution or temperature data, smart metering, photos reporting of road degradation, etc. [START_REF] Benamar | Routing protocols in Vehicular Delay Tolerant Networks: A comprehensive survey[END_REF] [START_REF] Abdelkader | A performance comparison of delay-tolerant network routing protocols[END_REF]. These applications will be able to tolerate data delays ranging from a few minutes to some hours. Typical ways to collect data using cellular networks and dedicated networks such as LPWAN might neither be sufficient nor efficient to handle the entire deluge of data, where the bandwidth limitation and the cost of subscription might be the limiting factors. Thus, alternative means to offload part of the delay-tolerant data will become increasingly necessary.

Simultaneously, in the landscape of vehicular technology, the capability of vehicles to communicate with each other (V2V) as well as to connected-objects in their surroundings (V2I) is becoming a norm. Vehicles will also have the capability to connect to the internet with current and future radio access networks. Furthermore, the regulatory body in Europe and US will soon make such communications capability compulsory for vehicles to support the safety-related applications [START_REF] Machardy | V2x Access Technologies: Regulation, Research, and Remaining Challenges[END_REF]. This exciting new development will pave the way for vehicles to participate actively in Smart Cities ecosystems.

Figure 1 illustrates a vehicle-based data collection architecture for smart cities, where stakeholders deploy wireless sensors and associated application servers. Each sensor knows to which application server it has to send the information it produces. The data collection service could be provided for free by the city to develop its IoT ecosystem. Data for delay-tolerant application generated by a set of wireless sensors across the city need to be delivered to its associated central server. The system utilizes Vehicular Delay-Tolerant Networks (VDTN) with its store-carry-forward mechanism to gather and deliver data to one of the wireless Internet Point-of-Presence (PoP) available in the city. Note that the goal of routing for the anticipated data collection is to reach one of the available fixed positioned PoP in the city as fast as possible. It is not needed to reach a specific destination, which could have even been a mobile destination. Thus, the routing algorithm could be far less complex than the existing protocols proposed in the literature. This paper aims to show that a simple algorithm could be efficient enough for most of the usages.

This work proposes and evaluates DC4LED (Data Collection for Low Energy Devices): a hierarchical VDTN routing, which sensibly takes into account several common features of mobility in smart cities (e.g., buses, taxis, and cars), and hierarchically defines their role in forwarding the data. We then evaluate the algorithm using a set of simulation scenarios to acquire its baseline performances for an application-agnostic vehicle-based smart city-wide data collection service.

II. RELATED WORKS

In the literature, there are a lot of legacy routing schemes; a survey of VDTN routing approaches is provided in [START_REF] Benamar | Routing protocols in Vehicular Delay Tolerant Networks: A comprehensive survey[END_REF]. However, most of them are too complex to be implemented in cars, not because of a lack of computational capabilities, but because such mechanisms need to be standardized in international bodies. The standardization environment of ITS (Intelligent Transportation System) is very complex, and if the mechanism to be standardized is too complex, it will take a while to reach a consensus, even if realizable. The data collection mechanism needs to be as simple and as generic as possible to cope with multiple service scenarios and serve a broad range of applications which have few wellidentified characteristics. Moreover, it will be possible to collect data from objects with minimum modification to their behaviors.

Previous work in [START_REF] Almeida | Forwarding Strategies for Future Mobile Smart City Networks[END_REF] described the use of DTN-based architecture implementing multiple technologies to collect various kinds of data in smart cities. Numerous types of mobile nodes are considered such as cars, bicycles, and drones. They applied and evaluated Epidemic Routing strategy for message forwarding. Their real-world evaluations highlighted the problems of the flooding approach which waste a lot of network resources and causes a lot of packet drops due to buffer size limits. Their results showed that Epidemic routing reached a better delivery ratio and a lower latency compared to their proposed strategies, while on the network overhead their approach was about 75% more efficient. In our work, we focus more on the utilization of vehicles to efficiently forward data without too much complexity. Through simulations, our work also allows a performance assessment over a larger city-wide area, as well as showing the effect of increases in vehicular mobility.

III. VDTN ROUTING

In this section, we elaborate on the need for having a simple VDTN routing scheme. Then we propose DC4LED (Data Collection for Low Energy Devices): a hierarchical VDTN routing scheme.

A. Routing Ecosystem

We focus on data collection in cities using VDTN routing. Such vehicular routing ecosystem is very heterogeneous. The data collection chain in a city starts from sensors and then the data is routed using cars, taxis, buses, etc., to an Internet PoP and then finally to Server. We argue that each of these steps in the routing chain has their complexities and constraints. For example, existing works assume routing using advanced strategies such as using multiple copies, GPS information, using an adaptive mechanism, or even social networking strategy among vehicles [START_REF] Ahmed | Vehicular Delay Tolerant Network (VDTN): Routing perspectives[END_REF] [START_REF] Zhu | A Survey of Social-Based Routing in Delay Tolerant Networks: Positive and Negative Social Effects[END_REF]. However, the feasibility and practicality of such adaptive strategies are arguably low. The adaptive mechanism may be implemented in sensors as well as servers which in turn are dedicated equipment of the service. However, as pointed out before, the standardization environment of ITS (Intelligent Transportation System) is very complex. Thus, it is difficult to assume that specialized as well as intelligent crosslayer approaches, such as multiple copies or using GPS information, will be easy to standardize in common cars. Additionally, we also discuss that the behavior and mobility pattern of each entity is different, which in turn has an impact on routing protocol design. Therefore, we review some of the entities below:

• Sensors: It has energy constraints, but can realize adaptive strategies such as forwarding multiple copies or adapting the routing according to the type of vehicle encountered by it.

• Cars: One may envision that only general and simple routing schemes will be standardized and implemented for common cars. Thus, we assume that cars will only provide simple forwarding such as single copy forwarding without necessarily using advanced information such as that of GPS. Also, we notice that cars will have mobility patterns corresponding to a very few trips per day, sometimes with very long stop times. Therefore, cars are not a very reliable entity for forwarding data.

• Taxis: As taxis are operator controlled, one can assume that it will be possible to implement some advanced routing schemes. Service provisioning business can act as motivation for taxi operators.

If we consider their mobility pattern, then taxis generally have multiple trips in a day. The places visited range from random places in the city to a more specific area of interests and particular points such as taxi stands. Thus forwarding data to taxis can have some advantages such that they are more likely to visit many points in a city during the day.

• Buses: As with taxis, buses are also operator controlled and thus advanced routing can also be assumed here. Concerning the mobility, buses have a very deterministic mobility pattern, unless there is congestion in the city. Therefore, buses are a reliable DTN routing option as they visit fixed places and their trajectory is very predictable.

• PoP: It is the internet access point. One cannot assume it to be dedicated for the data collection service. Hence, only general Internet routing may be assumed here.

• Server: As server will already be dedicated, and for now we assume that the data collection service is only one way, from the sensor to the server.

B. DC4LED: A Hierarchical VDTN Routing Algorithm

Most legacy routing approaches for VDTN such as Epidemic , Spray and Wait , MaxProp , and PRoPHET use the multiple-copy approach. However, for such schemes, the work in [START_REF] Abdelkader | A performance comparison of delay-tolerant network routing protocols[END_REF] underlined the high network overhead caused by the high number of redundant message copies. Thus, here we propose DC4LED: a hierarchical VDTN Routing scheme. For a baseline evaluation of our approach, we choose to use a singlecopy routing. Our approach sensibly takes into account common features of mobility in smart cities such as buses with fixed routes and stops as well as taxi services and cars. The fact that buses follow fixed routes makes it advantageous to deploy PoP along its path which ensures that data will be forwarded to the central server. Cars and taxis, on the other hand, can roam streets which are not passed by buses and can gather data from sensors located close to any streets in the city. The main difference between cars and taxis is in their stationary time during transit, where cars generally stop longer than taxis. Thus, we can assume that taxis are more reliable to deliver data than cars.

The Algorithm 1 describes the forwarding decisions of the simple Hierarchical Routing scheme. The idea is to statistically assign a level to the nodes in the city, instead of having complex routing decisions and metrics. This level is based on their reliability and capability to deliver the data to the Server. Note that a node does not forward the data to another node if that node's hierarchical level is inferior or equal to the current node. Thus, a bus or a taxi will not forward the data to a car, etc. There may be some missed forwarding opportunities due to this, but the idea is to keep forwarding decision as simple as possible. The performance will be extensively studied later in the paper. It illustrates a centralized air pollution monitoring in the city of Helsinki, where 37 wireless sensors were placed almost evenly in an area of about 9 km 2 . Each sensor positioned approximately 500 meters to each other which gave one air quality sample data from 37 locations in the city every 5 minutes. Cars, taxis, and buses equipped with V2X capabilities then opportunistically collect data from in-range sensors to be delivered to the first PoP it encounters, which in turn relays data to a central server. We used two bus routes in the city, bus routes 17 and 24, where a PoP is strategically placed at each end of bus routes. The last PoP is positioned at the city center where traffic usually converges, which in total makes 5 PoPs available to pick up data from cars, taxis, and buses. We evaluate the performance of the Hierarchical VDTN Routing algorithm by using the Opportunistic Networking Environment (ONE) simulator [START_REF] Keranen | The ONE Simulator for DTN Protocol Evaluation[END_REF].

We assess the performance with a fix number of buses (2 for each bus route) and taxis (9 taxis, correspond to taxi density of 1 per km 2 ), and an increasing number of cars roaming the city, from 9 to 54 with an increase of 9 each time, which correspond to car density of 1 to 6 per km 2 . We run each scenario ten times with different initial positioning of cars and taxis in the city, and accumulate all results to come up with convincing trends.

V. RESULTS AND DISCUSSION Fig. 3 and4 present the comparison of our DC4LED routing with two simple legacy VDTN routing approaches which represent the two extreme ends of the DTN routing family: First-Contact (FC) which is a single-copy strategy [START_REF] Jain | Routing in a Delay Tolerant Network[END_REF] and Epidemic (EP) which is a multiple-copy flooding strategy [START_REF] Vahdat | Epidemic Routing for Partially-Connected Ad Hoc Networks[END_REF]. Both can be categorized as blind forwarding approach. The only increased complexity of DC4LED as compared to both is the process of differentiating the node level to forward the messages hierarchically. The comparison are shown with increasing value of car density. It can be seen that in different scenarios, DC4LED performs better than FC and almost similar to Epidemic in terms of delivery probability. By examining the average latency, DC4LED has on average 4 minutes higher latency, as compared to Epidemic. The result is expected as Epidemic uses flooding, which however has a disadvantage in that it wastes network resources. Flooding also leads to the circulation of multiple redundant copies in the network which is apparent in Fig. 3. It shows the network overhead to be highest for Epidemic and lowest for the proposed DC4LED scheme, where the overhead for the DC4LED scheme only increased slightly with the increasing number of vehicles, in contrast to the two legacy routings. This fact is advantageous for the scalability of DC4LED's implementation.

Fig. 5 summarizes two Key Performance Indicators (KPI) for the DC4LED Routing: the message delivery probability and the average latency. It contains three separate graphs, where the first part shows that a high delivery probability of 0.92 could already be reached when car density is 1 per km 2 . The positive trend continues when the car density increases further. It also shows the decreasing trend of messages delivery latency from about 55 minutes with a car density of 1 per km 2 to about 47 minutes when car density increased to 6 per km 2 . Furthermore, the fact that we only include a fixed number of taxis, buses, and PoPs in the simulation points toward the high possibility to improve the KPI by increasing the number of involved taxis and buses for the data collection and by deploying more PoPs.

VI. CONCLUSION

Simulation results for DC4LED show that the hierarchical VDTN routing scheme has a low network overhead which is advantageous for its implementation scalability. It can also achieve high data delivery probability even in a low vehicles density. We argued that the complexity of existing VDTN routing schemes is not necessary for the kind of data collection service we envisioned in smart cities. Due to our strong but realistic assumptions for the service that we have in mind, our much simpler mechanism is sufficient for the data collection task.

Fig. 1 .

 1 Fig. 1. Vehicle-Based Data Collection Architecture for Smart Cities

Initialize

  Nodes: Server.Level = 6; PoP.Level = 5; Tram.Level = 4; Bus.Level = 3; Taxi.Level = 2; Car.Level = 1; Sensor.Level = 0; Input: Connected NeighbourNode while (CurrentNode.Level != 0) do if CurrentNode.Level < NeighbourNode.Level then forward message; end end Algorithm 1: DC4LED Routing Forwarding Decision.

Fig. 2 .

 2 Fig. 2. Simulation Overview IV. SIMULATION SETUPAn overview of our simulations is shown in Fig.2. It illustrates a centralized air pollution monitoring in the city of Helsinki, where 37 wireless sensors were placed almost evenly in an area of about 9 km 2 . Each sensor positioned approximately 500 meters to each other which gave one air quality sample data from 37 locations in the city every 5 minutes. Cars, taxis, and buses equipped with V2X capabilities then opportunistically collect data from in-range sensors to be delivered to the first PoP it encounters, which in turn relays data to a central server. We used two bus routes in the city, bus routes 17 and 24, where a PoP is strategically placed at each end of bus routes. The last PoP is positioned at the city center where traffic usually converges, which in total makes 5 PoPs available to pick up data from cars, taxis, and buses. We evaluate the performance of the Hierarchical VDTN Routing algorithm by using the Opportunistic Networking Environment (ONE) simulator[START_REF] Keranen | The ONE Simulator for DTN Protocol Evaluation[END_REF].We assess the performance with a fix number of buses (2 for each bus route) and taxis (9 taxis, correspond to taxi density of 1 per km 2 ), and an increasing number of cars roaming the city, from 9 to 54 with an increase of 9 each time, which correspond to car density of 1 to 6 per km 2 . We run each scenario ten times with different initial positioning of cars and taxis in the city, and accumulate all results to come up with convincing trends.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Comparison of delivery probability and overhead ratio

ACKNOWLEDGMENT This work is supported by a Doctoral Scholarship from the Indonesian Endowment Fund for Education (LPDP) of the Indonesian Ministry of Finance, under Contract Number PRJ-43/LPDP.3/2016.


M Gassoumi
email: mohamed.gassoumi@irt-jules-verne.frb

S Rolland

Du Roscoat

P Casari

P J J Dumont

L Orgeas

F Jacquemin
email: frederic.jacquemin@univ-nantes.fr

S Rolland Du Roscoat
email: sabine.rollandduroscoat@3sr-grenoble.frc

L Orgéas

Shear behavior of thermoformed woven-textile thermoplastic prepregs: an analysis combining biasextension test and X-ray microtomography

published or not. The documents may come

Shear behavior of thermoformed woven-textile thermoplastic prepregs: An analysis combining bias-extension test and X-ray microtomography

INTRODUCTION

Thermoplastic polymers reinforced by woven fabrics become attractive materials for automotive and aeronautic structural applications because of their good thermal stability, high toughness and damage tolerance. Their properties highly depend on their meso and microstructures that are drastically affected during thermoforming. Thermoforming consists of four stages. First, the pre-consolidated thermoplastic blank is preheated so that the matrix reaches its molten state. Then, the prepreg is automatically transported to a stamping/compression press, where it is stamped and then compressed in a mold. Finally, the mold is maintained closed under pressure at low temperature in order to cool down the composite, enhance its consolidation and avoid the formation of porosity. In case of woven thermoplastic prepregs, five main deformation mechanisms occur [START_REF] Friedrich | Composite Sheet Forming[END_REF][START_REF] Advani | Composite Sheet Forming[END_REF]: in-plane shear and extension, out-of-plane shear, compression and bending.

In-plane-shear is the predominant deformation mode [START_REF] Guzman-Maldonado | [END_REF] and is associated by the in-plane rotation of the yarns. The bias-extension test has been recognized as a proper mechanical loading to characterize this deformation mode (4)(5)(6). It is an off-axis tensile test where warp and weft directions are initially at with respect to the tensile direction. When the initial gauge length of the sample is larger than twice the sample width , several zones with three different deformation modes appear: a uniform sheared zone in the center of the gauge length, four half-sheared zones and a non-deformed zone. Assuming that (i) fibers are inextensible, (ii) no slip occurs in the sample and (iii) that three different zones exist in terms of shear deformation, an analytical estimate of the global in-plane shear angle as a function of fabric size and the current displacement can be obtained from geometrical considerations (7):

,

Where . However, other experimental studies (5) have shown that the true shear angle could differ from that predicted by Eq.1, with discrepancies that could exceed 5°. In addition, the existence of well-defined halfsheared zones can be questionable. At last, the out-of-plane deformation induced during the bias-extension test is rarely analyzed, as most of experiments consist of 2D observations of macroscopic and surface deformation mechanisms. Thus, using current experimental results, it is still not possible to propose fine constitutive equations for the mechanical behavior of thermoplastic prepregs during thermoforming that account for the complex interplay between the thermomechanical history, the matrix rheological properties the fabric architecture and the corresponding 3D deformation mechanisms at the microscopic (intra-bundle scale), mesoscopic (fiber bundle scale) and macroscopic (specimen scale) scales. For that purpose, bias-extension tests were performed on woven glass fiber (GF) fabrics impregnated with PA66 at several different temperatures. The mechanical results show the thermal dependence of the mechanical response of these thermoplastic prepregs. In addition, the evolution of several local structural descriptors (shear angle, fiber content and thickness variations) were investigated using 3D X-ray microtomography images of the deformed specimens to better characterized flow-induced mesostructrures.

MATERIALS AND METHODS

Materials

The tested thermoplastic prepregs were commercial pre-consolidated plates supplied by Solvay (Saint-Fons, France). They were made up of four stacked layers of twill 2-2 glass fiber (GF) fabrics impregnated with a PA66 thermoplastic matrix. Their initial volume fraction of fibers and thickness were and mm, respectively.

Bias-extension tests

Bias-extension tests were conducted using a specially designed setup. Rectangular specimen with in-plane initial dimension of mm 2 were placed in a furnace mounted on an electromechanical tensile testing machine (Zwick, load cell kN). Specially designed clamps were used to ensure a constant and homogeneous clamping force to minimize the sliding of specimens. During the tests, the temperature of specimens was controlled using four thermocouples inserted along the sample gauge length into small holes drilled into the specimens.

The thermal history of samples during thermoforming is a crucial factor with a large influence on the deformation mechanisms. Thus, specimens were subjected to a thermal heating cycle that mimicked the industrial conditions (Fig. 1a). In this cycle, the material was heated above its melting temperature (A). This stage is representative of the pre-heating stage. Then, the temperature was decreased (cooling stage) down to the testing temperature (B). This stage is representative of the transport stage. During this period, the temperature decreased significantly from approximately 30-40°C. The temperature was then homogenized in the entire specimen in order to reduce temperature gradients (C). Finally, the specimens were deformed (D). Experiments were performed at various testing temperatures (270-290°C) and by imposing displacement increments of mm (constant clamp relative displacement of mm.min -1 ) for a total displacement of mm. Between each displacement step, the clamps remained fixed for s to enable the specimen relaxation (Fig. 1b).

3D imaging of the sample mesostructures

Specimens were scanned using a laboratory X-ray microtomograph (3SR Lab, Grenoble, France, RX Solutions), with a voxel size of μm and a beam energy set to kV and μA. Using 1984 X-ray projections and suitable reconstruction algorithm, 3D images were obtained. These images were used to obtain several mesostructure descriptors such as the local orientation angle of fibre bundles, fibre bundle volume fraction, and local thickness of samples.

All these structural descriptors were obtained from three samples tested at C, C and C, respectively. For each specimen, three representative Zones were scanned (Fig. 2). These 3D volumes were divided into small unit cells, i.e., representative elementary volumes (REV) with in-plane dimensions of mm 2 (Fig. 2). In each of these cells, the orientation analysis was performed using the so-called structure tensor computed via the OrientationJ plugin implemented in ImageJ (9). This algorithm provided a local orientation for each voxel of the gray scale images (Fig. 4a-b). The orientation angle between the weft and warp directions was obtained by analyzing the orientation distribution function of the structure tensors in the deformed REVs. Assuming that in the initial state, the weft direction and the warp direction were perpendicular, i.e., , the local shear angle was computed as follows:

(2) Using X-ray microtomography images, 2D maps of the volume fraction of fiber bundles and the sample thickness maps were also obtained. For that purpose, the 3D images were first binarized to separate voxels belonging to the fibrous phase from a phase containing the matrix and pores. This was achieved using the Otsu thresholding algorithm implemented in Image J. Then, for each voxel position in the plane of the samples, the thickness variation of the sample was estimated, as well as the mean relative volume fraction of bundle in the thickness , as shown in Figs. 5a-c and 6a-c, respectively. Finally, these local values were averaged to obtain mean 2D value per REV, as shown in the graphs of Figs. 5d and6d.

Zone 1 Zone 2 Zone 3

MECHANICAL PROPERTIES

Fig. 3 shows the evolution of the loading force as a function of time for test temperatures of 270°C, 280°C and 290°C. Whilst increasing the displacement, the loading force gradually increased. However, after each displacement interruption, the force also exhibited a pronounced relaxation, revealing the viscoelastic behavior of the thermoplastic prepreg (3). Furthermore, the loading force decreased as the testing temperature increased (3). The overall behavior of this thermoplastic prepregs was typical of woven fabrics. At low tensile elongation , the macroscopic response is usually associated to free rotation of bundles: the shear resistance of the prepreg is low since only friction efforts between bundles occur. Then, the force gradually increases as fiber bundles progressively align along the tensile direction, come into lateral contacts and are subjected to transverse compression.

MICROSTRUCTURAL PROPERTIES

Shear angle

Figs. 4a and4b show a typical in-plane slice REV before and after the image treatment used to determine the local orientation map for the prepreg fabric, respectively. These figures show that bundles exhibited two principal directions related to the warp and weft directions. Fig. 4c shows the evolution of the shear angle along the length of the specimen, both in the middle of the sample (R23, averaged values of the REVs of lines 2 and 3) and near its lateral faces (R14, averaged values of the REVs of lines 1 and 4). We first proved (not shown here) that the shear angle did not depend on the considered layer of the samples. In addition, it is noticeable that the shear angle decreased whilst approaching the clamp (right hand-side of Fig. 2c). Lastly, in the middle of the sample, the measured shear angle is much lower than that predicted from Eq. ( 1), as already pointed out in previous works (3).

Fiber content and thickness maps

Figs. 5a-c shows maps of in each of the three aforementioned zones. These maps show large heterogeneities that were all the more important as the zone was located close to the clamps. This trend is confirmed by the evolution of the normalized fiber content along the specimen length, as shown in Fig. 5d. The decrease in fiber content was associated to an increase in the porosity of the specimens. Both were induced by in-plane and out-of-plane bending and local buckling fiber bundles, as well as intra-bundle deconsolidation. These phenomena also conducted to a large increase in the local thickness of the specimens, as shown in the thickness maps and profiles shown in Fig. 6.

Locally, the thickness increase ranged between 50% and 100% (Fig. 6d). Thus, the macroscale in-plane shear deformation induced the deconsolidation of the thermoplastic prepregs and resulted in large microstructural variations. Similar observations were done for all tested specimens. Fig. 7 shows that the profile evolutions for the shear angle, normalized fiber content, and normalized thickness were in general similar for all temperatures. However, some differences were noticed for the lowest testing temperature of 270°C. The decrease in the shear angle was slightly enhanced for this temperature whilst approaching the clamp (Fig. 7a). Again, the normalized fiber content was slightly affected and exhibited a larger decrease. The most noticeable effect was obtained for the variation in the normalized thickness, which was clearly enhanced for the specimen deformed at 270°C. The potential origins of these phenomena are complex and could be related for example to a variation in the in-plane and out-of-plane bending rigidity of fiber bundles accompanied with an increase in the local loading due to an increase in local inter-bundle interaction forces and moments.

CONCLUSION

The objective of this study was to investigate the mesostructural changes of thermoplastic prepregs induced during bias-extension. These tests were performed on a pre-consolidated multilayer woven GF-PA66 prepregs using testing temperatures and thermal cycles that were representative of the thermoforming process. Results also showed that the in-plane-shear resistance increased for the lowest testing temperatures, corresponding to complex local deformation mechanisms as revealed by the analysis of the local evolution of several mesostructural characteristics such as fiber orientation, fiber volume fraction, local thickness and bundle shapes. These experiments also revealed the effect of the testing temperature on the evolution of these descriptors. All this information will form a unique database for the multiscale modelling of thermoforming of thermoplastic prepregs.

FIGURE 1 :

 1 FIGURE 1: Bias-extension test, (a) Thermal cycle, (b) imposed clamp displacement to the specimen.

FIGURE 2 :

 2 FIGURE 2: Top: positions of the scanned specimens delimited (red boxes). Box 1 is shown on the left-hand side of the top image, whereas box 3 is shown on the right-hand side. Bottom: discretization of 3D volumes into REV cells used for the microstructural analyzes. Ri (i=1, 2, 3) are lines number. di (i=1,2,3) are distances between delimited boxes.

FIGURE 3 :

 3 FIGURE 3: Bias-extension test: load vs. time at different temperatures.

FIGURE 4 :

 4 280°C-specimen. (a) Typical REV. (b) Orientation map. (c) Evolution of the local shear angle as a function of the horizontal position of the REV along the tested specimen.

FIGURE 5 :FIGURE 6 :

 56 280°C-specimen. Fiber content maps of Zone 1 (a), 2 (b) and 3 (c). (d) Fiber content variation along the horizontal direction (referring to Fig. 2 in raws 1-4 and 2-3). 280°C-specimen. Normalized thickness maps of Zones 1 (a), 2 (b) and 3 (c). (d) Normalized thickness variation along the horizontal direction for raws 1-4 and 2-3 (referring to Fig. 2).

FIGURE 7 :

 7 FIGURE 7: Evolution of the thermoplastic prepreg mesostructure for different temperatures: (a) shear angle, (b) normalized fiber content, (c) normalized thickness.

ACKNOWLEDGEMENTS

This study is part of the COMMANDO-STAMP project managed by IRT Jules Verne (French Institute in Research and Technology in Advanced Manufacturing Technologies for Composite, Metallic and Hybrid Structures). The authors wish to associate the industrial and academic partners of this project, respectively CEMCAT, PSA Group, SAFRAN, SOLVAY, and 3SR Lab, GeM, LaMCoS, LTN.