Controlled Assembly of AIE Active Bolaamphiphilic Mac- romolecules into Luminescent Organic Nanoparticles Op- timized for Two-Photon Microscopy in vivo

The (Z) and (E) isomers of an extended tetraphenylethylene-based chromophore with optimized two-photon induced luminescence properties are separated and functionalized with water-solubilizing pendant polymer groups, promoting their self-assembly in physiological media in the form of small, colloidal stable organic nanoparticles. The two resulting fluorescent suspensions are then evaluated as potential two-photon luminescent contrast agents for intravital epifluorescence and two-photon fluorescence microscopy. Comparisons with previously reported works involving similar fluorophores devoid of polymer side chains illustrate the benefits of the later functionalization regarding the control of the self-assembly of the nanoobjects, and ultimately their biocompatibility towards the imaged organism.


INTRODUCTION
Since its invention in the late 1990's two-photon fluorescence microscopy has experienced impressive developments, 1-2 and has opened unprecedented perspectives in tissues imaging that have substantially benefited such diverse fields as neurobiology, 3 physiology, 4 embryology 5 and tissue engineering. 6 These developments have been achieved thanks to combined advances in the optical setups design and in the developments of increasingly sophisticated and selective endogenous (green fluorescent proteins) 7 and exogenous (synthetic organic chromophores and nanoparticles) fluorescent contrast agents. [8][9][10][11] In this framework, the conception of molecular probes or nanoobjects that combine sizeable two-photon induced emission properties with good solubility (or colloidal stability) in physiological media has been a major issue, on which we have been extensively working in recent years. [12][13][14] In previous reports, we have proposed chromophore functionalization with water solubilizing poly(hydroxyethylacrylate) (PHEA) polymeric side chains as a relevant approach to achieve high biocompatibility of molecular bioprobes. 15 We have shown that this functionalization could efficiently shield the molecule from interactions with its local environment, ensuring solubility of inherently lipophilic and prone-to-stacking fluorophores into aqueous media, and keeping luminescence efficiencies high. This strategy has been used in the making of fluoro-phores that have already found promising application for diverse bio-related applications such as intravital imaging of cerebral vascularization [16][17][18] or the detection of biomolecules (ds-DNA, 19 G-quadruplexes [20][21] and proteins 22 ) in complex physiological media. In parallel, we have recently reported on extended tetraphenylethylene (TPE) derivatives -a class of molecules classically used for their Aggregation-Induced Emission (AIE) properties [23][24][25] in particular for bioimaging and biosensing applications [26][27][28][29][30][31] -that had been specifically designed to maximize the two-photon emission properties of their aggregates. 32 In the course of this study, it turned out that, in spite of the excellent colloidal stability and excellent spectroscopic properties of the suspension obtained by nano-precipitation of TT OMe , only limited control over the assembly was achieved : as a result, mixtures of large (d>100 nm) and disperse nanoparticles were obtained with detrimental outcome on the long-term survival of the imaged zebrafish. It has already been established in previous literature reports that the self-assembly of fluorophores containing macromolecules (either in the form of grafted polymers or conjugated polymers) usually led to smaller and better controlled self-assembly compared to dye-loaded nano-objects obtained by conventional nano-precipitation methods. 33 In particular, direct self-assembly of fluorescent amphiphilic polymers upon dispersion in aqueous media has proved to be a remarkably effective approach for the synthesis of small (10-14 nm) monodisperse nanoparticles, well-suited for in vivo applications, 34 while larger nanoparticles (> 100 nm, typically) are rapidly cleared from the bloodstream upon internalization within macrophages and tend to induce more severe adverse effects such as acute inflammations. 35 In this work, wishing to further explore extended TPE derivatives and taking advantage of our above-mentioned long-time expertise in the functionalization of lipophilic fluorophores through controlled radical polymerization, we have synthesized and studied TT P a new two-photon absorbing, water-soluble and AIE active luminescent probe, that can be derived into two stereoisomers differing by the stereoconfiguration of their central double bond (Fig. 1). Through a combined study, involving DLS and DOSY NMR, we investigate the self-assembly process of both isomers and their mixture in aqueous suspension. In particular, we show that, owing to the amphiphilic macromolecular nature of the molecule, it is possible to obtain controlled small-sized (ca 10 nm) nano-assemblies of the chromophores under either their (Z) and (E) forms in both pure water and physiological media. The resulting colloidal suspensions combine significant two-photon absorption (2PA) cross sections and Aggregation-Induced Emission efficiencies, making them suitable for two-photon fluorescence microscopy in living biological systems. Comparison with our previously studied assemblies of pure nano-precipitated (E/Z)TT OMe chromophores 32 on several days after injection into zebrafish embryos clearly demonstrated the benefits of polymer functionalization and underlined the biocompatibility of these resulting nano-objects.

Synthesis
Syntheses were performed in oven-dried vessel purged with argon. Solvents were dried on 3 Å molecular sieves. The reactions were monitored by analytical thin layer chromatography (TLC) on Merck 60 F-254 precoated silica gel plate (0.2 mm thickness) and product were revealed at UV lamp. Purification by column chromatography was made using over Mercksilica gel 60 (0.040−0.063 mm).
All newly synthesized compounds were routinely characterized by 1 H and 13 C NMR, and high-resolution mass spectrometry (HRMS). Detailed synthetic protocols and characterization data, including NMR spectra, are available as supplementary material (S1 and S2, Fig. S2-S15) NMR studies NMR spectra were recorded at room temperature on a Bruker Avance 400 functioning at 400 MHz. Chemical shifts are reported as values (ppm) with reference to the peak of CDCl3, DMSO-d6, or D2O. Abbreviations for the 1 H NMR data are: chemical shift δ, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants J. Diffusion experiments were performed on a Bruker Avance III 400 MHz spectrometer equipped with a Prodigy Cryoprobe. The temperature was regulated at 298K. A bipolar pulse longitudinal eddy current delay (BPPLED) pulse sequence was chosen, and the diffusion time was set to 250 ms. Sine shaped pulse field gradient were used with a length of 2.5 ms and the gradient intensity was linearly incremented from 0.963 to 47.187 G cm-1 over 30 experiments. After each gradient pulse, a 200 ms delay was used as a recovery delay. DOSY NMR were generally processed by Mestrelab MNova using bayesian transformation. Polydispersity of the polymer was determined thanks to the maximum entropy methodology, processed on NMRNotebook. 36 Dynamic light scattering (DLS) was recorded with a Malvern Instruments Zetasizer nano series instrument (with solutions at constant 10 −5 M concentration). HRMS measurements were performed at the Centre Commun de Spectrométrie de Masse (CCSM) of Lyon (France).

Spectroscopic measurements
Absorption spectra (UV-Vis) were recorded with a Jasco 670 spectrometer. Fluorescence spectra were performed on a Horiba Jobin-Yvon Fluorolog-3 spectrofluorometer. Spectra were reference-corrected for both the excitation source light intensity variation (lamp and grating) and the emission spectral response (detector and grating). Emission spectra were recorded at 350 nm as excitation wavelength. For the comparison of emission spectra of compound in various water/DMSO mixtures, spectra were normalized of their respective absorbance. Absolute fluorescence quantum yields were measured with a calibrated integrative sphere (2π steradians covered with spectralon®, model G8 from GMP) as described in the literature 1 . Each sample was placed on a quartz tube at the concentration of 2.10 -5 M. Four measurements were made with constant excitation and emission to give four integrated intensities. E c and E a are integrated fluorescence spectra of direct excitation with and without the sample respectively. L c and L a are integrated excitation spectra of the sphere with and without the sample respectively. A density filter (f = 0.5 %) was used to reduce the intensity of excitation profile. Quantum yield is calculated according to the equation (1): Two-photon absorption cross sections measurements Two-photon excitation spectra of the studied compounds were measured by two-photon excitation-induced fluorescence technique in the 700-900 nm range. Excitation source was a tunable femtosecond Ti:sapphire laser (Chameleon Ultra I, Coherent). Laser beam was focused into a 10x10 cm quartz (QZS) cell using a 100 mm focal length lens. The cell containing the sample was positioned to minimize the optical path of the exciting laser and that of the emitted fluorescence in the sample thus minimizing inner filter effects and other artefacts. Emitted fluorescence was collected at 90° with respect to the excitation, filtered by adapted low-pass filters and was detected by a fiber optic spectrometer (AvaSpec-EVO, Avantes). Excitation power was measured by a thermal detector (S405C, Thorlabs). It was first verified that 2PA induced fluorescence showed quadratic excitation power dependence at 720 nm for all compounds. Then power was set to 40 mW for the measurement of the two-photon spectra. 2PA-induced fluorescence spectra did not show excitation wavelength dependence. Moreover, we checked in each case that the corrected fluorescence signal overlapped very well with the one-photon absorption (1PA) induced fluorescence spectra. The 2PA cross sections were calculated based on the integration of the fluorescence signal, by comparison with that of a fluorescein solution (10 -5 mol/L, pH 13) as a 2PA standard, taken from the most recent literature. 37
Cytotoxicity study MCF-7 cells were seeded in 96-well plate, incubated with increasing concentrations (from 2.5 to 150 µg.mL -1 ) of the different isomers for 3 days and the percentage of living cells was quantified by MTT assay. This consists in a colorimetric test performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Promega) where cells were incubated with 0.5 mg·mL −1 MTT for 4 h to determine mitochondrial enzyme activity. Then, MTT precipitates were dissolved in 150 µL ethanol/DMSO solution (in proportion 1/1) followed by 20 min of shaking and absorbance was read at 540 nm. Cells treated with the vehicle were served as control.

Imaging potential in vitro
The cellular uptake experiment was performed using confocal fluorescence microscopy on living cells. Cells were seeded (10 6 cells·cm −2 ) on culture dishes with a glass bottom (Fluorodish from World Precision Instruments, Stevenage, UK). After seeding, cells were incubated with 50 µg.mL -1 of (E) or (Z) isomers for 24 h. Cells were also loaded 15 min with CellMask TM (Invitrogen, Cergy Pontoise, France) at 5 µg·mL −1 , for membrane staining. Then, cells were washed twice with culture medium and fluorescence imaging was performed on living cells with a LSM780 LIVE confocal microscope (Carl Zeiss, Le Pecq, France), at 750 nm for isomers detection and 561 nm for cell membranes. All images were obtained with a high magnification (Plan-Apochromat 40x/1.3 Oil DIC).

Zebrafish breeding
Casper zebrafish embryos were purchased from Zebrafish International Resource Center (ZIRC) and raised to adulthood in circulating aquarium system at 28 °C and 14 h light/10 h dark cycle, in the Molecular Mechanisms in Neurodegenerative Dementia (MMDN) laboratory (Inserm U1198), Montpellier University. Experiments with zebrafish embryos are performed according to the EU Directive 2010/63/EU on the protection of animals used for scientific purposes.

Imaging on Casper zebrafish embryos
Casper zebrafish embryos of 72 h post fertilization (hpf) were intravenously injected with 10 nL of (Z) or (E) isomer at 1 mg mL -1 concentration (n = 7 for control, n = 9 for (Z) isomer, n = 10 for (E) isomer). One hour after injection, embryos were observed using one-photon excitation microscope at excitation wavelength of 340-380 nm, and after embryos were observed using two-photon excitation confocal microscopy at excitation wavelength of 750 nm.

Toxicity study on zebrafish embryos
After injections, embryos were then maintained 3 days in breeding conditions to analyze every day the biocompatibility of (Z) isomer and (E) isomer according to various parameters relating the good development of embryos, such as mobility, heart rate, malformations and deaths.

Synthesis
All molecules discussed in the following are presented in Figure  1. Synthesis of the (E/Z)TT In macroinitiator was adapted from the method previously published by us for the (E/Z)TT OMe molecule, 32 keeping here the phenolic function free from the beginning (Fig. S1, detailed synthetic procedures available as supplementary materials, S1). Thus, Buchwald-Hartwig coupling between 4-hydroxydiphenylamine and para-bromo-iodobenzene was achieved with a good yield to afford 4-bromo-N-(4-hydroxyphenyl)diphenylamine (1), thanks to a procedure that made it compatible with the presence of the free phenolic moieties. 38 Then, a palladium catalyzed Sonogashira cross-coupling with (triisopropylsilyl)acetylene yielded alkyne 2, which was in turn deprotected with TBAF. The instable free alkyne as obtained was directly engaged in Sonogashira coupling with TPE-Br to lead to (E/Z)TT OH . Finally, esterification between the (E/Z)TT OH diol and 2-bromo-2-methylpropionyl leads to the target macroinitiator (E/Z)TT In . This synthesis being non-stereoselective, separation of the macroinitiator diastereoisomers was required to afford stereopure versions of the chromophore. It was herein performed on amylose-based chiral stationary phase (Fig. S16), which efficiency has already been demonstrated by us on similar chromophores. 32 The controlled polymerizations of (E/Z)TT In as a (E/Z)-mixture, and also in stereopure form, were carried out in bulk 2hydroxyethyl acrylate (HEA) monomer, using 2,2'-bipyridine and copper(I) bromide as a catalytic system, as already described in previous reports for the elaboration of related polymer-chromophore adducts. [15][16][17][18] The characteristic signatures of the aromatic part of the 1 H NMR spectra confirm that stereopurity has been preserved after the ATRP reaction and purification (Fig. S13). In all polymerization cases reported herein, a rigorous control of the polymerization kinetics makes it possible to obtain a range of target chain lengths in a controlled manner with low polydispersity (vide infra) which is essential for the comparison of the systems.

Spectroscopic studies
Then, the spectral features of (E/Z)TT P were studied in DMSO. The absorption and emission spectra are presented in Figure 2, while main spectroscopic data are gathered in Table 1. First, we notice that the spectral signatures of (E/Z)TT P (Fig. 2) correspond well to those observed for their parent monomeric equivalent (E/Z)TT OMe which can be summarized as follows: (i) a structureless ICT band, typical of large π-conjugated system; (ii) large Stokes shift (ca 6500 cm -1 ) indicating large reorganization processes between ground and excited electronic states; (iii) an absorption band of the (Z)-isomer slightly blue-shifted (λabs = 357 nm) with respect to (E)-isomer (λabs = 374 nm), and a reversed behavior for the emission with λem = 543 nm for the (Z)-isomer, 530 nm for the (E)-isomer of TT P ; (iv) a similar two-photon absorption cross section σ2PA for the (Z)-isomer as for the (E)-isomer (140 vs. 100 GM) in the same range of wavelengths maxima (ca 710 nm). Consequently, functionalization of this chromophore with the polymeric PHEA chains does not markedly affect its main spectroscopic characteristics in DMSO solutions. Table 1. Spectroscopic properties of TT P isomers in solution in DMSO.
The fluorescence quantum yields Φf of TT P (stereopure Z and E form, and (E/Z) mixture) were also first estimated in DMSO solution, with the aim to highlight the influence of the polymerization degree on this parameter. Indeed, while the quantum yield of the macroinitiator (n = 0 in Fig. S17)   . 5 the addition of polymer chains leads to a moderate yet significant improvement to reach Φf = 0.07, a value that subsequently remained constant, within experimental error, for polymer chain lengths between 7 and 70 monomer units (Fig. S17). Thus, while the introduction of polymer chains seems to favor the emission (probably owing to steric-induced restriction of the rotation around the central double, a well-known mechanism in viscosity probes 39 ) the effect quickly saturates as the polymer chain is extended, and emission quantum yield subsequently becomes independent on the length of the PHEA chains. In the rest of this study, we chose to focus on (E/Z)TT P with an average polymer chain length n = 16 (Fig S14), which offers good dispersibility in aqueous media with a limited impact on the molecular weight of the chromophore. The spectroscopic properties of the PHEA polymers in aqueous media were then studied in DMSO/water mixtures with increasing water volumic fraction (Fig. 3). For a volumic fraction in water fH2O lower than 50%, emission properties of (E/Z)TT P remain virtually constant with Φf ≈0.07. For fH2O above 50%, a hypsochromic shift from 520 to 467 nm is noticed, consistent with that observed for the parent monomeric fluorophore in similar conditions; this is accompanied by a marked and progressive increase in Φf with further addition of water, to reach a maximal Φf = 0.35 for a fH2O of 80%. These two properties (hypsochromic shift and Φf increase) are compatible with the hypothesis of a restriction of the rotation phenomena around the central double bond 40 resulting from interchromophore aggregation in aqueous media. Thus, in contrast with previously reported similar chromophore polymer adducts, [15][16][17][18] the presence of the water-solubilizing chains do not suffice to avoid molecular aggregation of (E/Z)TT P at high water contents, and the molecules self-assemble into nano-aggregates. Similar evolution patterns are observed for the AIE properties of the pure (E) and (Z) isomers of TT P , although precipitation onset is shifted to fH2O ca 60%. At fH2O of 90%, the fluorescence quantum yields of (Z) and (E) isomers reach a maximal Φf = 0.38 and 0.37 respectively. These values, similar to those observed with the (E/Z) mixture of TT P , are however significantly lower than the values reached in their parent stereopure monomeric analogues in nano-suspensions. 32 This similarity of AIE properties between the stereopure versions and the mixture of (E/Z)TT P thus suggests that the nature of the supramolecular assemblies formed in the case of PHEA In other words, this suggests that, quite expectedly, the presence of the PHEA chain influences the chromophore packing in the nano-assemblies. NMR study of (E/Z)TT P To gain understanding on the nature of the interactions between (E/Z)TT P polymers as a function of fH2O, and the characteristic size of their assemblies, NMR experiments were then performed in different D2O/ DMSO-d6 mixtures. As a first notice, 1 H NMR spectra reveal a peculiar evolution of the signal belonging to the chromophoric part and the polymer chains upon increasing of fD2O (Fig. S15). Indeed, while the signals of the polymer chains remain constant in width and intensity, both the aromatic signals of the chromophore (between 6.80 and 7.45 ppm) and the signal of the two methyl end chains (1.24 ppm) broaden and decrease drastically with increasing the water fraction. At fD2O as low as 10%, these are already hardly distinguishable from the baseline and are no longer visible at fD2O = 50%. It would thus seem that aggregation phenomena between the hydrophobic parts of the core (chromophore and attached initiator group) take place in an aqueous medium, in agreement with the spectroscopic evolutions observed. Again, this evolution contrasts strongly with previous examples of similar polymer appended chromophores reported by us, 17 where the relative intensity of the different peaks was unchanged between DMSO-d6 and D2O media, characteristic of individual macromolecular objects in solution. Such a behavior is classically observed and has been studied in depth in the past during the self-aggregation process of conjugated polymer such as polythiophenes. 41 It indicates here that, like in the latter, π-stacking constitutes unsurprisingly the main driving force for the selfassembly of the polymer chains.
To investigate this aggregation phenomenon, and its dependence on medium composition and chromophore stereoconfiguration, 2D diffusion-ordered NMR spectroscopy (DOSY) was then used (available as Supplementary Materials S3, Fig. S18-S29). First, in pure DMSO-d6 solution, DOSY was used to verify that (E/Z)TT P molecules were present as individual polymer chains, and to determine their associated polydispersity index (Đ). thanks to the methodology initially developed by Delsuc et al. and tested in the past on similar polymers. 36,42 In the case of all samples studied herein, the NMR signals attributed to (E/Z)TT P belong to the same polymer species (with a single diffusion constant Fig, S18). Polydispersity index determination provides a value for Đ = 1.22 which, although in the high range of what is usually achieved by ATRP, appears satisfactory considering our previous results. [16][17] In each solvent, diffusion coefficient values D of a molecule, macromolecule or nano-object relates to the gyration radius of the latter, considered as a random coil: thus, comparison of the D values obtained for the (Z) and (E) isomers of TT P featuring similar polymer side chain lengths brings valuable information regarding the impact of stereochemistry on the characteristic dimensions of the associated coil. In DMSO-d6 solution, similar mean diffusion coefficients are monitored for the (Z) and (E) isomers (59 and 52 µm²/s respectively), characteristic of a comparable mobility and size for both solvated molecules ( Fig. S19 and S20). As expected, the average diffusion coefficients D are smaller in D2O, as a consequence of the aggregation process, yet similar for each isomer (25 for the (E) isomer and 26 µm²/s for (Z) isomer, Fig. S21 and S22). This indicates a similar nature for the small-sized nano-objects formed by molecular aggregation in the case of both stereoconfigurations. Wishing to get more insight into the aggregation processes taking place at high water contents, we then monitored in a next series of experiments the evolution of the D(E/Z)TT P coefficients as a function of fD2O (Fig. 4, Table S1). Since the spectroscopic studies had already underlined that the stereopurity had only little impact on the AIE properties, this study was limited to the (E/Z) mixture of TT P , available in larger quantities (as typically required for DOSY analyses) than its stereopure analogues. First, a global very noticeable decrease of D(E/Z)TT P values could be qualitatively observed upon increasing the water fraction from the initial pure DMSO sample, in agreement with the suspected aggregation process (Figs S23-S29). However, when comparing systems in different solvents compositions, diffusion coefficient of a given object does not solely depend on its radius, but also critically on the viscosity of the medium: it is well documented that the latter varies strongly in DMSO/water mixtures depending on the ratio of each solvent. In order to discriminate between both contributions, it is possible and beneficial to use a viscosity-corrected diffusion coefficient. Recent studies have indeed highlighted that, providing that polymer concentration was kept low in the medium (ca 1mg/mL), using such viscosity-corrected values for the diffusion signal allowed a precise evaluation and comparison of macromolecules dimensions even in solvents possessing very different intrinsic viscosities. 43 We thus considered in a next step the variations of a corrected diffusion coefficient defined, for each composition fD20 by equation 2: With the viscosity η (mPa.s) of the DMSO/water mixture of the considered sample, 44 the viscosity of pure DMSO.
Using this viscosity corrected diffusion coefficient we notice for fD20 > 10%, a progressive decrease in the corrected diffusion coefficient Dη as fD20 is increased (Fig. 4, Table S1). This confirms that a progressive aggregation process occurs, and that the average size of the resulting nano-aggregates gets bigger and reaches a maximal size in pure water, in agreement with the observed AIE evolution.
Stokes-Einstein equation relates the diffusion coefficient D of a well-defined spherical nano-object to its radius r, according to equation (3): Where kb is the Boltzmann constant (1.380 649 × 10 −23 J.K −1 ), T the temperature in Kelvins (here taken as 298 K) and η the dynamic viscosity, in Pa.s. The latter is well known and has been tabulated in the past for different DMSO/water mixtures. 44 Using these tabulated values, we calculated a nanoparticle radius (considered as rigid spheres) ca 7 nm in DMSO:water, 70:30 (v/v, corresponding to the onset of the self-assembly process). In pure water, it culminates to about 9 nm, i.e. a diameter of about 18 nm, in the spherical assumption.

Dynamic Light Scattering (DLS) study
Although DOSY NMR brings conclusive evidence for the formation of nanoparticles by self-assembly of (E/Z)TT P at high water content, extrapolation of the characteristic size of the nano-objects diffusion coefficient is inherently prone to error, specifically for non-rigid and non-perfectly spherical nano-objects. [45][46] In order to get additional and complementary insight on the dimension of the self-assembled nanoparticles, DLS experiments were thus undertaken on the aqueous suspensions of (E/Z)TT P . As expected, no scattering signal was obtained for the initial solution of the (E/Z) mixture of TT P in DMSO. In contrast, reliable DLS correlogram could not be obtained for both isomers ( This result indicates that, as expected, the bolaamphiphilic nature of our polymer appended chromophore favors the controlled formation of small and monodisperse nano-objects, which size seems ideally suited for bio-related applications.

Biological studies
In a previous report, we showed that suspensions of non-polymeric (E/Z)TT OMe were efficient in providing highly contrasted two-photon fluorescence microscopy images, both in cellulo and in vivo (after injection in the caudal vein of zebrafish embryos). However long-term following of the fate of the zebrafish embryos revealed that various malformations and premature death occurred within the days after injections, raising important issues regarding the biocompatibility of these nano-objects. Size and surface functionalization of nanoparticles are indeed two important parameters that largely determine their fate and biocompatibility in living systems, both of which turned out to be suboptimal in this initial works. Since our present study allowed us to underline several promising features of (E/Z)TT P as potential two-photon luminescent contrast agent, we decided to further investigate that point using similar setups and biological systems as in our abovementioned work. More specifically we aimed at establishing whether the adjunction of polymer side substituents and the resulting impact on the features of the self-assembled nano-systems could be beneficial in terms of biocompatibility. In a first step, we have studied the biocompatibility of such compounds on human cells in culture. The goal was to deter mine which maximal concentration could be used while avoiding any significant cell death. To that end, increasing concentrations of compound in its isomeric (Z) and (E) form, were incubated with MCF-7 cells for 3 days. The result reported in figure 6a suggested that, even at such high concentrations as 150 µg.mL -1 , both compounds were safe for the cell. In figure 6b, we can see that both compounds are highly luminescent (green dots) and efficiently internalized in the cancer cells, which plasmatic membranes are stained with cell mask (in red). In this experiment, isomers in living cells were revealed by two-photon excitation at 750 nm excitation wavelength. This imaging potential seems very similar to those obtained in our previous report on (E/Z)TT OMe . Then, to determine the possibility for biomedical use, the imaging potential was analyzed in vivo, in zebrafish embryos. After injection in the caudal vein, TT P , either in its E or Z form, provide high luminescence signal visible under UV-light excitation (pseudocolor green, Fig. 7) and exhibits a high solubility and bioavailability in all the organism. Importantly, the luminescence of these compounds was also investigated in two-photon microscopy conditions under pulsed laser excitation (Fig. 7b) strong capability of imaging was observed when excited at 750 nm, similar to that achieved in our aforementioned report. 32 In parallel, the biocompatibility of both isomers injected in the blood stream of embryos was studied. For 3 days, embryos development, rate heart and their mobility were daily monitored. The data reported in the table (Fig. 7c) demonstrate for both isomers that no toxicity could be detected in zebrafish embryos in the studied conditions, translating into no increase in the mortality of the said embryos. Survival and development of the zebrafish embryos, as monitored over several days, are indeed perfectly similar in the treated and control embryos. These results constitute a marked improvement in comparison with our recently published results with nano-precipated assemblies of TT OMe chromophores and highlight the strong potential of TT P for long term bioimaging of living organisms.

CONCLUSIONS
In this paper, we illustrate the benefits in functionalizing TPE based AIEgens, which structure is specifically optimized for two-photon induced emission properties at the solid state, with Figure 6. In cellulo studies a) Cytotoxic study of E and Z isomers. Increasing concentrations (from 2.5 to 150 µg.mL -1 ) of compounds were incubated with MCF-7 cells for 3 days and the percentage of living cells was quantified by MTT assay. b) Confocal microscopy imaging of living MCF-7 cells after 24 h incubation with 50 µg.mL -1 of E or Z isomers (green dots). Isomers were revealed by two-photon excitation at λexc = 750 nm (Chameleon, LSM 780) and cell membranes (red outlines) were stained with orange CellMask (λexc/λem = 554/567 nm). Scale bars = 10 µm.

Figure 7.
In vivo studies. Casper zebrafish embryos of 72 hpf were intravenously injected with 10 nL of TT P (isomer Z or isomer E in their pure form) at 1 mg.mL -1 concentration. One hour after injection, embryos were observed using a) one-photon excitation microscopy at 340-380 nm and b) two-photon excitation microscopy at 750 nm. c) Embryos were then maintained 3 days in breeding conditions to analyze every day the biocompatibility of Z isomer and E isomer according to the parameters reported in the table, with "+" for presence and "-" for absence.
hydrophilic side polymer chains. In DMSO, the resulting bolaamphiphilic macromolecular chromophores (E/Z)TT P exist as individual, low-emissive (φf ≈ 0.07) polymer chains. This is attested by DOSY NMR experiments, which indicate that, for both the (Z) and (E) isomers, a similar length of the polymer arms (n = 16 units) and a similar low polydispersity (Đ = 1.22) can be achieved, in agreement with the controlled nature of the radical polymerization.
In contrast, when dispersed in water, either from nano-precipitation of a concentrated solution of the molecule in DMSO or by direct mixing of the polymer powder in pure water or physiological solution, a characteristic AIE phenomenon is observed which correlates to the formation of small nano-bundles of several chromophore units of (E/Z)TT P . DLS analyses carried out on both the (Z) and (E) isomers confirm the presence of nanoaggregates at fH2O > 0.5, with a mean size ca. 10-15 nm and a low dispersity. To refine our understanding of the aggregation process dynamics, DOSY experiments over the whole range of fH20 gave additional insight on the formation of these aggregates. The latter is characterized by a progressive restriction in the measured diffusion coefficient for chemical shifts belonging to the polymer chain upon increasing fH20, to reach its lowest value in pure water, confirming that a self-assembly process drives the formation of the fluorescent organic nanoparticles. It is thereby confirmed that the strategy introduced herein is efficient in yielding ultra-bright, two-photon active organic nanoparticles with size optimized in regard with the target biological applications.
To illustrate the benefits of our approach in the specific framework of intravital bioimaging, the resulting aqueous suspension of both (Z) and (E) TT P (fH20 = 100%) were used as nano-sized fluorescent organic nanoparticle contrast agents in epifluorescence and two-photon fluorescence microscopy of the vascular systems of zebrafish embryos. Besides the favorable spectroscopic properties of the nano-objects, which afforded in all cases highly contrasted images, a distinctive advantage of the polymer functionalized two-photon AIEgens reported herein in comparison with their molecular analogues is their much-improved biocompatibility, which enables longer term studies on living systems and might constitute a keystone and determining feature of this strategy towards its possible future use in a biomedical context. .

ASSOCIATED CONTENT
Supporting Information. Detailed synthetic procedures, hardcopies of 1H and 13C NMR spectra for all new molecules and polymers, conditions for separation of chromophoric macroinitiators by preparative HPLC on chiral stationary phases and associated chromatograms, complementary fluorescence studies (chart) regarding the dependence on fluorescence quantum yield to polymer length, hard-copies of all DOSY NMR spectra used in the study.