Ultrabright Red to NIR Emitting Fluorescent Organic Nanoparticles Made from Quadrupolar Dyes with Giant Two-Photon Absorption (2PA) in the NIR Region. Confinement Effect on Fluorescence and 2PA and Tuning of Surface Properties

Ultrabright Red to NIR Emitting Fluorescent Organic Nanoparticles Made from Quadrupolar Dyes with Giant Two-Photon Absorption (2PA) in the NIR Region. Confinement Effect on Fluorescence and 2PA and Tuning of Surface Properties

Ultrabright red to NIR emitting fluorescent organic nanoparticles made from quadrupolar dyes with giant two-photon absorption (2PA) in the NIR region.

INTRODUCTION
Fluorescence microscopy is a powerful technique for imaging biological samples.Even though the majority of dedicated fluorophores absorb and emit in the UV-visible spectral region, the use of red to near infrared (R-NIR) light is particularly favorable for in depth imaging.The first optical transparency window has indeed been identified around 650-900 nm where the emission of endogenous fluorophores responsible for background noise 1 and the absorption and scattering by blood and fatty contents 2 are both at their lowest.Two-photon (2P) microscopy 3 is currently one of the most favored methods to achieve NIR excitation.First, because the 2P process allows excitation of fluorophores at about twice the wavelength necessary for 1P excitation (except in the case of symmetry-forbidden transitions), which usually means exciting at wavelengths higher than 700-800 nm.Second, because it displays an intrinsic sectional ability thanks to its non-linear nature.As 2P excitation only occurs at the focal spot of the objective, only the fluorophores localized in the focal plane become visible.2P microscopy thus greatly improves axial resolution while reducing photobleaching of the probes and photo-damage of the samples.The capacity of a fluorophore to be excited by the 2P process is described by its two-photon absorption (2PA) cross-section 2, and its most critical parameter is its brightness 2f, both expressed in Goeppert-Mayer (GM).Commercially available water-soluble organic fluorescent dyes exhibit low to modest 2P brightness values, rarely exceeding a few hundred GM and exceptionally reaching in the thousands 4 .0][11] Among various systems, molecular-based Fluorescent Organic Nanoparticles (FONs) 12,13 may prove to be good candidates to this aim.On the one hand, because their organic nature allows for the fine tuning of specific properties by molecular engineering, including morphological, 14,15 photophysical, 16,17 colloidal 18 and surface 19,20 properties.On the other hand, because nanoparticles (NPs) have had valuable developments in numerous biomedical fields that can benefit from 2P advantages, ranging from diagnosis 21 to photodynamic therapy 22 to localized photo-release of active molecules 23 to image guided surgery. 24In addition, nanoparticles are typically brighter than molecular dyes as long as their fluorescence is maintained.In the pursuit of obtaining bright FONs displaying large 2PA cross-sections and emitting at long wavelengths, two major aspects should be considered: tuning the photophysical properties of the composing dye and balancing the effects of nanoconfinement.In this respect, molecular dyes-based FONs represent the ultimate confinement, such that the chromophores are in direct interaction with each other, with no spacing polymeric or inorganic matrix.Thus, while the elevated content of dye subunits per nanoparticle should favor brightness and photostability, 25 intermolecular interactions may modulate the properties of the confined dyes, often to the detriment of fluorescence emission. 26Therefore, controlling the relative orientation of dyes upon aggregation within FONs is determinant.Moreover, multipolar (i.e.dipolar, quadrupolar or octupolar) dyes are preferred to achieve large 2PA responses.Therefore, by influencing the self-orientation upon aggregation and polarization of dye subunits, electrostatic interactions between dyes are expected to play a major role in determining the optical properties of the resulting NPs.As an example, we reported earlier the molecular engineering of tailor-made dipolar dyes that yield NIR-emitting FONs showing giant two-photon brightness (up to 5 10 5 GM). 27garding the applicability of FONs to biomedical use, it should be noted that the interplay between NPs and living tissues is not yet fully understood. 28A common practice towards controlling this parameter is to coat the surface of NPs, typically with polymers or zwitterionic moieties. 29Such coatings have several purposes.First, they lower the immediate colloidal and biocompatibility issues of, typically inorganic, water-insoluble NPs.While this configuration allows for a plethora of applications, it is difficult to envisage for clinical translation since the potential toxicity of the core remains unchanged.Consequently, even though systematic studies are still lacking in this field, organic nanoparticles are often seen as promising alternatives to their inorganic counterparts.Second, surface coating aims at modulating the fate of NPs, i.e. whether they are internalized by cells or remain in the extracellular space.On the one hand, NP internalization by target cells is required for localized intracellular action.Coating thus often serves as a backbone for conjugating targeting ligands to the NPs. 30On the other hand, long circulation times of NPs in vivo are favorable for long-term effects.Coating thus also aims at making overall stealth NPs, i.e.NPs that do not interact with cellular membranes. 31This rather contradictory role for the coating to mediate both targeted cellular uptake and otherwise stealth behavior complicates the design of biocompatible NPs.
Taking a different approach towards addressing nano-bio interactions, we recently reported on the use of a quadrupolar red fluorescent dye that yields spontaneously stealth FONs. 20We hypothesized that the quadrupolar structure of the dye, combined with its bulkiness and strong hydrophobic character could be in part responsible for the stealth behavior of its FONs.Adding to this interesting property, the quadrupolar design hints to the possibility that these FONs may also be strong 2P absorbers.In this paper, we present a series of related dyes tuned to emit in the R-NIR spectral region (up to ~ 700 nm).These dyes yield FONs displaying giant 2P brightness values (2f ~ 10 5 GM) thanks to aggregation-induced cooperative effects.Moreover, three of the four dye designs result in FONs also displaying a stealth behavior towards cellular membranes.To highlight that these FONs can be used for bioimaging, we further coated them with a positively charged polymer to induce their internalization in living cells.We thus achieve controlled cellular uptake of organic nanoparticles for NIR-to-NIR bioimaging.

METHODS
This section provides an overview of the preparation, characterization and use of FONs.
Extended experimental procedures with detailed methodology are provided as supplementary material.
Dye synthesis.The QBDF and QBDtF dyes were synthesized as in Li et al 32  FONs preparation and characterization.FONs were prepared by nanoprecipitation, a process consisting of the rapid addition of a minute amount of stock dye solution in spectral grade THF into a large volume of deionized water (1% v/v).When applicable, surface coating of the FONs was subsequently achieved by dropwise addition of an aqueous poly(allylamine hydrochloride) (PAH) solution (1% v/v).FONs morphology was observed by Transmission Electron Microscopy (Hitachi H7650) and their zeta potential determined on a Nano Particle Analyzer (SZ-100 Horiba).Lifetime measurements were performed in the time-correlated single photon counting (TCSPC) configuration on a Fluorolog spectrofluorometer (Horiba).
Spectroscopy.One-photon absorbance data was acquired on a UV/Vis spectrophotometer (Jasco V-670).Thanks to the emissive properties of our dyes, two-photon cross sections were determined using the Two-Photon Excitation Fluorescence (TPEF) method.Two-photon absorbance data was acquired on an in-house setup using a Ti:Sapphire oscillator (Coherent chameleon, Nd:YVO4, 140 fs, 80 MHz), a 10X objective (Provider, NA 0.25) and a compact CCD spectrometer (BWTek BTC112E).Emission data was acquired on a Fluoromax-4 spectrofluorometer (Horiba).Molar extinction coefficients, 2PA cross sections and brightness values provided in the following Tables and Figures refer either to the molecular characteristics (f, 2) or to the FONs characteristics (f.FONs, 2.FONs) depending on whether the dye concentration or the nanoparticle concentration is being considered.
Cellular imaging.FONs were incubated on Cos7 cells (1% v/v) for 24h in serumsupplemented DMEM and washed with PBS prior to imaging.One-photon imaging was achieved in scanning-confocal configuration while two-photon imaging was performed on the same setup using a pulsed Mai-Tai excitation laser and opened emission pinhole.

RESULTS AND DISCUSSION
Dye design.The chromophores designed to yield stealth, 2P-responsive, R-NIR emitting FONs share a quadrupolar scheme (D-π-A-π-D).This symmetry was rationally introduced for two reasons.First because it favors 2P absorption. 33Second because it allows for circumventing antiparallel arrangement of dipolar dyes driven by dipole-dipole interactions, which is deleterious to both fluorescence and 2PA. 34As previously described for the QBDF dye, 20 the quadrupolar dyes display a benzothiadiazole (BDTA) acceptor moiety at their center, propeller-shaped diphenylamine donor moieties at their ends and two alkylated fluorene motifs as hydrophobic, conjugated connectors linking the donors with the acceptor.The aim of choosing bulky propellershape donor end groups and introducing steric hindrance with the alkyl chains was to prevent close parallel -stacking of the chromophores (i.e.H-aggregates) within nanoparticles, which could lead to vanishing fluorescence.From this common template, we modulated the connecting π-bridges, aiming to modulate the dyes' emission properties.The fluorenyl -linkers were thus flanked with either ethynyl, vinyl or thienyl motifs, resulting in the QBDeF, QBDvF and QBDtF chromophores respectively (Scheme 1).The good solubility in THF and insoluble character in water of these dyes make them good candidates for nanoprecipitation. 35heme 1.Molecular design of 2-photon absorbing R-NIR emitting dyes.
FONs preparation.Upon nanoprecipitation, QBDF, QBDeF, QBDvF and QBDtF dyes all easily and rapidly yield transparent and colored aqueous colloidal solutions ranging from orange to pink.
We confirmed the presence of spherical nanoparticles by transmission electron microscopy (TEM) (Figure 1).Interestingly, each chromophore spontaneously and reproducibly yields very small FONs of fixed median diameters with low polydispersity, ranging from 12 nm for QBDeF, to (Table 1).Table 1.Structural properties of FONs made from quadrupolar dye subunits.Optical properties.The spectral properties of dyes and FONs were characterized to determine the influence of the molecular structure and the effects of nanoconfinement on the optical properties of the resulting nanoparticles.Results are provided in Table 2 and Figure 2.
Table 2. 1-and 2-photon optical properties of quadrupolar dyes in THF and as FONs subunits.Absorption maxima wavelengths under 1-photon excitation; b) Molar extinction coefficient of the dye at max 1P ; c) Emission maximum wavelength; d) Fluorescence quantum yield; e) Twice max 1P corresponding to the low-energy (ICT) absorption band; f) Absorption maxima wavelengths under 2-photon excitation; g) 2PA cross-section of the dye at max 2P Linear optical properties in solution.By selecting conjugated connectors of various natures, we manipulated the periphery-to-core intramolecular charge transfer (ICT) already present in QBDF in order to influence the photo-physical properties of the new dyes.Elongating the π-bridges aimed at favoring red-shifted absorption and emission.We indeed observe the anticipated bathochromic shift in absorption for all three novels designs (Figure 2A).Moreover, we notice a marked increase (~ +50%) in their molar attenuation coefficient values as compared to the original QBDF template (Table 2).Interestingly, QBDvF and QBDtF dyes also display the awaited red shift in emission and exhibit quantum yield values of 0.6 and 0.4, gradually decreasing in accordance with the red-shifted emission. 36QBDeF however behaves differently such that its emission in THF is much more red-shifted than anticipated and its quantum yield of 0.1 is surprisingly low.We hypothesized that this behavior may be due to the formation of a TICT state, 37 in accordance with the stronger sensitivity of this chromophore emission to the polarity of its environment.When dissolved in solvents of various polarities (Figure S1), none of the dyes display a shift in their ICT absorption band, underlying the neutral nature of the ground state of these symmetrical quadrupoles.In contrast, the emission of all four compounds is sensitive to polarity, in agreement with symmetry breaking occurring in in the excited state. 38,39We note from these experiments that QBDeF is twice as sensitive as the other chromophores to the tested solvents, corroborating the TICT hypothesis.Taken together, these dyes display interesting R-NIR emission upon visible excitation in solution, some of which with relatively high quantum yields.
Non-linear optical properties in solution.The four dyes display interestingly high 2 max values in the NIR region ranging from 1 to 3.10 3 GM in THF (Table 2, Figure 2).We note that these values are higher than those reported for similar derivatives bearing acceptor end-groups 40 instead of the strong donating end-groups used here, or for proquinoidal D-A-D or D-A-D-A-D derivatives built from BDTA units 41 .As expected from the quadrupolar nature of the dyes, the 2PA peaks are markedly blue-shifted as compared to twice the wavelength of the one-photon absorption peak (Table 2 and Figure S2).Yet we observe a shoulder in the 2PA spectra, indicating that the lowest one-photon excited state is also partly 2P allowed.This observation is related to the conformational flexibility and V-shape of the dyes, 42 which are not purely linear quadrupoles.This dipolar component of the 2PA signal results in sizeable cross-section values (300-500 GM) for the QBDF, QBDeF and QBDvF dyes at 880, 950 and 960 nm respectively.The pronounced V-shape of QBDtF even leads to the appearance of a definite 2PA band located at 980 nm and peaking at 950 GM, a promising property for 2P microscopy of thick biological samples.
Moreover, the extended conjugation induces a red-shift (from 840 nm in QBDF to 890 nm in the other dyes) and marked increase (from 1250 GM in QBDF to 1650, 1880 and 2400 GM in QBDeF, QBDvF and QBDtF respectively) in the 2PA peak corresponding to the strongly 2P-allowed excited state (quadrupolar component).Finally, we note the appearance of an additional, higher energy band in the 2PA spectra of extended dyes.Ideally located around the maximum excitation achievable with a Ti:Sapphire laser commonly used for 2P microscopy, this 820-840 nm band leads to 2PA responses about 2-fold higher in the elongated dyes than in QBDF at the same wavelength.
Confinement effects on linear optical properties.As dye molecules self-assemble into nanoparticles upon nanoprecipitation in water, specific optical properties may arise, depending on the nano-organization of the dyes and their interchromophoric interactions within aggregates.
Following the trend observed in solution under 1P excitation, elongating the conjugation bridge induces a bathochromic shift of the ICT absorption band in FONs (Figure 2C).Similarly, we note a comparable ~ +50%-100% increase in the molar absorption coefficient values of QBDeF, QBDvF and QBDtF dyes in FONs as compared to QBDF.
We also notice that these  max values decrease upon aggregation (~ -20%), with the exception of that of QBDvF which remains almost unchanged (Figure 3).Additionally, while the ICT bands of QBDF dyes in THF and in FONs have similar full-widths at half-maximum (FWHM), we note a slight flattening and broadening of the low energy ICT bands, with the appearance of a red shoulder in the case of QBDvF and QBDtF.This may originate from excitonic coupling but also result from intermolecular charge transfer interactions in relation with the quadrupolar D-A-D nature of the dyes. 46Regarding emission, contrary to what was observed in solution, the emission maxima of dyes in FONs follow the trend of the absorption bands.In other words, QBDeF now behaves as anticipated, with an emission maximum lying in between the peaks of the shorter QBDF dye and the more flexible QBDvF dye (Figure 2).This is consistent with TICT being impeded in the confined state in comparison to the dye dissolved in a medium-polarity, nonviscous solvent.We also note that all dyes do not show similar band shifts upon aggregation.
While QBDF and QBDeF dyes emission is blue shifted in FONs as compared to in THF, QBDvF dyes emit at the same wavelength and QBDtF dyes are red-shifted (Figure 3).This may be related to their length and flexibility, which modulate their nano-organization (i.e.modulation of the slip stacking of the dyes 43,44 ) within aggregates.We also note a marked decrease in the quantum yield of the dyes in FONs as compared to in solution which can be ascribed to both decrease of the radiative decay rate and increase in the non-radiative decay rate (Table S1).This loss may be ascribed to both interchromophoric interactions (leading to a reduction of the radiative rate) and additional non-radiative decay processes favored by water molecules bound to the FONs surface.
Yet, we stress that the fluorescence quantum yield values (2% to 30%) remain substantial for R-NIR emitting dyes in water in the absence of protective additives.Interestingly, while the quantum yield values of QBDF, QBDeF and QBDvF dyes decrease with conjugation, the quantum yield of QBDtF in FONs remains larger than that of the other extend dyes (i.e.8%).As a result, the FONs made from QBDtF dye show both the most red-shifted and most intense fluorescence of the elongated dyes, indicating that their relative positioning (slip stacking) within FONs is the most favorable to luminescence.2, Figure 2D).Similarly to what was observed in solution, the 2PA responses significantly increase with conjugation, with QBDF having the lowest peak 2PA cross-section, QBDtF the highest and QBDeF and QBDvF having intermediate values.Yet a more detailed observation shows that the nanoconfinement of the dyes into nanoparticles strongly affects their 2PA spectra, in a different manner depending on the structure of the dye (Figure 4).
Most interestingly, QBDF displays an increase of its 2 max value by over 40% along with a slight red-shift (from 820 to 840 nm).Interestingly its low energy 2PA band located at 880 nm corresponding to the lowest one-photon allowed excited state (dipolar component) is even more impacted with an increase by over 70 % (Table 1).This cooperative enhancement may be tentatively ascribed to electrostatic interchromophoric interactions whose effects depend on the relative positioning of the quadrupolar dyes within the aggregates and suggest a favorable slip stacking 45 .While QBDeF, QBDvF and QBDtF show a major reduction (50%, 40% and 30 % respectively) of their highest energy 2PA bands located at 820-840 nm, we note that their lower energy 2PA band (quadrupolar component located at 890 nm) is much less impacted upon confinement within FONs (only 12%-16% reduction in 2 value, Table 2).This effect is accompanied by a definite broadening of the 2PA bands and a pronounced increase of the 2PA response of the low energy sub-band located around 950 nm, most notably in FONs made from the most extended and flexible dyes (Figure 4).These marked effects may originate from both electronic, electrostatic, and vibronic contributions.As a result, QBDvF and QBDtF maintain high 2PA responses (>1000 GM) at 950 nm.From a practical point of view, this is of major interest for bioimaging purposes as it allows for improved collection and spectral discrimination of the fluorescence signal by operating at 2P excitation wavelengths away from the R-NIR emission.FONs brightness and stability.While studying the optical properties of dyes in solution and in FONs help us understand the effects of confinement, the properties of the FONs themselves, as a collection of a multitude of dyes, should be considered for applications.The brightness of FONs is described as the product of their absorption capacity ( or 2) and their emissive capacity (f).As shown in Table 3, FONs exhibit giant molar extinction coefficients (with FONs values in the 10 7 M -1 .cm - range) and 2PA cross-sections (with 2.FONs values in the 10 6 GM range), thanks to the high number of dyes that are accommodated per nanoparticle ranging from a few hundreds to a couple thousands (Table 1).Table 3. Optical properties of the FONs made from quadrupolar dye subunits.a) Absorption maximum wavelength corresponding to the low-energy (ICT) band under 1photon excitation; b) Emission maximum wavelength; c) Estimated molar extinction coefficient of FONs at max 1P considering N dye molecules per FON (see Table 1); d) Estimated one-photon brightness of FONs; e) Absorption maximum wavelength under 2-photon excitation; f) Estimated cross-section of FONs at max 2P considering N dye molecules per FON; g) Estimated two-photon brightness of FONs.
We had previously reported that the remarkable absorption and high quantum yield of QBDF let its FONs reach a giant one-photon brightness of 1.10 7 M -1 .cm - . 20We now report that these FONs also display a huge two-photon brightness of 6.10 5 GM, in relation notably to interchromophoric interactions resulting in the cooperative enhancement of the 2PA response of the individual QBDF dyes.Interestingly, the excellent properties of QBDtF FONs described above make them our second brightest probe, with the foremost advantage that both its absorption and emission are redshifted as compared to QBDF.In addition, QBDtF FONs retain huge two-photon brightness at 950 nm (2.10 5 GM) revealing the potential for NIR-to-NIR imaging in thick biological samples.
QBDeF and QBDvF display lower 1-and 2P brightness values, in relation with to their smaller size and lower fluorescence quantum yields (Figure S3).Overall, the bottom-up molecular engineering based on the use of quadrupolar dyes derived from the QBDF structure as FONs subunits proved to be even more successful than an earlier study based on dipolar dyes 27 , with notably QBDtF FONs reaching similar 2P brightness as NIR-emitting FONs reported earlier 27 despite being twice as small.For comparison, red-emitting inorganic quantum-dots, long considered the state-of-the-art in terms of probe brightness, display 10 6 M -1 .cm - 1P-brightness 47 and 10 4 GM 2P-brightness 48 values in water.Among organic nanoparticles, polymeric redemitting nanoparticles of small size (Ø < 30nm) rarely reach 10 6 M -1 .cm - 1P-brightness 49 and 10 5 GM 2P-brightness 50 values.Our FONs are thus closer to another family of all-organic, fluorescent nanoparticles referred to as AIE-dots.Most R-NIR emitting AIE-dots display 1Pbrightness comparable to QDs (and exceptionally up to 10 7 M -1 .cm - ) 51 and 10 5 GM 2Pbrightness values. 52However, the formulation of these dots require polymeric surfactants and the resulting nanoparticles are usually larger (Ø > 30nm).The FONs presented in this study therefore compare positively to other types of nanoparticles in terms of size, brightness and simplicity.
Finally, FONs should be stable in time to be easily usable.The highly negative zeta potential of the solution is indicative that these FONs may be colloidally stable, as electrostatic repulsion between particles helps prevent them from aggregating.We found that QBDF, QBDeF and QBDtF FONs absorption remains virtually unchanged over a month (Figure S4).Namely, we observe no setting and no appearance of a background scattering signal suggestive of the formation of microagglomerates, nor any decrease in the intensity of the ICT absorption band.Oppositely, QBDvF FONs appear surprisingly less stable, with a gradual decrease of the ICT band over time.This striking difference suggests that the structuration of the chromophores at the surface of the FONs markedly differs for this dye in such a way that nanoparticles are structurally much less stable at the interface with water.In other words, the interaction energy of a single QBDvF dye with the FONs surface appears as lowered compared to the other dyes, favoring either exchange with the bulk aqueous phase or with other nanoparticles upon contact.This finding emphasizes the critical point that a subtle change of the molecular structure of the dyes may significantly affect the colloidal stability of its FONs. 53Emission is slightly more affected than absorption for all chromophores, with QBDF and QBDeF FONs quantum yields decreasing by about 30% over a month, and that of QBDtF FONs by about 50%.This indicates that place exchange leading to surface defects 54 acting as fluorescence traps is a slow process and suggests that in comparison dyes QBDF, QBDeF and QBDtF show larger interaction energy with the surface.
FONs behavior in a cellular environment monitored by fluorescence imaging.Building upon the promising properties of these FONs, i.e. red shifted emission and high 1P and 2P brightness, we next investigated their usability for cellular imaging with two questions in mind: i) Do the three novel dye designs QBDeF, QBDvF and QBDtF yield stealth FONs as the QBDF dye does?And ii) Can stealth FONs be functionalized for controlled intracellular delivery?Moreover, we wanted to assess whether these FONs were compatible with two-photon microscopy, as their optical properties would suggest.
By incubating Cos7 cells with FONs for 24 h and imaging them under a one-photon confocal microscope, we confirmed that QBDF FONs do not internalize nor stick to membranes of living cells.This result is in accordance with their stealth behavior previously observed towards HeLa cells. 20Most interestingly, we observed the same lack of interactions for QBDtF FONs.QBDvF FONs, on the other hand, could readily be observed accumulated inside of cells.An intermediate behavior was observed for QBDeF FONs, which were detectably internalized but at lower levels than QBDvF FONs (Figure 5, upper panels).This suggests that our rational for dye design, i.e. elongated quadrupoles with pendant hydrophobic chains close to the end of the dyes, indeed tends to yield stealth FONs.Interestingly, other factors appear to come to play to rationally engineer this property and control its tunability.Notably, it appears that the rigidity of the dye backbone may be of importance, as the introduction of a flexible double bond in the QBDvF dye induces the internalization of QBDvF FONs.This again points to a different surface organization of the dyes as compared to the other chromophores, presumably responsible for both the uptake of these FONs by cells and their strikingly reduced colloidal stability.Further complementary studies dedicated to understanding these different organizations will be of the highest interest in view of fully controlling the nano-bio interface.
The original spontaneous stealth property of QBDF, QBDeF and QBDtF FONs opens the possibility of controlling their intracellular delivery via surface functionalization.We reasoned that shielding their surface would modulate their interactions with cells.We chose poly(allylamine hydrochloride) (PAH) as a coating polymer, both because its cationic nature would allow it to electrostatically interact with the negatively charged FONs and because it is known to enhance nanoparticle intracellular delivery. 55Coating was achieved by simple dropwise addition of PAH into the FONs solution under agitation and confirmed by observing the reversal of the solution's zeta potential (Table S2) Of note, PAH coating did not modulate the in vitro optical and colloidal properties of FONs (Figure S5).When incubated under the same conditions as bare FONs, all four PAH-coated FONs were strongly taken up by Cos7 cells (Figure 5, lower panels).
Importantly, QBDF@PAH and QBDtF@PAH FONs readily internalized although their bare counterparts did not.Notably, we observed no further increase in the internalization of QBDvF@PAH FONs as compared to bare QBDvF FONs.Oppositely, QBDeF@PAH FONs were clearly more internalized than bare QBDeF FONs.This suggests that cellular uptake of bare QBDvF FONs is already rapid and maximal whereas the slight uptake of bare QBDeF FONs is slow and can be enhanced by PAH coating.Finally, the strong signal originating from internalized FONs allowed us to confirm that all four types of FONs can be readily imaged under 2P excitation well within the optical transparency window.Our dye designs therefore yield a novel class of bright, 2P responsive, fluorescent nanoparticles.In this regard, the 890 nm to 950 nm-absorbing, 700 nm-emitting and very stealth QBDtF FONs appear as ideal candidates for NIR-to-NIR bioimaging with organic nanoprobes.

CONCLUSIONS
We have described and discussed the design, optical properties and bioimaging potential of a series of new R-NIR-emitting all-organic nanoparticles obtained by nanoprecipitation of quadrupolar dyes explicitly engineered to address two major properties relevant to biomedical applications: R-NIR absorption and emission, and reduced interactions with cells.
Starting from a previously described molecular template, recently shown to yield stealth nanoparticles, 20 we tuned the photo-physical properties of three dyes by modulating the nature, length and rigidity of conjugated π-connectors between bulky diphenylamine donor groups and a benzothiadiazole acceptor core.We show that these modulations have successfully induced a hyperchromic and bathochromic shift as compared to the original QBDF design.We also demonstrate that these quadrupoles all possess strong 2PA capacities, with the prototypical QBDF dye even displaying a definite aggregation induced enhancement of its 2PA cross sections in FONs.The obtained FONs therefore absorb and emit within the biological transparency window.Moreover, they display remarkable 1P and 2P brightness values.Interestingly, the present study also demonstrates that the nature of the conjugated linkers has a significant influence on the effect of nanoconfinement on the optical properties, indicative of the modulation of the relative positioning of the dyes within FONs by the length and rigidity of the -connectors.As a result, the QBDtF dye leads to NIR emitting FONs showing both the more red-shifted and more intense NIR emission, the largest brightness and enhanced 2PA in the 900-950 nm range.In contrast, the R-NIR emitting FONs made from the QBDvF dye show lower fluorescence and even more importantly strikingly reduced colloidal stability that reveals an additional role of the flexible bridges on the structuration of the FONs surface.Taken together, these properties allow FONs to compete with the current state of the art, especially for such small probes of no more than 20 nm in diameter.
In addition to these remarkable optical properties, we found that three of the four dyes derived from this template yield FONs that spontaneously minimally interact with cellular membranes.
Interestingly, the most fluorescent and colloidally stable FONs (i.e. made from orange/redemitting QBDF and NIR-emitting QBDtF) were also the most stealth FONs.As these first two parameters can be engineered by a bottom-up approach, this observation strongly suggests that controlling the nano-bio interface can be rationally addressed too.The present study therefore represents a first step towards this goal in the field of single component organic nanoparticles.
We hypothesize that the nano-organization of the dyes at the surface is key to addressing this issue and expect that detailed studies of the molecular arrangements occurring at the interface will be of the highest interest to further explore this concept.
following a 3-step procedure: a reactive donor-fluorene intermediate and an acceptor-bridge intermediate were first synthesized and then reacted together by Suzuki coupling.The QBDeF dye was synthesized via a similar route finalized by a Sonogashira coupling.The synthesis of the QBDvF dye followed a 2step procedure: a reactive donor-fluorene-bridge intermediate was first synthesized and then reacted onto the acceptor by Heck coupling.

Figure 1 .
Figure 1.Representative transmission electron microscopy images of FONs made from diameter determined by transmission electron microscopy; b) Number of dye subunits per nanoparticle calculated from ØTEM assuming a density of 1; c) Zeta potential.

Figure 2 .
Figure 2. Optical properties of the dyes in THF (Top) and in FONs (Bottom) A-C Overlapped

Figure 3 .
Figure 3. Linear properties of the dyes in THF (dashed lines) and in FONs (solid lines).

Figure 4 .
Figure 4. Non-linear properties of the dyes in THF (dashed lines) and in FONs (solid lines).

Figure 5 .
Figure 5. Interplay between FONs and living cells.For each type of FONs, representative one-

Finally
, we have shown that the surface of FONs can be functionalized with a positively charged polymer to induce their cellular internalization.Under these conditions, FONs were clearly visible under 2P excitation, confirming our in vitro results and pointing to a potential use for deep tissue bioimaging.In this context, we believe that QBDtF FONs represent the best candidates as promising all-organic, spontaneously stealth, NIR-to-NIR bioimaging nanoprobes.ACKNOWLEDGMENT This work was supported by the European Commission and received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under REA grant agreement no.607721.The work also received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement Nos.841379 to M.R. Funding from the Conseil Régional d'Aquitaine for a Chaire d'Accueil to MBD is also gratefully acknowledged.The project was also supported by the LAPHIA Cluster of Excellence.Electronic and optical microscopy were performed at the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux