General and Scalable Approach to Bright, Stable and Functional AIE

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Biological and Medical Applications of Materials and Interfaces

General and Scalable Approach to Bright, Stable and Functional AIE Fluorogen Colloidal Nanocrystals for in Vivo Imaging Xibo Yan, Maxime Remond, Zheng Zheng, Elsa Hoibian, Christophe Soulage, Stéphane Chambert, Chantal Andraud, Boudewijn Van der Sanden, Francois Ganachaud, Yann Bretonnière, and Julien Bernard ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07859 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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ACS Applied Materials & Interfaces

General and Scalable Approach to Bright, Stable and Functional AIE Fluorogen Colloidal Nanocrystals for in Vivo Imaging Xibo Yan,1 Maxime Remond,2 Zheng Zheng 2 Elsa Hoibian,3 Christophe Soulage,3 Stéphane Chambert,4 Chantal Andraud,2 Boudewijn Van der Sanden,5 François Ganachaud,1 Yann Bretonnière2* and Julien Bernard1* 1

Université de Lyon, Lyon, F-69003, France ; INSA-Lyon, IMP, Villeurbanne, F-69621, France ; CNRS, UMR 5223, Ingénierie des Matériaux Polymères, Villeurbanne, F-69621, France.

2

Univ Lyon, ENS de Lyon, CNRS UMR5182, Université Lyon 1, Laboratoire de Chimie, F-69342 Lyon, France.

3

Univ-Lyon, CarMeN laboratory, INSERM U1060, INSA Lyon, INRA U1397, Université Claude Bernard Lyon 1, F-69621 Villeurbanne, France.

4

Univ Lyon, INSA-Lyon, CNRS, Université Lyon 1, CPE Lyon, ICBMS, UMR 5246, Bâtiment Jules Verne, 20 avenue Albert Einstein, F-69621 Villeurbanne, France. 5

Intravital microscopy plateform, France Life Imaging, INSERM U1039 and University Grenoble Alpes: Unit Biomedical Radio-pharmaceutics, Medical Faculty, La Tronche, France

Corresponding authors: [email protected] or [email protected]

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ABSTRACT.

Fluorescent nanoparticles built from aggregation-induced emission-active organic molecules (AIEFONs), are emerging as powerful tools in life science research (for in vivo bio-imaging of organs, biosensing and therapy). However, the practical use of such bio-tracers has been hindered owing to the difficulty of designing bright nanoparticles with controlled dimensions (typically below 200 nm) and size dispersity, and long shelf stability. In this article, we present a very simple yet effective approach to produce monodisperse sub-200 nm AIE fluorescent organic solid dispersions with excellent redispersibility and colloidal stability in aqueous medium through combination of nanoprecipitation and freeze-drying procedures. By selecting polymer additives which act at the same time as stabilizers, promoters of amorphous-crystalline transition and functionalization/cross-linking platforms, we demonstrate straightforward access to stable nanocrystalline FONs that exhibit significantly higher brightness than their amorphous precursors and constitute efficient probes for in vivo imaging (of the normal - and tumor vasculature). FONs design principles reported here are universal, applicable to a range of fluorophores with different chemical structures and crystallization abilities, and are suitable for high throughput production and manufacturing of functional imaging probes.

Keywords: Organic nanocrystals, Aggregation-Induced Emission, Nanoprecipitation, Ouzo domain, in vivo imaging


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INTRODUCTION Recent advances in photoluminescent materials and nanotechnology have opened unprecedented possibilities in life sciences. The merger of these appealing approaches have paved the way to the conception of fluorescent nanoparticles (NPs) which constitute powerful tools for in vivo bio-imaging of organs, biosensing and therapy.1-5 Until now, this dynamic research area has been dominated by inorganic fluorescent NPs,6 e.g. quantum dots,7-9 upconverting NPs10-12 or silica-based NPs.13-17 These fluorescent probes show exceptional brightness and photostability, with respect to single organic fluorophores but, contrary to the latter, also suffer from severe drawbacks such as toxicity or lack of degradability18-22, de facto limiting their approval.23 In this context, Fluorescent Organic Nanoparticles (FONs) are purely organic, solid, fluorophore aggregated materials that represent an attractive avenue to bring together the best of both probe classes.24-27 The emergence of FONs has long been hampered by the photophysical behavior of many fluorophores that exhibit strong fluorescence in dilute solutions but are quenched at high concentration or in the solid state.28-29 In the early 2000s, the advent of a new class of molecules behaving precisely in an opposite manner, i.e. showing bright fluorescence in the aggregate state (notably owing to restricted intramolecular motions),30-33 provided strong impetus to the development of aggregation-induced emission (AIE)-active FONs.25, 33-38 The prevalent route to synthesize AIE-active FONs relies on the nanoprecipitation procedure.39-41 This is a straightforward and easy-to-handle, bottom-up strategy based on supersaturation of the hydrophobic dyes (initially dissolved in a water-miscible solvent) upon addition of water (as the non-solvent).33-34, 4243

However, the scope of this versatile solvent shifting process remains limited in regard to biomedical

applications by: i) the challenging control over the size and size distribution of the FONs, ii) the usual formation of amorphous particles, where the fluorophore molecular arrangement may not be always optimal for promoting fluorescence emission,44-46 and iii) the fact that such metastable dispersions endure stability issues owing to uncontrolled crystallization upon storage or temperature fluctuations.4748


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In this context, the establishment of simple, reproducible and general methods towards the fabrication of thermodynamically stable aqueous dispersions of bright crystalline FONs with sub-200 nm diameters and narrow size distribution, holds great promise and is still actively sought worldwide for imaging purposes. Transposing methodologies exploited for the conception of crystalline drug delivery systems with enhanced bioavailability and dissolution rates49 or semiconductor colloids,50 Guldi and Bottari,51 and more recently Tang and Liu,52 described the rare examples of highly emissive AIE-active nanocrystals through sonochemical transformation of amorphous nanoprecipitated FONs. An unprecedented level of control and brightness enhancement has been reached by Tang and Liu with TPE-FN as AIE-active molecule (where ~100 nm nanocrystals were prepared). Yet, these groups were confronted by the difficulties controlling the crystal growth with the other studied dyes, leading to substantial increase in size after ultrasound-induced amorphous/crystal transition. Herein, we describe a general, rapid and robust bottom-up approach which addresses the limitations of the current routes to bright nanocrystalline FONs. More precisely, we report on a simple and general methodology based on i) the establishment of AIE-active fluorophore/acetone (solvent)/water (nonsolvent) phase diagrams with identification of domains of composition where the design of FONs with optimal colloidal and emissive properties can be achieved, ii) the acquisition of crucial knowledge on the conditions of AIE-active fluorophores crystallization in the bulk (based on the determination of glass transition, crystallization and melting temperatures by DSC) and iii) a judicious choice of polymer additives (to be introduced in the aqueous phase prior to solvent shifting) acting simultaneously as stabilizers, crystallization-promoters and a functionalization and/or cross-linking platform. In doing so, we show that conditions can be conveniently set so that amorphous FONs in the 100-200 nm range are initially prepared through solvent shifting and subsequently inclined to undergo controlled nanoscale confined crystallization using routine freeze-drying procedures, without alteration of the size/dispersity of the final NPs and of their water-dispersibility (see Scheme 1). It is finally demonstrated that, independently from the nature of AIE-active fluorophore, this straightforward process is adaptable and


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scalable, and provides direct access to shelf-stable nanocrystalline FONs with enhanced brightness, whatever the considered fluorophore, making them ideal candidates for in-vivo imaging applications. Here, we first describe the procedure we set up for one example of red emitting fluorophore displaying slow rate of crystallization (F1, see Chart 1). In a second part, we explain how this technology allows us to generate nanocrystalline FONs with a large panel of probes. Finally, we report some subtle adjustments to drive this technology towards the generation of in-vivo biotracers and show the in-vivo proof of concept on a mouse tumor model.

Scheme 1. General strategy to prepare AIE-active nanocrystalline FONs.

RESULTS AND DISCUSSION A. Choice of Fluorophores For these studies, we selected four different push-pull probes (F1-F4, Chart 1) exhibiting interesting solid-state emission properties and most importantly different propensity to crystallize. The first two fluorophores F1 and F2 are based on two new electro-acceptor units derived from 4,5,5-trimethyl2(5H)-furanone ring, which we recently demonstrated the interest in the design of fluorophores emitting in the far-red in the crystal-state and also featuring AIE properties.53 The push-part, on the other hand, is made of more classical 4-(N,N-diphenylamino)phenyl- or the 9-ethyl-9H-carbazolyl- electro-donating ACS Paragon Plus Environment

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groups. F1 and F2 were obtained in one step through a Knoevenagel condensation between 4(diphenylamino)benzaldehyde or 9-ethyl-9H-carbazole-3-carboxaldehyde and the corresponding activated methylene compounds. The structures were confirmed by 1H,

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C NMR spectroscopies and

ESI-MS (see details in Supporting Information, section S2). The two solids exhibited intense orange-red fluorescence in their crystalline form with emission maxima at 660 nm (Φ=16%) and 596 nm (Φ=35%) for F1 and F2, respectively. The preparation of F3 was recently reported,54 but its solid-state fluorescence property and its crystal structure have not been described yet. F3 displayed an intense deep-red emission in the crystal-state with an emission maximum at 656 nm (Φ=20%). Interestingly, the three crystal packings, as revealed by X-ray diffraction on a single crystal, drew brickwork-like patterns created by single rows of molecules. Two neighboring rows in the same plane are either parallel (F1) or anti-parallel (F2 and F3). For F2, a slight tilting of one molecule with respect to the closest one also creates an undulation in the lines (see Figure S6). Such packing closely resemble what was observed for similar structural solid-state emitter.53,

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Finally, fluorophore F4 (λem=679nm, Φ=22% in the crystal

state) was chosen among a pool of solid-state and AIE emitters based on the 2-(3-cyano-4,5,5trimethylfuran-2(5H)-ylidene)malonitrile electron-withdrawing group.55 Besides the solid-state emission properties, the selection of the probes studied herein was based on the identification of the thermal transitions for the different fluorophores (as powders) by heat-cool-heat DSC scans at 10°C/min (see experimental conditions and thermograms in Supporting Information, sections S1 and S2, respectively). Such characterization is indeed useful to estimate each probes’ ability to crystallize during nanoprecipitation (vide infra). In Chart 1 we report the Tm and Tg detected over the course of the first heating and cooling ramp, respectively. Disparate crystallization behaviors were observed depending on the nature of the probes. The first cooling revealed a single crystallization peak for F4, not for F1 and F2. F3 partly crystallized on cooling and crystallized further during the second heating ramp. F2 showed a partial recrystallization in the second heating ramp contrary to F1. The different values of Tc (detected upon heating or cooling) are given in Figure S11. In summary, the


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selected probes exhibited disparate crystallization aptitudes: F4 was the fastest dye to crystallize, closely followed by F3. Crystallization of F2 was relatively slow whereas F1 remained amorphous (in the conditions of DSC analyses mentioned above).

F3

F2

F1

F4

Tg = 89°C

Tg = 76°C

Tg = 84°C

Tg = 175°C

Tm = 197°C

Tm = 208°C

Tm = 257°C

Tm= 285°C

Chart 1. Structure of the fluorophores with different crystallization abilities engaged in the design of functional FONs through nanoprecipitation.

B. Design principles to construct nanocrystalline FONs (exemplified with F1) 1. Nanoprecipitation and phase diagram Initial efforts were devoted to the identification of optimal conditions for the preparation of uniform bare nanometer-scale FONs dispersions through nanoprecipitation. The hydrophobic fluorophore F1 is highly soluble in water-miscible solvents such as THF or acetone but poorly soluble in water (Log P= 0.74). Evolution of its fluorescence spectrum with water mass fraction is given in Figure 1A. In this case, 75wt% of water was necessary to detect fluorescence, although nanoparticles were already formed above 40wt% water (see DLS measurements in Figure 1C). To account for such results, we established the phase diagram for F1/ acetone/ water ternary systems (see Figure 1B, details of the modus operandi given in the SI). Three different regions were distinguished on this composition map of F1/acetone/water. First, in the upper part of the diagram (i.e. the acetone-rich region), the fluorophore was fully dissolved, resulting in weak fluorescence emission.


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The limit of this monophasic region is represented by a straight line which linearly increases (from 60 to 90 wt% acetone) with the initial log (mass fraction) of F1 (from 10-6 to 10-4). Below this line, one enters a region where “mesoscale solubilization” is observed, the so-called surfactant-free microemulsion domain (SFME).56-60 In this domain, transparent dispersions of fluorophore aggregates were spontaneously formed. Accordingly, solvent shifting at 10-5 dye mass fraction and 50 wt% acetone (Condition 3, Figure 1C) led to the formation of swollen micelle-like aggregates with sub-100 nm dimensions (dz (DLS)=60 nm and dz (TEM)=53 nm, as given in Figures 1C and 1E). At these composition, with a final acetone mass fraction approximately varying between 20 to 70 wt% at 10-5 dye mass fraction, sizes ranged from 30 to 80 nm (Figure S12). Under such conditions, a non-negligible content of solute can be found in the main phase after solvent shifting. This leads to homogeneous nucleation through a conventional Ostwald ripening process and the growth of large crystalline needles is rapidly observed in the vial after nanoprecipitation, in addition to aggregation (Figure S13). The resulting fluorophore-rich aggregates contain a substantial fraction of acetone (enhancing mobility of F1 therein) which explains the absence of fluorescence enhancement (see vide infra and inset Figure 1A). These results advocate for definitively discarding this SFME region for the synthesis of bright size-controlled FONs. At higher water contents, one enters the Ouzo domain (see Figure 1B). Its frontier with the SFME domain is delimited by a straight line (on a semilog scale), the binodal curve, rising with fluorophore content, typically from 10 to 35wt% acetone mass fraction for F1 mass fraction from 10-6 to 10-4. Note that the so-called Ouzo limit61 has not been sought in this work (but could indeed be observed at higher fluorophore concentrations). As shown by DLS (Figures 1C), depending on the acetone mass fraction, nanoprecipitations in the Ouzo domain resulted in the formation of FONs with sizes ranging from 50 to 110 nm (Figure S12). In agreement with the literature, 49-50 one-shot solvent shifting experiments carried out in the Ouzo domain initially resulted in the formation of amorphous spherical FONs (see TEM pictures, Figures 1F and XRD analyses in Figure S5-A).


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Figure 1. A) Emission spectra of F1 (λexc =475 nm) in acetone (black curve) and of freshly prepared F1 dispersions in water/acetone mixtures (water mass fraction ranging from 0.2 to 0.99). Inset: relative emission intensities (Imax/Io) vs water mass fraction; B) Phase diagram of the F1/ acetone/water ternary system; C) z-average diameter and dispersity of freshly prepared F1 dispersions at 10-5 dye mass concentration and different water mass fraction from DLS measurements. D) Photographs of FONs dispersions prepared in conditions 1 (monophasic domain), 3 (SFME domain) and 8 (Ouzo domain) under natural light (up) and 365 nm light irradiation (down). E) TEM picture of FONs obtained in conditions 3 (SFME domain). F) TEM picture of FONs obtained in conditions 8 (Ouzo domain).

Consistent with AIE principles, fluorescence characteristics of freshly prepared solutions or dispersions of F1 (at 10-5 dye mass concentration) were expected to evolve dramatically with the composition of the ternary systems. We subsequently compared the emissive character of molecular solutions of fluorophores (fw =0 and 0.20, see condition 1 in Figure 1B) and of FONs dispersions (fw=0.20-0.99, see conditions 2-8 in Figure 1B) in the different regions of the phase diagram. Close examination of Figures 1A/1D for F1 clearly highlights that photoluminescence (PL) intensity did not significantly evolve in solution (acetone-rich domain) or in the SFME region. In contrast, operating in ACS Paragon Plus Environment

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the Ouzo domain through additional water enrichment caused a sharp enhancement of PL. In agreement with the formation of AIE-active aggregates, the PL intensity was 90 times greater in acetone/water mixtures at fw ~ 0.90 as compared to that at fw ~ 0.70 or in pure acetone. Interestingly, an increase in the solvent polarity (from pure acetone to water/acetone mixtures at fw ~ 0.70) was accompanied with bathochromic shift (from 675 to 694 nm) in agreement with the solvatochromism in emission usually associated with the fluorescence of push-pull dipolar dyes. A slight ipsochromic shift was observed when precipitation was undertaken at higher water fraction (from 694 nm to 656 nm for F1), suggesting a decrease in the polarity around the solid fluorophore.

2. Colloidal stability issues and how to tackle it The dimensions of the FONs tend to increase over time, leading to the eventual precipitation of the nanoparticles. We have shown in a previous study,62 that when the acetone content is fixed below 0.05 immediately after nanoprecipitation, Ostwald ripening is (at least partly) postponed. Nevertheless, twice-volume dilution with water and rapid evaporation of the acetone fraction did not inhibit this trend (Figure S14) suggesting that the solubility of F1 is not negligible in water. Note though that the diameter of the FONs levelled off at around 100 nm, which is suitable for bio-imaging applications. As a first trial, we then attempted to freeze-dry F1-based dispersions (generated in conditions 2-8) so as to produce water-dispersible powders that could be handled over the long term. However, such treatment inexorably resulted in the irreversible formation of large fluorophore clusters after re-dispersion, ruining the processability of the FONs (see Figure S15). To solve these colloidal instability issues, we next implemented the solvent shifting procedure in the presence of commercial non-ionic polymer surfactants, namely poly(vinyl alcohol) (PVA, 130 kg/mol), and Pluronic F68 (poly(PEG-co-PPG), 8.4kg/mol). (Note: other polymers, namely PVP and PVME, were tested as stabilizers and these results are discussed in Section S3). These polymer surfactants, initially added in the aqueous phase, are conventionally used in bio-related applications owing to their approval by the FDA. The following nanoprecipitations were thus performed in the Ouzo domain at 10-5 ACS Paragon Plus Environment

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mass fraction of fluorophore, 10-3 mass fraction of surfactant and fw = 0.95 (see Figure 2 and detailed procedure in SI). Under these conditions, aqueous dispersions of amorphous fluorophore nanoparticles with diameters between 70-150 nm were obtained (70 and 132 nm in the presence of F68 and PVA, respectively). Decrease of the surfactant quantity did not affect the size of FONs in the presence of PVA, contrary to F68 (Figure S16). The FONs grew larger overnight (16 h), but typically remained in the appropriate size range for injection. In the presence of polymer surfactants, aggregation of the particles was suppressed over the course of the freeze-drying procedure and the resulting powders could be effectively re-dispersed in water. Yet, colloidal stability was finally lost for such FONs dispersions after few hours, particularly for the PVA-stabilized particles (Figure S17). A further refinement of the process thus consisted in constructing a stable polymeric corona surrounding the hydrophobic core by crosslinking the chains, as previously reported for the preparation of glyconanocapsules.62 Nanoprecipitations proceeded in the presence of hydroxyl-rich PVA and isophorone diisocyanate (IPDI, introduced in the organic phase) to cross-link the shell through isocyanate-alcohol reactions. In this case, aqueous FON dispersions were efficiently regenerated from freeze-dried powders without loss of colloidal stability or alteration of dimensional/optical properties as compared to their precursors (vide infra). In summary, we found nanoprecipitation conditions to easily prepare amorphous F1-based FONs smaller than 200 nm in diameter and with good colloidal stability.

3. On demand crystallization The redispersed PVA and F68-stabilized FONs were then observed by TEM (Figure 2). Whereas the PVA-stabilized FONs remained amorphous, F68-stabilized FONs crystallized throughout the freeze drying process. Note that the relatively high value of F1‘s Tg excludes an amorphous-crystalline transition for bare FON solid dispersions in the freeze-drying chamber. We also considered other surfactants, namely poly(vinylpyrrolidone) (PVP) and poly(vinylmethylether) (PVME) (see properties in material section, supporting information). Crystallization of F1 did not occur upon freeze-drying in the presence of PVP (as observed with PVA), whereas, akin to F68, the use of PVME as stabilizer ACS Paragon Plus Environment

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endorsed the formation of nanocrystalline FONs under similar conditions. Importantly, all resulting powders re-dispersed easily in water.

Table 1: Main features for carrying out nanoconfined crystallization. Tg (surfactant)

Tg (mixture)a

Db

DFD c

(°C)

(°C)

(nm)

(nm)

F68

-62

-37

70

85

25

PVME

-25

-3

97

142

-

PVP

173

103

83

100

-

PVA

75

82

132

156

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Quantum Yield

a

Tg of 1:1 wt/wt of surfactant/F1 mixtures (DSC); b z-average diameter of freshly-prepared F1-based FONs (DLS in water at 1 mg/mL); c z-average diameter of FD and re-dispersed F1-based FONs (DLS in water at 1 mg/mL)

Figure 2. A. DSC scans (second heating ramp, 10°C/min) of nanoprecipitated and freeze-dried surfactant:F1 mixtures (1:1 wt/wt). Tg was maintained below room temperature for F68 or PVME/F1 mixtures, whereas higher Tg values were observed in the presence of PVA or PVP, precluding crystallization of the probe. B. XRD of main precursors in the solid state (PVA, F68 and F1) and of ACS Paragon Plus Environment

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aqueous dispersions of FD FONs (surfactant:F1 mixtures 1:1 wt/wt). C. Evolution of FONs size before and after FD for all dispersants (DLS in water at 1 mg/mL). D. Corresponding TEM photos. Note the presence of numerous tiny dark dots observed together with PVA or F68-stabilized FONs due to selfassembly of (excess) surfactant chains upon casting. Assisted-crystallization of actives with external agent is actually not new per se. For instance, Qian et al.63 previously reported that Pluronic surfactants can act as plasticizers for hydrophobic drugs and promote the formation of nanocrystalline drug-polymer solid dispersions through a spray-drying procedure at room temperature. The group of Frijlink64 also showed that freeze-drying is efficient in producing drug nanocrystals in presence of mannitol. These studies clearly support that the engineering of nanocrystalline FONs can be achieved through freeze-drying procedure, provided that additives display adequate miscibility with the fluorophores and ensure plasticization of the organic core. As F1 has a Tg ~ 89°C, crystallization during freeze-drying implies having recourse to low Tg surfactants (so that TFreezeDrying ˃ Tg

mixture)

to promote fluorophores mobility. Consistent with that, DSC curves of

polymer surfactants/fluorophore mixtures in 1:1 wt:wt ratio given in Figures 2 and S19 (see DSC curves of polymer surfactants in Figure S18 and Tg values in Table 1) showed that F68 or PVME/ F1 mixtures exhibit Tg’s far below the ambient temperature (see Table 1), whereas the Tg of F1:PVA and F1:PVP systems are both above 80°C. After freeze-drying, XRD patterns of the FONs (re-dispersed in water) displayed characteristic diffraction peaks of F1 crystallites attesting for the occurrence of an amorphous-crystalline transition (see Figure 2B, PVME-system in Figure S20). DLS studies on freshly prepared aqueous dispersions confirmed the production of FONs with dimensions and narrow size distribution in full agreement with those of the amorphous precursors (Figure 2C). TEM images highlighted the drastic changes of morphology within the dispersions, from homogeneous spherical NPs after nanoprecipitation to dense crystalline structures ~ 100 nm, i.e. the fluorophore aggregates, occasionally buried into larger crystalline chestnut burr-like polymer clusters after freeze-drying (Figure 2D). Note that these large ACS Paragon Plus Environment

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“chestnut burr”-shaped clusters disappear when TEM analyses of the dispersions are performed in situ in liquid water using the K-kit microchip technology,65 owing to the rapid dissolution of polymer crystals in water (see Fig S23). Another striking observation is the substantial impact of the amorphouscrystalline transition on the emissive properties of the FONs. Nano-confined crystallization of the fluorophore enhanced the emission of the nanoparticles by almost a factor of 5 vs amorphous PVAstabilized FONs, with an overall quantum yield reaching 25% for F1, whereas NPs without surfactant and PVA-stabilized amorphous NPs displayed 6% quantum yield (Table 1). In this case, the emission slightly shifted to the red with maxima over 650 nm. These data demonstrate that the amorphous patterns produced by nanoprecipitation efficiently serve as nanoscale templates for subsequent fluorophore crystal growth under nano-confinement.

4. Preparation of stable water-dispersible nanocrystalline FON solid dispersions Similar to the uncross-linked PVA or PVP-stabilized FONs described vide supra, an important decay of colloidal stability was observed for F68 or PVME-stabilized nanocrystalline FONs dispersions after several hours (data not shown). To yield nanocrystalline FONs possessing a perennial hydrophilic polymer shell, solvent shifting experiments were finally conducted in the presence of PVA (for shell cross-linking and functionalization purposes) and F68 (for crystallization purpose) as co-stabilizers (overall 10-3 mass fraction) using IPDI as a cross-linker. Different PVA/F68 compositions were investigated (PVA content at 10, 25 and 50 wt %). DLS and TEM analyses on freeze-dried FON aqueous dispersions clearly validated the construction of individual nanocrystalline particles, with dimensions in close agreement with those of the amorphous precursors (see Figure 3). Dimensions of the FONs were shown to gradually increase with PVA content (see Figure 3B) so that the FONs were preferentially prepared at 25wt% PVA (dz=119 nm, PDI=0.09). As assessed by XRD analyses (Figure S21), the freeze-drying procedure promoted amorphous-crystalline transition within the nanoparticles, indicating that co-addition of PVA does not hinder the crystallization process. The resulting shell-crosslinked FONs presented excellent dispersibility in water. Contrary to non-crosslinked freeze-dried ACS Paragon Plus Environment

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analogues, they exhibited excellent colloidal stability over months (see Figure S22). The optical properties were rather similar to those of non-crosslinked freeze-dried crystalline NPs with emission maxima at 656 nm and a remarkable enhancement of the fluorescence quantum yield that reached 36% in all conditions (Figure 3D and Table S3).

Figure 3. A. TEM pictures of PVA/F68-stabilized FONs dispersions at different PVA/F68 ratios after nanoprecipitation (top pictures) and after FD (bottom pictures); black arrows point out the presence of single FONs or FONs embedded in larger crystalline polymer clusters; B. Influence of PVA/F68 ratios on the average size of FONs after nanoprecipitation and after FD; C. DLS of the aqueous FONs dispersions prepared at 25% wt PVA (1 mg/mL). D. Fluorescence emission spectra of amorphous and nanocrystalline FONs obtained after FD (1 mg/mL in water). ACS Paragon Plus Environment

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C. Applicability and extension to the other probes To emphasize the general applicability of the proposed methodology, we further intended to construct colloidally stable nanocrystalline FONs from fluorophores F2-F4. F2 displays comparable crystallization aptitude and log P value as F1 (0.78 vs 0.74 for F1). The solubility curve was crossed for F2 between 40 and 70 wt% acetone vs 60 to 90 wt% for F1 (see Figure S24B). Fluorophore aggregates with dz

(DLS)=62

nm and dz (TEM)=52 nm were observed in the SFME

domain. The resulting dispersion weakly emitted fluorescence, suggesting that the F2-based aggregates formed in this domain of composition are not as swollen by acetone (and water) when compared to F1. The binodal curve was reached at similar fluorophore log (mass fraction) as F1, whereas the particle diameters are slightly larger on the full scale (70 to 120 nm) (Figure S24). A bathochromic shift, from 553 nm to 602 nm due to solvatochromism, was observed at 70 wt% water, followed by a slight ipsochromic shift at higher water fraction (from 602 to 598 nm, Figure S24B). Colloidal stability of F2 FONs was relatively poor, with a jump of the diameter from 100 to 500 nm within hours, unless diluting with water and removing acetone (Figure S25B). At 10-5 mass fraction of fluorophore, 10-3 mass fraction of surfactant and fw=0.95, amorphous nanoparticles with diameters 110, 100, 190 nm were constructed using respectively PVP, F68 and PVA as surfactants (Figure S26). After freeze-drying and cross-linking of PVA/F68 stabilized FONs (PVA 25%), F2-based FONs (dz=169 nm, PDI=0.13) showed an emission maxima at 607 nm and a fluorescence quantum yield of 16% (Figure S2 and Table S5). F3 is more prone to crystallization than F1 and F2. Here we only determined the binodal curve, located roughly at the same position as for the two other probes (Figure S28). Whereas the acetone solution of F3 emitted fluorescence, the photoluminescence totally disappeared in the SFME domain and rose again at ~50 wt% water fraction. Using F-68 and PVA (25%) as surfactants, nanoprecipitation of F3 and subsequent freeze-drying afforded uniform nanocrystalline probes of 170 nm in diameter (PDI=0.11), with a PL maxima at 655 nm and a quantum yield ~ 20% (Figure S29 and Table S5). ACS Paragon Plus Environment

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Finally, we selected F4 from a pool of red-emitting dyes displaying AIE properties. F4 rapidly crystallized in acetone/water solutions, making the generation of uniform and colloidally stable nanometer-scale F4-based FON dispersions particularly challenging through nanoprecipitation (See Figure S30). This is clearly illustrated by the rapid growth of bare FON’s dimensions after solvent shifting in the Ouzo domain (Figure S30C) and eventually the precipitation of the resulting structures. Accordingly, the fluorescence jumped up to fw = 0.80 and severely decreased at higher water content (Figure S30). In spite of these obstacles, the nanoprecipitation/ freeze-drying procedure enabled the preparation of water-dispersible F4-based nanocrystalline FONs with z-average diameters around 150 nm and a polydispersity index of ~ 0.13 (PVA 50%, see Figure S31 and Table S6), with a PL maxima at 584 nm and a quantum yield ~ 17% (Figure S31 and Table S6). Note that in contrast to F1-F3, amorphous shell-crosslinked F4-based FONs suffer from a lack of stability, whereas crystalline analogues can be stored as powders for several months and effortlessly re-dispersed in water without variation of the initial colloidal properties (not shown). We finally investigated the scalability of the nanoprecipitation/freeze-drying process. To do so, we intended to upgrade this method to gram-scale production with F1 (see protocol in Supporting Information and Figure S32). Resulting solid-state FON dispersions showed similar dimensions (zaverage diameter equal to 105 nm and dv/dn = 1.20), colloidal stability (facile re-dispersion in water) and optical properties as the materials described vide supra.

D. Towards biological applications 1. In-Vivo imaging with F1-FONs As a prerequisite to in vivo imaging applications, we first confirmed that shell-cross-linked amorphous or nanocrystalline FONs (0.1-4 mg/mL) do not elicit any noticeable cytotoxic effect on CACO-2 epithelial cells after 24h of incubation (see Figure S33). Then, we evaluated the potential of the functional nanocrystalline FON dispersions for imaging purposes.66-68 Aqueous dispersions of resuspended FON (0.2 mL at 45µg of F1 per mL, normal saline, 0.9%NaCl) were administered ACS Paragon Plus Environment

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intravenously (i.v.) via the caudal vein to healthy mice (n = 2) as well as mice bearing subcutaneous human gliomas (U87MG, n = 2) in the ear. Healthy mice were followed for 1 month after i.v. injection and showed no external signs of FON toxicity, such as weight and physical activity losses or dehydration. Two-photon images were acquired on mice of both groups using an excitation wavelength of 900 nm and the fluorescence was epi-collected using emission band pass filters of 630 ± 40 nm for the red channel and 542 ± 50nm for the green channel, see Figure 4. The FONs biodistribution in the main clearance organs, i.e. kidney, liver and spleen (see Figure S34), were analyzed for the mice bearing tumors. FITC-dextrans (70 kDa) were injected i.v. ten minutes before sacrificing the mice in order to delineate the vascular compartment (see green fluorescence signals in Figures 4A and B). This further permitted a clear distinction between intra- and extra-vascular FON distributions, because the red fluorescence signals of the FONs that were present in the blood became yellow after mixing with the green fluorescence signals of FITC-dextrans in the blood plasma, whereas the extracellular FONs maintained a red fluorescence signal. All clearance organs contributed to the evacuation of the FONs (see Figure S34) which resulted in short half-life times that varied between 485 and 572 s in the blood plasma. In the solid tumor, see Figure 4C, FONs accumulated in macrophages and/or mast cells at sites of peritumoral inflammation as well as in the tumor cells (small arrows in Fig 4B).

C

Figure 4. A. Two photon image of the normal vasculature (green fluorescence signal of FITC-dextran 70 kDa) and FONs distribution (red fluorescence signal) in a mouse ear, 2h30 after i.v. injection. The large red dots are endogenous fluorescence signals of sebaceous glands in the skin. The small red dots are due to FONs after phagocytosis by macrophages and/or mast cells. B. Two photon image of the ACS Paragon Plus Environment

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tumor vasculature (green signal) and FONs distribution (red fluorescence signal) in a mouse ear 2h30 after i.v. injection. Parts of the tumor vasculature are leaky and show a local uptake of the FITC-dextran (large arrows) or enhanced permeability retention. The red FONs accumulated inside the tumor, see small red dots (small arrows), and in macrophages at sites of peritumoral inflammation. The white dotted line delineates the tumor area. C. 3D two-photon image of the tumor showing the important uptake of the FONs in macrophages at sites of peritumoral inflammation.

2. Engineering of the F1-FONs As previously reported, the numerous hydroxyl groups (located herein on PVA-rich FONs shell) can be conveniently used as handles to decorate the periphery of the nanoparticles with bio-relevant moieties (see Figure 5).


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Figure 5. A. Route to nanocrystalline FONs functionalization. B. DLS of biotin-functionalized PVA/F68-stabilized F1-based FONs (PVA 25%) in water (1mg/mL) and avidin/biotin-functionalized PVA/F68-stabilized F1-based FONs aggregates. C. Dispersion of magnetic NPs functionalized PVA/F68-stabilized F1-based FONs. Functionalization of the FONs can indeed be achieved in the course of the nanoprecipitation through incorporation of the co-reactants in the aqueous phase before solvent shifting. To illustrate this point, amino-α-functionalized biotin, PEG chains (Mn = 2000 g.mol-1) or magnetic metal nanoparticles have been conveniently anchored on the shell of the F1-based FONs to confer additional properties to the probes (targeting, stealthness, magneto-responsiveness…). Decoration of the nanoparticle shells with amino-functionalized derivatives had no noticeable impact on the dimensions of the FONs and their dispersibility in aqueous medium (See experimental part and Fig S35-S37 in Supporting Information).

Conclusion In summary, we have reported a simple methodology to design bright nanocrystalline FONs from a panel of fluorophores using a nanoprecipitation/freeze-drying process. This method relies on the opening preparation of sub-200 nm colloidally stable amorphous FONs through solvent shifting procedure and followed by crystallization of the solute in a nano-confined environment upon freezedrying. We showed that the construction of tiny amorphous nanoparticle precursors is easily achieved through manipulation in the Ouzo domain (at high supersaturation of the solute), by stabilizing the nanoparticles through cross-linking of the surfactant located within the FON’s shell. We then demonstrated that crystallization aptitudes of the resulting FONs can be precisely regulated by playing with the nature and the relative proportions of the surfactants. Incorporation of plasticizing surfactants such as F68 or PVME, together with PVA to build a robust cross-linked shell, promotes a nano-confined amorphous-crystalline transition within FONs dispersions resulting in the formation of stable nanocrystals with similar dimensions but significantly higher brightness than their amorphous ACS Paragon Plus Environment

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precursors. Thanks to these features, aqueous FONs dispersions were efficiently applied for in vivo imaging of the normal - and tumor vasculature, as well as, tumor cells, macrophages and mast-cells in the tumor and at the site of peritumoral inflammation. Taken together, the universal character of the method, the facile control of the amorphous-crystalline transition, the versatile and straightforward access to surface functionalization, the enhanced brightness of nanocrystals dispersions, their suitability for long-term storage in the solid state and the convenient scalability of the process render the described process of great interest for bioimaging applications.

Methods Reactants. Pluronic F-68 (F-68, Poloxamer 188), poly(vinyl alcohol) (PVA, MW ~130,000), polyvinylpyrrolidone (PVP, MW ~30,000), poly (methyl vinyl ether) solution (50 wt% in water), methoxypolyethylene glycol amine (MW ~2000), isophorone diisocyanate (IPDI, 98%), water (HPLC grade) were purchased from Sigma Aldrich and used without further purification. Acetone (99.5%) was purchased from Carlo Erba. Amino-functionalized biotin was obtained from protonated biotin ethylenediamine hydrobromide (Sigma Aldrich, 95%) by adding 1.5 eq. of triethylamine (Sigma Aldrich, 99%). Turbo beads PEG amine was purchased from TurboBeads. Unless otherwise stating, other reactants were purchased from Sigma-Aldrich. In vitro cells. CACO-2 epithelial cells (Homo sapiens, Colorectal adenocarcinoma) were obtained from ATCC (ref HTB-37, LC-GC, Molsheim, France) and grown in 96-well plates in modified Eagle’s medium (MEM) (GlutaMAX, Invitrogen), supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 1% (v/v) nonessential amino acids and 10% (v/v) heat inactivated (30 min, 56°C) fetal bovine serum. Cultures were maintained at 37°C in a water-saturated atmosphere containing 5% (v/v) CO2. Animal model. Human glioma cells (U87MG GFP+, ATCC cell line, Teddington Middlesex, UK) were injected subcutaneously at a concentration of 108 cells/ml in the left and right ears of nude mice ACS Paragon Plus Environment

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(BALB/c, Charles River, Écully, France). The total injected volume was 20 µL: 10 µL cell suspension and 10 µL matrigel® (with growth factors, BD Bioscience, Europe, Erembodegem, Belgium). Mice were used for two-photon experiments at approximately 2–3 weeks after glioma cell injection. In accordance with the policy of Clinatec (permit number: B38-185 10 003) and the French legislation, experiments were done in compliance with the European Parliament and the Council Directive of September 22, 2010 (2010/63/EU) on the protection of animals used for scientific purposes. The research involving animals was authorized by the Direction Départementale des Services Vétérinaires de l’Isère – Ministère de l’Agriculture et de la Pêche, and the Ministère de l’Enseignement Supérieur et de la Recherche, France permit number: 2015051914157522_v1 (PI: Boudewijn van der Sanden, PhD, permit number 38 09 40 for animal experiences). All efforts were made to minimize the number of mice used and their suffering during the experimental procedure. Nude mice were housed in ventilated cages with food and water ad libitum in a 12 h light/dark cycle at 22 ± 1°C. Determination of the solubility curve. This limit corresponds to acetone/water compositions for which fluorophores are not soluble anymore. Mixing acetone solutions of fluorophores with water at different mass fractions, at a certain composition, big aggregates or crystals started being observed in the systems after overnight. This composition was identified as the solubility limit. Determination of the binodal curve. This equilibrium limit corresponds to acetone/water compositions in which biphasic emulsion start generating. The method consists in titrating acetonic solutions of fluorophores with water until the mixtures turn instantaneously milky or even phase separate, through the generation of droplets. Nanoprecipitation and Freeze-drying procedures. Typically, the fluorophore was dissolved into acetone (mass fraction of fluorophore: 2.10-4), whereas surfactants (F-68 and/or PVA) were fully dissolved into water separately (mass fraction: 10-3). Then, the aqueous solution (950 mg) was poured into the acetone solution (50 mg) all at once, final mass fraction of fluorophore was fixed at 10-5 and acetone mass fraction was 0.05. To freeze the growth of nanoparticles, additional water (2×950 mg) was ACS Paragon Plus Environment

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immediately added after the solvent shifting and acetone was then removed by evaporation. The aqueous dispersion was finally freeze-dried to obtain 1 mg of FON powder. Cross-linking. To prepare cross-linked nanoparticles, IPDI was dissolved into acetone phase prior solvent shifting, and final mass fraction of IPDI was fixed at 10 wt% of PVA. After 2 hours of reaction at room temperature, the dispersion was freeze-dried. The FONs solid dispersion was re-dispersed in water and further characterized by DLS and TEM. Scale up experiment. Fluorophore and IPDI were pre-dissolved in the acetone phase (50 g). The acetone solution was then poured into the aqueous solution (950 g) of PVA (25%) and F-68 (75%) and gently stirred. Final mass fraction of fluorophore, IPDI and polymers were 10-5, 10-4 and 10-3. Acetone mass fraction was 0.05. Right after nanoprecipitation, 2 liters of pure water were poured into the system. After removal of acetone by rotary evaporator, the solution was kept at room temperature for 2h. 1g of solid FON dispersion was finally obtained after freeze-drying. Cytotoxicity of the FONs. Cytotoxicity of FONs on CACO-2 cells was evaluated using Alarmar blue (AbD Serotec (Oxford, UK) according to the manufacturer’s recommendations. Briefly, sub-confluent CACO-2 cells were incubated for 24h with suspensions of FONs (0.1-4.0 mg/mL, suspended in culture medium). Medium containing FONs was discarded and cells were further incubated 4 hours with 5% (v/v) of Alamar blue. The optical density of each well was determined at wavelength at 570 nm and 600 nm on a microplate reader (Thermoscientific, France). Data were expressed as percent of the untreated controls (mean ± SD) and compared using one-way ANOVA. Differences were considered significant at the P

General and Scalable Approach to Bright, Stable and Functional AIE

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