Océane Florès

Jacques Pliquett

Laura Abad-Galan

Robin Lescure

Franck Denat

Olivier Maury

Agnès Pallier

Pierre-Simon Bellaye

Bertrand Collin

Sandra Même

Célia S Bonnet

Ewen Bodio

Christine Goze

Aza-BODIPY Platform: Towards an Efficient Water-Soluble Bimodal Imaging Probe for MRI and Near-Infrared Fluorescence

In this study, an original aza-BODIPY system comprising two Gd 3+ complexes has been designed and synthesized for magnetic resonance imaging/optical imaging application, by functionalization of the boron center. This strategy enabled to obtain a positively-charged bimodal probe, which displays an increased water-solubility, optimized photophysical properties in the near-infrared region, and very promising relaxometric properties. The absorption and emission wavelengths are 705 and 741 nm respectively, with a quantum yield of around 10 % in aqueous media. Moreover, the system does not produce singlet oxygen upon excitation, which would be toxic for tissues. The relaxivity obtained is high at intermediate fields (16.1 mM -1 .s -1 at 20 MHz and 310 K) and competes with that of bigger or more rigid systems. A full relaxometric and 17 O NMR study and fitting of the data using the Lipari-Szabo approach showed that this high relaxivity can be explained by the size of the system and the presence of some small aggregates. These optimized photophysical and relaxometric properties highlight the potential use of such systems for future bimodal imaging studies.

Introduction

Imaging and especially molecular imaging has gained an increasing interest during these last years. Indeed, the insights in this field facilitate an earlier and more reliable diagnostic, enable the monitoring of the efficiency of a therapy, and even assist the surgeon during operations. [START_REF] Barabino | Intraoperative Near-Infrared Fluorescence Imaging Using Indocyanine Green in Colorectal Carcinomatosis Surgery: Proof of Concept[END_REF][START_REF] Frangioni | In Vivo Near-Infrared Fluorescence Imaging[END_REF][START_REF] Kiyose | Molecular Design Strategies for Near-Infrared Ratiometric Fluorescent Probes Based on the Unique Spectral Properties of Aminocyanines[END_REF][START_REF] Yuan | Far-Red to near Infrared Analyte-Responsive Fluorescent Probes Based on Organic Fluorophore Platforms for Fluorescence Imaging[END_REF][START_REF] Zhu | Fluorescent Probes for Sensing and Imaging within Specific Cellular Organelles[END_REF][START_REF] Guo | Recent Progress in the Development of Near-Infrared Fluorescent Probes for Bioimaging Applications[END_REF] Therefore, molecular imaging can benefit to a large number of pathologies. Moreover, this imaging approach also permits the discovery of novel biological targets or biomarkers and help to understand mechanisms of different diseases. Each molecular imaging technique has its own advantages and drawbacks and synergy is expected by association of two complementary modalities in a single molecular probe. [START_REF] Zhao | Recent Developments in Multimodality Fluorescence Imaging Probes[END_REF] This is particularly true when combining the two non-ionizing techniques: Magnetic Resonance Imaging (MRI) with Optical Imaging (OI). Indeed, MRI has an excellent spatial resolution and offers 3D anatomical and also functional information, which is ideal for in vivo imaging, but it suffers from low sensitivity. On the other hand, OI exhibits high sensitivity and high microscopic resolution (<10 nm in the case of super resolved fluorescent microscopy), but suffers from poor tissue penetration. [START_REF] James | A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications[END_REF] Another advantage of OI is that it enables real time imaging. Moreover, more recently, despite its limited penetration depth, it has gained an increasing interest in the clinic for intra-operative fluorescence assisted surgery, where the use of fluorescence imaging particularly helps the surgeon to better delineate tumor margins, identify the lymph nodes invaded by cancer cells… [START_REF] Hernot | Latest Developments in Molecular Tracers for Fluorescence Image-Guided Cancer Surgery[END_REF] In order to perform bimodal MRI/OI, there is an increasing interest in the conception of new efficient multimodal probes. Indeed, combination of these two imaging modalities into a single molecule assures the identical biodistribution compared to the combination of two separate imaging agents, and thus enables to perform a much more accurate diagnosis. The design of such bimodal probes is a real challenge, mainly because of the difference of sensitivity between MRI and OI. Therefore, the majority of the reported MRI/OI agents are based on functionalized polymers or nanoparticles. However small molecular bimodal agents were also reported and proved to be very effective. These agents combine fluorine atoms for [START_REF] Wang | Brain Imaging with Near-Infrared Fluorophores[END_REF] F MRI or one, two, or more complexes, which are active in MRI, such as Mn 2+ or Gd 3+ complexes, and one fluorophore, which can be a dipyridophenazine, fluorescein, cyanine, rhodamine, quinoline, BODIPY... [START_REF] Wang | Visualization of Fluoride Ions In Vivo Using a Gadolinium(III)-Coumarin Complex-Based Fluorescence/MRI Dual-Modal Probe[END_REF][START_REF] Wang | A Gadolinium(III) Complex Based Dual-Modal Probe for MRI and Fluorescence Sensing of Fluoride Ions in Aqueous Medium and in Vivo[END_REF][START_REF] Harrison | Multimeric Near IR-MR Contrast Agent for Multimodal In Vivo Imaging[END_REF][START_REF] Ceulemans | Gadolinium(III)-DOTA Complex Functionalized with BODIPY as a Potential Bimodal Contrast Agent for MRI and Optical Imaging[END_REF][START_REF] Dong | Gadolinium(III)-Fluorescein Complex as a Dual Modal Probe for MRI and Fluorescence Zinc Sensing[END_REF][START_REF] Yamane | Method for Enhancing Cell Penetration of Gd3+-Based MRI Contrast Agents by Conjugation with Hydrophobic Fluorescent Dyes[END_REF][START_REF] You | Manganese Displacement from Zinpyr-1 Allows Zinc Detection by Fluorescence Microscopy and Magnetic Resonance Imaging[END_REF][START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF][START_REF] Stasiuk | Dual-Modal Magnetic Resonance/Fluorescent Zinc Probes for Pancreatic β-Cell Mass Imaging[END_REF] For in vivo optical imaging applications, the fluorophores need to absorb and emit in the spectral range, where biological tissues are more transparent and less scattering. This region, often called biological, transparency or theranostic window, corresponds to near infrared (NIR, as example NIR I corresponds to 650-900 nm). [START_REF] Wang | Brain Imaging with Near-Infrared Fluorophores[END_REF][START_REF] Hong | Near-Infrared Fluorophores for Biomedical Imaging[END_REF][START_REF] Hilderbrand | Near-Infrared Fluorescence: Application to in Vivo Molecular Imaging[END_REF] Development of NIR fluorophores is a hot topic because, despite the diversity of reported fluorophores, it seems that none of them is ideal for in vivo applications, and that most of them lack of chemical and photochemical stability, are poorly water soluble, low emissive, difficult to functionalize and their synthesis is time-consuming. [START_REF] Li | Recent Progress in Small-Molecule Near-IR Probes for Bioimaging[END_REF][START_REF] Martinić | Near-Infrared Emitting Probes for Biological Imaging: Organic Fluorophores, Quantum Dots, Fluorescent Proteins, Lanthanide(III) Complexes and Nanomaterials[END_REF][START_REF] Haque | Next Generation NIR Fluorophores for Tumor Imaging and Fluorescence-Guided Surgery: A Review[END_REF][START_REF] Garland | A Bright Future for Precision Medicine: Advances in Fluorescent Chemical Probe Design and Their Clinical Application[END_REF] Very recently, the aza-BODIPY family attracted considerable attention because it may be one of the best candidates to tackle all these issues. These very stable BODIPY derivatives display a lower brightness than wellknown NIR cyanines such as ICG and IRDye® 800CW (Figure 1), but can be rapidly synthesized in large scale and are easily functionalized. [START_REF] Ge | Azadipyrromethenes: From Traditional Dye Chemistry to Leading Edge Applications[END_REF] Figure 1: structure of some optical imaging clinically used contrast agents and of Wazaby (watersoluble aza-BODIPY), we reported previously [START_REF] Pliquett | A Promising Family of Fluorescent Water-Soluble Aza-BODIPY Dyes for in Vivo Molecular Imaging[END_REF] However, despite their potential, there are only very scarce reports of bimodal MRI/OI probes including (aza)-BODIPY. They have been used in combination with several fluorine atoms for [START_REF] Wang | Brain Imaging with Near-Infrared Fluorophores[END_REF] F MRI, [START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF] or with two GdDTPA complexes, [START_REF] Kim | In Vivo Tracking of Phagocytic Immune Cells Using a Dual Imaging Probe with Gadolinium-Enhanced MRI and Near-Infrared Fluorescence[END_REF] or GdDOTA complexes in a monomolecular system [START_REF] Ceulemans | Gadolinium(III)-DOTA Complex Functionalized with BODIPY as a Potential Bimodal Contrast Agent for MRI and Optical Imaging[END_REF] or in a polyrotaxane [START_REF] Fredy | Cyclodextrin Polyrotaxanes as a Highly Modular Platform for the Development of Imaging Agents[END_REF] . The sensitivity of [START_REF] Wang | Brain Imaging with Near-Infrared Fluorophores[END_REF] F MRI remains lower than that of 1 H MRI. GdDTPA complexes are not the best candidates for in vivo use (vide infra) and the relaxivity of the BODIPY-GdDOTA system (vide infra) can certainly be optimized. This leaves room for improvement of such systems. It should also be noted that a bisGdDTPA-aza-BODIPY system was recently reported by Kim, Long and coll. to track in vivo phagocytic immune cells by MRI/OI bimodal imaging, highlighting the potential of such systems. [START_REF] Kim | In Vivo Tracking of Phagocytic Immune Cells Using a Dual Imaging Probe with Gadolinium-Enhanced MRI and Near-Infrared Fluorescence[END_REF] Despite their interesting properties, the limited use of BODIPY in bimodal imaging can certainly be explained by their poor water solubility, which is problematic for their use in vivo, and particularly for MRI. [START_REF] Wu | PEGylated BF2-Azadipyrromethene (NIR-AZA) Fluorophores, for Intraoperative Imaging[END_REF][START_REF] Yang | A Benzoxazole Functionalized Fluorescent Probe for Selective Fe 3+ Detection and Intracellular Imaging in Living Cells[END_REF][START_REF] Bodio | BODIPYS and Aza-BODIPYS Derivatives as Promising Fluorophores for in Vivo Molecular Imaging and Theranostic Applications[END_REF] Several groups tethered water-solubilizing groups to the aza-BODIPY core, but even if sometimes it enabled in vitro and in vivo investigations, the resulting probes were still subject to significant aggregation phenomena (requiring the addition of surfactant). [START_REF] Wu | PEGylated BF2-Azadipyrromethene (NIR-AZA) Fluorophores, for Intraoperative Imaging[END_REF][START_REF] Daly | BF 2 -Azadipyrromethene NIR-Emissive Fluorophores with Research and Clinical Potential[END_REF][START_REF] Grossi | Lysosome Triggered Near-Infrared Fluorescence Imaging of Cellular Trafficking Processes in Real Time[END_REF] Very recently, some of us succeeded in designing fully water-soluble aza-BODIPY derivatives, called Wazaby (for watersoluble aza-BODIPY), thanks to the introduction of ammonium groups via boron functionalization (functionalizing boron induced aza-BODIPY faces hindrance, which prevented aggregation) (Figure 1). [START_REF] Pliquett | A Promising Family of Fluorescent Water-Soluble Aza-BODIPY Dyes for in Vivo Molecular Imaging[END_REF] These probes were bioconjugated onto antibodies, were very stable in physiological media, and presented better in vivo optical properties than the Cy5, used as a control.

Building on this success, we decided to further investigate the potential of these Wazaby dyes and to conceive MRI/OI MOnomolecular Multimodal Imaging Probes (MOMIP). For this aim, we decided to attach two gadolinium complexes onto the boron-functionalized arms of the Wazaby scaffold, thanks to the strategy developed previously (Figure 2). Concerning the aza-BODIPY core, we chose an aza-BODIPY bearing methoxy groups on the aromatic rings at the "south" of the molecule (also called proximal phenyl groups in aza-BODIPY chemistry) in order to shift the maximal absorption wavelength around 690-700 nm. To maximize this effect (maximal absorption wavelength >700 nm) by creating a greater "push-pull" effect, bromine atoms were introduced on the aromatic rings at the "north" of the molecule (distal phenyl groups). It has to be noted that this functional group would enable further functionalization of the molecule in the future (extension of conjugation or introduction of a bioconjugatable handle if vectorization is needed). In MRI, DOTAREM® is one of the most clinically used contrast agents due to its high thermodynamic stability and kinetic inertness, which is essential for the safe use of the probe. [START_REF] Brücher | Stability and Toxicity of Contrast Agents[END_REF] Indeed, free gadolinium ions are highly toxic, and recently, complexes with lower kinetic inertness have been shown to increase the risk of nephrogenic systemic fibrosis (NSF), especially for patients having renal failure. [START_REF] Wahsner | Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers[END_REF] As macrocyclic chelates are known to be more stable than linear ones, a DOTA derivative (DOTA monoamide or DOTAMA) was used for Gd 3+ complexation.

Results and Discussion Synthesis

Aza-BODIPY 2 was synthesized at the gram scale according to the method we previously reported, [START_REF] Pliquett | A Promising Family of Fluorescent Water-Soluble Aza-BODIPY Dyes for in Vivo Molecular Imaging[END_REF] starting by the synthesis of the aza-BODIPY 1, [START_REF] Loudet | Functionalized BF2 Chelated Azadipyrromethene Dyes[END_REF] followed by the substitution of the fluorine atoms on the boron center using the Grignard reagent of N,N-dimethylpropargylamine. The resulting intermediate platform was then alkylated by an excess of 4-(bromomethyl)benzoic acid in presence of NaHCO 3 (Scheme 1) to give Wazaby5. Special attention was paid to the base employed as proton scavenger. A stronger base led to the formation of small amount of ester side product -alkylation of the carboxylate -, leading to a tricky purification.

Scheme 1: synthetic route toward the bimodal probe Gd 2 -Wazaby6

Wazaby5 was reacted with a macrocyclic derivative (DO3A(tBu) 3 NH 2 ) in classic peptidic coupling reaction conditions and directly treated by HCl 3M in order to hydrolyze the protected tertiobutylester arms. The bis-DOTA derivative was then purified by semi-preparative HPLC to lead to the formation of pure Wazaby6. It is worth noting that the fluxionality of macrocycles implied to record NMR spectra at high temperature. A representation of variable temperature 1 H-NMR of Wazaby6 is displayed in Figure S2.

Gadolinium was introduced into the two macrocycle cavities by reacting 2.4 eq of Gd(CF 3 SO 3 ) 3 salt with Wazaby6 in water in presence of sodium hydroxyde. The resulting homobimetallic complex Gd 2 -Wazaby6 was characterized by high resolution mass spectrometry (HRMS), HPLC, elemental analysis and ionic chromatography but not NMR due to its paramagnetic character.

Photophysical studies

As highlighted in the introduction, aza-BODIPY fluorophores display very interesting properties for in vivo optical imaging. However, in general, their main drawback is their poor solubility and their tendency to aggregate in water and in physiological media, despite the introduction of solubilizing groups. Most of the time, imaging experiments are performed in the presence of surfactants like Cremophor EL or castor oil. [START_REF] Jing | Visualization of Nitroxyl (HNO) in Vivo via a Lysosome-Targetable near-Infrared Fluorescent Probe[END_REF][START_REF] Bhuniya | A Fluorescence off-on Reporter for Real Time Monitoring of Gemcitabine Delivery to the Cancer Cells[END_REF][START_REF] Kamkaew | Aza-BODIPY Dyes with Enhanced Hydrophilicity[END_REF][START_REF] Daly | BF2-Azadipyrromethene NIR-Emissive Fluorophores with Research and Clinical Potential[END_REF] Therefore, it was crucial for us to investigate the photophysical properties of our bimodal compounds in organic solvent (DMF, DMSO), as well as in PBS (Phosphate-Buffered Saline), without adding any surfactant. * reference: aza-BODIPY 3 (φ F = 36% in chloroform, 298 K, λ exc = 670 nm) [START_REF] Brouwer | Standards for Photoluminescence Quantum Yield Measurements in Solution (IUPAC Technical Report)[END_REF] The photophysical data of Wazaby5, Wazaby6, and the related bimetallic complex Gd 2 -Wazaby6 were studied in DMSO, DMF, and PBS, and results are depicted in Table 1, Figure 3, and Table S2. The three compounds display comparable absorption and emission profiles, with absorption wavelength at ≈705 nm and emission maxima at ≈740 nm. Interestingly, the introduction of the two macrocycles as well as the complexation of the two gadolinium atoms had almost no impact on the photophysical properties of the fluorophore.

When comparing Wazaby6 and Gd 2 -Wazaby6 in DMSO and PBS, we can notice that the absorption, emission and excitation profiles of the compounds are comparable in both media (Figure 3), even if we can observe that absorption band is slightly larger than excitation one between 600 and 650 nm in PBS. It suggests that very limited aggregation occurs in PBS. If these spectra are compared to the ones of Kim's and Long's system, [START_REF] Kim | In Vivo Tracking of Phagocytic Immune Cells Using a Dual Imaging Probe with Gadolinium-Enhanced MRI and Near-Infrared Fluorescence[END_REF] it clearly highlights the interest of boron-functionalization for limiting aggregation phenomenon, which is really frequent for aza-BODIPY fluorophores. Interestingly, the quantum yield efficiency remains comparable both in organic solvents (around 20%) and in PBS (around 10%). Such values are good for NIR emitters and are clearly enough to undertake future OI experiments.

It is well known that the presence of "heavy atoms" induces an increase of the spin-orbit coupling and therefore promotes the generation of singlet oxygen. [START_REF] Bodio | BODIPYS and Aza-BODIPYS Derivatives as Promising Fluorophores for in Vivo Molecular Imaging and Theranostic Applications[END_REF][START_REF] Karatay | The Effect of Heavy Atom to Two Photon Absorption Properties and Intersystem Crossing Mechanism in Aza-Boron-Dipyrromethene Compounds[END_REF] However, this effect is generally more substantial when the heavy atoms occupy a position in the core region of the aza-BODIPY. In the present study, a non-toxic probe is under scope, thus, the production of singlet oxygen needs to be controlled. In order to test if toxic singlet oxygen is generated, the probes were dissolved in a nonprotic solvent (DMF) for feasible observation of the phosphorescence band of singlet oxygen at 1270 nm, as protic solvents will efficiently quench its emission. 45 In every case this band was found to be absent, which confirms that neither Wazaby6 nor Gd 2 -Wazaby6 produce singlet oxygen, when irradiated (see supporting information for details). Moreover, preliminary antiproliferative tests on HepG2 cells (Human hepatocellular carcinoma, ATCC ® HB-8065 ™ ) confirmed that Gd 2 -Wazaby6 do not display significant toxicity (IC 50 at 24h > 100 µM). Additional experiments will be performed in the future to ascertain this aspect.

Wazaby6

Relaxivity studies

Given the good solubility of the system, relaxivity measurements could be undertaken to characterize the efficiency of the system as an MRI probe. To this aim, NMRD (Nuclear Magnetic Resonance Dispersion) profiles were measured in water at 0.64 mM at pH = 7.2 in the field range 40 kHz -400 MHz at two different temperatures (298 K and 310 K) as illustrated in Figure 4. Interestingly, the temperature dependence of the system shows that we are in an intermediate regime where the rotation of the system starts to limit relaxivity rather than the water exchange rate. Indeed, the 37°C relaxivities are very close and even higher than those at 25°C. Typically, when the rotation is the limiting parameter (as observed for small molecular systems), relaxivity is decreasing when increasing temperature, whereas when the water exchange rate is limiting, relaxivity is increasing with increasing temperature. This result is also in accordance with the characteristic hump of slowly-rotating species visible on the NMRD profiles at intermediate fields (20-80 MHz). As this system remains quite flexible, we decided to investigate the relaxivity as a function of the Gd 3+ concentration. Relaxivities were measured in the Gd 3+ concentration range 0.1 -4 mM (0.05 -2 mM for the system) both in water at pH = 7.2 and in PBS at pH = 7.4, and the results are presented in Figure 5. At the concentration used for measuring relaxivities (0.64 mM of Gd 2 -Wazaby6), we can see that some aggregation occurs both in water and PBS probably due to interactions between the aza-BODIPY units. This is in accordance with the photophysical studies, highlighting small aggregation, but which doesn't prevent water solubility. The aggregation is favored in PBS, which can be explained by additional electrostatic interactions between the Gd 2 -Wazaby6 cation (positive charge) and PBS (minus charge). This phenomenon certainly explains the temperature dependency of this otherwise quite flexible system. The relaxivity of the system at 0.64 mM in water and at 20 MHz, 310 K is 16.1 mM -1 .s -1 . As a comparison, and as expected due to the aggregation phenomenon, it is considerably higher than that of the mononuclear GdDOTA-BODIPY complex (3.9 mM -1 .s -1 ) [START_REF] Ceulemans | Gadolinium(III)-DOTA Complex Functionalized with BODIPY as a Potential Bimodal Contrast Agent for MRI and Optical Imaging[END_REF] and the relaxivity of the Bis-GdDTPAaza-BODIPY system is 13.3 mM -1 .s -1 at 60 MHz and room temperature [START_REF] Kim | In Vivo Tracking of Phagocytic Immune Cells Using a Dual Imaging Probe with Gadolinium-Enhanced MRI and Near-Infrared Fluorescence[END_REF] (see Figure S10 for structures). The relaxivity is in the same order of magnitude than a multimodal polyrotaxane comprising cyclodextrins bearing GdDOTAMA or BODIPYs (r 1 = 18.60 mM -1 .s -1 ) 29 or a porphyrinic system DPP-ZnP-GdDOTA (see Figure S10; r 1 = 17.9 mM -1 .s -1 ) presenting also some aggregation. [START_REF] Schmitt | A Theranostic Agent Combining a Two-Photon-Absorbing Photosensitizer for Photodynamic Therapy and a Gadolinium(III) Complex for MRI Detection[END_REF] Finally, the relaxivity compares well with those of nanosized objects such as MOFs [START_REF] Carné-Sánchez | Relaxometry Studies of a Highly Stable Nanoscale Metal-Organic Framework Made of Cu(II), Gd(III), and the Macrocyclic DOTP[END_REF] and micelles [START_REF] Nicolle | From Monomers to Micelles: Investigation of the Parameters Influencing Proton Relaxivity[END_REF][START_REF] Martins | Gd3+ Complexes Conjugated to Pittsburgh Compound B: Potential MRI Markers of β-Amyloid Plaques[END_REF] .

In order to gain more insight into the parameters governing relaxivity, [START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF] The reduced [START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF] O transverse relaxation rates first increase (up to ca. 318 K), then decrease with increasing temperature indicating that the complex is in the slow kinetic region at low temperatures and in the fast exchange region at higher temperatures. In the slow kinetic region, 1/T 2r is directly determined by the exchange rate constant k ex , whereas in the fast exchange region, it is determined by the transverse relaxation rate of the coordinated water oxygen, 1/T 2m , which is in turn influenced by the water exchange rate, k ex , the longitudinal electronic relaxation rate, 1/T 1e , and the scalar coupling constant, A/ħ. Due to the different concentrations used for the [START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF] O NMR (4.78 mM of Gd 3+ ) and NMRD samples (1.27 mM of Gd 3+ ) and the aggregation process previously evidenced, the [START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF] O NMR and NMRD data have been analysed separately according to the Solomon-Bloembergen-Morgan theory.

Table 2 : Best-fit parameters obtained from the independent fitting of the 1 H NMRD profiles (Figure 4) to the SBM theory, including the Lipari-Szabo approach to describe internal flexibility and the transverse [START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF] O NMR relaxation rates as a function of temperature (Figure 6) at 9.4 T. The number in brackets represents the error on the last digit.

Parameters Gd 2 -Wazaby6

[DPP-ZnP-GdDOTA] -c GdDOTAC 12 (micellar form) d Zn-LZF2Gd e r 1 (mM [START_REF] Nicolle | From Monomers to Micelles: Investigation of the Parameters Influencing Proton Relaxivity[END_REF] ; e From ref [START_REF] Isaac | MRI and Luminescence Detection of Zn2+ with a Lanthanide Complex-Zinc Finger Peptide Conjugate[END_REF] In the fitting procedure, the number of water molecules directly coordinated to Gd 3+ was fixed to 1 as it is expected for GdDOTAMA complexes. In the fit of the proton relaxivities, the water exchange parameters were fixed to values obtained in the [START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF] O NMR study. The rotational dynamics could be only described by applying the Lipari-Szabo approach. In this approach, that separates local and global motions, characterized by the local and global rotational correlation times, τ l 298 and τ g 298 , respectively, and a model independent order parameter, S 2 . It reflects the degree of spatial restriction of the local motion with respect to the global motion. Its value ranges from 0 to 1, with S 2 = 0 if the internal motions are isotropic, and S 2 = 1 if the internal motions are completely restricted. The fitting has been restricted to frequencies above 6 MHz for NMRD data as at low magnetic fields the SBM theory fails in describing electronic parameters and rotational dynamics of slowly rotating species. Details of the analysis and the equations used in the fit are given in the Supporting Information, and the most important parameters obtained are listed in Table 2. A complete list of the parameters fitted for our system is also given Table S1.

For comparison, three other systems have been presented within the table (Table 2): a porphyrinic system with a GdDOTAGA appended (DPP-ZnP-GdDOTA); a GdDOTAC 12 complex in its micellar form, and a 30 aminoacid peptides derived from a Zn-finger peptide and bearing a GdDOTAMA complex in a folded form (Zn-bound form; Zn-LZF2Gd). They have been chosen because: (1) they have been all analyzed using the Lipari Szabo approach; (2) the porphyrinic system has a similar molecular weight as our system and shows some small aggregation as well;

(3) the GdDOTAC 12 is in its micellar form which could "mimick" an aggregated state; (4) the peptidic system has a similar coordination sphere for Gd 3+ than our system and the relaxivity behavior as a function of temperature is similar (relaxivity limited by rotation rather than water exchange rate).

The fitting of the [START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF] O data yields a value of k ex of 2.81.10 6 s -1 , which is lower than that of GdDOTA (k ex = 4.1.10 6 s -1 ) as expected when a carboxylate function is replaced by an amide function, and in the same range than other GdDOTAMA complexes (ca. 2.10 6 s -1 ). [START_REF]The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging: Helm/The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging[END_REF] It suggests that the aggregation process, which certainly occurs through the BODIPY moiety, does not affect the water exchange rate of the system. The activation entropy calculated is also positive and in the same order of magnitude as that of complexes possessing similar coordination spheres. The water exchange mechanism has not been directly assessed (it is beyond the scope of this study), but the activation entropy calculated gives an indication of a dissociative mechanism as expected for nine-coordinated Gd 3+ complexes. In dissociative mechanism, the steric crowding is of primary importance as the determining step is the dissociation of the water molecule. A highly-crowded environment will favour this dissociative activation step. As carboxylate functions are more coordinating than amide function, they imply more constraints explaining why GdDOTA complexes have higher water exchange rates than GdDOTAMA complexes.

The local and global correlation times of the system are within the same range as those of similar systems giving rise to similar relaxivities as shown in Table 2. The low value of S 2 , comparable to those of DPP-ZnP-GdDOTA or the micellar system, shows that the motion of the Gd 3+ -coordinated water proton vector is largely decoupled from the overall motion of the aggregates. This can be explained by the quite flexible chain between the Aza-BODIPY core and the Gd 3+ complexes. If we look in more detail, the global correlation time of the system correlates well with that of the micellar system, or that of DPP-ZnP-GdDOTA having a similar molecular weight and presenting some aggregation. It is slightly higher than that of the peptidic system with a higher molecular weight but which is not in an aggregated form. All this confirms that some aggregation occurs for our system. The value of the local correlation time, consistent with that of a small Gd 3+ complex, corroborates the flexibility around the Gd 3+ complexes and shows that the aggregation certainly occurs through the aza-BODIPY core.

Finally, the temperature dependence of the relaxivity of our system is related to the low water exchange rate of the system together with a slow motion explained by the size of the complex and the presence of some aggregation. The same behavior is not observed with the porphyrinic system due to the difference in the water exchange rates (explained by the different Gd 3+ coordination spheres). On the contrary, the peptidic system presents the same (even exacerbated) temperature behavior, which is explained by a more rigid system with slightly lower water exchange rate, despite the same Gd 3+ coordination sphere. To sum up, the high relaxivity observed is due to the size of the system and the presence of some small aggregates.

Preliminary imaging studies on phantom and measurements in HSA

Phantom images of the system Gd 2 -Wazaby6 have been recorded both in MRI and in fluorescence to highlight its potential.

T 1 -weighted images were recorded at 7 T, and show a greater contrast compared to the commercial GdDOTA at the same concentration (150 µM) (Figure 7). Fluorescence images were recorded thanks to an optical imager for in vivo experiments (IVIS Lumina III, Perkin Elmer, Figure 8). Gd 2 -Wazaby6 was imaged at 150 µM (the concentration used for MRI imaging), 15 µM, and 1.5 µM in water; Aza-BODIPY 3 was imaged at 150 µM as a control. The first observation that can be made is the strong fluorescence of Gd 2 -Wazaby6 with respect to Aza-BODIPY 3 (3 is not fluorescent due to its insolubility in water). Additionally, it is worth noting that Gd 2 -Wazaby6 displays higher fluorescence at 15 µM than at 150 µM, which corroborates the fact that some aggregation phenomenon can occur at high concentration. Moreover, we were pleased to note that a significant signal can be observed up to 850 nm, especially when exciting the molecule at 700 or 720 nm, meaning that Gd 2 -Wazaby6 can be detected wherever we want in biological window. The relaxivity and the optical emission properties of the system have also been measured in the presence of 0.6 mM of Human Serum Albumin (HSA) (physiological concentration) in order to simulate biological environment. Concerning the relaxivity of Gd 2 -Wazaby6 (at 145 µM), a 14 % increase is observed in water at pH = 7.3, 60 MHz, and 25°C, in the presence of HSA. This increase is explained by the formation of a supramolecular adduct between the probe and HSA as commonly observed for hydrophobic systems. Concerning the optical properties, the emission of Gd2-Wazaby6 (at 2µM) increased by 230% in presence of HSA, probably due to the more lipophilic environment.

The improved properties of Gd2-Wazaby6 for both MRI and OI in presence of HSA are reassuring regarding its efficiency in real biological media.

These different preliminary studies are very promising for the future use of our probe for in vivo bimodal imaging.

Conclusion

For the first time, a monomolecular multimodal imaging probe for MRI and Near-Infrared Fluorescence (Gd 2 -Wazaby6) was synthesized thanks to a boron functionalization of an aza-BODIPY. It was fully characterized by usual spectroscopic techniques. The boron functionalization allows good solubility of the system both in water and PBS and enables full photophysical and relaxometric characterizations. Interestingly, the quantum yield of fluorescence of this bimodal system is around 10% in PBS, which is particularly high for a near infrared fluorophore. Moreover, this probe does not induce the production of singlet oxygen, which could be toxic for biological tissue. The relaxivity observed for Gd 2 -Wazaby6 is high and can be explained both by the size of the system and the presence of some small aggregation process, as also observed in the optical imaging studies. Despite these aggregates the system remains very soluble in water and in biological media, and could be studied without the presence of surfactant as often needed with BODIPY derivatives. Those small aggregates do not imply important fluorescence quenching and enable to achieve high efficiency in MRI (in future work, it will be worth studying whether these small aggregates are still present in vivo). The relaxivity could also be further optimized by rigidifying the system and modifying the coordination sphere of Gd 3+ , which is the subject of future studies. All together this suggests that these bimodal systems hold great promise as MRI and NIR probes in vitro and in vivo imaging.

TOC Graphic

Synthesis and characterization of an original aza-BODIPY system comprising two Gd 3+ complexes for magnetic resonance imaging (MRI)/optical imaging (OI) application, by functionalization of the boron center. This is the first aza-BODIPY based bimodal probe displaying a high brightness in near infrared fluorescence (λ em = 740 nm), an excellent relaxivity for MRI, and displaying good water solubility. The novel bimodal probe possesses all the requirements for in vivo application.

35

 35

Figure 2 :

 2 Figure 2 : Targeted bimodal probe and justification of the chosen functionalization

Figure 3 :

 3 Figure 3: Normalized absorption, emission, and excitation spectra of Wazaby6 and Gd 2 -Wazaby6 at 298 K (λ ex = 620 nm for emission spectra, detection at λ em = 820 nm for excitation spectra)

Figure 4 : 1 H

 41 Figure 4: 1 H NMRD profile of Gd 2 -Wazaby6 0.64 mM in water, pH = 7.2at 298 K () and 310 K ().The lines represent the best fit to the SBM theory.

Figure 5 :

 5 Figure 5: Relaxivity of the complex as a function of Gd 3+ concentration at 298 K, 20 MHz, in water at pH = 7.2 () and in PBS at pH = 7.4 ().

Figure 6 :

 6 Figure 6: Temperature dependence of the reduced 17 O transverse relaxation rates of Gd 2 -Wazaby6 (2.39 mM) at 9.4 T. The continuous curve represents the best fit to the experimental data points.

Figure 7 :

 7 Figure 7: T 1 -weighted MRI phantom images of Gd 2 -Wazaby6 (150 µM), GdDOTA (150 µM) and water (reference) at 7 T using spin-echo sequence with TE = 50 ms and TR = 500 ms.

Figure 8 :

 8 Figure 8: Phantom optical images of of Gd 2 -Wazaby6 (150 µM, 15 µM, and 1.5 µM), Aza-BODIPY 3 (150 µM), and water (reference) excitation at 660 nm and detection at 710 nm).

Table 1 :

 1 photophysical data of Wazaby5, Wazaby6, and Gd 2 -Wazaby6 in different solvents at 298 K at 2 µM (λ exc = 670 nm).

	Compound	Solvent	λ max, Abs [nm]	ε λmax [M -1 cm -1 ]	λ max, Em [nm]	φ F [%]*
	Wazaby5	DMSO	704	74,000	739	29
	Wazaby6	DMSO PBS	705 705	82,000 46,000	741 741	15 10
		DMSO	705	78,000	739	22
	Gd 2 -Wazaby6	PBS	705	41,000	741	9
		water	705	49,000	741	10

  O measurements as a function of temperature have been performed. Variable temperature 17 O T 2 measurements give access to the water exchange rate, k ex . The 17 O T 1 data are determined by dipole-dipole and quadrupolar relaxation mechanisms and provide information about the rotational correlation time, τ R . The 17 O chemical shifts give indication of the number of water molecules directly coordinated to Gd

3+

, q. Longitudinal, transverse

[START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF]

O relaxation rates and chemical shifts were measured as a function of the temperature on aqueous solution of Gd 2 -Wazaby6 at 2.39 mM, and on a diamagnetic reference (HClO 4 , pH 3.3) at 9.4 T. Due to the concentration used for the study,

[START_REF] Huynh | Small BODIPY Probes for Combined Dual 19 F MRI and Fluorescence Imaging[END_REF]

O chemical shifts and T 1 values were too close to those of the reference to yield reliable data. Only 17 O T 2 data were analyzed and are presented in Figure

6

.

  fixed in the fit; b at 60 MHz and 298 K; c From ref 46 ; d From ref

	-1 s -1 ; 20 MHz, 37°C)	16.1	17.9	17.9 b	15.8
	q a	1	1	1	1
	k ex 298 (10 6 s -1 )	2.81 [± 0.08]	4.1	4.9	1.6
	ΔH ≠ (kJ.mol -1 )	40 [± 1]	49.8	52	40
	ΔS ≠ (J.K -1 .mol -1 )	13 [± 3]	-	+ 67	-
	E l (kJ.mol -1 )	7 [± 4]	40	20	20
	τ l 298 (ps)	111 [± 9]	245	430	145
	E g (kJ.mol -1 )	5 ± [1.5]	14	35	13
	τ g 298 (ps)	1840 [± 70]	2640	1600	1160
	S 2	0.30 [± 0.01]	0.26	0.23	0.64

a

Acknowledgments

The Ministère de l'Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique (CNRS), the Conseil Régional de Bourgogne (PhD JCE grant # 2015-9205AAO033S04139 / BG0003203), and the French Research National Agency (ANR) via project JCJC ''SPID" ANR-16-CE07-0020 and project JCJC "WazaBY" ANR-18-CE18-0012 are gratefully acknowledged. This work is part of the projects ''Pharmacoimagerie et agents théranostiques" et ''Chimie durable, environnement et agroalimentaire" supported by the Université de Bourgogne and the Conseil Régional de Bourgogne through the Plan d'Actions Régional pour l'Innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs. It was performed within the Dijon's pharmaco-imaging consortium, a regional center of excellence in pharmacoimaging. This work was also supported by a French Government Grant managed by the French National Research Agency (ANR) under the program "Investissements d'Avenir" (reference ANR-10-EQPX-05-01/IMAPPI Equipex). Chematech® society is warmly thanked for providing DO3A-(tBu) 3 -NH 2 macrocycle. FrenchBIC, GDR AIM, and OncoDesign® are acknowledged for fruitful discussion. LAG acknowledged the French Research National Agency (ANR) for her grant (SADAM ANR-16-CE07-0015-02). Mr Soustelle, and Ms M.-J. Penouilh are gratefully acknowledged for HR-MS, NMR analyses, and Dr Myriam Laly for ionic chromatography.

Supporting Information Available

This section gathered all experimental details: synthetic procedures, analytical data ( 1 H, 13 C, 11 B, 19 F NMR, HRMS), 1 H-NMR spectra, analytical and semi-preparative HPLC conditions and chromatograms, ionic chromatography, protocol for determining photophysical properties, absorption, emission and excitation spectra, photophysical experiments with or without HSA and on phantoms, structures of the systems discussed in the article, details concerning relaxometric measurements, temperature dependent 17 O measurements, relaxivity measurements in the presence of HAS, phantom MRI images, full parameters used in the fitting procedure of NMRD and 17 O data, and equations used for the fits of 17 O NMR and 1 H NMRD data, report of singlet oxygen formation studies, and details of antiproliferative tests. References


Sophie Bou

Xinyue Wang

Nicolas Anton

Redouane Bouchaala

Andrey S Klymchenko

Mayeul Collot

Lipid-Core/Polymer-Shell Hybrid Nanoparticles: Synthesis and Characterization by Fluorescence Labeling and Electrophoresis

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

In the field of nanomaterials, lipid-based nanoparticles are drawing a growing interest especially as promising drug delivery systems, mainly due to the huge possibilities of lipophilic molecule loading they offer. 1,2 Among these nanoparticles, NanoEmulsions (NEs), 3,4,5 Solid Lipid Nanoparticles (SLNs) 6,7,8 and Nanostructured Lipid Carriers (NLCs) 9, 10 are composed of a lipid core that can serve as an efficient carrier of hydrophobic cargo such as drugs or contrast agents. Those lipid NPs are usually stabilized by monomeric surfactant or natural phospholipids with reduced possibilities of chemical modification thus limiting the modification of their physicochemical properties, their biodistribution, their circulation time in blood [START_REF] Mandal | Core-shell-Type Lipid-polymer Hybrid Nanoparticles as a Drug Delivery Platform[END_REF]12 or their targeting ability. Conversely, Polymeric Nanoparticles (PNPs) offer a wide diversity in their structures and functions due to accessible chemical modifications. 13,[START_REF] Elsabahy | Design of Polymeric Nanoparticles for Biomedical Delivery Applications[END_REF]15,16,[START_REF] Kamaly | Degradable Controlled-Release Polymers and Polymeric Nanoparticles: Mechanisms of Controlling Drug Release[END_REF] Consequently Lipid Polymer Hybrid Nanoparticles (HNPs) featuring the advantages of flexibility in the polymer design constitute promising new nanomaterials. [START_REF] Raemdonck | Merging the Best of Both Worlds: Hybrid Lipid-Enveloped Matrix Nanocomposites in Drug Delivery[END_REF] It is noteworthy that while polymeric core-lipid shell HNPs were extensively described, 19 20, 21, 22 those merging the advantage of an oily core with a polymeric shell, also called nanocapsules, remains poorly reported. 23,24,25 Therefore, a better comprehension on their formation and stability is required along with adapted analysis and characterization methods. Fluorescent labeling of lipidbased nanoparticles was used for bioimaging, 26,27 and for in cellulo 28 and in vivo [START_REF] Kilin | Counterion-Enhanced Cyanine Dye Loading into Lipid Nano-Droplets for Single-Particle Tracking in Zebrafish[END_REF] tracking of single particles. Additionally, it is also an efficient method of characterization using various fluorescence techniques including microscopy [START_REF] Kilin | Counterion-Enhanced Cyanine Dye Loading into Lipid Nano-Droplets for Single-Particle Tracking in Zebrafish[END_REF] and fluorescence correlation spectroscopy. 28,[START_REF] Klymchenko | Highly Lipophilic Fluorescent Dyes in Nano-Emulsions: Towards Bright Non-Leaking Nano-Droplets[END_REF] Although HNPs are generally characterized by dynamic light scattering (DLS) and by transmission electronic microscopy (TEM), these techniques do not provide fast and clear-cut information regarding their formation and composition. Conversely, electrophoresis relies on a simple and affordable setup and has already been reported as a reliable technique for analyzing and separating NPs of various sizes and shapes 31 32 and as a characterization method for nanomaterials including polymer wrapped inorganic nanoparticles [START_REF] Anton | The Universality of Low-Energy Nano-Emulsification[END_REF] and upconverting nanoparticles. 34 When combined to fluorescence detection, electrophoresis enables highly sensitive detection of fluorescently labeled nanomaterials [START_REF] Fernández-Argüelles | Synthesis and Characterization of Polymer-Coated Quantum Dots with Integrated Acceptor Dyes as FRET-Based Nanoprobes[END_REF] and nanoconjugates. 36 Consequently, fluorescence labeling of HNPs combined with electrophoresis analysis would enable a precise assessment of their assembly and integrity. Herein, we fluorescently labeled amphiphilic polymers in red and developed a green emitting fluorogenic oil tracker, thus allowing to independently monitoring the fate and colocalization of the polymer and the oil phase during the formulation of HNPs by nanoprecipitation. Electrophoresis revealed by fluorescence was successfully used as a monitoring method for the formation of HNPs and was found to be a valuable tool providing complementary information compared to DLS and TEM.

Results and discussion

Fluorescence labeling of HNPs constituents

Herein we aimed at formulating new hybrid nanoparticles (HNPs) based on the wrapping of a lipid droplet by an amphiphilic polymer (Figure 1C) as well as assessing their formation by fluorescence technique using simple methods. Firstly, an amphiphilic polymer was obtained from chemical modification of a commercially available alternating polymer: Poly (maleic anhydride-alt-1-octadecene) (PMAO), which is composed of a repeating unit consisting of a hydrophobic hydrocarbon chain (C16) and an amine reactive succinic anhydride function (Figure 1A). These alternating amphiphilic polymers are widely used to wrap nanocrystals to obtain water soluble inorganic nanoparticles [START_REF] Anton | The Universality of Low-Energy Nano-Emulsification[END_REF] and upconverting nanoparticles. 37 We also showed that PMAO, without any chemical modification, when incorporated at 1 wt % in oil can significantly modify the physicochemical properties of nanoemulsions. 24 PMAO can be readily reacted with Jeffamine, an amine-terminated polypropylene glycol/polyethylene glycol copolymer mainly composed of PEG. Vitamin E acetate (VEA) is a biocompatible and viscous oil that was chosen to constitute the non-polar core of our HNPs as we recently demonstrated its ability to form small and monodisperse fluorescent lipid droplets by spontaneous nanoemulsification. 28 In order to assess the formation of HNPs, both constituents, namely the amphiphilic polymer and the oil, were fluorescently labeled. To this endeavor, 1% of the reactive anhydride sites of PMAO were reacted with Rhodamine-NH2, a red emitting amino-rhodamine (Figure 1A), 37 while the oil phase was labeled using a newly developed fluorogenic oil tracker consisting of a lipophilic green emitting BODIPY, BDP-2C8. Indeed, this hydrophobic molecule displayed an impressive fluorescence enhancement at 507 nm from PBS to VEA (214-fold, Figure 1B), and also when incorporated in VEA based nanoemulsions (130-fold, Table S1, Figure S1). Thanks to its hydrophobicity, BDP-2C8 possesses a high affinity towards lipid oil, as its calculated CLogP was found to be close to the logP of VEA (11.2 and 9.95 respectively). Consequently, two complementary colors channels (green and red) can be used in fluorescence imaging to individually monitor both the oil phase and the polymer during the formulation of HNPs. Once all the constituents synthesized, two formulation methods were explored. 1) Rehydration of a dry organic film consisting in adding a non-solvent (PBS) on a film containing all the components (Oil/Polymer/BDP-2C8). 2) A "nanoprecipitation" approach consisting of quickly adding a non-solvent (PBS) to a concentrated solution of the components in dioxane (Figure 1C). While the rehydration approach led to flocculation and heterogeneous solutions, the nanoprecipitation method provided a visually clear nano-suspension and was thus adopted for the rest of our study.

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Formulation & Electrophoresis

In a first experiment, we used the amphiphilic polymer HP1 where all the anhydride functions were reacted with Jeffamine-1000 and 1% with Rhodamine-NH2 (see SI). This amphiphilic polymer thus displays hydrophobic hydrocarbon chains at one end, PEG chains at the other end and negatively charged carboxylate groups at the interface (Figure 1A). We first assessed the formation of HNPs by varying HP1/oil weight ratio (wt %). To this aim, a solution of VEA (2 mg/mL in dioxane) containing 1 wt % of BDP-2C8 (as oil tracker) and a solution of HP1 (2 mg/mL in dioxane) were mixed at various ratios, and PBS (95 Vol % of the final volume) was vigorously added for nanoprecipitation at room temperature. The formulations were then first analyzed by DLS. In absence of polymer large and polydisperse (510 ± 307 nm, PDI 0.35) pure oil droplets were obtained (Figure 2 A). Interestingly, at only 10 wt % HP1, dramatic effects were obtained as smaller size (103 ± 11 nm) and improved dispersity (PDI = 0.15) were obtained (Figure 2A). Upon increase of HP1 wt %, the sizes gradually decreased (Figure 2A) to finally reach pure polymer nanoparticles (PPNPs) with size of 10.6 ± 0.6 nm. Although DLS provided information regarding the average size of the bulk formulation, it did not inform on the nature of the formed NPs. In order to provide a deeper characterization of the NPs composition and taking advantage of our fluorescent labels, electrophoresis was performed, allowing for individual tracking of the oil and polymer, using the green and red channels, respectively (Figure 2B). Remarkably, the obtained gel displayed clear-cut and informative results. While in the absence of polymer the oil was unable to migrate to the anode, in the presence of 10 wt % HP1, the green signal was split up in two spots, one corresponding to the initial position (no migration), and the other one to a significant migration of the oil. At 20 wt % HP1, the pure oil spot was undetectable thus showing that this polymer/oil ratio was sufficient to uptake the entire amount of oil into new migrating species. At higher polymer wt %, the red and green fluorescence signals merged perfectly indicating that the new appearing spot might correspond to HNPs composed of both oil and polymer. Interestingly, the use of larger polymer/oil ratios revealed the appearance of a new red fluorescent spot corresponding to pure polymeric NPs (PPNPs). This was confirmed by the formulation in absence of oil that provided a red spot at the same migration distance (Figure 2A, 100 wt % HP1). Compared to DLS, which provides the size distribution of the whole population whatever its composition, this experimental approach appeared much more informative on the composition of the NPs suspension. The electrophoresis clearly discriminated the different populations and revealed the presence of hybrid NPs. According to these first results, 20 wt % polymer was sufficient to formulate HNPs with full uptake of the oil. In addition to this experiment, another serie of formulations was studied, fixing the amount of oil in order to keep a high green signal and the amount of polymer was gradually increased, ranging from 10 to 50 wt % (results reported in Figure S2). The electrophoresis revealed that, upon an increase of the polymer wt %, the spots corresponding to the HNPs displayed the same yellow color (merge of green and red) with no significant change in their migration distance, whereas the intensity of spots corresponding to PPNPs gradually increased. These results indicated that the formulation of HNPs required a minimum amount of 20 wt % of polymer and that that the additional use of polymer does not lead to its incorporation into HNPs but rather forms a second population of pure polymer nanoparticles (PPNPs). At that point of our study several controls were performed. First, we checked that the non-migrating and intense green signal obtained in absence of polymer was due BDP-2C8 dissolved in oil (Figure 3B, formulation 1) and not to BDP-2C8 itself in aqueous solution. Therefore, BDP-2C8 was formulated alone in PBS at the same concentration than used in the presence of oil (Figure 3, formulation 2). BDP-2C8 formulated alone did not provide any signal at the level of pure oil. Instead, a dim orange signal was obtained at a higher migration distance. It is known that pure fluorophores can form aggregates at the nanoscale with low brightness due to Aggregation-Caused Quenching (ACQ). [START_REF] Shulov | Fluorinated Counterion-Enhanced Emission of Rhodamine Aggregates: Ultrabright Nanoparticles for Bioimaging and Light-Harvesting[END_REF][START_REF] Trofymchuk | BODIPY-Loaded Polymer Nanoparticles: Chemical Structure of Cargo Defines Leakage from Nanocarrier in Living Cells[END_REF] Spectroscopic studies confirmed that BDP-2C8, when formulated in PBS form dispersed aggregates. First, both absorption and emission spectra dramatically broadened (Figure S1). This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins Then, emission spectra also revealed an important red shift. Indeed, while dissolved in oil BDP-2C8 emitted at 500 nm with high quantum yield (0.82), pure BDP-2C8 NPs emitted at 577 nm with a notable quantum yield of 0.19 (table S1), explaining the dim orange spot obtained in electrophoresis (Figure 3B, formulation 2). This phenomenon was already reported for BODIPY fluorophores and was attributed to the formation of red shifted emissive J-Aggregates. 40 In addition, DLS measurements corroborated that BDP-2C8, when formulated in PBS, aggregated as monodisperse NPs (121 ± 1 nm, PDI = 0.08, Figure 3A formulation 2). This experiment confirmed that the non-migrating green spot resulted from the oil droplets containing BDP-2C8. Then, the polymer (HP1) was formulated in the presence of BDP-2C8 and in the absence of oil (formulation 3) in order to verify whether the spots correspond to PPNs or overlap with HNPs (Figure 3, formulation 4 and 5, respectively). This control experiment showed that the migration spot of pure BDP-2C8 NPs (formulation 2) disappeared, giving rise to an orange signal (formulation 3) that appeared at the same level than PPNPs (formulation 4), with similar small sizes (20 ± 2 nm and 11 ± 1 nm for formulations 3 and 4, respectively). This experiment showed the ability of PPNPs to efficiently entrap BDP-2C8 providing a different spot than HNPs. Overall, these controls proved that an intense green signal is obtained only in the presence of the oil, and thus, that the colocalization of green and red signals (i.e. intense yellow spot), was correctly assigned to HNPs (Figure 3, formulation 5). Since electrophoresis was used to monitor and optimize the formation of HNPs, we hypothesized that it could also serve to assess their stability. To this end, HNPs composed of HP1 (20 wt %) were formulated and submitted to a temperature of 60°C over 4h. Every 30 min the size was measured by DLS and a small aliquot was kept for electrophoresis (Figure S3). On the one hand, we can note that although the size and the PDI only slightly increased over the time, the electrophoresis showed clear signs of degradation. Then, the migration distance of the spot corresponding to HNPs slightly decreased over the time along with its red color probably denoting a partial erosion of the polymer leading to the destabilization of the HNPs. At last, the green spots located at the deposition well, corresponding to pure oil, gradually appeared over the time. Overall, the electrophoresis clearly depicted a phase separation between the oil and the polymer over the time at 60°C and thus appeared as an efficient method to assess the stability of the fluorescently labeled HNPs.

Transmission Electronic Microscopy (TEM)

According to the obtained results, electrophoresis when correlated to DLS, provided evidences that HNPs can be obtained by simple nanoprecipitation using PMAO based amphiphilic polymer HP1. As complementary studies, HNPs composed of HP1 (20 wt %) were characterized by TEM. The images revealed the presence of 2 populations of particles (Figure 4A). The first population, present at a lower extent, displayed a spherical shape with a mean size of 503 ± 123 nm combined with a large polydispersity (Figure 4D). This was in accordance with the DLS measurements of pure VEA droplets (Figure 3A, formulation 1), probably resulting from the phase separation between oil and polymer during the preparation of the TEM sample at high vacuum. This type of images were already observed in TEM with nano-emulsions containing vitamin E-based compounds (or oils of similar viscosity), giving rise to spherical droplets that spread onto the carbon support. 41,42 The second population, present at a higher extent, displayed spherical shapes (Figure 4B) with an average size of 90 ± 14 nm (Figure 4E) and was attributed to HNPs; as their size was similar to the DLS measurements (Figure 3A, formulation 5). As a control, formulation involving only HP1 in absence of oil was imaged. TEM images revealed that PPNPs formed a dense layer of spread particles (Figure 4C) with an average size of 11 ± 1 nm (Figure 4F), which was again in accordance with DLS (Figure 3A, formulation 4).

Use of various amphiphilic polymers

We showed that fluorescent labeling of oil and polymer in combination with electrophoresis analysis could be used to monitor the formation and degradation of HNPs. We then chemically modified the amphiphilic nature of PMAO-based polymers to assess their ability to form HNPs and use this new experimental approach to disclose the impact of their chemical structure on the formulations. To this end, various PMAO-based amphiphilic polymers were synthesized (Figure 5A). Jeffamine-1000 and Jeffamine-2000 were used to modulate the PEG length and thus gave rise to HP1, HP1c, FP1 and HP2, HP2c and FP2 respectively. The negative charges brought by the remaining carboxylates groups (COO -) were capped either by coupling with an excess of short ethanolamine molecules to give HP1c, HP2c, or by coupling supplementary Jeffamine PEG chains giving rise to fully PEGylated polymers, FP1 and FP2. Accordingly to our previous results, the formulations were performed with 20 wt % amphiphilic polymers and 80 wt % VEA in PBS. These formulations were analyzed by DLS (Figure 5B) and by electrophoresis (Figure 5C). First, the electrophoresis showed that all of the polymers when used at 20 wt %, were able to form HNPs. The obtained sizes by DLS spanned within a small range, from 71 ± 2 nm (HP1c) to 117 ± 6 nm (FP2) with larger sizes obtained with the fully PEGylated polymers. The slight difference in DLS size might be explained by the difference of molecular weight especially between the half PEGylated and the fully PEGylated polymers. Indeed although the wt % was kept at 20%, the molar polymer/oil ratio is significantly smaller in the case of "heavy" polymers that led to larger HNPs, this is consistent with our previous observation (figure2A). Additionally, while full PEGylation led to slightly lower migration distances compared to half PEGylation, the use of large PEG-2000 systematically led to an important decrease of migration. It is noteworthy that charge capping by ethanolamine, while slightly enhanced the monodispersity of the obtained HNPs compared to the charged polymers, hardly influenced their migration distance and their size. Although the efficiency of the chemical reactions performed on the polymer was confirmed by 1 H NMR (see SI), the measured z potential displayed similar values (∼26 mV, Table S2). The correlation between electrophoretic migration and values of z potential from charged and capped polymers suggests that the particles still present a significant surface potential, which can come from various origins, PEG chain conformations, dipoles and interactions of the ions present in the media. Overall, it appeared that the nature of the amphiphilic polymer, more precisely its PEGylation rate, but also the length of the PEG This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins chains significantly influenced the migration distance of the HNPs. To further support this hypothesis, a fully ethanolaminecapped polymer was used to study the influence of PEG groups on the formulation of HNPs (Figure S4). Interestingly the electrophoresis showed that neither the polymer nor the oil migrated indicating that this less polar polymer failed to form HNPs at any polymer/oil ratio. These results underline the major role of PEG chains in the formulation of HNPs. Eventually, the electrophoresis appeared to be a method of choice also adapted to characterize the surface modifications of these HNPs.

Conclusion

Herein, we aimed at developing new lipid-core/polymer-shell hybrid nanoparticles (HNPs) by simple nanoprecipitation protocol. In order to facilitate the screening of the optimal conditions, both oil and polymer constituents were fluorescently labeled with distinct colors. To this end, the amphiphilic polymer was covalently labeled with a red emitting rhodamine and in parallel, an oil tracker was developed based on a hydrophobic fluorogenic BODIPY displaying a 214-fold fluorescence enhancement from PBS to oil. The obtained formulations were systematically analyzed by DLS and through an innovative experimental approach using dual color fluorescently revealed electrophoresis. We herein showed that, unlike DLS measurements and TEM imaging, electrophoresis provides unambiguous information by discrimination of the different nanomaterials that can be obtained during the formulation. Additionally, we showed that this technique could be used to assess the stability of the lipid nanomaterial, as well as to discriminate the relationships between particle composition and surface properties. We believe that, in the future, this method might facilitate the development of new lipid nanoparticles by accelerating the optimization of the formulation conditions. Finally, the herein presented hybrid nanomaterials present many advantages in terms of preparation and chemical modifications. Consequently, ongoing work is being performed by our group to develop functionnalizable hybrid particles based on this approach.

Experimental section

Materials

Poly (maleic anhydride-alt-1-octadecene) (PMAO, Average Mn 30,000-50,000), diisopropylethyl amine (DIEA), ethanolamine, tetrahydrofuran (THF) and dioxane were purchased from Sigma Aldrich. Jeffamine M-1000 (J-1000) and Jeffamine M-2070 (J-2000) polyetheramine (J-2000) were kindly offered by Huntsman Corporation (Texas, U.S.A.). Vitamin E acetate (VEA) was purchased form TCI Europe. The solvents were of analytical grade, Dimethylformamide (DMF) was anhydrous.

Synthesis

NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer. Mass spectra were obtained using an Agilent Q-TOF 6520 mass spectrometer. Spectra can be found in the supplementary information. cLogP value of BDP-2C8 was obtained using ChemDraw (Cambridge software).

BDP-2C8.

To a solution of acid BDP-COOH (200 mg, 0.598 mmol, 1 eq) in DMF (4 mL) under Argon, was added bis(2ethylhexyl)amine (0.270 mL, 0.897 mmol, 1.5 eq), followed by DIEA (0.313 mL, 1.790 mmol, 3 eq) and HATU (273 mg, 0.718 mmol, 1 .2 eq). The reaction mixture was allowed to stir for 1h. The excess of solvent was evaporated. The crude was extracted with DCM and washed with water (x2) and brine (x2). The organic phase was dried over anhydrous MgSO4, filtered and evaporated. The product was purified by chromatography column on silica gel using DCM/EtOAc (98/2). 159 mg of BDP-2C8 was obtained (Yield= 48%) as an orange powder. Rf= 0.25 (100% DCM). 1 H-NMR (400 MHz, CDCl3): δ 6.07 (s, 2H, H ), 3.29 (ddd, J = 14.0, 7.4, 1.6 Hz, 2H, 2 N-CH), 3.15 (d, J = 7.5 Hz, 2H, 2 N-CH), 3.05-3.01 (m, 2H, CH2), 2.53-2.47 (m, 14H, 4 CH3, CH2), 2.05-1.97 (m, 2H, CH2), 1.67 (s, 1H, CH), 1.60 (s, 1H, CH), 1.36-1.19 (m, 16H, 8 CH2), 0.94-0.86 (m, 12H, 4 CH3). 13 C-NMR (101 MHz, CDCl3): δ 171.9, 153.9, 145.7, 140.5, 131.5, 131.5, 121.6, 51.5, 48.9, 38.6, 37.0, 33.0, 30.6, 30.5, 28.8, 28.7, 28.7, 27.7, 27.4, 23.9, 23.8, 23.0, 23.0, 16.4, 14.4, 14.4, 14.4, 14.0, 14.0, 10.8, 10.6, 10.6. HRMS (ESI + ), calculated for C33H54BF2N3ONa [M+Na] + : 580.4426, found 580.4213. Calculated for C33H54BFN3O [M-F] + : 538.4344, found 538.4329. Calculated for C33H54BF2N3ONa [2M+Na] + : 1137.8554, found 1137.8528.

Rhodamien-NH2. We described the synthesis of rhodamine-NH2 elsewhere. 43 HP1. To a solution of PMAO (30 mg, 85.71 µmol of repeating unit see SI) in degassed anhydrous DMF (3 mL) was added a solution of a Rhodamine-NH2 (8.57 mol, 69 µL C=10 mg/mL in DMF) followed by DIEA (0.85 mmol, 200 mL, 10 eq) and a Please do not adjust margins Please do not adjust margins solution of Jeffamine-1000 (1 mL, C=100 mg/mL in DMF, 0.102 mmol, 1.2 eq). The reaction mixture was allowed to stir for 2 h before the addition of few drops of water. The solvents were evaporated. The crude was purified by size exclusion column using DCM/MeOH (1/1). 61 mg of HP1 was obtained as a pink sirup (Yield= 53%). The synthesis of the other polymers can be found in the supplementary information.

Spectroscopy.

For spectroscopy the concentration of BDP-2C8 was 1 µM and solvents were of spectroscopic grade. The water was milliQ water. Absorption and emission spectra were recorded on a Cary 400 Scan ultraviolet-visible spectrophotometer (Varian) and a FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon) equipped with a thermostated cell compartment, respectively. For standard recording of fluorescence spectra, the emission was collected 10 nm after the excitation wavelength. All the spectra were corrected from wavelength-dependent response of the detector. The quantum yields were determined by comparison with fluorescein [START_REF] Mcclements | Nanoemulsions versus Microemulsions: Terminology, Differences, and Similarities[END_REF] (in NaOH 0.1 M, f= 0.95) following equation (1):

Where QY is the quantum yield, I is the integrated fluorescence intensity, n is the refractive index, and OD is the optical density at the excitation wavelength. R represents the reference.

Formulation of the HNPs

Preparation of stock solutions: First, the oil (VEA) was labeled by dissolving BDP-2C8 at 1 wt %. The labeled oil was then dissolved in dioxane to reach a concentration of 2 mg/mL. Polymers were dissolved in dioxane at a concentration of 2 mg/mL. The HNPs were formulated using two different methods: 1) Rehydration of films: In a round-bottom flask of 10 mL, 50 µL of premixed solutions of polymer and oil (according to the chosen ratio) were added before being slowly evaporated until obtaining a thin film. Then, 950 µL of PBS were vigorously added using back and forth pipetting using a 1 mL micropipette.

2) Nanoprecipitation: In an Eppendorf tube, stock solutions of polymer and oil (according to the chosen ratio) were added and mixed to reach a final volume of 50 µL. Then, 950 µL of PBS were vigorously added using back and forth pipetting using a 1 mL micropipette. The solution was immediately vortexed for 10 seconds.

Dynamic Light Scattering (DLS), and z potential

After the formulation, size of HPNs were measured by Dynamic Light Scattering with the instrument Zetasizer® Nano ZSP (Malvern, UK) using the formulation solutions. Measurements were performed at 25°C. Refractive index were set to 1.33 for the continuous phase (water) and 1.49 for the dispersed phase (Vitamin E Acetate). Both size distribution and polydispersity index (PDI) were recorded. The z potentials were measured on diluted solution in distilled water (0.02 µg/mL) at a stable conductivity of 0.05 mS/cm.

Electrophoresis

Gel electrophoresis was prepared by dissolving agarose (0.5 wt %) in TAE (Tris Acetate 40 mM, EDTA 1 mM). 13 µL of the formulation solutions were carefully added in the wells. The electrophoresis was run at 125 V for 1h. The gels were visualized with an ImageQuant LAS 4000 (GE Healthcare Life Sciences) using Cy2 (excitation: 473 nm) and Cy3 (excitation: 532 nm) channels and by acquiring successive images with an exposition time of 10 seconds. The images were treated with ImageJ.

Stability assay

HNPs composed of HP1 (20 wt %) were formulated as described in 4.4 and placed in a 1.6 mL cuvette for DLS measurement. The cuvette was then closed by help of an adapted stopper and was warmed at 60°C in a water bath. At each time point, the solution was rapidly cooled down to RT; then a DLS measurement was performed and 30 µL was taken off for electrophoresis.

Transmission Electron Microscopy (TEM)

5 µL of the formulation solution was deposited onto carboncoated copper-rhodium electron microscopy grids that were used following amylamine glow-discharge. The grids were then treated for 1 min with a 2 % uranyl acetate solution for staining and observed with a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operating at 100 kV. Areas covered with NPs of interest were recorded on a Peltier cooled CCD camera (Model 794, Gatan, Pleasanton, CA). Image analysis was performed using the imageJ software.

Notes and references

QY = QY ! × !×!" ! ×! ! ! ! ×!"×! ! ! !
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Figure 1 .

 1 Figure 1. (A) Synthesis of fluorescently labeled amphiphilic polymer. (B) Development of fluorescent oil tracker based on BODIPY BDP-2C8 and its fluorescent spectra at 1 µM in PBS and in oil. (C) Formulation of HNPs by nanoprecipitation and their schematic representation.

Figure 2 .

 2 Figure 2. Formulation of HNPs with various polymer/oil wt %. (A) Mean sizes measured by DLS resulting from at least 3 independent formulations. Numbers above the bars are the mean PDI values. (B) Merged green and red channels of the electrophoresis image. Scale bar is 0.5 cm.

Figure 3 .

 3 Figure 3. Various formulations studied. (A) Mean sizes measured by DLS resulting from at least 3 independent formulations. (B) Merged green and red channels of the electrophoresis image. Scale bar is 0.5 cm. (C) Schematized structures assigned to the formulations. 1) VEA (100 µg/mL containing) + BDP-2C8 (1 µg/mL); 2) BDP-2C8 (1 µg/mL); 3) HP1 (100 µg/mL) + BDP-2C8 (1 µg/mL); 4) HP1 (100 µg/mL); 5) VEA (80 µg/mL containing) + BDP-2C8 (1 µg/mL) + HP1 (20 µg/mL).

Figure 4 .

 4 Figure 4. (A) TEM image of HNPs composed of HP1 (20 wt %). (B) is the zoomed region of interest in (A). (C) TEM image of PPNPs composed of HP1. D, E and F are the corresponding histograms of the measured particles' sizes.

Figure 5 .

 5 Figure 5. Formulation of 20 wt % polymer HNPs with different amphiphilic polymers. (A) Schematic representation of the polymers used for formulation. (B) Mean sizes measured by DLS resulting from at least 3 independent formulations. Numbers above the bars are the mean PDI values. (C) Merged green and red channels of the electrophoresis image. Scale bar is 0.5 cm.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

Acknowledgements

This work was supported by the European Research Council (ERC) Consolidator grant BrightSens 648528 and the French National Research Agency (ANR) BrightRiboProbes (ANR-16-CE11-0010). XW was founded by the China Scholarship Council PhD fellowship (CSC No. 201706240033). The authors would like to thank Dr. Delphine Garnier from the Analytical platform (PACSI) for her assistance in the LC-MS and RMN analyses. We also thank Huntsman Corporation for providing Jeffamine-M-1000 and Jeffamine-M-2070.

Conflicts of interest

The authors declare no conflict of interest.