The thesis is funded by the China Scholarship Council (CSC) and École Normale Supérieure de Lyon (ENS de Lyon), I would like to first of all express my gratitude to the CSC and ENS de Lyon.

During the three years PhD study in the chemistry laboratory of the ENS de Lyon, I have not only learned much professional knowledges and skills, but also received a lot of care and help from many people. Now, I would like to take the chance to thank them for living me so precious three-year experience.

Firstly, I would like to express my most sincere respect and thanks to my supervisors Dr. Chantal Andraud and Dr. Yann Bretonnière. I really appreciate Chantal Andraud for giving me a chance to work in her lab, and supporting my research and study. I wish you a happy and wonderful life in the future. I deeply thank Yann Bretonnière for guiding me three years in both my work and life. I really benefit a lot from his rigorous scientific attitude and professional knowledge. Thank you for encouraging all my ideas and leaving me a lot of constructive suggestions. I hope to be able to establish a cooperative relationship with you in the future if I continue research, and I wish you a happy life, happy work.

Besides my supervisors, I would like to warmly thank our collaborators Dr. Arnaud Favier from the Polymer Materials Engineering Laboratory of Lyon for their efforts in preparation of polymer nanoparticles; Dr. Olivier Hugon and Dr. Boudewin Van der Sanden at Liphy and TIMC-IMAG in Grenoble for photoacoustic imaging and two-photon fluorescence imaging. Then, I also want to thank many members from the laboratory: Fréderic Lerouge for giving me guidance on manipulating DLS and preparation of amine modified glass slides; Fréderic Chaput and Delphine Pitrat for helping me prepare sol-gel and PMMA films and synthesize intermediate products; Sandrine Denis-Quanquin and Laure Guy for training me NMR and mass; Christian Melkonian for fixing all kinds of computer problems; Maxime Rémond for his help during my first year. All the people outside ENS who have collaborated with us; and finally all the other colleagues and friends surrounding me: Tingting, Junchao, Qingyi, Shagor, Damien, Orsola, Caroline, Jean, Isis, Maher, Sylvain, Laura… I really enjoy the four-year life in France. I will always keep this memory in mind. I wish everybody here a happy life. Finally, I would like to thank the understanding and support from my family, they are forever my strongest backing.

II

Table of Content

General Introduction

This thesis is mainly devoted to develop D-π-A dipolar dyes and nanoparticles for in vivo fluorescence or photoacoustic imaging. Dipolar dyes involve an intramolecular charge transfer between an electrondonating group D and an electron-withdrawing group A linked by a conjugated π bridge. Many advantages such as large Stokes shifts, high two-photon absorption cross sections and ease of modulation on dye absorption and emission by molecular engineering make dipolar dyes promising for fluorescence imaging.

Micelle/silica nanoparticles originally developed for drug delivery present excellent potential as vehicles to load organic dyes for bioimaging thanks to their good biocompatibility, suitable size, and surface modifiable capability. Polymer nanoparticles are good candidates for improving dye concentration in photoacoustic imaging.

For this objective, this thesis is separated into two parts. The first part deals with the design and preparation of near infrared emitting dyes and micelle/silica nanoparticles for biphotonic in vivo fluorescence imaging, and the second part focuses on near infrared absorbing dyes and polymer nanoparticles for photoacoustic imaging.

In Chapter I, we will present the importance of near-infrared light for bioimaging, and give a detailed overview of dye engineering for red-shifting the optical properties to near-infrared, focusing on several dye families. Then we will detail the working mechanism of photoacoustic imaging technology and introduce the nanoparticles including micelle/silica nanoparticles and polymer nanoparticles for dye loading in fluorescence and photoacoustic imaging.

Chapter II concerns the synthesis of new dyes emitting in the far red/near infrared and the preparation of surface functionalized micelle/silica nanoparticles. In order to obtain a more red-shifted fluorescence as well as an enhanced molecular lipophilicity, we introduce bulky donor groups or strong acceptor groups on the dyes structure. The optical properties of the new dyes in solution and in the solid state will be presented and compared with those of dyes previously synthesized in our laboratory. For particles surface functionalization, we try to modify Pluronic F127 with different functions which enable the covalent linking with functional moieties. The preparation and characterization of the nanoparticles will be detailed. Finally, we will provide in vivo biphotonic imaging on living mice.

Chapter III will present our efforts on improving dye concentration for photoacoustic imaging, including the collaboration work on preparation of high-dye-loaded polymer nanoparticles and our try for loading VIII dyes on micelle/silica nanoparticles surface. The optical and physical properties of the new dyes and nanoparticles will be presented. The preliminary in vivo photoacoustic imaging will also be described in this chapter.

Finally, in Chapter IV we will apply our dipolar dyes for primary amine detection, focusing on two dyes bearing dicyanovinyl groups. The sensing behavior and mechanism both in solution and in solid films will be discussed, and a preliminary device for circle detection of primary amine vapor will also be presented.

This thesis will end up with a conclusion and perspective in Chapter V, followed by the experimental part in Chapter VI.

Chapter I. Introduction to NIR Dyes and Nanoparticles (NPs) for in vivo Optical Imaging

Bioimaging Technologies

Bioimaging technologies are power tools to obtain biological information in non-invasive ways. They can not only reflect the structural morphology of organisms, but also enable researchers to directly monitor different biological processes, such as proliferation and differentiation of cells, [1] growth and metastasis of tumors, [2] and even expression of specific genes. [3] Hence, bioimaging technologies have recently attracted increasing interest in disease diagnosis and drug development fields. [4][START_REF] Contag | Bioluminescent indicators in living mammals[END_REF][6][START_REF] Gibbs-Strauss | Nerve-highlighting fluorescent contrast agents for image-guided surgery[END_REF] Based on different excitation or detection methods, they are mainly divided into ultrasonic, magnetic resonance and optical imaging. In this thesis, we will only focus on two optical imaging technologies: fluorescence and photoacoustic (PA) imaging.

Biological Transparency Window

Figure 1 -1. (a) Excitation coefficients of water and Hb/HbO2 from 400 nm to 1000 nm; [START_REF] Kobayashi | New strategies for fluorescent probe design in medical diagnostic imaging[END_REF] (b) Absorption spectra of human skin presenting the three different biological transparency windows in the far-red/NIR. [9] Imaging depth and spatial resolution are two significant indicators for in vivo optical imaging. The former closely relates to the penetration ability of photons in tissues, the latter is heavily affected by biological interference. Organisms are composed by various organic and inorganic components, which absorb and scatter light in varied degrees, leading to the transmission hindrance of photons and strong background noise.

In this regard, light source is of vital importance for both fluorescence and PA imaging technologies.

On the one hand, light absorption in organisms derives from a sum of the light-absorbing components, including water, hemoglobin (Hb), oxyhemoglobin (HbO2), protein and lipids, etc. From Figure 1 -1, one can see that Hb and HbO2 highly absorb the light in blue and visible range, limiting the photon penetration to a few millimeters. [START_REF] Kobayashi | New strategies for fluorescent probe design in medical diagnostic imaging[END_REF] Thus, light from 245 to 550 nm is most used for surface imaging. Meanwhile, water has gradually increased absorption to light from 650 nm to 1000 nm, which permits a minimum whole extinction coefficient in wavelengths from 650 nm to 900 nm (NIR-I), this range is called the first biological transparency window. Given the greater transparency of biological tissues, wavelengths ranging from 1000 nm to 1300 nm (NIR-II) and from 1300 nm to 1700 nm (NIR-III) are called the second and the third biological windows, respectively (Figure 1 -1, b). d) NIR (725-775 nm/790-830 nm) filter set. [START_REF]In vivo near-infrared fluorescence imaging. Frangioni[END_REF] On the other hand, auto-fluorescence from organisms exerts strong background interference to fluorescence imaging.

Engineering of NIR-emitting Organic Dyes

In vivo fluorescence imaging technology mainly relies on contrast agents that can transfer the light energy into fluorescence signals to form the images of the biological tissues, organs or cells by simulating the distribution of the contrast agents. To fully exploit the potential of NIR light in in vivo imaging, extensive attention has been attracted to NIR contrast agents exhibiting spectroscopic properties in one of biological windows previously described. In the past decades, many NIR contrast agents have been developed, including biological molecules, inorganic materials and organic dyes, etc. [START_REF] Licha | Optical imaging in drug discovery and diagnostic applications[END_REF][START_REF] Sevick-Muraca | Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents[END_REF][13][START_REF] Ntziachristos | Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging[END_REF] Organic dyes are seen as good candidates for their merits of low cost, ease of preparation, moderate toxicity, high contrast and importantly, finely tunable optical properties by structural engineering.

Since the optical properties of organic dyes originate from the energy gap between the highest occupied and the lowest unoccupied molecular orbitals (HOMO and LUMO), any factors that can decrease the energy gap will shift the optical properties toward higher wavelengths. Generally, the modulations include the length of the π-conjugation, the molecule aromaticity and flatness, the introduction of electron-donating or/and electron-withdrawing substituents (Figure 1 -3). [START_REF] Pansare | Review of long-wavelength optical and NIR imaging materials: contrast agents, fluorophores, and multifunctional nano carriers[END_REF] Figure 1 -3. Possible modulations of fluorophores in order to red-shift the excitation and emission wavelengths. [START_REF] Pansare | Review of long-wavelength optical and NIR imaging materials: contrast agents, fluorophores, and multifunctional nano carriers[END_REF] Following these simple molecular engineering strategies, it is possible to tune the absorption of dyes up to NIR. However, this is often accompanied by a sacrifice on the fluorescence quantum yield. Indeed, the longer the fluorescence emission wavelength, the smaller the molecular bandgap, thus the stronger competition from non-radiative processes. For this reason, the development of novel NIR dyes has faced many challenges.

Extensive literature survey on the conception of NIR dyes for bio-imaging also makes us realize that the vast majority of studies and structures recently proposed are based on only few generic structures. The following section will therefore present a selection of molecular engineering strategies that have been

Rhodamine Dyes

Scheme 1 -1. General structure of rhodamine.

Rhodamine (Scheme 1 -1) is a generic name of an important family of synthetic cationic dyes characterized by a symmetrical π-electron system extending over a diaminoxanthene framework. Such structure confers interesting spectroscopic properties: high molar absorption coefficient, good photostability and, generally, high fluorescence quantum yield. These optical properties can be easily tuned by structural modification and since the 1990s, great efforts have been dedicated to improve the synthesis and to red-shift the optical properties of rhodamines to the far-red. This field has been extensively reviewed and rhodamine derivatives now offer a veritable palette of fluorophores that absorb and emit across the visible spectrum. [START_REF] Sun | Rhodamine-inspired far-red to near-infrared dyes and their application as fluorescence probes[END_REF][START_REF] Ikeno | Silicon-substituted xanthene dyes and their unique photophysical properties for fluorescent probes[END_REF] Beside modification of the two amino group substituents, substitution of the O atom of the xanthene core for other elements, such as C, Si, P or its oxide, S and its oxide, is the most commonly described structural variation. It induces significant red-shifted optical properties that can be ascribed to the stabilization of the LUMO energy level of the fluorophore by σ*-π* conjugation between the X-C (X=C or Si) σ* orbital and a π* orbital, or by intramolecular charge transfer between the conjugated π-system and an acceptor moiety (P-oxide or SO2). Thus, carbopyronine or Si-Rhodamines elicit a 50 nm to 90 nm bathochromic shift in comparison to simple rhodamines, [START_REF] Kolmakov | A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy[END_REF][START_REF] Fu | A design concept of long-wavelength fluorescent analogs of rhodamine dyes: replacement of oxygen with silicon atom[END_REF][START_REF] Koide | Development of an Si-rhodamine-based far-red to near-infrared fluorescence probe selective for hypochlorous acid and its applications for biological imaging[END_REF][START_REF] Sauer | Robust synthesis of NIR-emissive P-rhodamine fluorophores[END_REF] whereas the fluorescence emission wavelength of phosphoroussubstituted rhodamines (such as Nebraska Red) can reach about 750 nm with relative high fluorescence quantum yield (about 0.2 at 744 nm for NR744). [START_REF] Zhou | Nebraska Red: a phosphinate-based near-infrared fluorophore scaffold for chemical biology applications[END_REF] As for sulfone rhodamines (SO2R), strong σ*-π* interaction between the vacant σ* orbital from the sulfur atom and the π* system of the butadiene part (d*π* conjugation) further lowers the LUMO energy level of the fluorophore, absorption can therefore reach about 700 nm, and the fluorescence emission wavelengths are over 740-750 nm (Figure 1234). [START_REF] Liu | Sulfone-rhodamines: a new class of near-infrared fluorescent dyes for bioimaging[END_REF] One can also find Se and Te replaced Rhodamines absorbing above 600 nm, [START_REF] Manjare | Selenium-and Tellurium-containing fluorescent molecular probes for the detection of biologically important analytes[END_REF][START_REF] Deng | Heteroatom-substituted rhodamine dyes: Structure and spectroscopic properties[END_REF] and also one example of Bismuth rhodamine [START_REF] Hirayama | Bismuth-rhodamine: a new red light-excitable photosensitizer[END_REF] absorbing at 635 nm and emitting at 658 nm. However, restricted by heavy-atom effect, these compounds display very weak fluorescence quantum yields (about 0.01 for Se-rhodamine in Methanol, below 0.001 for Te-rhodamine in water and less than 0.04 for Bi-rhodamine in methanol). However, oxidation diminishes the heavy-atom effect and therefore enhances the fluorescence of TeO or SeOrhodamine. [START_REF] Koide | A reversible near-infrared fluorescence probe for reactive oxygen species based on Te-rhodamine[END_REF] Note that all these dyes are efficient singlet oxygen photosensitizers. [START_REF] Deng | Heteroatom-substituted rhodamine dyes: Structure and spectroscopic properties[END_REF][START_REF] Hirayama | Bismuth-rhodamine: a new red light-excitable photosensitizer[END_REF] Figure 1 -4. Tuning rhodamines to NIR by substitution of the xanthene core. [START_REF] Liu | Sulfone-rhodamines: a new class of near-infrared fluorescent dyes for bioimaging[END_REF] To give an example of practical application in imaging of such modified rhodamine, let's cite the work . [START_REF] Fu | A design concept of long-wavelength fluorescent analogs of rhodamine dyes: replacement of oxygen with silicon atom[END_REF][START_REF] Koide | Development of an Si-rhodamine-based far-red to near-infrared fluorescence probe selective for hypochlorous acid and its applications for biological imaging[END_REF][START_REF] Koide | Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging[END_REF][START_REF]Evolution of group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer[END_REF] Figure 1 -5. Chemical structures of SiR 680, SiR 700, SiR 720 dyes, and the in vivo tumor imaging of SiR700-labeled anti-tenascin-C antibody based on NHS modified SiR 700 (PBS at pH 7.4 as the solvent). [START_REF] Koide | Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging[END_REF] However, although these recent developments have improved the situation, higher wavelengths still seem out of reach with "simple" xanthene-like (i.e. 3-fused rings) structures without extension of the πconjugated system. This is why some rectilinearly π-extended rhodamine dyes with up to 5 fused conjugated rings were reported, such as TJ730 that displays emission peak at 730 nm or ECX that presents maximal absorbing and emitting wavelengths at around 880/915 nm (Figure 123456). [START_REF] Bright | stable, and biocompatible organic fluorophores absorbing/emitting in the deep near-infrared spectral region[END_REF][START_REF] Liu | A unique rectilinearly π-extended rhodamine dye with large Stokes shift and near-infrared fluorescence for bioimaging[END_REF] Figure 1 -6. Chemical structures of TJ730 and ECX (EtOH as the solvent).

Similarly, combining fragments of another chromophore (coumarin, cyanine, etc) with the xanthene ring is an efficient approach to increase the conjugation length. The terminology of "hybrid-rhodamine" dyes or hybridisation is sometimes used to describe this strategy (Figure 1 -7). The first example (maximal absorption at around 700 nm) Merocyanine A obtained from the reaction of a chloroheptamethine cyanine with resorcinol in basic condition, was reported by Czerney in 1996, [START_REF] Czerney | New near-infrared-absorbing non-symmetric and symmetric trimethinecyanine dyes containing a 7dialkylamino-1-benzopyrylium end group[END_REF] but fluorescence properties were not described at that time. The fluorophore has been taken over by Lin's group in 2012 who reported good farred emission in methanol (λem = 743 nm, Φ = 0.34). [START_REF] Yuan | A uniqueclass of near-infrared functional fluorescent dyes with carboxylic-acid-modulated fluorescence ON/OFF switching: rational design, synthesis, optical properties, theoretical calculations, and applications for fluorescence imaging in living animals[END_REF] Since, quite systematic structure modifications were performed on this class of fluorophores referred as Changsha dyes in order to improve their NIR optical properties. [START_REF] Chen | De novo synthesis of phenolic dihydroxanthene near-infrared emitting fluorophores[END_REF][START_REF] Chen | integration" strategy for the rational design of optically tunable near-infrared fluorophores[END_REF][START_REF] Wang | Hybrid rhodamine fluorophores in the visible/NIR region for biological imaging[END_REF] Worth noting that one example of rhodamine-coumarin hybrid show emission maxima up to 830 nm albeit low quantum yields (< 0.01). [START_REF] Yuan | Analogs of Changsha near-infrared dyes with large Stokes shifts for bioimaging[END_REF] To the best of my knowledge, there is only one example of a xanthene-based NIR-II fluorophore to date.

RhIndz Ethyl Ester (Figure 1234567d) was reported by the groups of Hammer, Delcamp and Scott in 2019. [START_REF] Rathnamalala | Donor-acceptor-donor NIR II rmissive rhodindolizine dye synthesized by C-H bond functionalization[END_REF] It is a Donor-Acceptor-Donor (D-A-D) fluorophore in which the 3 and 6 positions of the electron poor xanthene core are coupled with an electron rich indolizine ring (Pyrrolo[1,2-a]pyridine). RhIndz Ethyl Ester display outstanding long wavelength emission (λabs = 920 nm/λem = 1092 nm) for such a small structure, albeit a very weak quantum yield (Φ ∼ 0.03%).

BODIPY Dyes

Scheme 1 -2. General structure of BODIPY.

BODIPY (Scheme 1 -2) is characterized by two pyrrole units that are connected by a methene bridge and both pyrrole-nitrogen atoms are coordinated by the same boron atom. The π-electrons fully delocalize over the framework. BODIPY dyes have many interesting properties such as high molar absorption coefficients (generally above 80,000 M -1 cm -1 ), good fluorescence emission (Φ commonly above 0.5), as well as excellent photostability. However, parent BODIPY unit has absorption peak near 500 nm. The BODIPY dyes reported rarely emit over 650 nm by now. This limits drastically their potential usage for optical imaging. In this regard, great efforts are needed to tune the classical framework of BODIPY to the NIR.

As a matter of fact, recent reviews have shown growing interest in strategies that can improve the optical properties of BODIPY dyes to reach the biological transparency window. [START_REF] Boens | Postfunctionalization of the BODIPY core: synthesis and spectroscopy[END_REF][START_REF] Loudet | BODIPY syes and their derivatives: syntheses and spectroscopic properties[END_REF][START_REF] Zhao | The triplet excited state of Bodipy: formation, modulation and application[END_REF] Firstly, for the readily modifiable property of BODIPY extension of the π-electron delocalization is an important manner to tune the absorption and emission of this family of dyes toward NIR. Introducing four styryl units by Knoevenagel-type condensation with aromatic aldehydes leads to Tetrastyryl-Bodipy (Figure 1 -8, a). [START_REF] Buyukcakir | Tetrastyryl-BODIPY dyes: convenient synthesis and characterization of elusive near IR fluorophores[END_REF] It has approximately 200-300 nm red-shifted absorption and emission as well as improved absorption coefficient (up to 173,900 M -1 cm -1 ) compared with parent BODIPY. By the same idea of extending the πsystem, fusion of BODIPY with two furan derivatives at both pyrrole units afforded Keio Fluors (KFLs), which have absorption and emission reached the far-red (738 nm) when the meso position is substituted by a trifluoromethyl group. Moreover, a BODIPY dimer Anti-bis-benzobodipy, reported by Nakamurain 2012, [START_REF] Nakamura | π-Fused bis-BODIPY as a candidate for NIR dyes[END_REF] presented emission reached 868 nm beneficial from its further extended π-delocalization system, albeit much decreased quantum yield (0.04) was observed.

Substitution of the carbon atom in meso position by a nitrogen atom results in aza-BODIPY (Figure 1 - 9). In comparison to the normal BODIPY dyes, aza-BODIPY dyes have generally 100 nm red-shifts in both absorption and emission along with improved fluorescence quantum yields. Koch et al. [START_REF] Koch | Monofunctionalized 1,3,5,7-tetraarylazaBODIPYs and their application in the synthesis of azaBODIPY based conjugates[END_REF] reported last year a monofunctionalized dye Tetra aryl-azaBODIPY, in which a BODIPY core acting as a donor unit was linked to an aza-BODIPY as an acceptor unit, the formed cyanine-like structure with a push-pull framework achieved the emission peak at 708 nm with a high quantum yield of 0.46. On the same fashion, Phenanthrene-fused azaBODIPY [45] obtained from phenanthrene fusion by a palladium-catalyzed intramolecular C-H activation show outstanding absorption (λabs = 797 nm, ε > 160,000 M -1 cm -1 ) and emission (λem = 810 nm, Φ = 0.35) properties. In 2018, the same group further synthesized a Ten-ring substituted azaBODIPY core by Suzuki coupling between aza-BODIPY and two meta-methoxy phenyl, followed by regioselective oxidative ring-fusion. [START_REF] Sheng | Synthesis, crystal structure, and the deep near-infrared absorption/emission of bright azaBODIPYbased organic fluorophores[END_REF] The fully annulated structure exhibited nearly planar conformation and excellent optical properties (absorption and emission in toluene: λabs/λem = 878/907 nm, Φ = 0.13), large extinction coefficient (260,100 M -1 cm -1 ) and excellent photostability.

Given the improved optical properties of aza-BODIPY compared to BODIPY, more attention to red-shift the optical properties of BODIPY dyes toward NIR have been attracted to functionalization of the aza-BODIPY core. Godard et al. [START_REF] Godard | Water-soluble aza-BODIPYs: biocompatible organic dyes for high contrast in vivo NIR-II imaging[END_REF] synthesized recently a water-soluble dye SWIR-WAZABY-01 (Figure 1 -9, d) by substitution of the boron on aza-BODIPY with ammonium groups. It has emission peak in the NIR-II at approximately 1200 nm in plasma but with quantum yield that needs to be optimized.

Cyanine Dyes

Scheme 1 -3. General structure of cyanines.

Cyanines (Scheme 1 -3) can be represented as a D-π-D + cationic structure consisting in an odd number of carbon atoms and two nitrogen-containing heterocycles terminated both ends. The fully delocalized charges give a perfectly symmetric electron delocalization system with no significant bond alternation.

Therefore, cyanine dyes are characterized by narrow absorptions with high absorption coefficients (log ε > 5 M -1 cm -1 ). As a rule of thumb, every addition of vinylene unit (CH=CH) on the π-conjugated bridge induces an approximate 100 nm bathochromic shift, to a limit length of 9 to 11 as further extension of the chain cause severe distortion (Peierls-type) resulting in a loss of molecular symmetry. [START_REF] Tolbert | Beyond the cyanine limit: Peierls distortion and symmetry collapse in a polymethine dye[END_REF] Consequently, the most published NIR cyanine dyes are heptamethine cyanines (7 CH units in the π-bridge). Notably, the huge majority of the Cy7 dyes were developed based on Indocyanine Green (ICG), a dye authorized by the US "food & drug administration" (FDA) agency as a clinical agent in 1954 (Figure 1 -10). Consequently, ICG has been widely applied in biological researches, including sentinel lymph node mapping, [START_REF] Miyashiro | Detection of sentinel node in gastric cancer surgery by indocyanine green fluorescence imaging: comparison with infrared imaging[END_REF][START_REF] Kitai | Fluorescence navigation with indocyanine green for detecting sentinel lymph nodes in breast cancer[END_REF] assessment of lymphatic vascular structure and function, [START_REF] Unno | Quantitative lymph imaging for assessment of lymph function using indocyanine green fluorescence lymphography[END_REF][START_REF] Rasmussen | Lymphatic imaging in humans with near-infrared fluorescence[END_REF] vascular repair [START_REF] Holm | Intraoperative evaluation of skin-flap viability using laser-induced fluorescence of indocyanine green[END_REF][START_REF] Holm | Perfusion zones of the DIEP flap revisited: a clinical study[END_REF][START_REF] Holm | Monitoring free flaps using laser-induced fluorescence of indocyanine green: a preliminary experience[END_REF] and oncology imaging, [START_REF] Ishizawa | Fluorescent cholangiography using indocyanine green for laparoscopic cholecystectomy: an initial experience[END_REF][START_REF] Alacam | Pharmacokinetic-rate images of indocyanine green for breast tumors using near-infrared optical methods[END_REF] despite its limited quantum yield in aqueous solution, poor photo-stability and high tendency to combine with biological components.

By molecular engineering strategies, many ICG derivatives were developed with improved optical properties for bioimaging. Fixing the heptamethyne chain with a cyclopentyl or cyclohexyl ring can not only stabilize the π-bridge for an improved photostability, but also provide a reduced torsional movement that otherwise causes non-radiative decay. Hence, lots of researches are currently under development to further red-shift the optical properties of ring-locked heptamethyne-cyanines. Functionalization in the central C4′ position offers another possibility for tuning the optical properties by modification of the charge delocalization along the π-framework. [START_REF] Pascal | Expanding the polymethine paradigm: evidence for the contribution of a bis-dipolar electronic structure[END_REF] Heptamethyne-cyanines with alkyl-amino or aryl-amino group exhibit larger Stokes shift (> 140 nm) and enhanced fluorescence, while creation of a C-C(Ar) bond at the same position enhanced the stability. Attachment of C4′-O-aryl or C4′-S-alkyl linkers is by far the most commonly used approach for bioconjugation or introduction of various sophisticated groups for the design of stimuli responsive probes (pH, metal ion, anions, uncaging, and so on…). [START_REF] Changhong | Review on near-infrared heptamethine cyanine dyes as theranostic agents for tumor imaging, targeting, and photodynamic therapy[END_REF][START_REF] Siriwibool | Near-infrared fluorescent pH responsive probe for targeted photodynamic cancer therapy[END_REF] Such as dye IR800-CW (Figure 1 -11, a), it has been widely used to label numerous of biomolecules after functionalization with a NHS (IR800-CW-NHS Ester) or a maleimide reactive group (IRDye® 800CW Maleimide) (Figure 1 -11, b and c). [START_REF] Marshall | Single-dose intravenous toxicity study of IR-dye 800CW in Sprague-Dawley rats[END_REF][START_REF] Sampath | Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer[END_REF][START_REF] Sevick-Muraca | Quality analysis of in vivo near-infrared fluorescence and conventional gamma images acquired using a dual-labeled tumor-targeting probe[END_REF] Extension of the π-conjugation system remains the main approach for further red-shift, but few examples of higher length cyanines are reported. [START_REF] Bricks | Molecular design of near infrared polymethine dyes: a review[END_REF] ONITCP and ODNITCP (Figure 1 [START_REF]Fluorescence quantum yields of a series of red and near-infrared dyes emitting at 600-1000 nm[END_REF] Owing to the longer π-conjugated systems, the dyes showed red-shifted emissions up to 900 nm. However, their fluorescence quantum yields are low, for an enhanced internal conversion and a narrowed HOMO-LUMO energy gap.

π-extension can also be achieved by ring fusion taking a benzo[cd]indole motive instead of a benzo[e]indole for instance, such as the commercially available dyes IR1048, IR1050 and IR1051. In this way, Zhang et al. redesigned an old dye family by adding sulphonic groups for water solubility. Compound FD-1080 show a remarkable NIR-II emission (λabs/λem = 1064/1080 nm, Φ = 0.31%), together with excellent aqueous solubility. Though the quantum yield is low, it can be increased to 6% by forming the FD-1080-FBS complex with fetal bovine serum FBS. [START_REF] Li | An efficient 1064 nm NIR-II excitation fluorescent molecular dye for deep-tissue high-resolution dynamic bioimaging[END_REF] Figure 1 -12. Chemical structures of ONITCP, ODNITCP, FD-1080, Flav7 and Indz-OMe-C5 dyes.

Heterocycle modification is an alternate approach and represents a promising avenue toward stable NIRemitting polymethine dyes. Extending heterocycle conjugation, varying the heteroatom from nitrogen to oxygen to other chalcogens, or adding electron-donating groups (or combination with other moieties) have been shown to bathochromically shift polymethine dyes. Thus, modified flavylium (with oxygen heterocycles) or thioflavylium (with sulfur heterocycle) polymethine fluorophores, inspired by older dyes IR-26, IR-1048, IR-1051 or IR-1061, [START_REF] Kopainsky | Lifetime, photostability, and chemical structure of IR heptamethine cyanine dyes absorbing beyond 1 μm[END_REF] were recently reported for bio-imaging in the NIR-II window.

Flav7, a dimethylamino flavylium anthocyanin dye displays fluorescence emission wavelength peaking at 1045 nm for a molar extinction coefficient of 236,000 L . mol -1. cm -1 , and a remarkable 5% Φ in solution. [START_REF] Cosco | Flavylium polymethine fluorophores for near-and shortwave infrared imaging[END_REF] Finally, Delcamp's group reported recently the use of a novel donor group, pyrrolo[1,2-a]pyridine, to build new cyanine fluorophores of various bridge lengths, showing very red-shifted optical properties compared to the "classical" indo-cyanine. [START_REF] Gayton | Indolizine-cyanine dyes: near infrared emissive cyanine dyes with increased Stokes shifts[END_REF] Indeed, a pentamethine cyanine (Indz-OMe-C5), with an emission at around 900 nm is reported paving the way to a completely new molecular engineering.

Some Other Organic NIR-II-emitting Dyes

Scheme 1 -4. General structure of BBTD.

Despite the high interest of emission in the NIR-II window for bioimaging, very few fluorophores have been designed meeting the study in this wavelength range. It is only very recently that the first specific dyes have appeared and, to date, they are almost all based on the benzobisthiadiazole core (BBTD) (Scheme 1 -4) as conjugated π system and having a D-A-D structure. Such compounds were initially designed as NIR fluorophores for non-doped NIR OLEDs. [START_REF] Qian | Simple and efficient near-infrared organic chromophores for light-emitting diodes with single electroluminescent emission above 1000 nm[END_REF] Theoretical calculations have since shown that the existence of a hypervalent structure leads to a much lower LUMO level and plays a key role in the red-shift of fluorescence emission in the NIR-II region. [START_REF] Zhou | Theoretical exploitation of acceptors based on benzobis(thiadiazole) and derivatives for organic NIR-II fluorophores[END_REF] Figure 1 -13. Chemical structures of CH1055, CH1055-PEG and synthesis of CH1055-affibody (water as the solvent). [START_REF] Antaris | A small-molecule dye for NIR-II imaging[END_REF] Dai's group [START_REF] Antaris | A small-molecule dye for NIR-II imaging[END_REF] reported the NIR dye CH1055 and substantially increased its water solubility by introducing four polyether glycol (PEG) chains (Figure 1 Chemical structure of dye CH1055-4Glu-AE105 and its application on tumor surgery. [START_REF] Antaris | A small-molecule dye for NIR-II imaging[END_REF] Further shifting to higher wavelength (up to 1100 nm) was achieved through extension of the πconjugation system by adding an electron rich thiophene between the BBTD core and the nitrogen donor [START_REF] Sun | Novel bright-emission small-molecule NIR-II fluorophores for in vivo tumor imaging and imageguided surgery[END_REF] or using a fluorene to further substitute the N,N-diphenylamino group (Figure 1 -15). [START_REF] Yang | Rational design of molecular fluorophores for biological imaging in the NIR-II window[END_REF] Figure 1 -15. Chemical structures of Q4 and IR-FT dyes (DMSO as the solvent).

Interestingly, theoretical calculations predicted that further red-shift would be possible provided larger ring-fused (up to 5 rings) systems are used, but still encompassing hypervalent BBTD. This opens the way to future screening out of potential candidates for excellent NIR-II molecular fluorophores. What the authors do not mention, on the other hand, is the stability of these extended motives towards oxidation, which should not be very high. [START_REF] Zhou | Theoretical exploitation of acceptors based on benzobis(thiadiazole) and derivatives for organic NIR-II fluorophores[END_REF] 1.

Push-pull Dipolar Dyes

Scheme 1 -5. General representation of a dipolar fluorophore pattern.

Push-pull dipolar dyes are characterized by a D-π-A scaffold (Scheme 1 -5) in which an electron donor group (D) is linked to an electron acceptor group (A) by a π-conjugated framework. This kind of organic dyes have been widely used in material science such as optoelectronic, [START_REF] Dalton | Electric field poled organic electro-optic materials: state of the art and future prospects[END_REF] functional polymers [START_REF] Cho | Recent progress in second-order nonlinear optical polymers and dendrimers[END_REF] and devices based on nonlinear optical effects, [START_REF]Nonlinear optical polymeric materials: from chromophore design to commercial applications[END_REF] etc.

Dipolar dyes have a particular photophysical characteristic: the absorption of a photon by the chromophore is accompanied by a charge transfer from the donor to the acceptor, namely intramolecular charge transfer (ICT). Optical properties will therefore depend on the strength of the D/A pair, the length and the type of the conjugated bridge. Thus, spectroscopic modulation is quite easy to perform by changing the strength of the D/A pair or the conjugated bridge. Normally, increasing the strength of the D/A pair, or elongating the bridge will induce a red-shift in the optical properties. [START_REF]Fundamental aspects of property tuning in push-pull molecules. Bureš[END_REF][START_REF] Harris | Dual-use chromophores for photorefractive and irreversible photochromic applications[END_REF][START_REF] Würthner | Solvent effect on color, band shape, and charge-density distribution for merocyanine dyes close to the cyanine limit[END_REF] Another characteristic of dipoles is their strong Stokes shift which results from the large difference between the ground state (neutral) and the excited state (zwitterionic). In addition, the Stokes shift is increased in polar solvents by solvatochromism. Comparatively to the other dyes families presented before, dipolar fluorophores often have moderate (molar absorption coefficient between 20,000 and 60,000 L . mol - 1. cm -1 ) but broad absorptions, so it can in theory be difficult to obtain high brightness (B = Φ . ε). However, a large Stokes shift avoids the reabsorption of higher energy photons emitted by neighboring fluorophores.

The larger the Stokes shift, the easier it is to distinguish a photon from the excitation source from a photon emitted by the fluorophore and thus to collect the entire fluorescence spectrum. A large Stokes shift also means that the emission is considerably shifted into the red or even the near-infrared for an absorption in the visible.

Finally, owing to the strong ICT dipolar dyes generally possess good two-photon absorption (2PA)

properties. The most characteristic feature in the 2PA properties in non-centrosymmetric D-π-A molecules is that excited states that are one-photon allowed states are also two-photon allowed. Consequently, the maximum 2P occurs at wavelengths exactly twice the maximum absorption wavelength, so NIR (800-1200 nm) for an absorption in the visible (400-600 nm), making it possible to use far-red/NIR for excitation by 2PA. a). [START_REF]Potentiometric dyes: imaging electrical activity of cell membranes. Loew[END_REF][START_REF] Zochowski | Imaging membrane potential with voltage-sensitive dyes[END_REF] The same fluorophores, because of their high non-linear optical (NLO) properties, were later used for fast-response voltage-sensitive second-harmonic generation microscopy combined to 2P microscopy (Figure 12345678910111213141516b). [START_REF] Obaid | Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis[END_REF][START_REF] Wuskell | Synthesis, spectra, delivery and potentiometric responses of new styryl dyes with extended spectral ranges[END_REF] In order to improve the fluorophore NLO properties, but also to red shift absorption and emission maxima to the NIR, considerable molecular engineering has been performed, naturally by playing on the nature and extent of the system-conjugated, incorporating groups such as amino-oligothiophene or porphyrine cycles. [START_REF] Reeve | Dyes for biological second harmonic generation imaging[END_REF] In the group, we designed amphiphilic far-red emissive dipolar fluorophore based on the strong electronacceptor group dicyanoisophorone (Figure 1 -17) for cell membrane imaging. Using copper(I) catalyzed alkyne-azide cycloaddition, we introduced saccharide moieties as water solubilizing groups to create the amphiphilicity. The position and the number (di-or trisaccharide) of saccharide structures were varied and very interesting fluorophores results were obtained with emission up to 694 nm in water. [START_REF] Redon | Red emitting neutral fluorescent glycoconjugates for membrane optical imaging[END_REF] Figure 1 -17. Chemical structures of the amphiphilic far-red emissive dipolar fluorophore dyes reported by our group (water as the solvent). [START_REF] Redon | Red emitting neutral fluorescent glycoconjugates for membrane optical imaging[END_REF] Moerner and Twieg used the strong 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (TCF) acceptor group in small push-pull structures (DCDHF), in which it is separated from the donor (a dialkylamino group) by different π-rich conjugated networks. Like this, they design different series of far-red emitting compounds for single-molecule imaging in cells, amongst which some photo-activable probes were used for super-resolution microscopy. [START_REF] Lord | DCDHF fluorophores for single-molecule imaging in cells[END_REF][START_REF] Lord | A photoactivatable push-pull fluorophore for single-molecule imaging in live cells[END_REF] Recently, using a 2-(4-methoxyaryl)-indolizine unit as the electron donor, the TCF group as the electron acceptor and a alkene-thiophene spacer as the conjugated π-bridge, Delcamp et al. [START_REF] Mcnamara | Donor-acceptor-donor thienopyrazines via Pd-catalyzed C-H activation as NIR fluorescent materials[END_REF] synthesized a push-pull dye AH25 (Figure 1 -18, b) with an unprecedented strong ICT.

For this reason, AH25 has an absorption peak at 925 nm and emission in the NIR-II (λem = 1050 nm) with a Stokes shift of 180 nm.

The dipolar structure is also particularly interesting for the design of solid-state emitting fluorophores and for fluorophores displaying aggregation-induced emission (AIE) properties. Indeed, interactions between permanent dipoles can force particular aggregation patterns necessary to observe a solid-state emission.

Also, the large Stokes shift characterizing most often the fluorescence of these compounds means that the spectral overlap between absorption and emission is minimal, which is an advantage for solid-state emission because it limits reabsorption phenomena and de-excitations by Förster-type energy transfer. Ben Zong Tang's group is more and more interested in dipolar fluorophores presenting an interesting fluorescence in aggregates, especially for applications in biology and imaging. [START_REF]Fumaronitrile-based fluorogen: red to near-infrared fluorescence, aggregation-induced emission, solvatochromism, and twisted intramolecular charge transfer[END_REF][START_REF] Wang | Facile synthesis of red/NIR AIE luminogens with simple structures, bright emissions, and high photostabilities, and their applications for specific imaging of lipid droplets and image-guided photodynamic therapy[END_REF][START_REF] Zhang | A photostable AIE luminogen with near infrared emission for monitoring morphological change of plasma membrane[END_REF][START_REF] Wang | Highly efficient photosensitizers with far-red/near-infrared aggregation-induced emission for in vitro and in vivo cancer theranostics[END_REF] Most of the fluorophores they reported are based on the N,N-diphenylamino-group as electron-donor part. Such group is called by the authors AIEgen, i.e. inducing AIE properties. Various electron-acceptor part and π -bridge were introduced affording fluorophores having emission up to 700 nm in solution and in the aggregate-state, and also increased 2PA cross-section between 800 and 1000 nm (DCX, Figure 1 -19, a). Similarly, Guo, Zhu and co-workers obtained small dipole QM emitting in the solid up to 721 nm using a quinoline-malononitrile electron-acceptor motive and a thiophene bridge. [START_REF] Shao | Far-red and near-IR AIE-active fluorescent organic nanoprobes with enhanced tumor-targeting efficacy: shape-specific effects[END_REF] Before Ben Zong Tang, Blanchard-Desce's group reported very similar fluorophores encompassing a bis-thiophene or thienothiophene units in the π-conjugated bridge and thiobarbiturate or dicyanovinylidene end pull group. Emission up to 800 nm in NPs (3-HIFONs) was obtained which is very interesting for in vivo imaging despite a low quantum-yield (Φ = 2%). [START_REF] Shao | Far-red and near-IR AIE-active fluorescent organic nanoprobes with enhanced tumor-targeting efficacy: shape-specific effects[END_REF][START_REF] Campanini | Amplified two-photon brightness in organic multicomponent nanoparticles[END_REF][100] Besides triphenylamine, other donor groups include dimethylamine or N-ethylcarbazole. Singh et al.

synthesized charge-transfer fluorophores with these two groups and various electron-acceptors. [START_REF] Singh | Tuning solid-state fluorescence to the near-infrared: a combinatorial approach to discovering molecular nanoprobes for biomedical imaging[END_REF] These chromophores showed intense fluorescence in aggregates reaching a 20% efficiency at 740 nm (CbV9).

Sekar and Lanke also use the N-ethylcarbazole group in association with the dicyanoisophrone (NECDCIP). [START_REF] Lanke | Aggregation induced emissive carbazole-based push pull NLOphores: synthesis, photophysical properties and DFT studies[END_REF] Finally, some dipolar boron complexes of 2'-hydroxychalcones and hemicurcuminoide (HBD) were described by D'Aléo et al. (the -OBF2O-unit is a strong electron-acceptor group). [START_REF] D'aléo | Boron difluoride hemicurcuminoid as an efficient far red to near-infrared emitter: toward OLEDs and laser dyes[END_REF] Emission wavelengths beyond 700 nm and even up to 855 nm have been observed in the solid-state for some of these remarkable compounds, although quantum yields are not very high (Φ = 1-3%).

Following its initial work on dicyanoisophorone chromophores, our group has been working on the development of different series of small push-pull dipolar dyes displaying emission properties in solution, but also in the solid state (crystal and aggregates due to aggregation-induced properties). So far, several families of far-red-to NIR-emitting dyes have been developed. [START_REF] Massin | Near-infrared solid-state emitters based on isophorone: synthesis, crystal structure and spectroscopic properties[END_REF][START_REF] Liao | Solid state red biphotonic excited emission from small dipolar fluorophores[END_REF][START_REF] Redon | Tuning the solid-state emission of small push-pull dipolar dyes to the far-red through variation of the electron-acceptor group[END_REF] In particular, we focused on the investigation of new electron-withdrawing groups.

During the course of his PhD, Zheng Zheng designed and synthesized two series of NIR dyes (a1-a11, Figure 1 -20) with 9,9-diethyl-9H-fluorene or fluorene as the conjugated π bridge, which was connected to either a diphenylamine or a carbazole donor group. Varying the electron acceptor part allows us to reach emission up to 800 nm in solution in apolar solvent and in the solid state (λem = 595~807 nm, Φ from <0.01 to 0.2). Significantly, all these dyes showed good 2PA properties between 900 and 1200 nm, thus are able to be excited by light in the NIR-II. [START_REF]Development of far-red / near-Infrared luminescent chromophores and nanoparticles for in vivo biphotonic applications[END_REF][108][109] Zheng Zheng also encapsulated the most promising fluorophore a10 in silica-pluronic NPs (vide infra part Dye-loaded F127-SiO2 NPs). He showed that the emission properties in this kind of NPs were the same as those obtained in apolar solvent. So, NPs with very brightness (Φ = 39 % at 650 nm) were obtained and in vivo 2P microscopy performed. [108] For the solid-state emissive fluorophores, Zheng Zheng obtained nanoaggregates NPs of roughly 80-100 nm by solvent shifting reprecipitation and stabilization by pluronic F127. The preparation method was later optimized by Maxime Rémond during his PhD. [START_REF]Développement de nouveaux chromophores dipolaires pour l'imagerie de fluorescence et l'imagerie photoacoustique[END_REF]111] By replacing the fluorene unit for a bithienopyrrole moiety, and using a triphenylamine group as the electron donor, Maxime Rémond obtained another series of NIR-emitting dyes (BTN-based dyes) with more red-shifted absorption and emission, as well as much higher quantum yields compared to the fluorenebased dyes (Figure 1 -21). Nevertheless, all the BTN dyes are prone to form non-emissive aggregates in both aqueous and organic solvents due to the poor solubility, as a consequence, they could hardly be incorporated efficiently into NPs. [START_REF]Développement de nouveaux chromophores dipolaires pour l'imagerie de fluorescence et l'imagerie photoacoustique[END_REF] Figure Principle of PA imaging. [START_REF] Wang | A practical guide to photoacoustic tomography in the life sciences[END_REF] PA imaging technology based on optical excitation and acoustic detection is of merits from both fluorescence imaging and ultrasonic imaging technologies. On the one hand, the acoustic signal scattering in biological tissues is two to three orders of magnitude lower than that of optical signal, the PA imaging depth is thus only limited by the penetration depth of the excitation. A 5-6 cm of depth can be reached while keeping the laser power harmless. Consequently, PA imaging technology possesses a greater imaging depth in comparison to fluorescence imaging. [113,[START_REF] Esenaliev | Sensitivity of laser opto-acoustic imaging in detection of small deeply embedded tumors[END_REF] On the other hand, the variation in the form of the input and output signals allow a minimized background interference, which is beneficial for higher contrast than pure ultrasonic imaging.

Dyes for PA Imaging

Innovation of the PA imaging instrument is by no means easy; this makes optimization of the PA contrast agents more significant in PA imaging. Hemoglobin as a biogenic component has played an essential role in development of PA imaging technic. However, it is restricted by short absorption wavelength and discontinuity of blood vessels. [115][116][117][118][119] Accordingly, various exogenous contrast agents with strong absorption in NIR region have been used in PA imaging over the past decades, including small NIR organic dyes, gold and polymer nanostructures, etc. (Figure 1 -23). As far as organic dyes are concerned, most of them that have been successfully used for PA imaging are old known compounds, such as ICG and Evans blue (BE).

[111, [120] Indeed, to our best of knowledge, no specific dye engineering for PA has been done yet as the link between molecular structure and PA signal generation remains quite opaque. In light of the following examples, we can just say that dyes with strong absorption in NIR and weak fluorescence emission may be promising for PA imaging. The relationship between the absorption and PA signal of ICG revealed its good PA response to NIR excitation (Figure 1 -24, a). [121] However, ICG can hardly be directly used for in vivo PA imaging due to its short blood half-life of a few minutes and high tendency to bind with many proteins in plasma. [122] This promoted the generation of many ICG-labelled polypeptides [123,124] and antibodies. [125][126][START_REF] Wilson | Intraoperative resection guidance with photoacoustic and fluorescence molecular imaging using an anti-B7-H3 antibody-indocyanine green dual contrast agent[END_REF][128] Furthermore, the fluorescent and PA properties make the use of ICG possible for bimodal imaging. Wang et al. imaged the sentinel nodes of a mouse through both fluorescence and PA imaging (Figure 1 -24, b and c). [120] Particularly, by comparison of the lateral resolution of the two imaging techniques as a function of the imaging depth, PA image presented more than ten times higher resolution than fluorescence image at a depth of a few millimeters.

BE is a biological dye with high affinity to albumin (Figure 1 -25). The BE-albumin complex as a PA contrast agent in serum presented higher sensitivity than hemoglobin without the issue of blood discontinuity. [120] Specifically, by tracing the diffusion of the free BE molecules in vascular system after injection, it is possible to monitor blood-brain barrier, assess potential leaks caused by head trauma as well as measure drug transfer efficiency. [129] Figure 1 -24. (a) optical (triangles) and acoustic (rounds) absorption of ICG [121] ; (b) depth-depended bimodal imaging of sentinel nodes (SLNs) using ICG as contrast agents and PA images and the corresponding three 1D profiles taken from along the dotted lines in the maximum amplitude projection (MAP) images; (c) fluorescence images and the corresponding three 1D profiles taken from along the dashed lines. [130] Figure 1

-25. (A) Chemical structure of Evans blue; (B) Combined PA imaging of hemoglobin (in red, excitation 570 nm) and

Evans blue (in blue, excitation 610 nm) immediately after injection; [131] (C) PA imaging following diffusion of Evans blue into the extravascular system after injection. [120] Cyanine dyes with strong absorptions in NIR region are considered promising for PA imaging. Onoe et al. [132] reported a symmetric cyanine IC7-1-Bu and compared it to ICG in terms of in vivo fluorescence Based on this study, the same group further developed a series of asymmetric cyanines IC-n-T. [133] A 4nitrobenzyl alcohol derivative as a triplet-state quencher (TSQ) was connected to IC7-1-Bu, leading to the enhanced photostability of IC-n-T dyes since the production of singlet-oxygen was inhibited. Additionally, neither the introduction of the 4-nitrobenzyl alcohol derivative nor the increase of the alkyl chain length exerted significant influence on other optical properties of the dyes. With the highest affinity to albumin, which helps improve the solubility and targeting ability of dyes in in vivo imaging, IC-5-T presented efficiently the mouse tumor by PA imaging.

NIR dye-loaded NPs for fluorescence or PA imaging

NIR organic dyes are promising candidates for preparation of optical contrast agents, however, they are restricted by poor water solubility in the real applications. [START_REF] Koide | Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging[END_REF] Structural modifications are widely used methods to overcome this issue, for example, on can introduce water-soluble groups such as ionic groups (anionic or cationic), [START_REF] Kohl | Towards highly fluorescent and water-soluble perylene dyes[END_REF][START_REF] Alexander | Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates[END_REF] polar segments (polyether chain or polyethylene ethanol) [START_REF] Williams | Perylene polyether hybrids: highly soluble, luminescent, redox-active dyes[END_REF]137] and watersoluble peptides, [START_REF] Zhu | Highly water-soluble, near-infrared emissive BODIPY polymeric dye bearing RGD peptide residues for cancer imaging[END_REF][START_REF] Heyer | Highly fluorescent and water-soluble diketopyrrolopyrrole dyes for bioconjugation[END_REF] or functionalize them with biomolecules especially when a specific targeting is required. [140] Nevertheless, these approaches often face difficulties in dye purification and functionalization processes.

Inspired by the drug-loaded vehicles for drug delivery, dye-loaded NPs have been developed to improve the water solubility of organic dyes, particularly for in vivo optical imaging. [141,142] Normally, NPs are prepared by encapsulation or covalent linkage of the dyes to water-soluble polymers. By doing so, in addition to an enhanced water-solubility, the dye-loaded NPs may obtain many other advantages, such as excellent brightness or PA intensity because of improved dye concentration, enhanced photo-stability due to blocking of the polymers that helps prevent the dyes from contacting with some reactive species (i.e.

Reactive Oxygen Species), as well as potential targeting capability if taking advantage of the particle surface modifiability.

On the contrary, by forming water soluble NPs, some dyes may suffer fluorescence quenching due to ππ stacking, particularly for the NIR dyes characterized by a long π-conjugation. For this reason, different kinds of dye-loaded NPs have been developed to meet the properties of the dyes for optical imaging. In the following section we will mainly present two kinds of dye-loaded NPs that we used in our research: micelle/silica encapsulated (F127-SiO2) NPs and polymer NPs

Dye-loaded F127-SiO2 NPs

F127-SiO2 NPs are NPs formed by self-assembly of the commercial amphiphilic polymer Pluronic F127 (F127) stabilized by a cross-linking induced by growth of a silica crust within the hydrophobic core of the micelle. F127 is a non-ionic triblock polymer having a central hydrophobic poly-propylene-oxide (PO) core capped by two hydrophilic poly-ethylene-oxide (EO) entity. With a EO100-PO65-EO100 constitution, F127 can encapsulate hydrophobic organic dyes within the NP core during micellization and simultaneously, leave the dyes separated by the PO segments. As a result, the encapsulated dyes present photophysical properties similar to the dyes dissolved in the mixture of chloroform and toluene (v/v=1/1). The synthetic process of micelle/silica encapsulated NPs was first proposed by Huo et al. [START_REF] Zanarini | Iridium doped silica-PEG nanoparticles: enabling electrochemiluminescence of neutral complexes in aqueous media[END_REF] , as shown in Figure 1 -27, a certain amount of F127 and dyes were premixed together, followed by micellization, silica growth and dialysis to form the NPs monodispersed in aqueous solution. The size of the silica core was measured to be 10 nm by transmission electron microscopy (TEM) and whole size about 25 nm by dynamic light scattering (DLS). [START_REF] Huo | A new class of silica cross-linked micellar core-shell nanoparticles[END_REF] Figure 1 -27. General process of preparation of F127-SiO2 NPs. [START_REF]Development of far-red / near-Infrared luminescent chromophores and nanoparticles for in vivo biphotonic applications[END_REF] Indeed, the F127 micelles were widely used as drug delivery carriers for their long lifespan in blood due to the stealth given by the highly biocompatible PEGs surface, as well as the ease of dissociation under critical micelle concentration (CMC). But for fluorescence imaging, the dissociation of the NPs would cause dye leakage and bring tremendous negative impact on the imaging process, thus a rigid silica shell is needed.

By addition of tetraethyl orthosilicate (TEOS), the formed Si-O-Si network can not only fix the NPs to prevent the dye from leakage, but also improve the photostability of the dyes by protecting them from possible quenchers, such as molecular dioxygen.

In [145] and a NPs concentration in the suspension before dialysis of 67 μM. [START_REF] Valenti | A versatile strategy for tuning the color of electrochemiluminescence using silica nanoparticles[END_REF] Figure 1 -28. Depict of Rampazzo's study: energy transfer on F127-SiO2 NPs based on the covalently linked dye R. [145] At last, the PO segment of the Pluronic counts for the dyes encapsulation and the core size of the NP (by TEM), the EO segment has influence on the whole size of the NP (by DLS), thus it is possible to optimize the NP size and dye loading capacity by changing the units of the PO and EO segments of Pluronic.

Biffi et al. [START_REF] Biffi | Multiple dye-doped NIR-emitting silica nanoparticles for both flow cytometry and in vivo imaging[END_REF] prepared a F127-SiO2 NPs based on Cy5.5 (Figure 1 -29). Triethoxysilane-modified Cy5.5

was grafted onto the NPs during hydrolysis and condensation of the silica precursor TEOS. The NPs presented high stability and maximum absorption and emission at 700 nm and 750 nm, respectively. The NPs were successfully used for in vivo tumor imaging. Notably, with a neutral and hydrophilic surface formed by the external PEG chains, micelle/silica NPs can not only escape from the uptake of the mononuclear phagocyte system to have a long half-life in blood circulation, but also be grafted with some specific species or ligands for further applications. [148] As shown in Figure 1 -30, Soster et al. [START_REF]Long-circulating near-infrared fluorescence core-cross-linked polymeric micelles: synthesis, characterization, and dual nuclear/optical imaging[END_REF] used the mixture of normal F127 and carbonyl-modified F127 as encapsulation matrix prepared the micelle/silica NPs with carbonyl groups on their surface, then peptides were covalently linked with the NPs through condensation reaction. The formed NPs were able to visualize sub-millimetric metastases. imaging. [START_REF] Biffi | Multiple dye-doped NIR-emitting silica nanoparticles for both flow cytometry and in vivo imaging[END_REF] Figure 1 -30. General structure of CGIYRLRS and CGVYSLRS peptides functionalized F127-SiO2 NPs. [148] Our group previously prepared a10@F127-SiO2 NPs based on the fluorene dye a10, with high brightness (Φ = 39 % at 650 nm) and monodispersed size around 22 nm in saline. The NPs were successfully used for mouse tumor imagination by two-photon excitation (Figure 1 -31). [START_REF]Development of far-red / near-Infrared luminescent chromophores and nanoparticles for in vivo biphotonic applications[END_REF]108] These NPs proved to be perfect blood-pool contrast agents staying in the vascular system for a long time, without leakage of the NPs near a tumor. Two-photon vasculature imaging of a10@F127-SiO2 NPs. [START_REF]Development of far-red / near-Infrared luminescent chromophores and nanoparticles for in vivo biphotonic applications[END_REF]108] In the general protocol of the first strategy, polymers and dye monomers are pre-dissolved in a watermiscible organic solvent (acetone, acetonitrile, etc.), followed addition of water to the mixture to induce the aggregation of the dye monomers through rapid diffusion of the organic solvent into the aqueous phase.

Dye-loaded Polymer NPs

Then the dye polymerization will give rise to a rigid dye-polymer core incorporated by the water-soluble polymers on its surface. By doing this, the polymer NPs formed are homogeneously dispersed in water, thus the particle suspension can be directly used for bio-imaging. The dye monomers are either small polymerizable segments, such as pyrrole, thiophene, or normal dyes which are modified by polymerizable groups (Figure 1 Chemical structures of several organic polymers used in PA studies. [150][151][152][START_REF] Chen | Highly absorbing multispectral near-infrared polymer nanoparticles from one conjugated backbone for photoacoustic imaging and photothermal therapy[END_REF][START_REF] Zhu | D-A polymers for fluorescence/photoacoustic imaging and characterization of their photothermal properties[END_REF] Zha et al. [150] obtained the PPy NPs with monodisperse size of about 46 nm through oxidation polymerization of pyrrole using FeCl3 as an initiator and polyvinyl alcohol (PVA) as a stabilizer. The strong absorption around 800 nm made it possible for PPy NPs to visualizeby PA microscopy the chicken breast muscles at depth up to 4.6 cm, as well as a higher clarity in a mice brain imaging in comparison to hemoglobin (Figure 1 -35). SP1 and SP2 are well known semiconductor polymers in photovoltaics with strong absorptions in NIR region. Pu et al. [131] mixed the ROS sensitive dye IR775S with SP1 to form the hybrid polymer NP that is able to detect in vitro the ROS by PA imaging (Figure 1 -36, a and b). Liu et al. [152] designed a non-radiative NPs with a wide absorption between 700 nm and 850 nm by synthesized the PFTTQ polymer and then coprecipitated it with DSPE-PEG2000. These NPs are found much more resistant to photobleaching than the gold nanobatons under excitation at 800 nm with a fluence of 15 mJ/cm². The polymer DPP-DT with absorption around 770 nm was prepared by Chen et al. [START_REF] Chen | Highly absorbing multispectral near-infrared polymer nanoparticles from one conjugated backbone for photoacoustic imaging and photothermal therapy[END_REF] After co-precipitation with the PEGylated polystyrene, the stabilized NPs were successfully used in photothermal therapy to treat tumors (Figure 1 -36, c). [131] (C) Thermal and PA imaging of a tumor containing DPP-DT. [START_REF] Chen | Highly absorbing multispectral near-infrared polymer nanoparticles from one conjugated backbone for photoacoustic imaging and photothermal therapy[END_REF] The second strategy relies on the preparation of dye-loaded polymers. Synthetic design of the polymers that bear functions to allow dye loading is the first step, then the correspondingly modified dyes will be grafted to form the dye-loaded polymers. Generally, the dye-polymer conjugation is performed in organic solvent, thus the formed polymers have to be extracted and saved in form of a solid state. Finally, selfassembly of the polymers in water leads to the dye loaded polymer NPs. Additionally, the size of the NPs can be regulated from several nanometers to 100 nm by simply control of the length of polymer. [155][START_REF] Fery-Forgues | Fluorescent organic nanocrystals and non-doped nanoparticles for biological applications[END_REF][START_REF] Kandel | Incorporating functionalized polyethylene glycol lipids into reprecipitated conjugated polymer nanoparticles for bioconjugation and targeted labeling of cells[END_REF][START_REF] Kumar | Near-infrared phosphorescent polymeric nanomicelles: efficient optical probes for tumor imaging and detection[END_REF][START_REF] Lee | Facile synthesis of fluorescent polymer nanoparticles by covalent modification-nanoprecipitation of amine-reactive ester polymers[END_REF][START_REF] Li | Highly emissive PEG-encapsulated conjugated polymer nanoparticles[END_REF][START_REF] Liu | Red-emitting DPSB-based conjugated polymer nanoparticles with high two-photon brightness for cell membrane imaging[END_REF][162][163][START_REF] Wu | Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting[END_REF][START_REF]Long-circulating near-infrared fluorescence core-cross-linked polymeric micelles: synthesis, characterization, and dual nuclear/optical imaging[END_REF][START_REF] Zhang | Fabrication of aggregation induced emission dye-based fluorescent organic nanoparticles via emulsion polymerization and their cell imaging applications[END_REF] Figure 1 -37. (a) Schematic illustration of fabrication of PEG-PLD-IR825 NPs and (b) the in vivo optical imaging. [START_REF] Pan | Turning double hydrophilic into amphiphilic: IR825-conjugated polymeric nanomicelles for nearinfrared fluorescence imaging-guided photothermal cancer therapy[END_REF] Pan et al. [START_REF] Pan | Turning double hydrophilic into amphiphilic: IR825-conjugated polymeric nanomicelles for nearinfrared fluorescence imaging-guided photothermal cancer therapy[END_REF] primarily prepared IR825 linked amphiphilic block copolymer PEG-PLD-IR825 by [168] Through connection of the polymer poly(maleic anhydride-alt-1-octadecene) (C18PMH) with a long PEG-amine (5 kDa) and a short biamine-PEG (324 Da), Gong et al. [168] obtained the amphiphilic block polymer C18PMH-PEG 5k/PEG 324-NH2 modified with amine. Chlorin e6 (Ce6) were then conjugated to C18PMH-PEG 5k/PEG 324-NH2 by amide groups, and the formed dye-linked polymer C18PMHPEG-Ce6 further encapsulated IR825 to fabricate the IR825@C18PMH-PEG-Ce6 by self-assembly. Finally, containing photothermal agents IR825 photosensitive Gd 3+ chelating agents Ce6, the IR825@C18PMH-PEG-Ce6 can be utilized for fluorescence, PA and magnetic resonance (MR) imaging and PTT and PDT of mice tumors (Figure 1 -38).

Yuan et al. [169] developed a doxorubicin (DOX) delivery system AIE-NPs/DOX with DOX encapsulated by an amphiphilic polymer TPETP-TK-PEG through self-assembly (Figure 1

Objectives

As reviewed above, NIR light is promising for bio-imaging for merits of strong penetration in organisms that favors a deeper imaging, low interference from auto-fluorescence that benefits for a high special resolution, as well as low energy input to minimize bio-damage. Hence, NIR organic dyes as contrast agents are extremely desired for fluorescence or PA imaging. In this regard, the main objective of this thesis is to synthesize high-performance NIR organic dyes.

Push-pull dipolar scaffold is an ideal candidate for design of NIR dyes, given its ease of HOMO-LUMO gap modulation by varying the D/A pairs or the conjugated π bridge. In this context, we will synthesize NIR dyes based on dipolars.

Meanwhile, the problem with water solubility of organic dyes for biological application can be overcame by forming water soluble NPs. On the one hand, F127-SiO2 NPs encapsulating organic dyes within its F127 micelle core can not only provide the dyes excellent water-solubility, but also allow a high brightness for improved bio-imaging sensitivity. Thus, the F127-SiO2 NPs encapsulation strategy will be utilized on NIR emitting dyes for fluorescence bio-imaging. Furthermore, taking advantage of the modifiable surface of F127-SiO2 NPs, we manage to further modify the dye-loaded F127-SiO2 NPs with different moieties. On the other hand, in order to meet the requirement for high-dye-concentration of PA imaging, polymer NPs with organic dyes loaded by dye polymerization or dye grafting, and F127-SiO2 NPs modified by organic dyes on their surface will be developed to maximize the concentration of the organic dyes for PA imaging.

Chapter II. Synthesis of NIR dyes and dye-loaded F127-SiO 2 NPs for in vivo fluorescence imaging 1. Introduction

Previous Work

Dye a10 with good optical properties was previously chosen by our group for fluorescence imaging.

Silica encapsulation strategy was developed to improve the water-solubility of the dye. Finally, the a10@F127-SiO2 NPs with monodispersed size around 22 nm in saline and great brightness successfully imaged a mouse tumor by two-photon excitation. Nevertheless, the same strategy failed for all BTN dyes for their high tendency of forming non-emissive dye aggregates during the F127 micellization process, while their more in red absorption and excellent brightness would be very promising for fluorescence imaging.

Objectives

In light of these observations, we aim at further optimizing the dyes for in vivo fluorescence imaging.

Given the great potential of fluorene dyes@F127-SiO2 NPs for two-photon fluorescence imaging, we will use the molecular engineering strategies that we mentioned before to realize a more red-shifted emission for the fluorene dyes. Meanwhile, taking advantage of the modifiable surface of the F127-SiO2 NPs, bioactive moieties will be grafted to endow the dye-loaded F127-SiO2 NPs potential tumor targeting capability.

Besides, BTN dyes possessing high brightness in NIR region, however, are hardly to be encapsulated by F127 due to poor solubility. In this regard, we will improve their lipophilicity by molecular modification to enable them for preparation of dye-loaded F127-SiO2 NPs for in vivo fluorescence imaging.

Synthesis of TCBZ dyes and TCBZ@F127-SiO2 NPs for fluorescence imaging

Synthetic Design

Accordingly, four steps are needed to obtain a surface functionalized F127-SiO2 NP with more red-shifted fluorene-dye loaded. Firstly, different electron-withdrawing groups will be introduced to modulate the ICT of the dyes for more red-shift emission. Then, normal F127 will be modified by different functions to allow a surface modified F127-SiO2 NPs. After forming the surface modifiable F127-SiO2 NPs with dye loaded, further conjugation with different functionalized moieties will give rise to the final surface-functionalized F127-SiO2 NPs.

Scheme 2 -1. Chemical structures of TCBZ dyes.

Through comparison of the fluorene dyes our laboratory synthesized before, it was found that substitution of the diphenylamine unit by the carbazole unit benefits a higher brightness. For this reason, carbazole was chosen by us for configuration of the new series of dyes. As shown in Scheme 2 -1, different electronwithdrawing groups including the b, c, d, g, f previously applied in the fluorene dyes and the a and c recently developed in our laboratory were used as the acceptors of the new series of dyes. Additionally, the introduction of two tert-butyl groups to the carbazole units may increase the lipophilicity of the dyes, which favors the dye encapsulation process by F127.

As shown in Scheme 2 -2, F127 has two hydroxy groups at both ends of its polymer chain readily to be transformed into carboxyl or azido groups. Using the modified pluronic F127-COOH and F127-N3 as the encapsulation matrices, the formed F127-SiO2 NPs will be modified by carboxyl or azido groups to be able to conjugate with moieties containing amine or alkynyl groups via amidation in presence of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC . HCl) and N-hydroxysuccinimide (NHS) or Cu(I) catalyzed alkyne-azide cycloaddition ("click") reaction.

Scheme 2 -2. Chemical structures of F127, F127-COOH and F127-N3.

As shown in (Scheme 2 -3), two different moieties encompassing an alkyne function were used for the preparation of the surface functionalized F127-SiO2 NPs: a propagyl modified moiety X provided by Dr. S.

Chambert from ICBMS / INSA Lyon (the structure of X will not be disclosed until a patent is issued) and triphylphosphine (TPP) bearing a positive charge.

Scheme 2 -3. Chemical structures of alkynyl modified X and TPP.

Synthesis and Photophysical Properties of TCBZ Dyes

Synthesis of TCBZ Dyes

The synthetic route to TCBZ dyes is depicted in Figure 2 -1. Firstly, the 2-bromo-9,9-diethyl-7-iodo-9Hfluorene 3 and the tert-butyl modified carbazole derivative 4 were synthesized according to our previous protocol from carbazole and tert-butyl chloride with ZnCl2 as the catalyst. [START_REF]Development of far-red / near-Infrared luminescent chromophores and nanoparticles for in vivo biphotonic applications[END_REF] In presence of CuI, 1,10-

Photophysical Properties of TCBZ dyes 2.2.2.1. Absorption and Emission

After obtaining the TCBZ1-TCBZ8 dyes, we firstly studied their absorption and emission properties in chloroform, a solvent of intermediate polarity. All data are gathered in Table 2 -

Sample λabs

[a] ε [b] λem [c]
Φ (%) [d] ν [a] Absorption maxima in nm.

[b] Absorption coefficients in 10 -4 L/mol/cm [c] Emission maxima in nm. [d] Quantum yields determined using rubrene in methanol (Φ = 27 %) as the standard. [170] [e] Stokes shifts in cm -1 . By comparison of the TCBZ dyes with the corresponding fluorene dyes bearing the same acceptors (TCBZ2-a1, TCBZ3-a2, TCBZ6-a4 and TCBZ8-a3), showed in Table 2 -1 using the same color to emphasize, one can notice that the TCBZ dyes have more red-shifted absorptions (around 20 nm) and emissions (around 50 nm), while keeping the very similar extinction coefficients (ε) and quantum yields (Φ). This result indicated our success in improving the optical properties of the dyes by molecular modification strategy.

Solvatochromism

Then the optical properties of TCBZ1-TCBZ8 in solvents of various polarities (cyclohexane, toluene, chloroform and DMSO) were measured. As these dyes are weakly emissive in polar solvent DMSO, the emission spectra are only shown for apolar cyclohexane and toluene in Figure 23. For each compounds of TCBZ1-TCBZ8, only a slight red-shift was observed in absorption with the increasing of the solvent polarity, which is in perfect agreement with previous observation on similar dyes. [START_REF]Development of far-red / near-Infrared luminescent chromophores and nanoparticles for in vivo biphotonic applications[END_REF] As shown in Figure 2 -3, except TCBZ6 which is not emissive in all the tested solvents, all the other dyes exhibited interesting solvatochromic effect in emissions. By varying the solvent from toluene to chloroform, the ICT effect produced distinctively bathochromic-shifted emissions from 76 nm (1880 cm -1 ) for TCBZ1 to 173 nm (4070 cm -1 ) for TCBZ4.

AIE property

AIE refers to the phenomenon which leads a dye solution to become more fluorescent under the aggregation. Given the interesting AIE properties of the fluorene dyes a1-a3, we also studied our new dyes in this context.

AIE measurement is commonly carried out by monitoring the fluorescence variations of the dye solution with a continuous addition of a poor solvent to induce the aggregation of the dye. In our test, water as the poor solvent was added to the solution of TCBZ dyes in acetone, with dye concentration fixed at 5×10 -5 mol.L -1 and water fraction (fw) increasing from 10% to 90%, the absorption and emission spectra of the mixtures were recorded at every fw increase of 10%.

From the results of Figure 234, except TCBZ6 which is weakly emissive in aggregated state, which is not surprising given dye a4, all the dyes showed interesting AIE behaviors in water/acetone mixtures.

Taking dye TCBZ1 as an example, initial addition of water induced a slightly decreased absorption and emission, which can be attributed to the increased solvent polarity by addition of water. Then a sudden decline of absorption was observed when the fw reached 50%, indicating the formation of TCBZ1- To evaluate the AIE properties of TCBZ1-TCBZ8, we collected the photophysical properties of these nanoaggregates into Table 2 -2. All the TCBZ-nanoaggregates showed emission peaks in NIR region with large Stokes shifts above 6000 cm -1 . The quantum yield Φ was determined to be 1%-19% using rubrene in methanol (27%) as the standard. [170] As we expected, TCBZ2, TCBZ3 and TCBZ8 have more red-shifted absorptions and emissions with improved quantum yields compared with those of a1-a3 aggregates.

nanoaggregates
Additionally, the relatively lower fw values (see fw (%) [a] in Table 2 -2), at which the dyes start aggregation, illustrating the increased lipophilicity of our new dyes in comparison to a1-a3.

Sample fw (%) [a] fw (%) [b] λabs [c] λem [d] Φ (%) [a] Water to acetone fractions starting inducing aggregation of the TCBZ dyes in solution [b] Water to acetone fractions at which the highest emissions of the TCBZ dyes in solution appeared.

[c] Absorption maxima in nm. [d] Emission maxima in nm.

[e] Quantum yields determined using rubrene in methanol (Φ = 27 %) as the standard. [170] [f] Stokes shifts in cm -1 .

Table 2 -2. Photophysical properties of TCBZ1-TCBZ8 aggregates in acetone/water mixtures (5.0×10 -5 mol L -1 ).

Solid state fluorescence

We also measured the emissions of TCBZ1-TCBZ8 in powder. The fluorescence spectra are shown in Figure 2 -5, the corresponding data are collected in Table 2 -3. Firstly, all the dyes were found emissive in solid state. Among them, TCBZ2 and TCBZ4 showed the most red-shifted emissions, which is consistent with their performances both in solution and in nanoaggregates. TCBZ7 possessing Φ up to 42% is promising for preparation of NIR solid luminescent materials. Furthermore, our new dyes are of more redshifted emissions and relatively higher Φ compared with a1-a3, thanks to the introduction of two tert-butyl groups that may exerted impact on the dye aggregation in solid. NPs brightness with the dye loading amount (from 0.1 to 0.8% weight ratio).

Preparation and Characterization of TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs

The general procedure for preparation of F127-SiO2 NPs is depicted in Figure 23456. F127 and the appropriate amount of dyes are pre-dissolved in dichloromethane, followed by argon blow to form a thin film. 0.85 N hydrochloric acid is then added to induce the micellization of F127 along with the encapsulation of the dyes within the F127-micelles. TEOS as the silica precursor is added to the suspension to start the silicate cross-linking on the micelles, which is terminated by further addition of diethoxydimethylsilane (DEDMS) to form a silica shell. The hydrochloric acid and impurities are lastly removed by dialysis.

Figure 2 -7 showed the absorption of TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs steadily increase as the dye-loading, the optimal brightness is reached at dye loading of 0.3% and 0.5%, respectively. Then we investigated the properties of the dye-loading optimized TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs (0.3% for TCBZ2 and 0.5% for TCBZ5). As shown in Figure 2345678, TCBZ2@F127-SiO2 NPs showed absorption peak at 475 nm and emission peak at 665 nm with Φ of 8%.

TCBZ5@F127-SiO2 NPs presented absorption and emission maxima at 428 nm and 605 nm, respectively, with Φ of 27%. In dynamic light scattering (DLS) measurements, both TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs exhibited monodispersed sizes around 22 nm in water and saline (Figure 23456789).

According to the particle concentration of 67 μM before dialysis from literature [START_REF] Valenti | A versatile strategy for tuning the color of electrochemiluminescence using silica nanoparticles[END_REF] and the dye concentration of 242 μM for TCBZ2 and 432 μM for TCBZ5, the average dye numbers for each TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs are 3 and 6, respectively (Table 234).

Sample wt% [a]

λabs [b]] λem [c] Φ (%) [d] ν [a] Weight percent of dyes to F127.

[b] Absorption maxima in nm.

[c] Emission maxima in nm. [d] Quantum yields determined using rubrene in methanol (Φ = 27 %) as the standard. [170] [e] Stokes shifts in cm -1 .

[f] Diameters measured by DLS in nm.

Table 2 -4. Photophysical properties of TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs in water. The synthetic routes of F127-COOH and F127-N3 are shown in Figure 2 -10. In order to modify F127

Preparation of Surface-functionalized TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs for in vivo Fluorescence Imaging

Synthesis of F127-COOH and F127-N3

with carboxyl, we firstly introduced a succinic acid moiety by opening succinic anhydride. However, the formed ester groups suffered hydrolysis in the silicate cross-linking process as it is done in acidic condition, so we changed to the second way. Still using commercially available F127, its hydroxyl groups were directly oxidized into carboxyl groups by the catalytic system of 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), KBr and NaClO, [171] F127-COOH was obtained in 60% yield.

Azido modified F127 (F127-N3) was synthesized by substitution of F127-OTs in NaN3 in yield of 94%.

F127-OTs was prepared by substitution of F127 with TsCl in 86% yield. All the modified F127 polymers were carefully purified by precipitation and fully characterized by IR spectrometry and NMR.

2.4.2. Preparation of TCBZ2/5@F127-COOH-SiO2, TCBZ2/5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2 NPs

By simply using the mixture of F127-COOH or F127-N3 with the normal F127 as the dye encapsulation matrices, the formed F127-SiO2 NPs will obtain carboxyl or azido groups on their surface. Followed conjugation with functional moieties will allow them to be further functionalized (Figure 2 -11). It's well known that surface chemistry is critical to NPs properties, especially stability. Rampazzo and Prodi found that it is possible to use up to 50 wt% of F127-COOH to F127 for preparation of F127-COOH-SiO2 before having particle aggregation. [172] We thus firstly studied the effects of surface modification on Rampazzo and Prodi. [172] It is probably due to the difference in the modification degree of F127-COOH we were using. Finally, 10% of F127-COOH was chosen for preparation of TCBZ5@F127-COOH-SiO2 NPs.

In the other hand, no obvious particle growth or aggregation was observed by varying the mass ratios of F127-N3 from 0% to 100% (Table 23456). This demonstrated the negligible influence of azido groups on the particle stability. Therefore, 50% of F127-N3 was used for preparation of TCBZ5@F127-N3-SiO2 NPs. TCBZ5@F127-N3-SiO2 NPs with azido groups modified on the surface are ready to be grafted by functional moieties containing alkynyl groups by click reaction. Thus, by direct addition of the functional moieties (the alkynyl-X or alkynyl modified triphylphosphine TPP) and the suitable "click" catalysts (CuSO4, sodium ascorbate and the THPTA ligand) into the stock solution of TCBZ5@F127-N3-SiO2 NPs (Figure 2 -12), we obtained the target functionalized F127-SiO2 NPs, TCBZ5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2 NPs. Dialysis was performed to remove the catalysts at the end. TCBZ5@F127-SO 2 TCBZ5@F127-COOH-SO 2 TCBZ5@F127-X-SO 2 TCBZ5@F127-TPP-SO 2 Φ (%) [c] ν [d] Size [e] ζ-potential (mv) TCBZ5@F127-X-SiO2

Characterization of Surface-functionalized TCBZ5@F127-SiO2 NPs

24

-0.7 ± 0.3

TCBZ5@F127-TPP-SiO2 23
2.2 ± 0.6

[a] Absorption maxima in nm.

[b] Emission maxima in nm.

[c] Quantum yields determined using rubrene in methanol (Φ = 27%) as the standard. [170] [d] Stokes shifts in cm -1 .

[e] Diameters measured by DLS in nm.

Table 2 -7. Properties of TCBZ5@F127-SiO2, TCBZ5@F127-COOH-SiO2, TCBZ5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2 NPs in water.

We then studied and compared the optical and physical properties of the surface functionalized NPs, TCBZ5@F127-SiO2, TCBZ5@F127-COOH-SiO2, TCBZ5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2. From Figure 2 -13, all the F127-SiO2 NPs presented monodispersed sizes around 22 nm, which are in good agreement with the size previously measured in our laboratory, indicating neither the loading of TCBZ5 nor the modifications of the NPs surface has significant influence on the particle size. Besides, the changes in ζ-potential proved us the successful conjugation of the functional moieties to the particle surface.

As we expected, the modifications of the NPs surface did not alter the optical properties of the TCBZ5@F127-SiO2 NPs, either (Figure 2 -14). Finally, all the four TCBZ5@F127-SiO2 NPs displayed absorption peaks at 428 nm and emission peaks at 605 nm with quantum yields around 27% (Table 234567).

TCBZ5@F127-SiO2 and TCBZ5@F127-X-SiO2 NPs for Biphotonic in vivo Fluorescence Imaging

We thus performed biphotonic in vivo fluorescence imaging using TCBZ5@F127-SiO2 and TCBZ5@F127-X-SiO2 NPs. The NPs suspensions were diluted 3 times by distilled water after dialysis (dye concentration ~ 140 μM) and then sodium chloride was added (0.9 wt%) to form the target solutions for imaging.

Two white CD-1 IGS mice (Écully, France) were sacrificed for this experiment. They were anesthetized by a gas mixture of 1% isoflurane in air with body temperature maintained at 36-37°C using an electric heating pad. 0.1 mL of NPs solution was injected through the caudal vein using a catheter (BD Neoflon TM , 16GA 0.6 x 19mm, Becton Dickinson, Helsingborg Sweden). A commercially available blood pool agent FITC-dextran (70 kDa) (0.01 mL) was also used as reference for blood vessels visualization and preliminary clearance experiments. FITC-dextran is indeed rapidly excreted through the kidneys. FITCdextran was injected either right after the NPs or 10 minutes before animal sacrifice.

A bright fluorescence showing the whole blood vasculature network of a mice ear is visible by a 900 nm excitation immediately after injection of TCBZ5@F127-SiO2 NPs ('red' channel without emission filter other than the dichroic mirror, collecting all wavelengths > 560 nm) and FITC-dextran (green channel, band pass filter 522 ± 50 nm). The fluorescence persists for a long time, as shown in From the two-photon microscopic images of the excised organs (Figure 2 -16), the NPs clearance seems the same for both TCBZ5@F127-SiO2 and TCBZ5@F127-X-SiO2 NPs: the dye is mainly in the spleen and in lower concentration in the liver or kidney. This again explained a long circulation time (at least 2 hours and more) of the NPs in the blood. To further explain, we take the kidney as an example. As shown in pictures b and f, the green channel shows the fluorescence signals from FITC-dextran in the kidney tubules whereas fluorescence from NPs is still in the vascular system (red channel).

Synthesis of EBTN dyes and EBTN@F127-SiO2 NPs for

Fluorescence Imaging

Synthetic Design of EBTN Dyes

Scheme 2 -4. Chemical structures of EBTN dyes.

To optimize the BTN unit, we intended to modify it by two butoxy groups, as it is shown in Scheme 2 -4. On the one hand, the introduction of two alkyl chains can increase the lipophilicity of dyes to make them easier to be wrapped by F127. On the other hand, alkoxy groups on the triphenylamine can strengthen its electron-donating ability and further push the spectra of the dyes forward longer wavelength. Furthermore, in order to retain the great photophysical properties of the previous BTN-based dyes, the same acceptors h, a, and b were chosen by us for the construction of the new series of dyes. At the same time, acceptor i with relatively weak electron-withdrawing ability, and d which was recently developed in our laboratory were used to enlarge the new dye system.

Synthesis and Photophysical Properties of EBTN Dyes

Synthesis of EBTN Dyes

Photophysical Properties of EBTN Dyes

Absorption and Emission

The absorption and emission spectra of EBTN1-EBTN5 were recorded in chloroform (Figure 2345678910111213141516171819).

The relevant data are collected in From the absorption spectra, EBTN1-EBTN5 with different acceptors showed their characteristic ICT bands from visible to NIR region (Figure 2 -19, left). Among them, EBTN4 and EBTN5 possessing relatively stronger acceptors thus more forceful ICT presented more red-shifted absorptions and emissions (λabs/λem > 700/850 nm). In Table 2 -8, by variation of the acceptors from the weakest i to the strongest b, the corresponding EBTN1 and EBTN4 showed absorption maxima red-shifted from 555 nm to 710 nm, accompanied by the emission maxima red-shifted from 725 nm to 885 nm. All the dyes are characterized by large Stokes shifts from 2520 cm -1 for EBTN5 to 4220 cm -1 for EBTN1 thanks to their ICT properties. In addition, all the EBTN dyes exhibited moderate to good quantum yield (Φ) in chloroform from Φ = 1 % for EBTN4 to Φ = 11 % for EBTN2, which is much higher in toluene from Φ = 17 % for EBTN3 to Φ = 62 % for EBTN2 (Table 23456789).

Notably, given the lack of reliable standards having large Stokes shifts in the deep red range, quantum yields for all dyes were determined by multiple confirmations. For instance, using styryl 9M (Φ = 24 % in CHCl3) [START_REF]174Rhodamine 800 as reference substance for fluorescence quantum yield measurements in deep red emission[END_REF] as a reference, the Φ of EBTN3 in CHCl3 was determined to be Φ = 8 %. This value was confirmed by using Rho 800 (Φ = 25 % in EtOH) [174] and EBTN1 (Φ = 10 % in CHCl3, in turn determined using cresyl violet, CV, Φ = 55 % in MeOH) [START_REF] Magde | Absolute luminescence yield of cresyl violet. A standard for the red[END_REF] as additional references. That's to say, we are relatively confident about the quantum yield values measured. To be more rigorous, we are planning to build a chain of standards to go to NIR, starting from well characterized Rho 6G (em = 552 nm in MeOH) and using compounds such as LDS 698 (abs/em = 508/677 nm in CHCl3) and LDS 798(abs/em = 596/753 nm in CHCl3). This work would be done in the near future to support all the NIR dyes we have synthesized in the lab.

In comparison with the previous BTN dyes (the dyes with the same acceptors are shown in the same color in Table 2-1), our new EBTN dyes present 20-30 nm more red-shifted absorptions and 30-60 nm more redshifted emissions, indicating their relatively stronger ICT due to the enhanced electron-donating ability of the butoxy modified triphenylamine unit.

Dyes in chloroform λabs

[a] ε [b] λem [c]
Φ (%) [d] ν [a] Absorption maxima in nm. [b] Absorption coefficients. [c] Emission maxima in nm. [d] Quantum yields of dyes in chloroform determined using CV in ethanol (Φ = 55%) [START_REF] Magde | Absolute luminescence yield of cresyl violet. A standard for the red[END_REF] as the standard for EBT1, styryl 9M in CHCl3 (Φ = 24%) [START_REF]174Rhodamine 800 as reference substance for fluorescence quantum yield measurements in deep red emission[END_REF] for EBT2-EBTN5.

[e] Stokes shifts in cm -1 .

Dyes in toluene λabs [a]

λem [c] Φ (%) [d] ν [c] Emission maxima in nm. [d] Quantum yields of dyes in toluene determined using CV in ethanol (Φ = 55%) [START_REF] Magde | Absolute luminescence yield of cresyl violet. A standard for the red[END_REF] as the standard for EBT1, styryl 9M in CHCl3 (Φ = 24%) [START_REF]174Rhodamine 800 as reference substance for fluorescence quantum yield measurements in deep red emission[END_REF] for EBT2-EBTN5.

[e] Stokes shifts in cm -1 . Note that, consistently to what we already observed for the BTN dyes, EBTN dyes are not emissive in solid state.

Preparation of EBTN@F127-SiO2 NPs for in vivo Fluorescence Imaging

Preparation of EBTN@F127-SiO2 NPs

During the preparation of the dye solutions, all the EBTN dyes were found to be of higher solubility in organic solvents in comparison to the BTN dyes, which indicated their improved lipophilicity and high potential to be used for preparation of F127-SiO2 NPs for in vivo fluorescence imaging. Absorbance Wavelength (nm) 0,003% 0,005% 0,01% 0,02% 0,03% 0,05% 0,07% 0,1% EBTN5@F127-SiO 2 As we have already observed, the previous BTN dyes are prone to assemble into non-emissive aggregates, leading to heterogeneous NPs and fluorescence quenching in preparation of the BTN@F127-SiO2 NPs.

Hence, we firstly took EBTN5 as the representative to study the relationship between the dye loading and the NPs properties. From Figure 2 -21, as increasing of the dye loading from 0.003% to 0.1%, the particle size slightly increased but no aggregates were observed. However, the absorption and emission spectra of EBTN5@F127-SiO2 NPs, as shown in Figure 2 -22, showed the steadily increased absorption is accompanied by the emission declined after reaching the maximum at the dye loading of 0.03%. The gradually decreased emission was accompanied by a slight red-shift, indicating the aggregation and fluorescence quenching of EBTN5 in F127-SiO2 NPs took place. All the EBTN dyes present an ACQ effect in the solid state, loading amount of 0.02% was thus chosen for preparation of the EBTN@F127-SiO2 NPs.

Following the general procedure, we obtained EBTN1-EBTN5@F127-SiO2 NPs.

Characterization of EBTN F127-SiO2 NPs

Sample in water λabs

[a] λem [b] Φ (%) [c] ν [d]
Size [a] Absorption maxima in nm.

[b] Emission maxima in nm.

[c] Quantum yields of EBT@F127-SiO2 NPs in water determined using CV in ethanol (Φ = 55%) [START_REF] Magde | Absolute luminescence yield of cresyl violet. A standard for the red[END_REF] as the standard for EBT1@F127-SiO2 NPs, styryl 9M in CHCl3 (Φ = 24%) [START_REF]174Rhodamine 800 as reference substance for fluorescence quantum yield measurements in deep red emission[END_REF] for EBT2-EBTN5@F127-SiO2. [d] Stokes shifts in cm -1 .

[e] Diameters measured by DLS in nm Then, we investigated the general properties of the EBTN@F127-SiO2 NPs. The size distributions are presented in Figure 2 -23, the absorption and emission spectra are shown in Figure 2 -24, and the relevant data are collected in Table 2 -10.

From Figure 2 -23, all the suspensions of the EBTN@F127-SiO2 NPs showed monodispersed size of 22 nm by DLS, which is in good agreement with the F127-SiO2 NPs we synthesized before. This proved the improved abilities of all the EBTN dyes to be encapsulated by F127 thus our success in BTN dyes modification. In Figure 2 -24, all the EBTN@F127-SiO2 NPs presented absorption and emission maxima between those in chloroform and in toluene, it is consistent with our previous finding that the environment of the dyes in F127-SiO2 NPs is similar to the mixture of chloroform and toluene. As shown in Table 2 -10, EBTN1@F127-SiO2 and EBTN2@F127-SiO2 NPs have Φ around 60%, which is much higher than those of the reported red/NIR NPs. [172,[START_REF] Gao | Biocompatible nanoparticles based on diketo-pyrrolo-pyrrole (DPP) with aggregation-induced red/NIR emission for in vivo two-photon fluorescence imaging[END_REF][START_REF] D'aléo | Two-photon excited fluorescence of BF2 complexes of curcumin analogues: toward NIR-to-NIR fluorescent organic nanoparticles[END_REF] Furthermore, even EBTN4@F127-SiO2 and EBTN5@F127-SiO2

NPs showed Φ falling to around 12%, this value is still considered great in the given wavelengths (above 800 nm). The NIR emissions along with the high Φ make all the EBTN@F127-SiO2 NPs promising for in vivo fluorescence imaging. Nevertheless, it is at present impossible to test in in vivo microscopy the most red-shifted EBTN3@F127-SiO2 -EBTN5@F127-SiO2 NPs because the detectors mounted on the microscope in the in vivo microscopy platform in Grenoble do not allow to collect at such wavelengths.

Current detectors cannot detect above at best 700 nm.

Conclusion

In this chapter, we synthesized two new series of push-pull dipolar dyes TCBZ1-TCBZ8 and EBTN1-EBTN5 based on the fluorene and BTN dyes previously synthesized in our laboratory. With improved optical and physical properties, they were allowed to be loaded by F127-SiO2 NPs for two-photon fluorescence imaging.

TCBZ1-TCBZ8 are synthesized by introduction of two tert-butyl groups to the carbazole unit. With different acceptors thus different ICT, they presented emissions red-shifted from 648 to 745 nm as well as interesting solvatochromism and large stokes shifts. All the TCBZ dyes are emissive in solid states due to AIE, particularly for TCBZ7 whose Φ up to 42% makes it candidate for NIR solid luminescent materials.

In addition, TCBZ2 and TCBZ5 with either the most red-shifted emission or relatively high Φ were chosen for preparation of F127-SiO2 NPs. The optimal dye loading for TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs was determined to be 0.3% and 0.5%, respectively.

For functionalization of the TCBZ@F127-SiO2 NPs with functional moieties for active targeting, carboxyl and azido modified F127 were prepared. Then TCBZ5@F127-COOH-SiO2 and TCBZ5@F127-N3-SiO2 NPs were afforded using 0.5% dye loading with 10% F127-COOH and 50% F127-N3, respectively.

Followed conjugation of the N3 modified NPs with alkynyl modified TPP and X, the final TCBZ5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2 were obtained. The same size of around 22 nm and the similar optical properties of these surface functionalized F127-SiO2 NPs indicated the negligible impacts of the surface functions to the particle optical and physical properties. In in vivo fluorescence imaging, TCBZ5@F127-SiO2 and TCBZ5@F127-X-SiO2 NPs successfully imaged the blood vessels and different organs of the mice by two-photon excitation, and they were demonstrated a slow clearance from the mice circulatory system.

New dyes EBTN1-EBTN5 obtained by introducing two butoxy groups to the triethylamine unit of the BTN dyes maintain interesting solvatochromism and large stokes shifts due to ICT. Among them, EBTN4

and EBTN5 with relatively strong electron-withdrawing acceptors showed emissions above 850 nm.

Moreover, thanks to the improved lipophilicity of EBTN dyes, they were successfully encapsulated by F127

to form the EBTN@F127-SiO2 NPs. To achieve a highest brightness, EBTN1-EBTN5@F127-SiO2 NPs were prepared using 0.02% dye loading. Lastly, all these EBTN@F127-SiO2 NPs are of high brightness in NIR region benefiting from the apolar environment and isolated environment provided by the particle core.

Complete measurement of the 2P cross-section is also missing. However, if cross-section measurements in the range 600-1000 nm is well described, this is not the case for higher wavelengths (> 1000 nm). At these wavelengths, solvent absorption has to be taken into account. Such measurements are not possible for the moment in the laboratory, but works are in progress in that direction.

Chapter III. Design of NIR Dyes and NPs for in vivo PA Imaging 1. Introduction

Previous Work

Figure 3 -1. Photoacoustic microscope (PAM). [START_REF]Développement de nouveaux chromophores dipolaires pour l'imagerie de fluorescence et l'imagerie photoacoustique[END_REF] The photoacoustic microscope (PAM) available for us is a homemade device assembled by our collaborators Olivier Hugon and Boudewin Van der Sanden at Liphy in Grenoble. The working principle diagram is shown in Figure 3 -1.

Four laser diodes (415 nm, 445 nm, 660 nm and 808 nm) are so far accessible, this means contrast agents with absorptions around these wavelengths are candidates for PA imaging. Indeed, modulation of the frequencies of the laser diodes through a sinusoidal function makes it possible to excite in a dual or multiple mode that facilitates the subsequent comparison of imaging generated by different contrast agents. For example, if the 415 nm and 660 nm laser diodes work together, their frequency will be set differently at one of 5 MHz and another of 5.3 MHz. The two laser beams are firstly merged into one by the reflection of motorized mirrors. In the 2-axis scan head, the beam is then extended to cover the entire entrance of an x20 lens. The objective with an average power of a few milliwatts at the exit helps focus the light on the sample.

After absorption of the light, the ultrasonic waves produced by the PA contrast agents are detected by an electric transducer. Finally, the electrical signal is amplified and demodulated to form the PA images of each wavelength. Therefore, with this combination it is possible to image the sample by two contrast agents with absorptions around 415 nm and 660 nm and compare their PA effects to each other.

Furthermore, two supports are used to pre-evaluate the PA effect of the contrast agents before in vivo imaging: a piece of paper on which the contrast agent is adsorbed, to simulate the PA signal, and a microfluidic circuit to simulate the microvasculature of mice.

Our laboratory recently tried two push-pull hemicyanine dyes CPO1 and CPO2 for in vivo PA imaging (Figure 3 -2). They were previously synthesized in our laboratory for second order non-linear optics and electro-optical modulation, as well as optical limitation in telecommunication wavelengths utilities. [178] With the indoline derivative as electron donor and TCF derivative as electron acceptor, these two dyes present good absorptions at 808 nm or 660 nm in chloroform, thus inspired us to try them for PA imaging. The first experiments performed on microfluidic circuits showed that a high dye concentration (at least 0.1 mM) is needed to visualize the PA signals. For the purpose of increasing the dye concentration in mice vessels, we tried to form dye nanoaggregates by nanoprecipitation. Each NPs indeed contains a high number of dyes.

We also managed to obtain very concentrated nanoaggregates suspensions by further rotary evaporation of water. In the final imaging of the vasculature of a mouse, the concentrated nanoaggregates of CPO1 and CPO2 showed excellent PA response to 660 nm excitation. However, due to excessively high concentrations as well as low colloidal stability of the nanoaggregates in blood vessels, which was proved by the obvious size increases of the nanoaggregates in water over time, the blockage of the certain capillaries took place.

Objectives

While the hemicyanine dyes CPO1 and CPO2 presented great potential for PA imaging beneficiating from their high absorptions at 660 nm or 808 nm, their nanoaggregates seemed far from ideal in in vivo application due to the blocking effect on the vascular system. Hence, our goal here was to find more stable and biocompatible nanoparticles to load our dyes for in vivo PA imaging.

Dye-loaded polymer NPs (PNPs), as we introduced in the first chapter, are good candidates for loading dyes for bioimaging. Therefore, we cooperated with Arnaud Favier from the Polymer Materials Engineering Laboratory of Lyon (IMP Lyon 1, UMR 5223), who has been working on developing polyacrylate polymers, to prepare the high dye-loaded PNPs for in vivo PA imaging.

Two strategies were envisaged:

-Grafting dyes on a pre-formed amphiphilic polymer through substitution, then forming the PNPs via self-assembly. -Directly using dyes as monomer to form the polymer which can be stabilized by another water-soluble polymer.

Accordingly, we were mainly responsible for the modification of the hemicyanine dyes to allow them for the two strategies and Arnaud Favier helped us prepare the PNPs.

Besides, F127-SiO2 NPs with limited dye encapsulation ability have been restricted in PA imaging. In this regard, we intended to take advantage of the modifiable surface of F127-SiO2 NPs for dye conjugation to improve their dye loading.

This chapter presents the preliminary results we have obtained in this field, essentially the preparation and the beginning of the characterization if the different nanoparticles. If this work is far from being complete, this is due to the very particular situation we encountered in 2020 because of the Covid and the resulting lockdown. We hope to be able to continue and complete this work as soon as possible.

Preparation of CPO-SO3 PNPs for PA Imaging

Synthetic Design of CPO-SO3 Polymer

Arnaud Favier developed a polyacrylate polymer with multiple activated carboxylic acid functions (NHS ester) on its side chain (Scheme 3 -1). [START_REF] Duret | Fluorescent RAFT polymers bearing a nitrilotriacetic acid (NTA) ligand at the α-chain-end for the site-specific labeling of histidine-tagged proteins[END_REF] Dyes containing amine groups are therefore easy to be grafted on the polymer via covalent linkage. Loading ratio can be varied by adjusting the stoichiometry. Hydrolysis or capping of the remaining acid functions with 4-(2-aminoethyl) morpholine (AEM) or various amines (we used decylamine, C10) make it possible to control the solubility or hydrophobicity of the formed dye-loaded polymer.

Statistical linear copolymer

X= AEM, COOH or C10 = CPO-SO3

Polyacrylate polymer

Scheme 3 -1. Polymers developed at IMP and proposal for grafting CPO-SO3 for PA imaging. [START_REF] Duret | Fluorescent RAFT polymers bearing a nitrilotriacetic acid (NTA) ligand at the α-chain-end for the site-specific labeling of histidine-tagged proteins[END_REF] Hence, on dye synthesis, we planned to replace the piperidine unit of CPO2 by a piperazine unit to obtain the amino group for dye grafting. Simultaneously, we will substitute the alkyl side chain carried by the 3,3-dimethyl-2,3-dihydro-1H-indolidene group for a sulfonate functionalized chain to endow the dye with good aqueous solubility. The synthetic route of CPO-SO3 is depicted in Figure 3 -3. The sulfonate functionalized indolium derivative 13 and the hemicyanine precursor 14 were synthesized according to the literature. [178,[START_REF] Grichine | Millisecond lifetime imaging with a europium complex using a commercial confocal microscope under one or two-photon excitation[END_REF] In presence of catalytic amount of pyridine they transformed into 15 in 78 % yield. Then, the chlorine atom is substituted for piperazine via a SNR1 reaction. This afforded CPO-SO3 with a good yield of 81 %. The dye was carefully purified and fully characterized by mass spectrometry and NMR.

Synthesis and Characterization of CPO-SO3 PNPs

Synthesis of CPO-SO3

Synthesis of CPO-SO3 Polymers and CPO-SO3 PNPs

Figure 34. Preparation of CPO-SO3 polymer. [START_REF] Duret | Fluorescent RAFT polymers bearing a nitrilotriacetic acid (NTA) ligand at the α-chain-end for the site-specific labeling of histidine-tagged proteins[END_REF] As illustrated in Figure 34, the preparation of CPO-SO3 polymer was started by reversible additionfragmentation chain transfer (RAFT) polymerization to form the polyacrylate polymers, then CPO-SO3 was added into the polymer solution to be grafted by substitution of the NHS ester of the polymer. Followed by addition of excess of AEM or a borate buffer (pH = 9) into the mixture, the afforded CPO-SO3 polymer were modified by AEM or carboxylic acid groups on its side chain. The CPO-SO3@AEM was purified by precipitation in ether and dried under a vane pump vacuum, and the CPO-SO3@COOH was purified by dialysis against distilled water and then dried by freeze-drying. Two specifications of polyacrylate polymers with average molar mass of 29600 or 59900 were preobtained. Then, 5 different CPO-SO3 polymers were obtained with varied stoichiometry of CPO-SO3 and post functionalization (

Dye coupling

Post-functioanalization water was found not very easy. By a long-time ultrasonication, 5 CPO-SO3@COOH (1.0 mg) was finally dissolved in water (0.5 mL) to afford the CPO-SO3 PNPs suspension with the dye concentration calculated to be 0.4 mM (stock solution).

Characterization of CPO-SO3 PNPs

CPO-SO3 PNPs for in vivo PA Imaging

To the CPO-SO3 PNPs stock solution was added salt to form the 0.9% saline for in vivo PA imaging.

The PA imaging was carried out on a CD-1 IGS mouse (Charles River, Écully, France). In the experiment, the mouse was anesthetized using the gas mixture of 1 % isoflurane in air followed by injection of 200 μL of CPO-SO3 PNPs solution through its caudal vein. Then, the mouse ear was cleaned with a hair-removing cream and 70% ethanol, and was then positioned on the focal plane of the objective with an ultrasound gel painted. The PA signal was collected by a focused piezoelectric transducer (NDT System, IBMF054), then amplified and demodulated by two lock-in amplifiers. NIH ImageJ software was used to process the formed image.

Lastly, after injection of CPO-SO3 PNPs, the vasculature on the mouse ear (in red) was successfully imaged with the 660 nm excitation (Figure 3 .

Preparation of CPOP PNPs for PA Imaging

Synthetic Design of CPOP Polymers

Linear block copolymer Linear dye polymer In order to obtain uniformly dispersed and easily re-dissolved PNPs, we designed another two dye-loaded polymers: the linear block copolymer (Scheme 3 -2, left) and the linear dye polymer (Scheme 3 -2, right).

The former polymer composed by a polyacrylate segment for dye grafting and a hydrophilic block NAM chain for micellization in aqueous solution. The later one was designed with dye as polymerization monomer to achieve a high dye loading, and the water solubility can be obtained by wrapping with other water-soluble polymers.

Accordingly, CPOP1 with a piperazine unit for covalent bonding with the polyacrylate polymer, CPOP2

with an acrylamide that facile polymerization was designed.

Synthesis of CPOP PNPs

Synthesis of CPOP1 and CPOP2 dyes

Started from CPO1 synthesized according to the literature, [178] substitution of the chlorine atom by piperazine form CPOP1 in 90 % yield. Further reaction with acryloyl chloride afforded CPOP2 in yield of As shown in Figure 3 -9, still through RAFT polymerization, a linear block polyacrylate copolymer with one side of the statistical linear copolymer and the other side of a hydrophilic block NAM chain was pre-obtained. Then in the presence of N,N-diisopropylethylamine (DIPEA), CPOP1 was grafted onto the statistical linear copolymer side, leading to the formation of an amphiphilic CPOP1 linear block copolymer.

Synthesis and Characterization of CPOP1 PNPs

Synthesis

Different post-treatments of the activated esters were then applied, as they may alter the final morphology of the polymers or of the micelles in solution: i) (COOH) hydrolysis in borate buffer pH=9; ii) (AEM) coupling with excess aminoethylmorpholine; iii) (C10) coupling with excess decylamine The hydrolyzed polymers (COOH) were purified by dialysis against distilled water and then dried by lyophilization. The AEM and C10 polymers were purified by precipitation in ether then dried by vacuum. The amphiphilic CPOP1 linear block copolymer with average molar mass of its hydrophobic segment of 23000 and its hydrophilic segment of 12000 was pre-synthesized. Then by three different post-treatments, the linear block copolymers CPOP1@COOH, CPOP1@AEM and CPOP1@C10 loaded by CPOP1 with average number of 13 were obtained (Table 3 -2).

Since the direct redissolution of these polymers by water was found difficult, DMSO with relatively low biological toxicity was used as a co-solvent to re-solubilize these polymers before redispersion in aqueous medium. Among them, CPOP1@COOH showed the best solubility in water and is therefore more favored for preparation of CPOP1 PNPs. CPOP2 polymer was synthesized by directly using CPOP2 as monomer to perform the RAFT polymerization with n-butyl acrylate (AIBN) and chain transfer reagent SCS-R in 84% conversion (Figure 3 -13). Finally, the CPOP2 PNPs composed by a hydrophobic core (CPOP2 copolymerized with AIBN) and a hydrophilic shell of PNAM were prepared. The hydrophilic PNAM polymer blocks of 11000 g/mol not only provided the CPOP2 PNPs good water-solubility, but also made it possible to limit the non-specific biological interactions. And after purification, the PNPs size measured by DLS was 60 nm with approximately 260 CPOP2 sequestered in the particle core (Table 3 -3). As shown in Figure 3 -15, CPOP2 PNPs presented absorption and emission spectra very close to that of the separated CPOP2 in DMF. This indicated the isolated state of the CPOP2 dye molecules in the PNPs by the barrier effect of AIBN.

Synthesis and Characterization of CPOP2 PNPs

Synthesis

Characterization

However, the CPOP2 PNPs solution that Arnaud Favier gave us is not concentrated enough (judged by naked eye from the pale-blue color of the solution), the synthetic procedure needs to be developed.

Preparation of F127-CPO-SO3-SiO2 NPs for PA Imaging

Synthetic Design of F127-CPO-SO3-SiO2 NPs

Scheme 3 -3. Proposal for grafting CPO-SO3 on F127-SiO2 NPs for PA imaging.

In the second chapter, surface-functionalized F127-SiO2 NPs were successfully synthesized by conjugation of surface modified F127-N3-SiO2 by click reaction with the alkynyl modified functional moieties. Such strategy can be applied here for preparation of the surface-dye-loaded F127-SiO2 NPs (Scheme 3 -3). Thanks to the big surface of the F127-SiO2 NPs, the dye loading was expected to be boomed up to meet the requirement for PA imaging. To do this, we intended to modify CPO-SO3 with an alkynyl group. The formed CPO-SO3-alkyne can then react with the azido group by click thus be grafted on the particle surface. For loading as much as possible the CPO-SO3 to the NPs surface, 100 % of F127-N3 was used to prepare the F127-N3-SiO2 NPs. The particles in this ratio were proved to be stable in the previous study. Then, by gradually increasing the concentration of CPO-SO3, big nanoaggregates were observed after the loading (mass ratio of CPO-SO3 to the F127-N3-SiO2 solution) above 0.05 %. This is probably due to the decreased stability of F127-CPO-SO3-SiO2 NPs as increasing of the CPO-SO3 grafted.

Synthesis and Characterization of CPO-SO3-F127-SiO2 NPs

Synthesis of F127-CPO-SO3-SiO2 NPs

Synthesis of F127-CPO-SO3-SiO2 NPs

Characterization of CPO-SO3-F127-SiO2 NPs

Sample in water λabs

[a] λem [b] ν [c]
Size [d] Dye/NP

F127-CPO-SO3-SiO2 646 875 4051 26 12
[a] Absorption maxima in nm.

[b] Emission maxima in nm.

[c] Stokes shifts in cm -1 . [d] Diameters measured by DLS.

Table 3 -4. Collective information of F127-CPO-SO3-SiO2

NPs in water.

F127-CPO-SO3-SiO2

NPs with mass ratio at 0.05 % presented monodispersed size of 26 nm and good stability in water (Figure 3 -18). According to the F127-N3-SiO2 NPs concentration (around 50 μM), CPO-SO3-alkyne concentration (614 μM), the average number of dyes per particle was calculated to be 12.

What's more, due to aggregation of CPO-SO3 molecules on the same particle, the dye absorption in water blue-shifted from 735 nm in isolated state to 646 nm, accompanied by the mostly quenched fluorescence emission. Lastly, the CPO-SO3 F127-SiO2 NPs with an absorption peak very close to 660 nm as well as a very weak emission were very promising for PA imaging (Figure 3 -19 and Table 3 -4).

F127-CPO-SO3-SiO2 NPs for in vivo PA Imaging

However, in the pre-test of in vivo PA imaging, out of our expectation, F127-CPO-SO3-SiO2 NPs did not show any PA signals on immersed paper fibers. Yet, the total dye concentration was estimated to be 0.55 mM, which is already higher than what is reported in the literature. [132] As the dye we use is also very similar in structure to what is used in the literature, this is rather surprising. Moreover, dye loaded F127-SiO2 NPs have also being used in PA imaging. Therefore, the reason for lack of PA signal is still not clear to us.

Conclusion

In this chapter, for the purpose of obtaining high dye-loaded NPs for PA imaging, we cooperated with IMP to synthesize three kinds of dye-loaded polymers (CPO-SO3, CPOP1 and CPOP2 polymers) by means of dye grafting or dye polymerization.

By adjusting the stoichiometry of the dyes and varying the post-processing of the remaining acid functions of the polymers, CPO-SO3 polymer and CPOP1 polymer with different dye loadings and functionalization were firstly obtained. Redissolution of CPO-SO3@COOH and CPOP1@AEM, which both containing the correspondingly highest dye densities in polymers afforded the CPO-SO3 PNPs and the CPOP1 PNPs. The

CPO-SO3

PNPs with dye concentration of 0.4 mM as well as maximum absorption at 668 nm successfully imaged a mouse vasculature system by the 660 nm laser diode. But due to their uneven size distribution the CPO-SO3 PNPs were fast cleared by the mouse liver and kidney. The CPOP1 PNPs monodispersed in water with size of 71 nm, good absorption at 660 nm, as well as high dye concentration of 0.44 mM are very promising for long-time in vivo PA imaging.

Through one-pot dye polymerization, CPOP2 PNPs were obtained with monodisperse size of 60 nm in water, and each PNP has approximately 260 molecules. Probably due to the blocking of AIBN to CPOP2 in the particle core, the CPOP2 PNPs presented similar absorptions to that of the dye in solution. With comparable absorptions at 660 nm and 808 nm, CPOP2 PNPs are of potential to be excited by the 808 nm laser diode for in vivo PA imaging.

Besides, a surface-loading strategy was developed to break the dye-loading limitation of F127-SiO2 NPs for PA imaging. By conjugation of the alkyne modified CPO-SO3 to the azido-modified F127-N3-SiO2 NPs developed in the second chapter via click reaction, F127-CPO-SO3-SiO2 grafted NPs with dye concentration of 0.55 mM was prepared. Unfortunately, these NPs failed for PA imaging probably due to the low sensitive PAM we are using.

Chapter IV. Dicyanovinyl Dyes for Primary Amine Detection

In parallel with the work presented before, we studied the stability and reactivity of the dipolar dyes in the presence of nucleophiles, especially primary amines.

Background

General Context

The dipolar dyes that we developed in chapter 2 contain various strong π-acceptors, which, by definition, remove electron density from the adjacent π-systems, hence create partial positive charges in their vicinity.

These electron deficient positions act as electrophiles and are prone to bonding with electron-rich nucleophiles. By the reaction that breaks the conjugation, the overall π-electron density of the chromophores will be heavily modified, resulting in consequent optical changes (absorption and/or emission). A recurrent and legitimate question when designing new chromophores bearing such electron-withdrawing groups is therefore their stability in presence of strong nucleophiles. As a matter of fact, given the huge optical changes induced of the reaction, nucleophilic addition to a chromophore electron deficient carbon center, and in particular to ICT chromophores, is a widely used approach to design optical (fluorescent) probes for nucleophiles, including primary amine, [START_REF] Li | Highly sensitive built-in strain sensors for polymer composites: fluorescence turn-on response through mechanochemical activation[END_REF][START_REF] Shi | A mild and catalyst-free conversion of solid phase benzylidenemalononitrile/benzylidenemalonate to N-benzylidene-amine and its application for fluorescence detection of primary alkyl amine vapor[END_REF][START_REF] Saravanakumar | A colorimetric and ratiometric fluorescent sensor for biogenic primary amines based on dicyanovinyl substituted phenanthridine conjugated probe[END_REF] thiol, [START_REF] Yuan | A ratiometric fluorescent probe for specific detection of cysteine over homocysteine and glutathione based on the drastic distinction in the kinetic profiles[END_REF][START_REF] Jung | Coumarin-based thiol chemosensor: synthesis, turn-on mechanism, and its biological application[END_REF] cyanide [START_REF] Orrego-Hernández | Synthesis of dicyanovinyl-substituted 1-(2-pyridyl)pyrazoles: design of a fluorescent chemosensor for selective recognition of cyanide[END_REF][START_REF] Jeong | Michael addition-based colorimetric and fluorescence chemodosimeters for the nanomolar-level tracking of cyanide ions in aqueous-organic media[END_REF][188] or else hydrazine (Scheme 4 -1). [189] Scheme 4 -1. Illustration of nucleophilic addition on electron deficient center of D-π-A structure.

This prompted us to test whether or not the push-pull dipolar dyes that we developed possessing a strong ICT and reactive sites for nucleophilic addition, are good candidates for nucleophile sensing, especially for primary amine. Started by the EBTN series, we simply tested their stability in presence of a large amount of a simple nucleophilic primary amine, n-propylamine, by monitoring the absorption and emission changes. The results are summarized in Figure 4 -1.

Preliminary Tests

As shown in Figure 4 -1, a and b, addition of n-propylamine to EBTN1-EBTN5 in DMSO induced considerable spectral changes. All the solutions had their colors (from red to deep blue) immediately faded away, becoming clear transparent (except EBTN1 for which a slight yellow solution was observed).

Absorption spectra show a complete disappearing of the ICT bands above 500 nm, which are substituted for new bands around 400 nm. This strong blue shift clearly implied the breaking of all the conjugated π-systems by n-propylamine.

Fluorescence spectra also showed considerable changes (Figure 4 -1). Immediately after addition of npropylamine, the far-red emission of EBTEN2 completely vanished. In turn, the weak far-red emissions of EBTN3-EBTN5 were completely replaced by the strong blue fluorescence (485 nm), whereas for EBTN1

an intense yellow fluorescence (556 nm) was observed. Interestingly, after several hours, EBTN2 started emitting the yellow fluorescence observed for EBTN1, while the fluorescence of EBTN3-EBTN5 changed from blue to the same yellow (Figure 4 -1, e and f) as well, albeit of lower intensity. This seems to indicate that at least one similar final (fluorescent) adduct was generated in all the mixtures.

As noted above, using a nucleophilic addition on the electrophilic position induced by the presence of an electron-withdrawing group in the dye is a commonly used approach to design optical probes. As far as amines as concerned as nucleophile, the electrophilic functional groups mostly used are aldehyde, [202][203][204][START_REF] Klockow | Tunable molecular logic gates designed for imaging released neurotransmitters[END_REF][206] or ketone, [207] but also trifluoroacetyl [208,209] or tricyanovinyl. [START_REF] Mohr | Chromo-and fluororeactands: indicators for detection of neutral analytes by using reversible covalent-bond chemistry[END_REF] The end product of the reaction between the amine and the sensor is an imine or an iminium in the case of aldehyde or ketones, or a direct adduct in the electron-withdrawing group in the case of trifluoroacetyl and a 1,2 adduct in the case of tricyanovinyl. Note that for trifluoroacetyl the reaction is reversible but for tricyanovinyl (Figure 4 -2).

To the best of our knowledge, for the electron-withdrawing groups that we have used in our EBTN dyes, only TCF and dicyanovinyl are reported to have response to primary amine and the others have not been really studied till now.

For TCF, Jen et al. described the reversible addition of an amine on the β carbon of the double bond (Michal addition) in a constraint polymer. The author pointed out that the product obtained was thermodynamic unstable so that a reversible elimination of the nucleophile by a low level mechanical force takes place to switch back to the starting dye, at least in the very specific system of the constraint polymer. [START_REF] Li | Highly sensitive built-in strain sensors for polymer composites: fluorescence turn-on response through mechanochemical activation[END_REF] On the other hand, Yang et al. who used the very same chromophores in solution for fluorescent and colorimetric (di)amine sensing, did not mention reversibility. The end product described just result from a Michael addition on the β carbon of the double bond. Surprisingly, a strong fluorescence at 600 nm (Φ = 0.32) is observed which is clearly in contradiction with the proposed structure. However, a closer on the Previously, Mohr et al. described a colorimetric read-out sensor based on push-pull azo dye and therefore working at wavelengths compatible with low-cost light sources and detectors. [START_REF] Mastnak | Design and characterization of dicyanovinyl reactive dyes for the colorimetric detection of thiols and biogenic amines[END_REF][START_REF] Mastnak | Indicator layers based on ethylene-vinyl acetate copolymer (EVA) and dicyanovinyl azobenzene dyes for fast and selective evaluation of vaporous biogenic amines[END_REF] A turn-on fluorogenic probe F3 (Figure 4 -5) for detecting 3,4-Methylenedioxymethamphetamine (MDMA) from ecstasy tablets [START_REF] Moreno | A turn-on fluorogenic probe for detection of MDMA from ecstasy tablets[END_REF] and probe NA-3T-CN for dual-mode detection of aliphatic amines and hydrazine by fluorescence [START_REF] Liu | Dual-mode photonic sensor array for detecting and discriminating hydrazine and aliphatic amines[END_REF] were also reported built upon a bis-diarylurea-dicyanovinyl push-pull fluorophore and a dicyanovinyl-substituted oligothiophene derivative associated with zinc tetraphenylporphyrin, respectively.

However in all these works, the end product was not characterized or simply postulated as being the 1,2

adduct. [START_REF] Mastnak | Indicator layers based on ethylene-vinyl acetate copolymer (EVA) and dicyanovinyl azobenzene dyes for fast and selective evaluation of vaporous biogenic amines[END_REF] Finally, Sathiyanarayanan et al. reported the design of a ratiometric fluorescence sensor for primary amine based on a dicyanovinyl substituted phenanthridine derivatives and they clearly identified the end product as being an imine resulting from the nucleophilic addition of the primary amine to the dicyanovinyl group followed by loss of a malononitrile molecule (Figure 456). [START_REF] Saravanakumar | A colorimetric and ratiometric fluorescent sensor for biogenic primary amines based on dicyanovinyl substituted phenanthridine conjugated probe[END_REF] Figure 4 -6. Mechanism for imine formation from dicyanovinyl provided recently. [START_REF] Mastnak | Indicator layers based on ethylene-vinyl acetate copolymer (EVA) and dicyanovinyl azobenzene dyes for fast and selective evaluation of vaporous biogenic amines[END_REF] The formation of an imine makes sense. The reaction of hydrazine on dicyanovinyl indeed yield hydrazone, a much weaker electron-withdrawing group and is well documented. [START_REF] Gupta | A sensitive TICT probe exhibiting ratiometric fluorescence repose to detect hydrazine in solution and gas phase[END_REF] This results in a decreased intramolecular charge transfer between the donor and the acceptor end of the chromophore and significant spectral changes (blue shift and generally fluorescence enhancement) exploited for hydrazine probes design.

So, given the relatively fast response of EBTN1 to n-propylamine, the dicyanovinyl group was considered more reactive than the other electron-withdrawing groups in the EBTN dyes. Therefore, we decided to start this work from the simplest dicyanovinyl group to deeply study its sensing mechanism to primary amine.

To do so, in addition to our dicyanovinyl containing dye EBTN1, we also used another dicyanovinyl pushpull dipolar dye PTTCN (Figure 4567) which was previously tested for NIR-PA bioimaging. and hydroxybenzotriazole (HOBt). Then the cyclization reaction with Lawesson's reagent [217] afforded 2d, which was formylated by n-BuLi and DMF to form PTTAld in 78% yield. Finally, PTTCN was obtained by the Knovenagel condensation of PTTAld and malononitrile in 64% yield. fading and the yellow-green fluorescence were observed in most of the mixtures (Figure 456789c), demonstrating the kinetic of the reaction is solvent-depended, or the reaction may be reversible.

Primary Amine Sensing: Solution Studies

Synthesis of PTTCN

Solvent Test

Meanwhile, as can be seen in Figure 4 Therefore, DMSO and DMF were considered more favorable for this reaction in comparison to other solvents, and DMSO was chosen for the following experiments.

Spectroscopic Response

Although addition of a large excess of n-propylamine to the solution of PTTCN or EBTN1 in DMSO induced an immediate color change of the mixture from violet to yellow-green that comes along with the appearance of an intense yellow-green fluorescence, the reaction was found slow for low dye concentration.

To allow enough time for the reaction to process, all the following spectra measurements were performed 14 h after addition of amines.

Upon stepwise addition of n-octylamine to PTTCN, the characteristic ICT band of PTTCN at 575 nm was gradually substituted by a new absorption peak at 412 nm, which left an isobestic point at 462 nm indicating that a single chemical reaction happened (Figure 4 -11, a). The same was observed for EBTN1.

Under stepwise addition of n-octylamine, the characteristic ICT band of EBTN1 at 530 nm disappeared while the violate colour of the dye solution faded away with the concomitant appearance of a new peak with a maximum absorbance at 410 nm and an isobestic point at 460 nm (Figure 4567891011c).

Accordingly, when exciting at the wavelength of the isobestic point (412 or 460 nm), the weak far-red emission of PTTCN (em = 685 nm) and of EBTN1 (em = 896 nm) are steadily replaced by intense emission at 535 nm or 556 nm, respectively, with increasing addition of n-octylamine (Figure 4 The same experiments in low concentration of amines (below 100 μM) allow estimating the theoretical detection limits, as the increase of the fluorescence intensity at the maxima (λex: isobestic point) are varying linearly with the quantity of amine added. So for both PTTCN and EBTN1, there is a linear variation (Figure 4 -12, b and d) between the fluorescence intensity at 535 and 556 nm vs amine concentration, respectively. The slopes of the straight line κ give the limits of detection applying the formula 3σ/κ, [218] where σ refers to the standard deviation observed in the intensity of fluorescence measured at the wavelength of detection by 10-times continues scans. Theoretical detection limits of 32 nM for PTTCN and 0.16 μM for EBTN1 were finally obtained. These figures are considered good in comparison with other amine probes published. [START_REF] Mallick | A ratiometric fluorescent probe for detection of biogenic primary amines with nanomolar sensitivity[END_REF] For instance, Longstreet [220] reported a primary amine probe with detection limit of 25 μM in 2014, Yang [START_REF]A reaction-based sensing scheme for volatile organic amine reagents with the chromophoricfluorogenic dual mode[END_REF] developed a probe tht possesses limit from 11.8 nM to 2.3 nM to different primary amines in 2020, and the most recently reported dicyanovinyl dye whose detection limit to n-butylamine is 15.8 nM.

Reversibility

We put in evidence the reversibility of the reaction. Indeed, for both dyes, further addition of malononitrile to the n-propylamine-dye adduct solution immediately induced another color change, with a smoke-like violet (PTTCN) or red (EBTN1) color spreading all over the solution (Figure 4 -13, A).

Homogenously violet solutions corresponding to the ones of the initial dyes (recovered) were obtained after a while.

More interestingly, the recovery of the starting dye was also observed when the excess n-propylamine from the end point of the titration experiments was removed by evaporation under vacuum (Figure 4 -13, B), proving the total reversibility of the reaction. We then evaluated the sensitivity and selectivity of PTTCN and EBTN1 to primary amine. Various nucleophiles including primary amines (putrescine, tyramine, cadaverine, octylamine, propylamine, histamine, tryptamine, spermidine, 1,3-diamine), secondary amines (diethylamine, diisopropylamine), tertiary amine (triethylamine), aromatic amine (pyridine, aniline), thiol (ethanethiol) and hydrazine (hydrazine and dimethylhydrazine) were tested in this study. As shown in Figure 4 -14 a and c, strong fluorescence enhancements were observed for all the primary amines, but not for either the secondary, tertiary, aromatic amines or thiol under the given condition. More importantly, the followed addition of noctylamine to these mixtures induced the characteristic yellow-emission (Figure 4 -14, b and d). This demonstrated the good sensitivity and selectivity of PTTCN and EBTN1 to primary amine over the other kinds of amine or thiol we used here. Indeed, no fluorescence enhancement on PTTCN and EBTN1 by addition of ethanethiol was out of our expectation, maybe it is a question of kinetics.

Sensitivity and Selectivity

NMR Studies

To understand the sensing mechanism of PTTCN and EBTN1 with primary amine, but also to clarify and confirm the structure of the final product, we then carried out the 1 H NMR analysis. In observed with thiols [START_REF] Mastnak | Design and characterization of dicyanovinyl reactive dyes for the colorimetric detection of thiols and biogenic amines[END_REF] or the cyanide anion [188,221] would otherwise lead to a drastic upfield shift of the proton originally at the β-position as well as those of the closest thiophene ring due to a disruption of the πconjugation from the donor to the acceptor end. The imine structures were further confirmed by mass spectrometry PTT-imine ([M+H] + calculated to be 319.1297, found at 319.1299), and EBTN-imine ([M+H] + calculated to be 812.3914, found at 812.3896).

PTTCN Films for Primary Amine Vapor Detection

In recent years, great efforts have been put to amine vapor detection, especially in food security field. [START_REF] Gao | Fluorescent light-up detection of amine vapors based on aggregation-induced emission[END_REF][START_REF] Alam | A highly sensitive bimodal detection of amine vapours based on aggregation induced emission of 1,2dihydroquinoxaline derivatives[END_REF] Jia et al. [START_REF] Jia | Amine-responsive cellulose-based ratiometric fluorescent materials for real-time and visual detection of shrimp and crab freshness[END_REF] prepared a cellulose-based ratiometric fluorescent material by covalently linking the amine-sensitive dye to the cellulose substrate, for detection of shrimp and crab freshness. In our case, having proved PTTCN as a high-performance primary amine sensor, we managed to use it for primary amine vapor detection. As the reaction of PTTCN with n-propylamine in DMSO is reversible, this feature can be used to prepare PTTCN film that is able to detect primary amine in a recyclable way. Nevertheless, the reacted films were

EBTN1-glass for Primary Amine Vapor Detection

Objective

In spite of the excellent sensing behavior of PTTCN sol-gel film to primary amine vapor, the film cannot take advantage of the reversibility of the reaction in solution to be recycled by vacuum off the excess of amine. Therefore, we proposed to covalently link the dye to a device such as glass to enable the dye-modified device to be recovered by rinsing with malononitrile solution after exposing to amine vapor. However, as PTTCN has no modifiable site on its structure, EBTN1 bearing a modifiable methoxy group would be allowed to be grafted onto a substrate for recycling detection of primary amine vapor (Scheme 4 -3).

Scheme 4 -3. Depict of reactive sites of EBTN1 for preparation of recyclable amine vapor device.

Glass slide is a commonly used material in preparation of DNA and protein chips, due to its low cost, corrosion resistance, low fluorescence background and ease of surface modification. All these advantages promoted us to use it as the substrate for preparation of the dye-loaded device for primary amine vapor detection. As shown in Figure 4 -23, EBTNAld functionalized by a NHS group is able to covalently link with the amine group modified on the glass surface via coupling. Followed treating with malononitrile, the EBTNAld molecules on the glass surface will transform into EBTN1, to form the EBTN1-loaded glass slide. It is worth noting that, the amine on the glass surface is a primary amine, which doesn't have reaction with EBTNAld in the absence of catalyst. Finally, upon exposure to primary amine vapor, the EBTN1 molecules on the glass surface will transformed into EBTN-imine to present strong fluorescence, most importantly, which can be recovered by rinsing with malononitrile solution taking advantage of the reversibility of the reaction.

Preparation of EBTN1-grafted Glass Slide

For preparation of the EBTN1-grafted glass slide, the first step was to prepare the amine modified glass slide. As shown in Figure 4 -25, normal glass slide was carefully washed with detergent to expose the hydroxy groups on its surface, followed by silicate cross-linking with APTES, the amine groups were fixed on the glass surface. After wash and dry, the amine modified glass slide was immersed into the solution of the EBTNAld-NHS to allow the grafting of EBTNAld onto the glass surface. Then further immersing of 106 the EBTNAld-loaded glass in the malononitrile solution enabled the formation of the EBTN1-grafted glass slide.

Preliminary Tests on EBTN1-glass

From the colored edges of the glass slides shown in Figure 4 -26 a, it can be seen that EBTNAld and EBTN1 were successfully grafted on the surface of the normal glass. However, given the single layer of EBTN1 coated insufficiently reduced the glass transparency, it's by no means easy to follow the color or fluorescence changes of the glass with naked eye in its process of detection of primary amine vapor. However, limited by the loading amount of EBTN1 molecules on the glass surface, the slide was weakly absorptive and emissive. Hence, other substrates with higher dye-loading ability were expected to overcome this problem.

Conclusion

In this chapter, based on nucleophilic addition and ICT mechanism PTTCN and EBTN1 were utilized for primary amine detection.

Addition of an excess of primary amine to the solution of PTTCN/EBTN1 in DMSO induced an immediate color change from violet to yellow (575 to 412 nm/530 to 410 nm) coming along with the apparition of an intense yellow fluorescence (535 nm, Φ = 1/556 nm, Φ = 0.75), which left an isobestic point at 462 nm/460 nm and the 1160-fold/112-fold fluorescence intensity enhancement. In addition to the good sensitivity and selectivity to primary amine, the detection limit was calculated to be 32 nM/0.16 μM for n-octylamine. Significantly, the reaction was found reversible by removing the n-propylamine added from the solution or by further addition of malononitrile. The imine structure of the sensing adducts were confirmed by NMR and mass spectrometry.

Furthermore, PTTCN/sol-gel and PTTCN/PMMA films were prepared to detect primary amine vapor.

Thanks to the strong start emission of the films (675 nm, Φ = 23% for PTTCN/sol-gel and Φ = 13% for PTTCN/PMMA), they are able to detect primary amine vapor in a ratiometric way (emission blue-shifted to 500 nm, Φ = 43% for PTTCN/sol-gel and Φ = 57% for PTTCN/PMMA). Both films have quick response (in 10 seconds) to primary amine vapor, and the sensing process can be easily followed by naked eye.

In order to take advantage of the reversibility of the reaction between dicyanovinyl and primary amine to prepare a recyclable device for primary amine vapor detection, EBTN1 functionalized glass slide was preliminarily designed. Upon exposure to primary amine vapor, the EBTN1-glass had absorption blueshifted from 542 nm to 420 nm coming along with its fluorescence shifted from 666 nm to 530 nm.

Dramatically, by immersing with malononitrile solution the glass was successfully restored, proving our objective of making recyclable primary amine sensing device is feasible.

Chapter V. Conclusion and Perspectives

To sum up, our main goal in this thesis was to synthesize novel organic contrast agents for in vivo fluorescence or PA imaging, focusing on small push-pull dipolar dyes and F127-SiO2 or polymer NPs.

In the first part, we design and synthesized two series of NIR-emitting dyes TCBZ1-TCBZ8 and EBTN1-EBTN5 based on the fluorene and bisthienopyrrole dyes previously synthesized in our group.

TCBZ dyes have tert-butyl substituted carbazole as the electron donor, which was connected to different electron acceptors by a fluorene unit, presenting absorptions and emissions from 433 to 512 nm and 648 to 745 nm, respectively, as well as interesting solvatochromism and large Stokes shifts. In comparison to the previous carbazole-fluorene dyes, the new dyes showed more red-shifted optical properties while keeping good solid-state emissions. EBTN dyes were synthesized using butoxy modified triphenylamine as the electron donor, which was linked by the bisthienopyrrole unit (BTN) to different electron-withdrawing

groups. An increased ICT effect made it possible to further push the emissions of the dyes toward longer wavelengths. Especially for EBTN4 and EBTN5, with relatively strong electron-withdrawing acceptors exhibited emission peaks above 850 nm in chloroform with excellent quantum yields, albeit low quantum yield (1 to 2 %). They, however, have a much higher quantum yield in toluene (30 and 47 % respectively) for an emission above 780 nm. More importantly, all the EBTN dyes presented much improved lipophilicities compared to the previous ones. This allows us to form F127-SiO2 NPs by dye encapsulation by F127. These NPs display very high brightness in NIR region, thanks to the apolar environment of the F127-SiO2 core that is similar to a toluene / chloroform mixture (1/1 v/v).

On the other hand, for the purpose of functionalization of the F127-SiO2 NPs surface with different moieties, carboxyl and azido modified F127 were firstly prepared. Then, the maximum surface modification that permits a good particle-stability and the dye loading ratio that allows a highest particle-brightness were optimized. Using 10 % F127-COOH and 50 % F127-N3 with 0.5 % of TCBZ5, TCBZ5@F127-COOH-SiO2 and TCBZ5@F127-N3-SiO2 NPs were obtained. Followed grafting of the NPs with alkynyl modified TPP or X via "click" reaction afforded other surface-functionnalized NPs: TCBZ5@F127-X-SiO2 or TCBZ5@F127-TPP-SiO2. TCBZ5@F127-SiO2 and TCBZ5@F127-X-SiO2 NPs were then tested in in vivo biphotonic fluorescence imaging. They successfully imaged the vasculature system of the mice while presented a long circulation time within the mice vessels.

In the third chapter, different strategies were developed to increase the dye-loading of the NPs, aiming to overcome the problem of insufficient generation of PA signal in biological tissues. On the one hand, through collaboration with IMP we designed and synthesized three kinds of dye-loaded polymer NPs, involving dye-polymer conjugation and dye polymerization methods. Polyacrylate polymer developed by Arnaud Favier with multiple NHS ester units was able to conjugate dyes containing amine groups, thus we synthesized CPO-SO3 and CPOP1. For polymerization, we introduced an acrylamide group to form the dye monomer CPOP2. Benefiting from the hemicyanine structures, all the dyes showed strong absorptions in NIR region with moderate fluorescence emissions. Among these polymer NPs, CPO-SO3 NPs was successfully used to image a mouse vasculature with a 660 nm excitation. However, the accumulated signals in liver and kidney indicated the fast clearance of the NPs from a mouse body. CPOP1 NPs and CPOP2 NPs with improved water solubility and homogeneity were expected for a long-time in vivo PA imaging.

On the other hand, based on the surface-functionalization of F127-SiO2 NPs developed in the second chapter, a surface dye-grafting strategy was applied to improve the particle dye-loading capacity. Through "click" reaction in presence of CuSO4, sodium ascorbate and the ligand tris(hydroxypropyltriazoly)methylamine and THPTA, CPO-SO3-alkyne was covalently linked to the F127-N3-SiO2 NPs. Limited by the particle stability, maximum dye loading was determined at 0.55 mM, about 10 dyes per particle according to the concentration of the particle provided by literatures. However, the concentration was still not enough for the PAM we are using.

The fourth chapter preliminarily investigated the response of EBTN dyes we obtained to primary amines.

It was found that all the dyes with different acceptors can form a same structure but EBTN1 with a dicyanovinyl acceptor presented the relatively fast kinetic. For this reason, PTTCN and EBTN1 were used for primary amine detection. Based on ICT mechanism, PTTCN and EBTN1 both showed drastic fluorescence "turn-on" response to primary amine in DMSO (1160-fold/112-fold fluorescence enhancement for PTTCN/EBTN1) with high sensitivity, selectivity as well as good detection limit (32 nM/0.16 μM for n-octylamine of PTTCN/EBTN1). The reaction was also found reversible by removing the amine or further addition of malononitrile from/to the adduct solution. PTTCN sol-gel and PMMA films were prepared for detection of primary amine vapor. In 10 seconds, the films in saturated n-propylamine changed color from violet to yellow-green with fluorescence changed from red to green. The quick response and the obvious color changing make it possible to follow the sensing process by naked eye. To take advantage of the reversibility of the reaction for circle detection of primary amine vapor, EBTN1 was modified by a NHS group for preparation of a dye-grafted glass slide. However, limited by the loading ratio, it is hard to follow the detection process by naked eye. To tackle this issue, other substrates or grafting methods that can improve the dye loading is required.

For in vivo optical imaging, firstly, the two-photon cross-section of all the dyes will be measured once the instrument is accessible. At the same time, the biphotonic in vivo imaging experiments based on NPs (TCBZ5@F127-COOH-SiO2) as well as PA (CPOP1 NPs) will be performed in the near future.

For primary amine detection, in order to improve the dye loading of the substrates, we manage to further modify EBTN1 with a triethoxysilane group which would allow the dye to be directly grafted onto various substrates. Additionally, preliminary work revealed that all the other EBTN dipolar dyes besides EBTN1 have similar fluorescence responses to primary amine, but with more complicated two-stages sensing processes and slower kinetics. Thus, in the following study, we will study the mechanism of all the other acceptors.

purchased respectively from Aldrich and Acros Organic. Absolute fluorescence quantum yields in solid Φ S were measured using a calibrated integrative sphere collecting all the emission (2π steradians covered with spectralon®), model G8 from GMP (Renens, Swiss). For each sample, four measurements were made using the same excitation and emission monochromatic band pass (slits opening) to give four integrated intensities.

Quantum yield are therefore given by equation ( 2):

Φ 𝑺 = 𝑬 𝒄 -𝑬 𝒂 𝑵𝑫×(𝑳 𝒂 -𝑳 𝒄 ) (2) where Ec and Ea are the integrated fluorescence resulting of a direct excitation of the sample and the integrated fluorescence without any sample (the background of the sphere), La is the integrated excitation profile with the empty sphere, and Lc is the integrated excitation profile with the sample inside the sphere.

For the determination of La and Lc, a neutral density filter (0.5 %) was used to reduce the intensity without changing the excitation profile. The ND value is wavelength dependent and is determined for exc.

Precursors a-h, 2-iodo-7-bromo-9,9-diethyl-9H-fluorene 3, 3,6-di-tert-butyl-9H-carbazole 4 and 4-(4methoxyphenyl)-4H-dithieno[3,2-b:2',3'-d]pyrrole 7 were previously synthesized in our team. [START_REF]Development of far-red / near-Infrared luminescent chromophores and nanoparticles for in vivo biphotonic applications[END_REF]111] Compound 10 was prepared according to the literature. [START_REF] Mátravölgyi | Synthesis and Investigation of Solar-Cell Photosensitizers Having a Fluorazone Backbone[END_REF][START_REF] Planells | Energy levels, charge injection, charge recombination and dye regeneration dynamics for donoracceptor π-conjugated organic dyes in mesoscopic TiO2 sensitized solar cells[END_REF] The alkynyl modified X was provided by our collaborator. In a 100 mL round-bottom flask, 3,6-di-tert-butyl-9H-carbazole (4) (4.30 g, 15.38 mmol), copper iodide (0.21 g, 1.10 mmol), 1,10-phenanthroline (0.49 g, 2.72 mmol) and K2CO3 (5.00 g, 36.18 mmol) were added to a solution of 2-iodo-7-bromo-9,9-diethyl-9H-fluorene 3 (5.56 g, 13.02 mmol) in DMF (56 mL) under argon atmosphere. After stirring at 120°C overnight, DMF was removed by evaporation under reduced pressure. Then the crude oil was purified by column chromatography on silica using pentane as the eluent to afford 5 as a white solid (3.10 g, yield: 41 %). 1 To a solution of 7 (0.50 g, 1.75 mmol) in anhydrous DCM (20 mL) was added anhydrous DMF (280 μL, 3.60 mmol) under argon atmosphere. After cooling down to 0°C using an ice-bath, POCl3 (500 μL, 5.10 mmol) was added dropwise to the mixture. After stirring at 0°C for 1h then at room temperature overnight, the reaction was quenched with 1M NaOH (5 mL). The organic phase was separated and washed with water and brine, then was dried over MgSO4 and evaporated under reduced pressure. Followed column chromatography on silica gel with DCM as eluent to afford 8 as a yellowish solid (360 mg, yield: 66%). To a solution of 8 (170 mg, 0.54 mmol) in THF (10 mL) was added NBS (103 mg, 0.58 mmol) at 0°C. The mixture was then stirred at room temperature for 3h. DCM and water were added to the mixture, followed by extraction of the aqueous phase separated using DCM. The combined organic phases were washed with saturated Na2CO3 and water. Finally, evaporation under reduced pressure gave 9 as an offwhite solid (200 mg, yield: 94%). 1 H NMR (400 MHz, DMSO-d6, ppm) δ 9.88 (s, 1H), 8.10 (s, 1H), 7.61 (d, J = 8.9 Hz, 2H), 7.47 (s, 1H), 7.16 (d, J = 8.9 Hz, 2H), 3.86 (s, 3H). 13 General procedure for Knoevenagel condensation. Synthesis of EBTN2-EBTN5.

Synthesis and Characterization of Dyes

In 10 mL microwave tubes, 12 (100 mg, 0.14 mmol) and the corresponding acceptors h, a, d and b (0.28 mmol) were dissolved in anhydrous DMF (5 mL), followed by addition of one drop of piperidine. The tubes were sealed and heated at 130°C by microwave irradiation After cooling down to room temperature, the mixtures were transferred into round-bottom flasks and evaporated under reduced pressure. Then the crude products obtained were purified by column chromatography on silica. 158.6, 156.0, 150.9, 150.5, 149.3, 142.0, 140.3, 139.9, 137.4, 135.5, 134.4, 134.1, 131.7, 127.0, 126.5, 126.0, 124.7, 122.5, 122.4, 119.6, 115.4, 115.3, 114.8, 105.6, 68.0, 55.7, 31.4, 19.3, 13. pressure to afford the crude product. Further purification by column chromatography on silica gel using DCM as eluent gave 14 as a yellow solid (1.20 g, yield: 96 %). 1 H NMR (400 MHz, CDCl3, ppm) δ 9.87 (s, 1H), 7.69 (s, 1H), 7.44 (d, J = 8.9 Hz, 2H), 7.15 (s, 1H), 7.08 (d, J = 8.9 Hz, 2H), 4.20 (q, J = 7.1 Hz, 2H), 4.12 (t, J = 6.1 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 2.27 -2.13 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H). 13 Under argon atmosphere, Pd(PPh3)4 (35 mg, 0.03 mmol) and 1M K2CO3 solution (0.2 mL) were added to a solution of 14 (38 mg, 0.07 mmol) and 5 (30 mg, 0.06 mmol) in THF (2 mL). The mixture was refluxed overnight. After cooling down to room temperature, DCM was added. Then the solution was filtered on celite to remove the Pd catalyst. The organic phase was washed three times with water then dried over MgSO4. The solvent was evaporated under reduced pressure to give a crude solid which was purified by column chromatography on silica (eluent: DCM:PE=1:1, v/v). 15 was obtained as an orange solid (45 mg, yield: 76%). 1 H NMR (400 MHz, CDCl3, ppm) δ 9.81 (s, 1H), 7.65 (s, 1H), 7.48 (d, J = 8.9 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.14 (s, 1H), 7.08 (d, J = 8.9 Hz, 6H), 6.92 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.9 Hz, 4H), 4.20 (q, J = 7.1 Hz, 2H), 4.12 (t, J = 6.1 Hz, 2H), 3.97 (t, J = 6.5 Hz, 4H), 2.59 (t, J = 7.2 Hz, 2H), 2. Wavenumber (cm -1 ) 2100 cm -1

Preparation of Dye-loaded F127-SiO2 NPs

General procedure of preparation of dye-encapsulated F127-SiO2

In a 5 mL round bottom flask, F127 and dye were dissolved in 1-2 mL of DCM, followed by evaporation of DCM by a gentle flow of argon at room temperature to form a film. NaCl (65 mg) and 0.85 N hydrochloric acid solution (3.1 mL) were added to the F127/dye mixture, not until an optical solution was generated, TEOS (360 μL) was added to start the silica cross-linking. After stirring for 105 min, the suspension was added DEDMS (30 μL) to terminate the silica growth, and was further stirred at room temperature for 24h.

The mixture was transferred into a dialysis bag (cut-off 14,000 g/mol) to remove acid, free F127 and unreacted additives through dialysis against distilled water for 3 days. Finally, the F127-SiO2 NPs solution was passed through a 0.2 μm filter (regenerated cellulose).

dwell time of 150 µs. The position of the target is set by a motorized three-axis translation stage. The PA signal is collected through an ultrasound transmission gel by a focused piezoelectric transducer (NDT Systems, IBMF054). The electric signal of the transducer is finally amplified and demodulated by two lockin amplifiers respectively running around f1 and f2 to recover both the amplitudes of the PA signals generated at 1 and 2. Écully, France). All efforts were made to minimize their number. They were housed in ventilated cages with food and water ad libitum in a 12 h light/dark cycle at 22 ± 1°C. For the experience, mice were anesthetized using a gas mixture of 1% isoflurane in air/30% O2. Their body temperature was maintained at 36-37° C using an electric heating pad with a feedback system. In experiments with dye infusion, mice were Image processing was performed with the NIH ImageJ software. Acquisitions were averaged or summed to give the images (sums gave a better image brightness). Background was subtracted (rolling ball radius of 20 pixels). Finally, the signal of CPO-SO3 PNPs was (660 nm) colorized in green and in red for hemoglobin and melanin (415 nm).

Diode\laser

EBTN1-glass

Glass slides were firstly cleaned by detergent, water, acetone and methanol in sequence using ultrasonication for 15 min, respectively, then, they were placed in the solution of APTES (5% in methanol)

to stand at room temperature for 45-60 min. After being washed with methanol and dried at 120°C for 30min the slides were immersed into the solution of EBTNAld (100 μM) at 45°C overnight. Followed by rinsing with methanol and natural drying, the EBTNAld-loaded glass slides were immersed in malononitrile solution (10 mM) at room temperature for 6h. By rinsing with methanol the EBTN1-glass was finally formed.

Other Experiments

TCBZ1-TCBZ8 nanoaggregates

TCBZ1-TCBZ8 stock solutions (1 mM) were prepared using acetone as a good solvent. In a volumetric flask (5 mL) was added dye solution (250 μL, 2.5 10 -4 mmol). Followed addition of water/acetone mixture (water fraction fw% from 0 to 90) to the volume of the flask gave the final dye concentration (5×10 -5 mol L - 1 ).

NMR titration

To a normal NMR tube was added PTTCN (6.0 mg, 18. The UV/vis and fluorescence spectra were measured all at once.

X-ray Crystallography

  ABBREVIATIONS ..................................................................................................................................................... V GENERAL INTRODUCTION ............................................................................................................................... VII CHAPTER I. INTRODUCTION TO NIR DYES AND NANOPARTICLES (NPS) FOR IN VIVO OPTICAL IMAGING .....................................................................................................................................................................

  AND PERSPECTIVES ....................................................................................... CHAPTER VI. EXPERIMENTAL SECTION .....................................................................................................

4 . 6 -

 46 AbbreviationsA Electron acceptor ACQ Aggregation caused quenching AEM 4-(2-Aminoethyl)morpholine AIBN Azobisisobutyronitrile AIE Aggregation-induced emission BBTD Benzobisthiadiazole C10 n-Decylamine CMC Critical micelle concentration CV Cresyl violet D Electron donor DCC N,N'-Dicyclohexylcarbodiimide DCM Dichloromethane DEDMS Diethoxydimethylsilane DFT Density functional theory DIPEA N,N-Diisopropylethylamine DLS Dynamic light scattering DMF N,N-Dimethylformamide DMSO Dimethylsulfoxide EBTN 4H-Dithieno[3,2-b:2',3'-d]pyrrole EDC•HCl N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride F127 Pluronic F127 FRET Förster resonance energy transfer HRMS High-resolution mass spectrometry HOMO Highest Occupied Molecular Orbital Hb Hemoglobin HOBt 1-Hydroxybenzotriazole I Light intensity ICG Indocyanine Green ICT Intramolecular charge transfer ISC Intersystem crossing J Scalar or J coupling constant LUMO Lowest Unoccupied Molecular Orbital

Figure 1 - 2 .

 12 Figure 1 -2. Auto-fluorescence images of a mice body. (a) Mice exposed under white light. Auto-fluorescence recorded by three different excitation/filter sets: (b) blue/green (460-500 nm/505-560 nm) filter set; (c) green/red (525-555 nm/590-650 nm) filter set and (d) NIR (725-775 nm/790-830 nm) filter set.[START_REF]In vivo near-infrared fluorescence imaging. Frangioni[END_REF]

Figure 1 -

 1 2 depicts the fluorescence variation of a mouse body by changing the excitation/emission filter sets. With a blue excitation, a strong green fluorescence appeared spreading all over the mouse skin, gallbladder, small intestine and bladder (Figure1 -2, b). The auto-fluorescence is greatly reduced using a green excitation (Figure1 -2, c), and is completely inhibited with a NIR filter set. This highlighted the low biological interference of NIR light for in vivo fluorescence imaging.

  developed over the recent years to obtain NIR dyes, focusing on three broad families of rhodamine, BODIPY and cyanine dyes. A paragraph will be dedicated to the rare and very recent examples of specific engineering of organic fluorophores emitting in the NIR-II window. Finally, we will end this presentation by introducing dipolar fluorophores, which although much less studied than the previous examples, are a very interesting route to NIR fluorophores.

by

  Koide et al., who synthesized a series of NIR-I-emitting Si-rhodamine dyes through extension of the xanthene ring (Figure 1 -5). Then, SiR 700 was modified with a NHS group to connect with the antitenascin-C antibody for in vivo targeted tumor imaging (Figure 1 -5)

Figure 1 - 7 .

 17 Figure 1 -7. Chemical structures of Merocyanine A, Changsha, Rhodamine-coumarin and RhIndz Ethyl Ester dyes.

Figure 1 - 8 .

 18 Figure 1 -8. Chemical structures of Tetrastyryl-Bodipy, KFLs and Anti-bis-benzobodipy dyes (CHCl3 as the solvent).

Figure 1 - 9 .

 19 Figure 1 -9. Chemical structures of Tetra aryl-azaBODIPY, Phenanthrene-fused azaBODIPY, Ten-ring substituted azaBODIPY and SWIR-WAZABY-01 dyes.

Figure 1 - 10 .

 110 Figure 1 -10. Chemical structure of ICG and general structure of Ring-locked heptamethine cyanine.

Figure 1 - 11 .

 111 Figure 1 -11. Chemical structures of IR800-CW, IR800-CW-NHS Ester and IRDye® 800CW Maleimide.

  -13). The formed CH1055-PEG presented high pharmacokinetics, fast renal clearance and good bioimaging properties. Meanwhile, the modification of the carbocylic acid group by a NHS makes it possible for CH1055 to connect to affibody. The CH1055affibody with a high biocompatibility as well as a good targetability was used for the image-guided surgery in a mouse body. Additionally, another CH1055 based dye CH1055-4Glu-AE105 designed by Kubegovic et al. (Figure 1 -14) was successfully used for an image-guided brain surgery.

Figure 1 -

 1 Figure1 -14. Chemical structure of dye CH1055-4Glu-AE105 and its application on tumor surgery.[START_REF] Antaris | A small-molecule dye for NIR-II imaging[END_REF]

  Dipolar fluorophores are ideal structures to design environment sensitive fluorescent probes because they show strong solvatochromic properties. Indeed, most of fluorescent polarity probes or viscosity probes used in bioimaging, membranes staining probes that show fluorescence in the rigid environment of cell membranes, fluorescent probes for cell membrane tension, are dipolar fluorophores.[81-84] In the years 1990-2000s, a great deal of work has been dedicated to the design of efficient fluorescent fast-response voltage-sensitive optical probes showing good affinity for cell membrane. These works focused on charged dipolar fluorophores incorporating nitrogen as electron donor groups (mostly dialkylamino groups) and phenyl-(or naphthyl-) styryl-pyridinium moiety has electron-acceptor part (Figure 1 -16,

Figure 1 - 16 .

 116 Figure 1 -16. Chemical structures of Di-2-ANEPEQ and Di-4-ANEPDHQ dyes (EtOH as the solvent).

Figure 1 - 18 .

 118 Figure 1 -18. General structures of DCDHF dyes and AH25 (toluene as the solvent).

Figure 1 - 19 .

 119 Figure 1 -19. Chemical structures of DCX, QM, 3-HIFONs, CbV9, NECDCIP and HBD dyes (in aggregates in water except for HBD).

1 - 20 .

 120 Figure 1 -20. Chemical structures of fluorene-based dyes previously synthesized in our laboratory (in solid state).

Figure 1 - 21 . 21 1. 3 .

 121213 Figure 1 -21. Chemical structures of BTN-based dyes previously synthesized in our laboratory (CHCl3 as the solvent).

Figure 1 -

 1 Figure 1 -22.Principle of PA imaging.[START_REF] Wang | A practical guide to photoacoustic tomography in the life sciences[END_REF]

Figure 1 - 23 .

 123 Figure 1 -23. Extinction coefficients of different PA contrast agents.[116] Abbreviations: SWNT (single-layer carbon nanotubes), CP NP (conductive nanoparticles) and GNR (gold nanorods).

IC7- 1 - 1 -

 11 imaging and in vitro PA imaging (Figure1-26). From the fluorescence imaging following the duration of IC7-1-Bu or ICG in mice body after injection and the PA intensity of IC7-1-Bu or ICG in aqueous solution, Bu showed a much longer biological half-life and 2.3-fold higher PA signal than ICG. Finally, IC7-Bu provided a PA image of a mouse tumor with the ratio of approximately 2.5 of tumor to background.

Figure 1 - 26 .

 126 Figure 1 -26. Chemical structures of IC7-1-Bu,IC-n-Tand their using for PA imaging.[133]

  addition, covalent linking of the organic dyes to the NPs can completely block the leakage of the dyes from NPs. Rampazzo et al. functionalized rhodamine with a triethoxysilane group that allows the dye R to covalently bind with the micelle/silica NPs through Si-O-Si bonds, consequently, no dyes leakage was observed in the process of NPs purification (dialysis and ultrafiltration) (Figure 1 -28). By Förster resonance energy transfer (FRET) strategy, they estimated an approximate number of dye molecule per NPs to be around 10

Figure 1 -

 1 Figure 1 -29. Fabrication of F127-SiO2 NPs using triethoxysilane modified Cy5.5 and the in vivo fluorescence tumorimaging.[START_REF] Biffi | Multiple dye-doped NIR-emitting silica nanoparticles for both flow cytometry and in vivo imaging[END_REF]

Figure 1 -

 1 Figure1-31. Two-photon vasculature imaging of a10@F127-SiO2 NPs.[START_REF]Development of far-red / near-Infrared luminescent chromophores and nanoparticles for in vivo biphotonic applications[END_REF]108]

Figure 1 - 32 .

 132 Figure 1 -32. Schematic illustration of self-assembly NPs. [149] Dye-loaded polymer NPs characterized by high dye loading have attracted great interest in optical imaging. Basically, two main strategies are applied for their preparation: emulsion polymerization of dye monomers and self-assembly of preformed dye-loaded polymers (Figure 1 -32).

  -33). The polymers are usually various amphiphilic surfactants such as the PEG containing lipids, mPEG-DSPE, phosphoethanolamine-N-(polyethylene glycol) (DSPE-PEG2000), DSPE-PEG5000mal and DSPE-PEG5000-folate (Figure 1 -34).

Figure 1 -

 1 Figure1 -33. Chemical structures of several organic polymers used in PA studies.[150][151][152][START_REF] Chen | Highly absorbing multispectral near-infrared polymer nanoparticles from one conjugated backbone for photoacoustic imaging and photothermal therapy[END_REF][START_REF] Zhu | D-A polymers for fluorescence/photoacoustic imaging and characterization of their photothermal properties[END_REF]

Figure 1 - 34 .

 134 Figure 1 -34. Chemical structures of normally used amphiphilic surfactants: mPEG-DSPE, DSPE-PEG2000, DSPE-PEG2000-Mal and DSPE-PEG5000-folate.

Figure 1 -

 1 Figure 1 -35. (a) fabrication and (b) PA imaging of PPy NPs.[150]

Figure 1 -

 1 Figure 1 -36. PA imaging with semiconductor polymers. (A) ROS ratiometric probe based on polymer SP1/IR7755 and the PA spectra of the NPs alone or in the presence of ROS; (B) in vivo PA imaging without (left) and with (right) zymosan treatment to produce ROS;[131] (C) Thermal and PA imaging of a tumor containing DPP-DT.[START_REF] Chen | Highly absorbing multispectral near-infrared polymer nanoparticles from one conjugated backbone for photoacoustic imaging and photothermal therapy[END_REF]

  conjugating the amine-functionalized IR825 to the double hydrophilic block copolymer PEG-PLD which containing 10 carboxyl groups in the PLD segment and then obtained the nanomicelles by self-assembly of PEG-PLD-IR825 in aqueous solution. With IR825 trapped in the NP core and the PEG segments distributed on the NP surface, the PEG-PLD-IR825 NPs presented excellent biocompatibility, high tumor accumulation due to enhanced permeability and retention effect, and great promise for cancer theranostics (Figure 1 -37).

Figure 1 -

 1 Figure 1 -38. Schematic illustration of fabrication of IR825@C18PMH-PEG-Ce6 and its in vivo fluorescence, PA and magneticresonance MR imaging.[168]

  photosensitive and AIE active TPETP, a hydrophilic PEG chain and a reactive ROS cleavable thioketal linker (TK), the TPETP-TK-PEG polymer can encapsulate a high amount of DOX to form a strong fluorescence emitting NPs in water, and disassembly upon light irradiation to release the DOX molecules, as a result, the drug delivery and release process in MDA-MB-231/DOX cells was successfully followed by fluorescence imaging.

Figure 1 -

 1 Figure 1 -39. (a) schematic illustration of fabrication of AIE-NPs/DOX and its disassembly upon light irradiation to release Dox in cells;(b) characterization and (c) cell fluorescence imaging of AIE-NPs/DOX.[169]

  phenanthroline and K2CO3, 3 and 4 afforded compound 5, in 41% yield. Lithiation by a stoichiometric amount of n-BuLi, followed by reaction with DMF, 5 transformed into the aldehyde precursor 6 in 82% yield. With a catalytic amount of piperidine, 6 afforded dyes TCBZ1-TCBZ8 by Knoevenagel condensation with the acceptors a-h (presented in Scheme 2 -1). All these dyes and intermediate products were carefully purified and fully characterized by NMR and mass spectrometry.

Figure 2 - 1 .

 21 Figure 2 -1. Synthetic routes of TCBZ1-TCBZ8.

1 .Figure 2 - 2 .

 122 Figure 2 -2. Normalized absorption (a) and emission (b) spectra of TCBZ1-TCBZ8 in chloroform.

Figure 2 - 3 .

 23 Figure 2 -3. Normalized absorption (left) and normalized emission (right) spectra of TCBZ1-TCBZ8 in different solvents.

Figure 2 - 4 .

 24 Figure 2 -4. (Left) absorption and (middle) emission changes of TCBZ1-TCBZ8 in acetone/water mixtures with increasing of water fraction; (Right) plot of fluorescence peaks intensity vs fw.

Figure 2 - 5 .

 25 Figure 2 -5. Emission spectra of TCBZ1-TCBZ8 in solid state.

Figure 2 - 6 .

 26 Figure 2 -6. General procedure of preparation of dye-loaded F127-SiO2 NPs.Wavelength and brightness are both important factors for F127-SiO2 NPs in in vivo fluorescence imaging, thus TCBZ2 and TCBZ5 with either the most red-shifted emission or a relatively high quantum yield in far-red region among the TCBZ series were chosen for preparation of the dye-loaded F127-SiO2 NPs. In consideration of the possible fluorescence quenching caused by π-π stacking of dyes in high-dye-loaded F127-SiO2 NPs, and to optimize the dye loading, we firstly investigated the relationship of the F127-SiO2

Figure 2 - 7 .Figure 2 - 8 .

 2728 Figure 2 -7. (Left) absorption and (right) emission changes of TCBZ2@F127-SiO2 (up) and TCBZ5@F127-SiO2 (down) by varying the dye to F127 mass ratio from 0.1 % to 0.8 %.

Figure 2 - 9 .

 29 Figure 2 -9. Particle size distributions of TCBZ2@F127-SiO2 (a) and TCBZ5@F127-SiO2 (b) in water, measured by DLS.

Figure 2 - 10 .

 210 Figure 2 -10. Synthetic route of F127-COOH and F127-N3.

Figure 2 - 11 .

 211 Figure 2 -11. General procedure of preparation of surface functionalized F127-SiO2 NPs.

F127-

  COOH-SiO2 and F127-N3-SiO2 NPs stabilities. The experiments were carried out on dye-absent NPs to exclude any possible interference from dye. The results are collected in

  Weight percent of F127-COOH to total F127 polymers. [b] Diameters measured by DLS in nm.

Figure 2 -

 2 Figure 2 -12. Preparation of TCBZ5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2 NPs from TCBZ5@F127-N3-SiO2 NPs.

Figure 2 - 13 .

 213 Figure 2 -13. Particle size distributions of TCBZ5@F127-SiO2 (a), TCBZ5@F127-COOH-SiO2 (b), TCBZ5@F127-X-SiO2 (c) and TCBZ5@F127-TPP-SiO2 (d) NPs in water, measured by DLS.

Figure 2 -

 2 Figure 2 -14 (a) absorption and (b) emission spectra of TCBZ5@F127-SiO2, TCBZ5@F127-COOH-SiO2, TCBZ5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2 NPs in water

Figure 2 -

 2 15 (a and b), taken 1 h after injection. Even after 2 h of circulation, the bright fluorescence can still be seen in the blood vasculature network, which indicated a long circulation of the NPs in mice body. Imaging near the skin reveals sebaceous glands (auto-fluorescence in the green channel). The shadows beneath the sebaceous glands (Figure 2 -15 a and b) indicated the absorption of NPs fluorescence by the biological tissue, and indicted that the imaging depth is less than 100 µm. It's worth noting that the yellow color of the blood vasculature came from the overlap of the red from TCBZ5@F127-SiO2 NPs and the green from FITCdextran. In the case of TCBZ5@F127-X-SiO2 NPs, as shown in Figure 2 -15 c and d, without injection of FITC-dextran only red was observed in the vasculature system.

Figure 2 -

 2 Figure 2 -15. Two-photon microscopic images of the circulation of CBZ5@F127-SiO2 (a, b) and CBZ5@F127-X-SiO2 (c, d) NPs in the mouse ear 1h after injection. The images are a z-projection of the standard deviations of fluorescence intensities: 50 slices, step-size 2 µm = thickness of 100 µm. exc = 900 nm, green channel: 542 ± 50 nm, red channel: > 560 nm, yellow = red + green. The green channel shows endogenous fluorescence signals from sebaceous glands and FITC-dextran. These glands are at the surface of the skin, indicating that the imaging depth is less than 100 µm.

Figure 2 -

 2 Figure 2 -16. Two-photon microscopic images of ear vasculature system (a, e), kidneys (b, f), livers (c, g) and spleens (d, h) 2hafter injection of CBZ5@F127-SiO2 (up) and CBZ5@F127-X-SiO2 (down) NPs. exc = 900 nm, green channel: 542 ± 50 nm, red channel: > 560 nm, yellow = red + green. 0.01 mL FITC-dextran was injected 30 min before dissection of the second mouse.The only difference we observed between the two kinks of NPs in the imaging experiment is from the liver images. As shown in Figure2-17 b, a zoomed picture of Figure g, CBZ5@F127-X-SO2 NPs seem stuck against the vasculature wall (endothelial cells), leading to a hollow lumen of the vessels. It is probably caused by the X moieties on the particle surface, but the specific mechanism is currently unknown.

Figure 2 -

 2 Figure 2 -17. Two-photon microscopic images of excised livers 2h after injection of CBZ5@F127-SiO2 (a) and CBZ5@F127-X-SiO2 (b) NPs. exc = 900 nm, green channel: 542 ± 50 nm, red channel: > 560 nm, yellow = red + green. 0.01 mL FITC-dextran was injected 30 min before dissection of the second mouse. Red and yellow dots are probably NPs in Kupfer cells (macrophages).

Figure 2 - 18 .

 218 Figure 2 -18. Synthetic routes of EBTN1-EBTN5.

Figure 2 -

 2 Figure 2 -19. (a) Normalized absorption and (b) emission spectra of EBTN1-EBTN5 in chloroform.

Figure 2 -

 2 Figure 2 -20. (Left) absorption and (right) emission spectra of EBTN1-EBTN5 in different solvents. (1.0×10 -6 mol L -1 ).Then we investigated the solvatochromic effects of BTN1-BTN5 by recording their absorption and emission spectra in toluene, dichloromethane, chloroform and DMSO., solvent of increasing polarity

Figure 2 - 21 .

 221 Figure 2 -21. Particle size distributions of EBTN5@F127-SiO2 NPs in water with increasing of the mass ratio of EBTN5 to F127 from 0.1% to 0.8%, measured by DLS.

Figure 2 -

 2 Figure 2 -22. (a) absorption and (b) emission changes of EBTN5@F127-SiO2 NPs with varied mass ratio of EBTN5 to F127 (0.1% to 0.8%).

Figure 2 - 23 .

 223 Figure 2 -23. Particle size distributions of EBTN1@F127-SiO2 -EBTN5@F127-SiO2 NPs in water, measured by DLS.

Figure 2 -

 2 Figure 2 -24. (a) absorption and (b) emission spectra of EBTN1-EBTN5@F127-SiO2 NPs in water.

Figure 3 - 2 .

 32 Figure 3 -2. Chemical structures of CPO1 and CPO2 (up); (down) in vivo PA imaging of CPO1(left) and the microfluidic circuit of CPO2 (right).[START_REF]Développement de nouveaux chromophores dipolaires pour l'imagerie de fluorescence et l'imagerie photoacoustique[END_REF]

Figure 3 - 3 .

 33 Figure 3 -3. Synthetic route of CPO-SO3.

Figure 3 - 5 .Figure 3 - 6 .

 3536 Figure 3 -5. Size distribution of CPO-SO3 PNPs in water, measured by DLS.

Figure 3 -

 3 Figure 3 -6 presented the absorption and emission spectra of CPO-SO3 PNPs in water and CPO-SO3 in DMF. By comparison, CPO-SO3 PNPs showed absorption band slightly blue-shifted and widened as well as emission band greatly attenuated due to the aggregation of the CPO-SO3 dye molecules in the formed

  -7, a), which indicated the strong PA response of CPO-SO3 PNPs in the mouse blood vessels. And after 30minutes of circulation, strong PA signals appeared in the tissue sections of liver and kidney (Figure 3 -7, b and c), which revealed the accumulation of CPO-SO3 PNPs in these organs and reflected their fast clearance from the mouse body.

Figure 3 - 7 .

 37 Figure 3 -7. In vivo PA imaging of CPO-SO3 PNPs in a normal mouse ear (a) as soon as injection, liver (b) and kidney (c) after 30 min circulation. Red color comes from the PA signal generated by hemoglobin and melanin (415 nm) and green color by CPO-SO3 PNPs (660 nm).

75 77 %

 77 (Figure 3 -8). CPOP1 and CPOP2 were carefully purified and fully characterized by mass spectrometry and NMR.

Figure 3 - 8 .

 38 Figure 3 -8. Synthetic routes of CPOP1 and CPOP2.

Figure 3 - 9 .

 39 Figure 3 -9. Preparation of CPOP1 linear block copolymer.

Figure 3 -Figure 3

 33 Figure 3 -10. Formation of CPOP1 PNPs.

Figure 3 - 11 .Figure 3 -

 3113 Figure 3 -11. Size distribution of CPOP1 PNPs in water, measured by DLS.

Figure 3 - 13 .

 313 Figure 3 -13. Preparation of CPOP2 polymers.

Figure 3 - 14 .

 314 Figure 3 -14. Formation of CPOP2 PNPs.

Figure 3 -

 3 Figure 3 -15. Absorption (solid) and emission (dot) spectra of CPOP2 in DMF (black) and CPOP2 PNPs in water.

Figure 3 -

 3 Figure 3 -16. Synthetic route of CPO-SO3-alkyne.

Figure 3 -

 3 Figure 3 -17. Preparation of F127-CPO-SO3-SiO2 NPs. Same as the preparation of the surface functionalized F127-SiO2 NPs in the last chapter, F127-N3-SiO2 NPs were pre-formed by using F127-N3 as the only amphiphilic co-polymer. F127-CPO-SO3-SiO2 NPs were obtained via click reaction by simply adding the alkynyl modified dye CPO-SO3-alkyne and the suitable Cu(I) catalyst into the stock solution (Figure 3 -17). Finally, the catalysts and unreacted dyes were removed by dialysis.

Figure 3 -Figure 3 -

 33 Figure 3 -18. Size distribution of F127-CPO-SO3 -SiO2 NPs in water, measured by DLS.

Figure 4 - 1 .

 41 Figure 4 -1. (a) absorption and emission spectra of EBTN1-EBTN5; (b) after addition of excess of n-propylamine for few minutes; (c) after addition of excess of n-propylamine for several hours. (6.0 μM in DMSO).

Figure 4 - 2 .

 42 Figure 4 -2. Illustration of interactions of aldehyde, ketone, trifluoroacetyl and tricyanovinyl with amines.

Figure 4 - 3 .

 43 Figure 4 -3. Illustration of Michael addition of amine on the β carbon of the double bond.

Figure 4 - 7 .

 47 Figure 4 -7. Chemical structure of PTTCN.

Figure 4 - 8 .

 48 Figure 4 -8. Preparation of PTTCN. Reaction conditions: (I) Succinic anhydride, AlCl3, DCM; (II) DCC, HOBt, piperidine, DCM; (III) Lawesson's reagent, toluene; (IV) n-BuLi, DMF, THF.As shown in Figure4 -8, PTTCN was synthesized starting from thiophene. The piperidine unit was introduced by amide coupling on the acid unit of 2b in presence of N,N'-dicyclohexylcarbodiimide (DCC)

Firstly, we investigatedFigure 4 - 9 .

 49 Figure 4 -9. Photographs of PTTCN in different solvents (a) before, (b) after addition of 50 eq. of propylamine for 5 minutes, (c) overnight; (Left) under sun light, (Right) under UV lamp (365 nm). 10 μM. toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), chloroform (TCM), ethyl acetate(EA), acetone (DMC) acetonitrile (CAN), ethanol(EtOH), methanol (MeOH), dimethyl formamide (DMF), dimethyl sulfoxide(DMSO).

Figure 4 - 10 .

 410 Figure 4 -10. Absorption and emission spectra of PTTCN in different solvents (a) before and (b) after addition of 50 eq. of npropylamine overnight (10 μM).

  -10 b showing the absorption and emission spectra of the mixtures after one night, it is only for the DMF and DMSO solutions that the absorption bands of PTTCN at 570 nm) completely disappeared, replaced by a blue-shifted band at 410 nm. In other solvents, two absorption bands at around 570 and 410 nm indicate the presence of at least one new compound along with starting PTTCN.

  -11, b and d). The newly formed compounds are highly emissive with quantum yields values closed to unity (Φ = 1 and 0.75, respectively). As PTTCN or EBTN1 are barely emissive in DMSO, real "turn-on" fluorescence responses are observed, giving an almost 1160-fold emission fluorescence enhancement in the case of PTTCN and 112-fold for EBTN1 (Figure 4 -12, a and c).

Figure 4 - 11 .

 411 Figure 4 -11. Absorption changes of PTTCN (a) and EBTN1 (c) by increased addition of n-octylamine. Insert: photograph of PTTCN or EBTN1 in the absence (left) and presence (right) of excess of n-octylamine under the ambient light. Fluorescence changes of PTTCN (b) and EBTN1 (d) by increased addition of n-octylamine. Insert: photograph of EBTN1 in the absence (left) and presence (right) of excess of n-octylamine under UV lamp (365 nm λex: isobestic point. 10 μM in DMSO.

Figure 4 - 13 .

 413 Figure 4 -13. Photographs of PTTCN (up) and EBTN1 (down) solutions. Treating processes: (a) addition of excess of npropylamine, (b) addition of malononitrile, (c) vacuum.

Figure 4 -

 4 Figure 4 -14. (a) Fluorescence intensity of PTTCN (at 535 nm) and EBTN1 (at 556 nm) upon addition of various amines (0-blank, 1-putrescine, 2-tyramine, 3-cadaverine, 4-octylamine, 5-propylamine, 6-histamine, 7-tryptamine, 8-spermidine, 9-1,3-diamine, 10-diethylamine, 11-diisopropylamine, 12-trimethylamine, 13-pyridine, 14-aniline and 15-ethanethiol respectively, 50 eq). (b) Fluorescence intensity of PTTCN (at 535 nm) and EBTN1 (at 556 nm) upon addition of various amines (black bars: 1-blank, 2-diethylamine, 3-diisopropylamine, 4-trimethylamine, 5-pyridine, 6-aniline, and 7ethanethiol respectively, 50 eq). Red bars represented the corresponding fluorescence intensities with subsequent addition of n-octylamine (50 eq). λex: isobestic point. 10 μM in DMSO.

Figure 4 -

 4 Figure 4 -15. (a) absorption and (b) emission spectra of PTTCN upon addition of excess of ammonia, hydrazine or dimethylhydrazine. 10 μM in DMSO. On the other hands, it is worth noting that PTTCN also has fast fluorescence-response to ammonia and hydrazine compounds. From Figure 4 -15, upon addition of ammonia, hydrazine or dimethylhydrazine to PTTCN solutions, the color violet immediately faded away (disappearing on the 575 nm absorption band) and a new absorption band around 400 nm appeared. The formed adducts are also emissive, but less fluorescent than the n-propylamine-PTTCN adduct: PTTCN/ammonia (543 nm, Φ = 0.6), PTTCN/hydrazine (516 nm, Φ = 0.3) PTTCN/dimethylhydrazine (535 nm, Φ = 0.05). This would allow PTTCN to further distinguish hydrazine from other nucleophiles.

  [d6]-DMSO, addition of n-propylamine immediately transformed PTTCN (resp. or n-octylamine to EBTN1) to only one another new compound. All the aromatic protons shifted upfield for both PTTCN and EBTN1 (Figure 4 -16). The vinylidene proton of both molecules also moved upfield, a new singlet integrating for one proton at 8.33 ppm for PTTCN and 8.43 ppm for EBTN1 appeared (Figure 4 -16). This is in perfect agreement with an imine structures (Figure 4 -17 and Figure 4 -20) in which the push-pull character is preserved, but a weaker electron-acceptor group is present. Indeed, a simple Michael addition of n-propylamine, as 99

Figure 4 -

 4 Figure 4 -16. 1 H NMR changes upon addition of (left) n-propylamine to PTTCN or (right) n-octylamine to EBTN1. To further confirm the Schiff-base structures, PTT-imine was independently synthesized from PTTAld and n-propylamine (Figure 4 -17). By comparison of the 1 H and 13 C NMR spectra of the PTTCN/npropylamine adduct to these of PTT-imine (Figure 4 -18), the imine structure was unambiguously verified.

Figure 4 - 17 . 100 Figure 4 -

 4171004 Figure 4 -17. Synthesis of PTT-imine.

Figure 4 - 19 .

 419 Figure 4 -19. Reversibility of PTTCN to n-propylamine proved by 1 H NMR.

Figure 4 - 20 .

 420 Figure 4 -20. Sensing mechanism of PTTCN and EBTN1 to primary amine.

  By spin-coating of PTTCN/sol-gel or PTTCN/poly(methyl methacrylate) (PMMA) mixtures onto a glass slide, a homogeneous PTTCN/sol-gel or PTTCN/PMMA film was formed on the glass surface. To our surprised, although PTTCN is not emissive in solid state, both films show very strong red fluorescence (675 nm, Φ = 23% for PTTCN/sol-gel and Φ = 13% for PTTCN/PMMA). This is probably due to the rigidification as well as the isolation of PTTCN molecules within the films. More importantly, this property endows the films a ratiometic fluorescence response to primary amine vapor. As shown in Figure4-21 a, upon exposure to a saturated n-propylamine vapor, the PTTCN/sol-gel film changes from violet to palegreen with absorption peak blue-shifted from 565 nm to 400 nm. Under UV 365 nm light, a significant 102 emission variation from red to green (500 nm, Φ = 43%) was observed by naked eye. PTTCN/PMMA film presented the same sensing behaviour to primary amine vapor with 57% quantum yield at the end. (Figure4-21, b). Coming along with the quick response (in 10 seconds), PTTCN films are excellent for real-time primary amine vapor detection.

Figure 4 - 21 .

 421 Figure 4 -21. Absorption and fluorescence changes of (a) PTTCN/sol-gel and (b) PTTCN/PMMA films upon exposure to n-propylamine vapor. Insert: Photographs of PTTCN sol-gel film before (red) and after (green) exposure to n-propylamine vapor under ambient light and UV lamp (365 nm).

  failed to be recovered by vacuum, indicating the irreversibility of the reaction in the films. The reason could be attributed to the dilution of the imine and the malononitrile molecules by the films (Figure 4 -22).

Figure 4 - 22 .

 422 Figure 4 -22. Sensing mechanism of PTTCN films to primary amine vapor.

103

 103

Figure 4 - 23 . 4 . 1 . 1 . 1 .. 105 Figure 4 - 24 .

 4234111105424 Figure 4 -23. Synthetic design of EBTN1-glass for recyclable primary amine vapor detection.4.1.1.1. Synthesis of EBTNAld-NHSThe synthesis of EBTNAld-NHS was depicted in Figure4-24. Firstly, in the presence of BBr3, compound 9 transformed into 13 through demethylation in 93% yield. Then a functional lateral chain bearing an end ethyl ester group was introduced by nucleophilic substitution of 13 with ethyl 4bromobutanoate. Followed by Suzuki cross-coupling, compound 15 was formed in 76% yield. The ethyl ester group of 15 was then hydrolyzed in acidic condition to afford compound 16. Lastly, the EBTNAld-NHS was obtained by addition of 1-hydroxypyrrolidine-2,5-dione under the catalysis of EDC-HCl in yield of 82%.

Figure 4 - 25 .

 425 Figure 4 -25. Procedure of preparation of EBTN1 grafted glass. Conditions: (I) Detergent/water/acetone/methanol, ultrasonication; (II) APTES (5% in methanol), 45~60min; (III) Wash with methanol, dry for 30min at 120°C; (IV) Immerse in EBTNAld solution (100 μM) overnight, rinse with methanol; (V) Immerse in malononitrile solution (10 mM) for 6h, rinse with DCM.

  Benefited by the strong emission property of EBTN-imine, EBTN1-glass slide in n-propylamine vapor atmosphere showed bright blue fluorescence (Figure 4 -26, b).

Figure 4 -

 4 Figure 4 -26. (a) Photographs of normal, EBTNAld and EBTN1-glass slides; (b) Photographs of EBTN1-glass slide in saturated propylamine vapor under ambient light (left) and UV lamp (365 nm, right).

Figure 4 -

 4 Figure 4 -27. (a) absorption and (b) emission changes of EBTN1-glass slide in the cycle test of n-propylamine vapor.

Figure 4 -

 4 Figure 4 -27 showed the absorption and emission changes of EBTN1-glass slide in its cycle test of propylamine vapor. From the spectra, EBTNAld modified glass slide with absorption and emission peaks at 430 nm and 555 nm red-shifted to 542 nm and 666 nm, respectively after soaking in malononitrile solution overnight, which indicated the conversion of EBTNAld to EBTN1 on the glass slide. It's worth noting that EBTN1 on the glass slide presented similar absorption but enhanced fluorescence compared to itself in DMF, this can be attributed to the separation of EBTN1 molecules on the glass surface. By placing the glass in saturated n-propylamine vapor for 5 min, its absorption band at 542 nm blue-shifted to 420 nm with a corresponding new fluorescence peak at 530 nm appearing, this demonstrated the colorimetric detection of EBTN1-glass slide for primary amine vapor. Then, immersing the glass slide into the malononitrile solution

9-( 7 -

 7 bromo-9,9-diethyl-9H-fluoren-2-yl)-3,6-di-tert-butyl-9H-carbazole (5)

  H NMR (400 MHz, CDCl3, ppm):  8.18 (s, 2H), 7.88 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.59-7.48 (m, 6H), 7.38 (d, J = 8.8 Hz, 2H), 2.07 (q, J = 7.4 Hz, 4-(4-methoxyphenyl)-4H-dithieno[3,2-b:2',3'-d]pyrrole-2-carbaldehyde (8)

  1 H NMR (400 MHz, CDCl3, ppm) δ 9.88 (s, 1H), 7.73 (s, 1H), 7.50 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 5.4 Hz, 1H), 7.15-7.04 (m, 3H), 3.92 (s, 3H). 13 C NMR (101 MHz, CDCl3, ppm) δ 183.0, 158.6, 148.6, 144.0, 140.6, 131.8, 128.8, 124.7, 124.4, 120.6, 116.3, 115.2, 111.9, 55.7. HR-MS (ESI-QTOF) m/z: [M+H ]+ Calcd for C16H12NO2S2, 314.0309; found, 314.0296.

2-(( 6 -

 6 (4-(bis(4-butoxyphenyl)amino)phenyl)-4-(4-methoxyphenyl)-4H-dithieno[3,2-b:2',3'-d]pyrrol-2-yl)methylene)-1H-indene-1,3(2H)-dione (EBTN2) Black solid (66 mg, yield: 57 %), eluent DCM:PE=3:1, v/v. 1 H NMR (400 MHz, CD2Cl2, ppm):  8.26 (s, 1H), 7.91 (m, 3H ), 7.72 (m, 2H ), 7.54 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 8.7 Hz, 2H), 7.14 (m, 3H) 7.09 (d, J = 8.9 Hz, 4H), 6.92 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.9 Hz, 4H), 3.97 (t, J = 6.5 Hz, 4H), 3.92 (s, 3H), 1.87-1.72 (m, 4H), 1.52 (m, 4H), 1.01 (t, J = 7.4 Hz, 6H). 13 C NMR (101 MHz, CDCl3, ppm):  190.8, 189.7,

  9. HR-MS (ESI-QTOF): m/z [M] + calcd for C51H44N2O5S2, 828.2692; found, 828.2688. 123 (E)-2-(4-(2-(6-(4-(bis(4-butoxyphenyl)amino)phenyl)-4-(4-methoxyphenyl)-4H-dithieno[3,2-b:2',3'd]pyrrol-2-yl)vinyl)-5,5-dimethyl-3-(phenylthio)furan-2(5H)-ylidene)malononitrile (EBTN3) Black solid (73 mg, 53%), eluent DCM. 1 H NMR (400 MHz, CDCl3, ppm) δ 7.53-7.45 (m, 3H), 7.42 (d, J = 8.7 Hz, 2H), 7.39-7.31 (m, 2H), 7.28-7.22 (m, 4H), 7.15-6.99 (m, 7H), 7.03 (d, J = 16.2 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.9 Hz, 4H), 3.97 (t, J = 6.5 Hz, 4H), 3.92 (s, 3H), 1.81 (s, 6H), 1.80-1.72(m, 4H), 1.52 (m, 4H), 1.01 (t, J = 7.4 Hz, 6H).13 C NMR (101 MHz, CDCl3, ppm) δ 180.5, 170.1, 158.6, 155.9, 149.1, 148.6, 147.9, 144.5, 140.0, 138.3, 136.7, 134.5, 131.7, 129.6, 127.8, 127.0, 126.9, 126.3, 126.2, 124.8, 122.0, 120.0, 116.8, 116.3, 115.4, 115.2, 114.8, 114.1, 113.0, 112.8, 105.8, 95.1, 68.0, 56. 0, 55.1, 31.4, 27.0, 19.3, 14.0. HR-MS (ESI-QTOF): m/z [M] + calcd for C58H52N4O4S3, 964.3145; found, 964.3151.

  C NMR (101 MHz, CDCl3, ppm) δ 183.0, 173.1, 158.1, 146.1, 143.0, 140.9, 131.3, 124.8, 124.0, 120.4, 116.2, 115.8, 115.2, 67.3, 60.6, 30.7, 24.6, 14.3. HR-MS (ESI-QTOF) m/z: [M+H] + Calcd for C21H19BrNO4S2, 491.9939; found, 491.9929. Ethyl 4-(4-(2-(4-(bis(4-butoxyphenyl)amino)phenyl)-6-formyl-4H-dithieno[3,2-b:2',3'-d]pyrrol-4yl)phenoxy)butanoate (15)

  To a solution of 15 (150 mg 0.22 mmol) in EtOH (2 mL) was added a solution of piperazine (116.3 mg, 1.35mmol) in DMF (1 mL) under argon atmosphere. The mixture was stirred at room temperature overnight.Then the solvent was evaporated under reduced pressure, and the obtained crude product was purified by column chromatography on silica (eluent: MeCN:MeOH:H2O=90:9:1, v/v/v) to afford COP-SO3 as a dark blue solid (130 mg, yield: 81%).1 H NMR (400 MHz, DMSO-d6, ppm) δ 7.91 (s, 1H), 7.65-7.52 (m, 2H), 7.47-7.34 (m, 2H), 7.21 (t, J = 7.4 Hz, 1H), 6.20 (d, J = 6.8 Hz, 1H), 5.83 (d, J = 6.8 Hz, 1H), 4.31 (t, J = 7.0 Hz, 2H), 3.88-3.64 (m, 4H), 3.28-3.06 (m, 4H), 2.75-2.65(m, 2H) 2.11-1.95 (m, 3H), 1.83 (t, J = 12.4 Hz, 1H), 1.63 (d, J = 6.8 Hz, 6H), 1.52 (d, J = 8.5 Hz, 6H), 1.39-1.21 (m, 2H), 0.99 (s, 9H). 13 C NMR (101 MHz, DMSO-d6, ppm) δ 177.0, 172.2, 170.4, 162.8, 144.5, 142.7, 141.4, 137.4, 129.1, 128.7, 125.

Figure 6 - 2

 62 Figure 6 -2 Characterization and comparison of F127-OTs (red) and F127-N3 (blue) with F127 (green).

  cannulated in a caudal vein (BD Neoflon TM , 16GA 0.6 x 19mm, Becton Dickinson, Helsingborg Sweden) and connected to a 1 ml syringe with a target volume of 200 μL of CPO-SO3 PNPs. Before positioning the ear on the lid of a petri dish (Ø=10mm) in the focal plane of the objective with ultrasound gel, ears were cleaned with a hear-removing cream (VEET, France) and rinsed with 70% ethanol. In accordance with the French legislation, experiments were done in compliance with the European Parliament and the Council Directive of September 22, 2010 (2010/63/EU). 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: B. van der Sanden, permit number 38 09 40 for animal experiences).

.

4 .

 4 Preparation of Solid Device for Primary Amine VaporDetectionPTTCN sol-gel and PTTCN-PMMA filmFirstly, the solutions of PTTCN (2.0 mg) in THF (1mL) and DCM (1mL) were prepared. Followed addition of MTEOS&TEOS (2g, 54 wt% in THF) and PMMA (1.5 g, 10 wt% in DCM) to the PTTCN solutions formed the PTTCN sol-gel and PTTCN-PMMA mixtures. Then by spin-coating at speed of 2000 rpm, the mixtures were spread homogeneously on a glass surface. After heating at 90°C for 1h, the films were cooled down and protected from light.

  4 μmol) or EBTN1 (6.0 mg, 8.0 μmol) and deuterated DMSO (0.45 mL). N-propylamine (400 μL) or n-octylamine (100 μL) was diluted using deuterated DMSO to form a 2 mL amine solution (N-propylamine: 2.4 M; n-octylamine: 1.34 M). Then theUV/vis and fluorescence titrationStock solution of PTTCN (1.0 mM) or EBTN1 (0.5 mM) was prepared by dissolving PTTCN (1.63, 5.0 μmol) or EBTN1 (1.87 mg, 2.5 μmol) into DMSO in a volumetric flask (5 mL). N-octylamine (8.2 μL) was dissolved in DMSO in a volumetric flask (50 mL) to form the n-octylamine stock solution (1.0 mM). In 11 volumetric flasks (5 mL) was added the same amount of stock solution of PTTCN(50 μL) or EBTN1(20 μL). Then, different and increased volume of amine stock solution was added into each flask (0, 50, 100 μL, and so on). The mixtures were diluted to volume of flask with DMSO, and preserved in dark place for 14 h.

CHAPTER II. SYNTHESIS OF NIR DYES AND DYE-LOADED F127-SIO2 NPS FOR IN VIVO FLUORESCENCE IMAGING

  Biological Transparency Window ................................................................................................................. .2.1. Synthesis of TCBZ Dyes .......................................................................................................................................... 2.2.2. Photophysical Properties of TCBZ dyes .................................................................................................................. III 2.4.1. Synthesis of F127-COOH and F127-N3 ..................................................................................................................2.4.2.Preparation of TCBZ2/5@F127-COOH-SiO2, TCBZ2/5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2 NPs .......2.4.3. Characterization ofSurface-functionalized TCBZ5@F127-SiO2 NPs .................................................................... 2.4.4. TCBZ5@F127-SiO2 and TCBZ5@F127-X-SiO2 NPs for Biphotonic in vivo Fluorescence Imaging ..................... 3. SYNTHESIS OF EBTN DYES AND EBTN@F127-SIO2 NPS FOR FLUORESCENCE IMAGING 55 3.1. Synthetic Design of EBTN Dyes .................................................................................................................. 3.2. Synthesis and Photophysical Properties of EBTN Dyes .............................................................................. 3.2.1. Synthesis of EBTN Dyes ......................................................................................................................................... 3.2.2. Photophysical Properties of EBTN Dyes ................................................................................................................. 3.3. Preparation of EBTN@F127-SiO2 NPs for in vivo Fluorescence Imaging ................................................ 3.3.1. Preparation of EBTN@F127-SiO2 NPs ................................................................................................................... 3.3.2. Characterization of EBTN F127-SiO2 NPs.............................................................................................................. SiO2 NPs ........................................................................................... 4.2. Synthesis and Characterization of CPO-SO3-F127-SiO2 NPs .................................................................... 4.2.1. Synthesis of F127-CPO-SO3-SiO2 NPs ................................................................................................................... 4.2.2. Synthesis of F127-CPO-SO3-SiO2 NPs ................................................................................................................... General Context .......................................................................................................................................... 1.2. Why Primary Amine? .................................................................................................................................. 1.3. Preliminary Tests ........................................................................................................................................ Synthesis of PTTCN ..................................................................................................................................... 2.2. Solvent Test ................................................................................................................................................. 2.3. Spectroscopic Response .............................................................................................................................. 2.4. Reversibility ................................................................................................................................................

	1. BIOIMAGING TECHNOLOGIES 1.1. 3. OBJECTIVES 4. PREPARATION OF F127-CPO-SO3-SIO2 NPS FOR PA IMAGING 1.1. 2. PRIMARY AMINE SENSING: SOLUTION STUDIES 4.1. Synthetic Design of F127-CPO-SO3-1. BACKGROUND 2.1.	1 32 80 84 91

1.2. Engineering of NIR-emitting

Organic Dyes .................................................................................................. 1.2.1. Rhodamine Dyes ....................................................................................................................................................... 1.2.2. BODIPY Dyes ........................................................................................................................................................... 1.2.3. Cyanine Dyes .......................................................................................................................................................... 1.2.4. Some Other Organic NIR-II-emitting Dyes ............................................................................................................. 1.2.5. Push-pull Dipolar Dyes ...........................................................................................................................................

1.3. NIR Organic Dyes for in vivo PA Bioimaging ............................................................................................ 1.3.1. PA Bioimaging ........................................................................................................................................................ 1.3.2. Dyes for PA Imaging ............................................................................................................................................... 2. NIR DYE-LOADED NPS FOR FLUORESCENCE OR PA IMAGING 24 2.1. Dye-loaded F127-SiO2 NPs ........................................................................................................................ 2.2. Dye-loaded Polymer NPs ............................................................................................................................ ................................................................................................................................. 1. INTRODUCTION 34 1.1. Previous Work ............................................................................................................................................. 1.2. Objectives .................................................................................................................................................... 2. SYNTHESIS OF TCBZ DYES AND TCBZ@F127-SIO2 NPS FOR FLUORESCENCE IMAGING 35 2.1. Synthetic Design .......................................................................................................................................... 2.2. Synthesis and Photophysical Properties of TCBZ Dyes .............................................................................. 22.3. Preparation and Characterization of TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs ......................... 2.4. Preparation of Surface-functionalized TCBZ2@F127-SiO2 and TCBZ5@F127-SiO2 NPs for in vivo Fluorescence Imaging ............................................................................................................................................ 4. CONCLUSION 64 CHAPTER III. DESIGN OF NIR DYES AND NPS FOR IN VIVO PA IMAGING ........................................... 1. INTRODUCTION 66 1.1. Previous Work ............................................................................................................................................. 1.2. Objectives .................................................................................................................................................... 2. PREPARATION OF CPO-SO3 PNPS FOR PA IMAGING 69 2.1. Synthetic Design of CPO-SO3 Polymer ....................................................................................................... 2.2. Synthesis and Characterization of CPO-SO3 PNPs .................................................................................... 2.2.1. Synthesis of CPO-SO3 ............................................................................................................................................. 2.2.2. Synthesis of CPO-SO3 Polymers and CPO-SO3 PNPs ............................................................................................. 2.2.3. Characterization of CPO-SO3 PNPs ........................................................................................................................ 2.3. CPO-SO3 PNPs for in vivo PA Imaging ...................................................................................................... 3. PREPARATION OF CPOP PNPS FOR PA IMAGING 74 3.1. Synthetic Design of CPOP Polymers ........................................................................................................... 3.2. Synthesis of CPOP PNPs ............................................................................................................................ 3.2.1. Synthesis of CPOP1 and CPOP2 dyes ..................................................................................................................... 3.2.2. Synthesis and Characterization of CPOP1 PNPs ..................................................................................................... 3.2.3. Synthesis and Characterization of CPOP2 PNPs ..................................................................................................... 4.2.3. Characterization of CPO-SO3-F127-SiO2 NPs ........................................................................................................ 4.3. F127-CPO-SO3-SiO2 NPs for in vivo PA Imaging ...................................................................................... IV 5. CONCLUSION 83 CHAPTER IV. DICYANOVINYL DYES FOR PRIMARY AMINE DETECTION .......................................... 2.5. Sensitivity and Selectivity ............................................................................................................................ 2.6. NMR Studies ................................................................................................................................................ 3. PTTCN FILMS FOR PRIMARY AMINE VAPOR DETECTION 101 4. EBTN1-GLASS FOR PRIMARY AMINE VAPOR DETECTION 103 4.1. Objective ................................................................................................................................................... 4.2. Preliminary Tests on EBTN1-glass ...........................................................................................................

Table 2 -

 2

1. Photophysical properties of TCBZ1-TCBZ8 in chloroform.

Table 2 - 3

 23

. Photophysical properties of TCBZ1-TCBZ8 in solid state.

Table 2 -

 2 5 and Table 2 -6. Asshown in Table2-5, increasing the mass ratio of F127-COOH resulted in a more negative surface potential (ζ-potential) of the formed F127-COOH-SiO2, which is consistent with more carboxyl groups on the particle surface. Meanwhile, no obvious size increase or particle aggregation problems happened during dialysis until the mass ratio of F127-COOH reached 15%, this value is much lower than 50% reported by

Table 2 -

 2

5. Properties of carboxyl modified F127-SiO2

NPs with varied mass ratios of F127-COOH to F127.

Table 2 -

 2

6

. Properties of azido modified F127-SiO2 NPs with varied mass ratios of F127-N3 to F127.

Table 2

 2

-8.

Table 2 -

 2

8. Photophysical properties of EBTN1-EBTN5 in chloroform.

Table 2 -

 2

	3.2.2.2.	Solvatochromism
			EBTN1				Toluene		1600000	EBTN1				Toluene
		0,06					DCM							DCM
							CHCl3							CHCl3
							DMSO							DMSO
									1200000
	Absorbance	0,02 0,04						PL Intensity (a.u.)	800000
									400000
		0,00							0
			450	500	550	600	650	700		600	650	700	750	800	850	900
					Wavelength (nm)						Wavelength (nm)

9. Photophysical properties of EBTN1-EBTN5 in toluene.

Table 2 -

 2

10

. Properties of EBTN1-EBTN5@F127-SiO2 NPs in water.

Table 3 -

 3

1. Properties of CPO-SO3 polymers.

Table 3

 3

		Mn polymer
	Sample		nc*	X	solvent
		(g/mol)
	1 CPO-SO3@AEM	29600	1.7	AEM	Water
	2 CPO-SO3@COOH	29600	1.7	COOH	Water
	3 CPO-SO3@COOH	59900	5.8	COOH	Water
	4 CPO-SO3@COOH	59900	12.5	COOH	Water
	5 CPO-SO3@COOH	59900	14.5	COOH	Water

-1). Among them, 5 CPO-SO3@COOH with the highest dye density of 14 per polymer was tried for making CPO-SO3 PNPs. Actually, the redissolution of 5 CPO-SO3@COOH by nc*: Estimated average number of CPO-SO3 per polymer

Table 3 -

 3

2. Properties of CPOP1 polymers.

Table 3 -

 3 3. Properties of CPOP2 PNPs.

	Sample	Mn bloc PNAM (g/mol) Dh (DLS) (nm)	nc*	solvent
	CPOP2 PNPs	11000	60	≈ 260	Water

nc*: Estimated average number of CPOP2 per NP

  Figure 4 -12. Fluorescence spectra comparison of PTTCN (a) or EBTN1 (c) before (black) and after (red) addition of 140 or 500 µM of n-octylamine. (d) Plot of the fluorescence intensities of PTTCN (b) at 535 nm and EBTN1 (d) at 556 nm as a function of concentration of n-octylamine added. λex: isobestic point. 10 μM in DMSO.

	EBTN1
	Equation					y	=	a	+	b*x
	Plot						B
	Weight					No	Weighting
	Intercept					-20116,74426	±	16002,770
	Slope					4791,61334	±	238,11468
	Residual	Sum	of	Squares	3,9689E8
	Pearson's	r			0,9951
	R-Square	(COD)	0,99022
	Adj.	R-Square			0,98777

  C NMR (101 MHz, DMSO-d6, ppm) δ 185.0, 158.7, 145.7, 142.7, 141.2, 131.1, 125.2, 123.6, 123.1, 116.6, 116.3, 116.2, 115.7, 56.0. HR-MS (ESI-QTOF) m/z: [M+H] + Calcd for C16H11BrNO2S2, 391.9415; found, 391.9409. 6H). 13 C NMR (101 MHz, CDCl3, ppm) δ 158.9, 156.1, 151.5, 151.4, 150.4, 149.6, 144.4, 139.8, 132.0, 131.1, 128.1, 127.1, 126.6, 125.5, 124.9, 119.5, 115.5, 115.4 (2C), 114.9, 114.4, 105.5, 71.0, 68.0, 55.7, 31.4, 19.3, 13.9. HR-MS (ESI-QTOF) m/z: [M] + Calcd for C45H40N4O3S2, 748.2542; found, 748.2524.

  1, 124.7, 122.9, 118.6, 117.2, 116.3, 111.8, 103.4, 99.6, 94.6, 51.4, 49.2, 48.5, 44.9, 44.0, 43.5, 33.0, 29.1, 28.3, 27.5, 27.3, 26.7, 23.8. HR-MS (ESI-QTOF) m/z: [M+H] + Calcd for C41H50N6O4S, 723.3687; found, 723.3651. In DMF: λabs (706 nm), ε (53400 L/mol/cm), λem (856 nm), Φ (7%).

					1746 cm -1
	4000 4000	3500 3500	3000 3000	2500 2500	2000 2000	1500 1500	1000 1000	500 500
			Wavenumber (cm -1 )
					399,6748 399,6748 78,4434 78,4434	76,513	76,513
					400,1569 400,1569 77,2449 77,2449	81,5948 81,5948
					400,639 400,639	78,3775 78,3775	85,9886 85,9886
				135 401,1211 401,1211 81,8917 81,8917 136 137	88,6265 88,6265
					401,6032 401,6032 87,5149 87,5149	89,2379 89,2379
					402,0854 402,0854 94,334 88,2344 94,334 88,2344
					402,5675 402,5675 100,894 100,894	86,54	86,54
					403,0496 403,0496 105,782 105,782	85,3106 85,3106

Figure 6 -1 Characterization and comparison of F127-COOH (red) with F127 (green).

  Animal model. PA experiments were performed on the ears of white CD-1 IGS mice (Charles River,

	power	10%	20%	30%	40%	50%	60%	70%	80%	90%	100%
	415nm	0,7	1,5	2,3	3	3,7	4,2	4,7	5,2	5,7	6,1
	660nm	0,8	1,7	2,7	3,6	4,2	5	6,2	7	7,8	7,2

  Single crystals of TCBZ8 and PTTCN were prepared by diffusion of a non-solvent (pentane or EtOH) in a concentrated solution of dye (in CHCl3) at room temperature. The crystallographic data and refinement details are summarized in the following table.

	Dcalc./g cm -3	1.206	1.366
	Crystal system	Monoclinic	Monoclinic
	Colour	Orange	Dark violate
	μ [mm -1 ]	0.072	0.336
	T [K]	150.00 (10)	150.01 (10)
	Space group	P21/c	P21/c
	a [Å]	34.383 (5)	16.4065 (16)
	b [Å]	12.0537 (12)	6.4682 (6)
	c [Å]	17.729 (3)	15.0664 (17)
	α[°]	90	90
	β[°]	100.401 (13)	98.286 (9)
	γ[°]	90	90
	V [Å 3 ]	7226.8 (17)	1582.2 (3)
	Compound Z	TCBZ8 8	PTTCN 4
	Z'	1	1
	Chemical structure Wavelength/Å	0.71073	0.71073
	θrange (°)	2.838-29.716	3.031-26.620
	total no. data	23036	21500
	no.unique data	8501	4126
	Crystal structure
	no. params refined	5166	3829
	R1	0.1778	0.0537
	Cristal growth wR2	CHCl3/Pentane (diffusion) 0.2509	CHCl3/EtOH (diffusion) 0.1270
	Empirical formula GOF	C47H45NO2 0.940	C17H15N3S2 1.073
	Formula weight	655.84	325.44

[a] Weight percent of F127-N3 to total F127 polymers. [b] Diameters measured by DLS in nm.

H NMR spectra were performed after every addition of n-propylamine (7.6 μL, 18.4 μmol) or n-octylamine solution (6 μL, 8.0 μmol) for 30 s.

Acknowledgements

NMR spectra in the SI of this article show the presence of at least another unidentified compound beside the Michael adduct (Figure 4 -3). [START_REF]A reaction-based sensing scheme for volatile organic amine reagents with the chromophoricfluorogenic dual mode[END_REF] Figure 4 -4. Mechanism of forming an imine from a dicyanovinyl group.

For the dicyanovinyl group, Shi et al proposed in 2014 that reaction with a primary amine form an imine resulting from the nucleophilic addition of the primary amine to the dicyanovinyl group followed by loss of a malononitrile molecule (Figure 4 -4). [START_REF] Shi | A mild and catalyst-free conversion of solid phase benzylidenemalononitrile/benzylidenemalonate to N-benzylidene-amine and its application for fluorescence detection of primary alkyl amine vapor[END_REF] The imine being much more emissive than the starting dye, they used that to design "turn on" amine vapor sensor of very sensitivity. Chapter VI. Experimental Section 1. Synthesis

General Information

All commercially available solvents and reagents were used without further purification. Anhydrous THF was obtained by distillation over sodium and benzophenone, toluene was dried over Al2O3 column.

Anhydrous DMF, DCM and acetonitrile were purchased from Acros under AcroSeal™. Column chromatography was performed on Merck silica gel (40-63 μm). Microwave syntheses were conducted in 20 mL or 5 mL sealed tube on a Biotage Initiator 2.5 single-mode reactor using external IR temperature control. NMR spectra were recorded at ambient temperature on Bruker Advance 300 or 400 operating at 300.1 or 400.0 MHz for 1 H and 75.0 or 101.0 MHz for 13 C, respectively. Chemical shifts are given in parts per million (ppm) with the residual solvent peaks as internal standard (7.26 ppm and 77.2 ppm for chloroform, 2.50 and 39.5 ppm for DMSO). 1 H NMR splitting patterns are described as singlet (s), doublet (d), triplet (t) or multiplet (m) and coupling constants are given in hertz (Hz). High-resolution mass spectrometry (HRMS) were measured by ESI-TOF (Bruker Daltonics Micro TOF-Q II) at the Centre Commun de Spectrométrie de Masse (UCBL, Villeurbanne, France).

All solvents used in optical measurements are of spectrophotometric grades. UV/Vis absorption spectra were measured using a dual beam Jasco 670 spectrometer. Fluorescence spectra in diluted solution and in solid were recorded on a Horiba Jobin-Yvon Fluorolog-3® spectrofluorimeter equipped with red-sensitive Hamamatsu R928 or water-cooled R2658 photomuliplier tubes. Spectra were reference corrected for both the excitation source light intensity variation (lamp and grating) and the emission spectral response (d etector and grating). Quartz cuvettes with thickness of 1 cm or 1mm were used as sample cells. The quantum yield relative to the reference is given by equation ( 1):

where S is the slope obtained by plotting the integrated area under the fluorescence emission spectrum vs the absorbance at λexc and nd the refractive index of the solvents. Superscript Ref and s correspond to the reference and the sample respectively. For each experiment 5 points were recorded, all corresponding to an absorbance at λexc below 0.1. Fluorescence standards Rubrene, Cresyl violet perchlorate and Styryl 9M were

Synthesis and Characterization of Modified F127

(F127-COOH) F127 (2.0 g, 0.16 mmol) was dissolved in H2O (50 mL), TEMPO (1.0 mg, 6.4 μmol), KBr (38.0 mg, 0.32 mmol) and NaClO (10 mL, 4.8%) were then added at 0℃. The mixture was stirred for 1h at 0℃ then 6h at room temperature, and the pH of the mixture was controlled around 10 by addition of 0.5 N NaOH in this period of time. After quenching by EtOH (1mL), 4N HCl was added to adjust the pH of the mixture to 3.

The mixture was then extracted by DCM, washed with water (250 mL×2) and concentrated by evaporation under reduced pressure. Followed by precipitation with diethyl ether and filtration, 1.2 g of F127-COOH as white powder was afforded. (Yield: 60%) (F127-OTs) F127 (5.0 g, 0.4 mmol) was dissolved in DCM (25 mL), TsCl (305 mg, 1.6 mmol) and TEA (5 mL) were then added. The mixture was stirred for 16h at room temperature. Then the organic solvent was evaporated under reduced pressure. 1M HCl (50 mL) was added, followed by extraction using DCM and washing twice with water (250 mL×2). The organic phase was collected and concentrated. After precipitation with diethyl ether and filtration, 4.35 g of F127-OTs was obtained as white powder. (Yield: 86%) (F127-N3) F127-OTs (1.5 g, 0.12 mmol) and NaN3 (46 mg, 0.71 mmol) were dissolved in DMF (20 mL). After stirring at 80℃ for 6h, DCM (50 mL) was added. The mixture was washed with water (100 mL×3) and extracted by DCM. The organic phase was collected and concentrated, followed by precipitation with diethyl ether and filtration to form 1.40 g F127-N3 as white powder. (Yield: 94%).

TCBZ5@F127-SiO2, TCBZ5@F127-COOH-SiO2, TCBZ5@F127-N3-SiO2 and TCBZ5@F127-X-SiO2 NPs

TCBZ5@F127-SiO2 and TCBZ5@F127-COOH-SiO2 NPs were synthesized following the general procedure of making dye-encapsulated F127-SiO2 NPs. For TCBZ5@F127-X-SiO2 and TCBZ5@F127-TPP-SiO2 NPs, a TCBZ5@F127-N3-SiO2 NPs solution was firstly prepared by doubling the amount of all the reagents used in the general procedure. After passing through a 0.2 μm filter (regenerated cellulose), the TCBZ5@F127-N3-SiO2 NPs was obtained with volume around 8.4 mL. Then the solution was diluted into 9 mL and separated into two parts with every part of 4.5 mL (particle concentration around 50 μM). Then, CuSO4 (7.5 μmol), sodium ascorbate (2.5 μmol) and THPTA (12.5 μmol) were added to the mixtures followed by addition of the functional moieties (2.5 μmol). After gentle stirring for 3h, the mixtures were purified by dialysis for 3 days.

EBTN1-EBTN5@F127-SiO2 NPs

Sample Dye (mg) F127 (mg)

EBTN1@F127-SiO2 NPs EBTN1 (0.04 mg) 200

EBTN2@F127-SiO2 NPs EBTN2 (0.04 mg) 200

EBTN3@F127-SiO2 NPs EBTN3 (0.04 mg) 200

EBTN4@F127-SiO2 NPs EBTN4 (0.04mg) 200

EBTN5@F127-SiO2 NPs EBTN5 (0.04 mg) 200 TCBZ5@F127-SiO2 and TCBZ5@F127-X-SiO2 NPs.

Preparation of F127-CPO-SO3-SiO2 NPs

In vivo Microscopy

Two-Photon imaging

Two-photon microscopy was performed using a Trimscope II (LaVision BioTec, Bielefeld, Germany) equipped with a 16x water-immersion objective (NA 0.8, Nikon, France) and Imspector 2015 software. The excitation wavelengths of the pulsed infrared laser (Insight DeepSea, Spectra-Physics, Evry, France) were 900 nm for both NPs.

PA imaging

The experimental setup is shown in