Fabien Lucas

Lambert Sicard

Olivier Jeannin

Joëlle Rault-Berthelot

Emmanuel Jacques

Cassandre Quinton

Cyril Poriel
email: cyril.poriel@univ-rennes1.fr

[4]Cyclo-N-ethyl-2,7-carbazole: Synthesis, structural, electronic and charge transport properties

Keywords: nanoring, organic field effect transistor, organic semiconductor, charge transport, bridged cyclo-oligophenylenes, structure-properties relationship

Nanorings, which are macrocycles possessing radially directed -orbitals have shown a fantastic development in the last ten years. Unravelling their unusual electronic properties has been one of the driving forces of this research field. However, and despite promising properties, 10.1002/chem.201901066

Introduction

With the synthesis of the first cyclo-para-phenylene (CPP) in 2008, [1] hoop-shaped πconjugated macrocycles possessing radially directed -orbitals (so called nanorings or nanohoops) have been the subjects of intense research worldwide. [2][3][4][5][6] Compared to their linear counterparts, nanorings present singular electronic and structural characteristics. [3][4][5][6][7] In addition, studies of the influence of the nanoring size and of the building units on the structural, electronic, physical, chiral and encapsulating properties have already shown the diversity of this field. [3][4][5][6][7][8] The growing interest in nanorings chemistry and physics has led to recent applications such as imaging tools in biology [9] or complexation of single-walled carbon nanotubes. [10] In 2016, Nuckolls and coworkers have shown the several advantages of cyclic oligomers over corresponding linear counterparts in electronics devices. [11] In 2019, theoretical calculations have predicted the strong potential of CPPs and Donor-Acceptor CPPs as high mobility materials. [12,13] However, and despite the fantastic development of organic electronics technology in the last twenty years, [START_REF] Bredas | The WSPC References on Organic Electronics: Organic Semi-Conductors[END_REF] only very few examples of nanorings incorporation in 10.1002/chem.201901066 Accepted Manuscript organic electronic devices have been reported so far. [START_REF] Liu | [END_REF]16] Bridging the gap between organic electronics and nanorings appears hence as an appealing challenge. In this context of organic electronics, mobility of charge carriers is a central concept, [17] which drives the performance of the three types of devices, namely Organic Light-Emitting Diode (OLED), Organic Field-Effect

Transistor OFET and solar cell. However, the charge transport properties of nanorings are almost unknown and the literature only reports one example of a nanoring, namely 10cycloparaphenylene (10[CPP]), for which an electron mobility value of 4.5 × 10 -6 cm 2 V -1 s -1 has been measured in an electron-only device using space charge limited current technique (SCLC). [16] In order to further incorporate nanorings in efficient electronic devices in the future, knowledge of their charge carriers motilities is a mandatory step. As far as we know, no example of a nanoring in an OFET has ever been reported. In the present work, we report the synthesis, the electronic properties and the incorporation in an OFET of a cyclic tetracarbazole linked at para positions C2 and C7, namely [4]cyclo-N-ethyl-2,7-carbazole ( [4]C-Et-Cbz). The structural, photophysical and electrochemical properties have notably been compared to those of structurally related analogues [4]cyclo-9,9-diethyl-2,7-fluorene [4]C-diEt-CF (with carbon bridges) and [8]-cycloparaphenylene [8]CPP (without any bridge, see molecular structures in chart S1) in order to shed light on the impact of the bridging in nanorings. OFET characterization of [4]C-Et-Cbz provides a hole mobility μFE of 1.1 ×10 -5 cm² V -1 s -1 , a threshold voltage VTH of 24 V, a subthreshold swing SS of 2.2 V/dec and an on/off ratio of the drain current (IDon/IDoff) of 4.26 10 4 . This work not only shows that nanorings can be used as active layer in OFETs but also provides the first OFET characteristics benchmark for nanorings.

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Results and discussion

Scheme 1. Synthesis of [4]C-Et-Cbz

The carbazole as its 'all carbon' structural analogue fluorene belongs to the family of 'bridged biphenyls', which are both key building units in the design of organic semi-conductors for electronics. [18][19][20][21][22] In linear organic semi-conductors, the bridge has a significant impact on the structural properties (by notably rigidifying and flattening the molecular structure) which in turn modify the electronic properties. [20,[23][24][25] In the case of carbazole, the effect of the nitrogen bridge is strong. For example, this fragment displays an electron-rich character, which has been advantageously used for hole transport in organic semi-conductors. However, in nanorings, these effects have not been described to date. In 2006, Müllen and coworkers have reported a cyclododeca-2,7-carbazole around a porphyrin template. [26] The first examples of highly strained 2,7-cyclocarbazoles possessing a radial-conjugation have only been reported ten years later, in 2016, by Yamago and coworkers. [27] These molecules, constituted of four carbazole units substituted on the nitrogen atoms by either a methyl or a phenyl group, were designed to investigate the inner region of nanorings by 1 H NMR studies and their electronic properties, despite being recorded, were not discussed.

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The synthetic pathway to [4]C-Et-Cbz reported herein is inspired by the works of Yamago and coworkers [28] and Isobe and coworkers (Scheme 1). [29] On first attempt, [4]C-Et-Cbz was obtained with a relatively low yield of 6% from the di-pinacolborane carbazole 1 in two steps (1. Pt(cod)Cl2/CsF at the reflux of THF for 24h, 2. P(Ph)3 in toluene 30 min at rt and then 24h at 100°C, Table S1). This yield is half of that reached in the case of its structurally related tetracyclofluorene analogue [4]cyclo-9,9-diethyl-2,7-fluorene [4]C-diEt-F (12%), under strictly identical conditions. [30] As the synthesis of nanorings is a central point in the field and particularly if an electronic application is envisaged, synthetic investigations (scouting the impact of temperature, solvent and reaction time, see Table S1) for both the transmetallation and reductive elimination steps were performed. The best conditions (Scheme 1) found are described below. The intermediate 2, based on four square-shaped tetranuclear platinum complexes, was obtained by stirring the dipinacol carbazole 1 with Pt(cod)Cl2 and cesium fluoride in a refluxing THF solution for 24 h. Then, the carefully dried crude containing 2 was treated with triphenylphosphine in o-dichlorobenzene, 1 h at rt and 48 h at 180°C to give [4]C-Et-Cbz with an improved yield of 27% from the dipinacolborane carbazole 1. Thus, the reductive eliminations step being unfavoured because of the strain energy, it can be helped by increasing both the temperature (by using o-dichlorobenzene as previously reported by Isobe and coworkers for cyclonaphthylene nanohoops [31] ) and reaction time. Calculations of the strain energy of [4]C-Et-Cbz (73 kcal.mol -1 , see SI for details on calculations, Figure S15) appears almost identical to that previously described for [4]C-diEt-F (72.4 kcal.mol -1 ), [30] showing the similar impact of the C and N bridge (substituted herein with ethyl chains) on this parameter.

In the end, the global yield of the formation of [4]C-Et-Cbz from its corresponding dibromocarbazole (ca 22%) has been increased threefold using the present optimized conditions compared to that described by Yamago and coworkers via a distanylated carbazole (yield of 10.1002/chem.201901066

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7% for a N-methyl-substituted cyclocarbazole). [27] This is an interesting feature for the future incorporation of nanorings in electronic devices. A NOESY experiment allows to discriminate between the two rotamers exclusively showing the through-space coupling between protons H 1 and H 3 Figure 1, right. This indicates that we are in presence of the all anti conformer . As the  conformer was also obtained following the same approach in the case of the fluorene analogue [4]C-diEt-F, [30] this result indicates that the nature of the bridge (N vs C) does not modify the formed rotamer. The variable-temperature NMR studies from 223 K to 373 K do not show any significant modifications of the 1 H spectrum (Figures S5 andS10), confirming the presence of a single isomer, and not a time-averaged dynamic structure, at room temperature. These variable temperature NMR studies also indicate that the carbazole units cannot flip and that the other rotamers (αααβ, αααα and ααββ isomers) cannot be obtained by thermal input (until 373 K) As exposed above, the hydrogen atom H 1 appears to be the most shielded due to the ring current of the macrocyle to which it is exposed. [27] It suggests the carbazole units are tilted the bridge side towards the inside of the ring (as also observed in single-crystal X-Ray crystallography, see below). The degree of tilt of the two other [4]-cyclocarbazoles [27] reported in literature (substituted on the nitrogen atom by either methyl or phenyl units) seems to be the same since they display similar H 1 chemical shifts of 6.45-6.51 ppm in 1 H NMR. Thus, in the case of the [4]-cyclocarbazole family, the size of the pending substituent seems to have no influence on the arrangement of the nanoring in solution, unlike recent results reported for the [4]-cyclofluorene family. [30] X-Ray diffraction of single crystals of [4]C-Et-Cbz (Figure 2), which were obtained by vapor diffusion of hexane in a concentrated solution of chloroform, confirms the αβαβ conformation assigned by NMR. [4]C-Et-Cbz crystallizes in a C2/c group and the length of the C/C bonds of the carbazole units (around 1.40 Å) indicates that the benzenoid character is reasonably conserved, such as its non-bridged analogue [8]CPP as reported by Jasti and coworkers (CCDC reference : 871414). [32] [4]C-Et-Cbz forms a distorted ellipsoidal nanoring with axes of 11.1 and 10.6 Å Figure 2, top-right), which is different than its fluorene analogue [4]C-diEt-F, which forms a perfect circle (diameter=11.0 Å, CCDC reference:1580867). [30] As shown in the

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NMR part, X-ray diffraction confirms that the upper part of each carbazole (including the nitrogen atom) is tilted towards the centre of the nanoring. The shortest distance between two cofacial hydrogen atoms is hence measured between the two H 1 atoms (mean distance H 1 -H 1 : 8.05 Å, Figure S16). Then, the corresponding distance between two cofacial hydrogen atoms gradually increases from H 3 -H 3 (mean distance: 10.15 Å, Figure S17) to H 4 -H 4 (mean distance: 11.87 Å, Figure S18). Due to this inclination, the cofacial nitrogen atoms are also relatively

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As the main factors at the origin of the specific electronic properties of the nanorings are the deformations of the constituted units and their relative arrangement, [7] those of the present cyclocarbazole [4]C-Et-Cbz deserve to be precisely stressed out. Four structural parameters should be considered: the out-of-plane distortion, the bending angle, the displacement angle and the torsion angle (see definitions and detailed calculations in SI, Figure S21-S26). First, the distance C2-C7 of a carbazole unit can give an idea of its out-of-plane distortion. For [4]C-Et-Cbz, this distance is short (around 6.56 Å, varying from 6.51 to 6.59 Å depending on the carbazole unit, Figure 2-right), clearly showing the strong deformation of the carbazole units when compared to differently substituted carbazoles (the C2-C7 distance is evaluated at around 6.8 Å, Figure S31). [33][34][35] Furthermore, the bending angle is measured at around 129° for the carbazole units in [4]C-Et-Cbz (Table S8), whereas it is found at around 178° (Table S11) for the previously mentioned carbazoles, [33][34][35] close to planarity (180°). Thus, the short C2-C7 distances and small bending angles measured for [4]C-Et-Cbz show the high deformation of the present carbazole fragments. Nevertheless, it is important to note that the mean displacement angle [3] measured for [4]C-Et-Cbz, 7.20° (Table S9), is smaller than that of nonbridge analogue [8]CPP, 9.58° (Table S10), [32] and almost identical to that of carbon-bridged analogue [4]C-diEt-F, 7.10°, [30] showing that the bridge (either carbon atom in fluorene or nitrogen atom in carbazole) tends to flatten the biphenyl unit. In addition, the mean internal torsion angle between two phenyl units of the same bridged biphenyl is small, evaluated around 1.34° (ranging from 0.09° to 2.87°) for [4]C-Et-Cbz (Figure S26). This value is higher than that observed in [4]C-diEt-F for which the angle is null. [30] Thus, in Accepted Manuscript torsion angle between two adjacent carbazole units is also an important parameter as it is notably involved in the stabilization/destabilization of the frontier molecular orbitals. [7] The mean torsion angle appears herein as high as 40.6° (values comprised between 38.38 and 42.35°, Figure S24), which is similar to that measured in [4]C-diEt-F (41.5° between two fluorene units) [30] and significantly higher than that of [8]CPP (24.3° between two phenyl units, Figure S29). [32] Thus, one can conclude that the presence of a bridge induces higher torsions between the constitutive units than in non-bridged CPP. To conclude, bridged-oligophenylene nanorings display a strong deformation of the constitutive units and a high torsion between them. These parameters seems to be one of the structural particularities of bridgedoligophenylene nanorings and will have important repercussions in the electronic properties described below.

The packing diagram of [4]C-diEt-Cbz

shows that the nanorings form a tube-like structure along the b axis and are stacked along the a axis with a particularly short C-C distance of 3.31 Å, inferior to the sum of their van der Waals radii (dC-C=3.4 Å [36] , Figure 2-bottom and Figure S27). For a given nanoring (coloured in red in Figure 2 To precisely compare the photophysical properties of [4]C-Et-Cbz with those of the two other nanorings of interest in this study, they have been investigated in different solvents, in chloroform (to compare the absorption properties with the ones of [8]CPP, [28] see Figure S33), in cyclohexane (to compare the absorption properties with the ones of [4]C-diEt-F, [30] see Figure S32) and in THF (to compare the fluorescence properties of the three nanorings, [37] Figure 3

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(Figure 3, top, black solid line). In addition and as for the two other nanorings, [8]CPP [28] and [4]C-diEt-F, [30] [4]C-Et-Cbz displays a band tail at around 400 nm which is due, according to TD-DFT calculations, to a symmetry forbidden (f=0) HOMOLUMO transition (Figure 3, bottom). Indeed, as observed for the nanorings in general, the two orbitals are delocalized on the whole nanoring with a radial geometry. On the other hand, the band at around 337 nm is much more intense with a molar absorption coefficient  of 7×10 4 mol.L -1 .cm -1 . According to TD-DFT calculations, this band is due to six transitions (almost degenerated two by two): H-1LUMO and H-2LUMO (th=361 nm), HOMOL+1 and HOMOL+2 (th=354 nm) and H-5LUMO and H-6LUMO (th=340 nm). The H-1, H-2, H-5 and H-6 orbitals are all delocalized on the whole nanoring, including the nitrogen atoms, and are thus specific of chromophore [4]C-Et-Cbz. However, the HOMO density resembles that of [8]CPP, [38,39] with no participation of the nitrogen atom, whereas the LUMO density resembles the one of the molecule of carbazole Cbz itself, with no participation of the carbon atom in para position of the nitrogen atom. The L+1 and L+2 orbitals are similar to the ones of [8]CPP [39] in the way that they are mainly localized on two opposite carbazole units, but the repartition of the lobes seems specific to [4]C-Et-Cbz. Thus, despite the involvement of molecular orbitals specific to [4]C-Et-Cbz, the presence and the nature of the bridge seem to have no impact on this absorption band at ca 340 nm which is also observed in [8]CPP (=340 nm, =3×10 4 mol.L - 1 .cm -1 in CHCl3) [28] and in [4]C-diEt-F (=347 nm, =9×10 4 mol.L -1 .cm -1 in cyclohexane), Table S12. [30] On the other hand, the band observed at 292 nm in cyclohexane for In emission spectroscopy (Figure 3, top, blue solid line and Figures S32-S38), [4]C-Et-Cbz displays an unresolved spectrum (in the three solvents) with a maximum at 490 nm (in THF).

The emission spectrum of [4]C-Et-Cbz resembles that of [4]C-diEt-F (max=492 nm in THF,

Figure S38) and is highly blue-shifted compared to that of [8]CPP (533 nm in THF). [37] Thus, the presence of a bridge linking two phenyl units (in both smaller Stokes shift in the former than in the latter. This can be explained by a weaker conjugation because of both a decrease of the displacement angle and an increase of the torsion angle between biphenyl units, such as for larger CPPs. [40] Thus, the emission of bridged cyclooligophenylenes occurs in a different range than that of non-bridged analogues and the effect of the bridging is different to that of linear analogues.

Furthermore, the quantum yield of [4]C-Et-Cbz in THF is measured at 16% and thus is twice that of [8]CPP (8%), [37] which also indicates a bigger partial levelling of the excited state (due to the bridge) which induces an enhanced self-trapping of the exciton and thus a higher transition dipole moment for [4]C-Et-Cbz compared to [8]CPP. [40] Note that, if the nature of the bridge seems to have no influence on the shape of the spectrum, it impacts the intensity of the fluorescence since the quantum yield measured for S37) provides a lifetime of 5.8 ns, which is shorter than that of [8]CPP reported at 17.6 ns. [37] The radiative rate constant kr of [4]C-Et-Cbz is calculated to be 2.8×10 7 s -1 , being six times higher than that of [8]CPP (4.8 ×10 6 s -1 ), whereas the non-radiative constant In thin film (Figure 3, top, dashed lines), the most relevant differences compared to solution are surprisingly observed for the absorption spectra and not for the emission spectra. In absorption, one can note a small red shift and an increase of full width at half maximum of the bands and an important band tail at high wavelengths. In emission, only a small extension of the bandwidth is observed with no shift of the maximum between solution and thin film (= 492 nm). This feature allows a high control of the emission wavelength in the solid state, key point for future optoelectronic applications. Furthermore, the absolute quantum yield of thin film of [4]C-Et-Cbz was measured at 15%, a value identical to that obtained for the solution of [4]C-Et-Cbz in THF (16%). This shows the great stability of the emission in the solid state. It should be noted that this is the first time that a nanoring displays the same quantum yield both in solution and in solid state (for example, the one of [4]C-diEt-F is decreased by a factor 2 in thin film compared to solution [30] ). To sum up, the emission properties of [4]C-Et-Cbz (both shape of spectrum and quantum yield) in diluted solution are kept in spin-coated film. This finding appears appealing for the future design of carbazole nanorings for solid state optoelectronics.

Electrochemical analyses of [4]C-Et-Cbz were performed in DMF (for reduction) and in

CH2Cl2 (for oxidation). Cyclic voltammetry (CV) recorded between 0.0 and -2.65 V presents two irreversible reduction waves with two maxima (or shoulder) at -2.40 (sh) and -2.55 V vs SCE (Figure S39). In oxidation, three successive processes with maxima at 0.86, 1.40, and 2.03 (sh) (vs SCE) were recorded (Figure 4, top left). The HOMO and LUMO energies calculated Accepted Manuscript from the onset potential of the first oxidation and reduction (Eonset ox : 0.77 V and Eonset red = -2.18 V) were respectively evaluated at -5.17 and -2.22 eV leading to a HOMO/LUMO gap of 2.95 eV. The first oxidation of [4]C-Et-Cbz is therefore significantly shifted compared to that of its structurally related cyclofluorene analogue [4]C-diEt-F (Eonset ox = 0.86 V, HOMO= -5.26 ev). [30] and even more shifted compared to that of [8]CPP (Eonset ox = 0.93 V, HOMO= -5.33 ev). [41] This feature translates the more electron-rich character of [4]C-Et-Cbz. Despite the three molecules being constructed on the cyclic association of eight phenyl units, the different first oxidation potential shows the important role played by the bridge (C vs N) on the electrochemical properties (and more precisely herein on the HOMO energy level). One can note that the trend is similar to that of their building units (HOMOs decrease from Nethylcarbazole [42] to 9,9'diethylfluorene [43] to biphenyl [44] ).

When scanning between 0 and 1 V, one can observed (i) that the first wave is not fully reversible and (ii) that there is a slight shift and a current decrease after recurrent sweeps in the same potential range signing the reactivity of the radical cation formed (Figure 4, top right). This instability of [4]C-Et-Cbz .+ is due to the high reactivity of the C3 and C6 positions of the carbazole units which can rapidly be involved in C-C couplings (see below). [45] The evolution of the CVs is even accentuated when cycling between 0.2 and 1.86 V (Figure 4, bottom left) with a complete disappearance of the first oxidation wave at 0.86 V, in favour of the appearance and the regular growth of a new redox system centred at 1.5/1.3 V and of the covering of the electrode surface with an insoluble deposit. The presence of a deposit on the electrode surface indicates an anodic electrodeposition process. If anodic coupling is a well-known technique to generate -conjugated oligomers and polymers from various linear building blocks, [46] [51,52] as far as we know, this strategy has never been examined for nanorings. In the present case, this anodic coupling seems to be efficient as at the tenth scan, the current intensity at 1.5 V is more than seven times that of the first scan. The electrochemical behaviour of the electrodeposited shows a reversible redox process with a maximum at 1.5 V and a Eonset ox at 1.02 V (HOMO= -5.42 eV). Thus, there is surprisingly a HOMO energy level lowering of 0.25 eV (from -5.17 to -5.42 eV) between the monomer [4]C-Et-Cbz and the material formed at the electrode, indicating that the -conjugation is not extended (and even restricted) in the latter which is different from what is commonly observed for other linear -systems. [47][48][49][50][51][52][53] This particularity can be first explained by the carbon atoms involved in the coupling. Indeed, as previously shown with various carbazole oligomers or polymer, [46] such type of anodic couplings take place at the C3 and C6 carbon atoms (as for classical electrophilic aromatic substitutions), [54] which are both in meta position of the biphenyl linkage leading therefore, from this point of view, to an electronic decoupling. Leclerc and coworkers have indeed reported that the oxidation of a linear poly(2,7-carbazole) leads to coupling reactions at these C3/C6 positions. [55] Secondly and considering that the carbazole units in [4]C-Et-Cbz are connected from their C2 and C7 positions to form the nanoring, one can suppose that linking two nanorings by their C3 and/or C6 positions would provide a sterically hindered molecular system, resulting in a perturbation of the -electron delocalization within the nanoring and hence a lower HOMO energy level (Figure S40). Thus, we believe that both electronic and steric considerations can be invoked to understand the HOMO lowering observed for the present electrodeposited material compared to its monomer [4]C-Et-Cbz. architecture (device structure [56,57] in Figure S41). This BG-BC structure, in which the active layer is evaporated in the last step, avoids any structural effect resulting from the deposition of other layers. First, it should be mentioned that [4]C-Et-Cbz can be easily evaporated (230°C, 2.10 -7 mbar). OFET characterization provides a mobility μFE of 1.1 ×10 -5 cm² V -1 s -1 , a threshold voltage VTH of 24 V, a subthreshold swing SS of 2.2 V/dec and an on/off ratio of the drain current (IDon/IDoff) of 4.26 10 4 , Figure 5. Despite this hole mobility being obviously low in regards to the state of the art, [58] this work displays the first incorporation of a nanoring in a non-optimized OFET and hence the first example of a transfer characteristic and a FE mobility.

It is important to mention that SS (capability to switch between on and off state) is particularly understand OFET behaviour in an electronic circuit, which is the final goal of these components. [59,60] Indeed, two working modes could be employed for an OFET. The first one is the use of OFETs in a digital circuit. In this case, the OFET is used in two distinct polarization points. In the 'on' regime, Gate-Source voltage VGS should be high and equal to the supply voltage of the circuit (VGS=VDD) when Drain-Source voltage VDS is close to 0 V. This regime is called linear regime and Drain-Source resistance RDS has to be low (RDS=RDSon). In the 'off' regime, voltages are inverted with VGS equal to ground value (VGS=0V) and VDS equal to the supply voltage of the circuit (VDS=VDD). This regime is called saturated regime with a high value of RDS (RDS=RDSoff). In the case of a digital circuit, the RDSoff/RDSon ratio has to be high to discriminate the two regimes. The slopes in the saturated and linear regimes for the present case of [4]C-Et-Cbz are different enough (RDSoff/RDSon=33.8) to differentiate these two regimes (Figure 5, bottom-left), which is interesting for future applications.

The second working mode is the use of OFETs in an analog circuit. In this case, OFET is supplied with only one polarization point where Drain current ID is independent of VDS and a small change in VGS value (small signal regime) has a high impact on ID value. This polarization point is fixed with the crossover of output characteristics and resistance characteristics used to polarize the OFET (Figure 5, bottom-right). In the case of [4]C-Et-Cbz, the dependence of ID to VDS is low and we can therefore imagine using this OFET in an analog circuit to amplify small input signal. We are working in this direction. Thus, [4]C-Et-Cbz-based OFET displays some interesting characteristics such as a low dependence of ID to VDS. possesses a high HOMO energy (-5.17 eV) and that the emission properties are kept in solid state (in term of wavelength and quantum yield). We believe that these appealing emission properties can be used in the future in optoelectronic devices. [4]C-Et-Cbz has been successfully incorporated as active layer in a p-type channel OFET with a particularly low SS of 2.2 V/dec possessing a bottom-gate bottom-contact architecture. The FE mobility extracted, 1.1×10 -5 cm 2 .V -1 .s -1 appears as the first value measured for a nanoring and shows the possibility to use such macrocyles with radially directed -orbitals in organic electronic devices increasing therefore the molecular diversity of organic semi-conductors. With a more accurate molecular design (alkyl chain engineering for example), this performance will be surely improved in the future. We are currently working in this direction. Thus, these results not only show that nanorings can be used as active layer in OFETs but also provides a first benchmark value.

  10.1002/chem.201901066

The 1 H

 1 NMR of [4]C-Et-Cbz displays in the aromatic region an upfield shifted singlet corresponding to the hydrogen atom H 1 (=6.51 ppm) and two deshielded signals assigned by 2D NMR spectroscopy to H 3 (=7.49 ppm) and H 4 (=7.92 ppm), Figure 1, left. The presence of only three signals for the aromatic region is in accordance with a symmetrical molecule possessing either anor an arrangement (relative position of the nitrogen bridges).

Figure 1 .

 1 Figure 1. Left: 1 H NMR spectrum of [4]C-Et-Cbz in CDCl3. Right: selected part of the NOESY spectrum in CDCl3showing the H 1 /H 3 through space correlation (full spectra can be found in the SI, FigureS42).

  close to each others, dN-N=9.51 and 9.04 Å (Figure 2, top-left). Thus, the solid state molecular arrangement of [4]C-Et-Cbz is similar to that found for [4]C-diEt-F, with the bridges pointing towards the centre of the ring.

Figure 2 .

 2 Figure 2. X-Ray crystal structure representations (Top) and packing diagram (Bottom) of [4]C-Et-Cbz with distances in Ångström.

  [4]C-Et-Cbz, the presence of a nitrogen bridge between two phenyl rings flattens the structure of the carbazole unit compared to a biphenyl in [8]CPP but nevertheless in a less efficient manner than the carbon bridge of [4]C-diEt-F. It can be hence concluded that the nitrogen bridge provides more structural flexibility than the carbon bridge in a nanoring constituted of four units. Finally, the 10.1002/chem.201901066

  -bottom), which is surrounded by six other nanorings, many short C-C distances comprised between 3.31 and 3.49 Å are detected. It should be mentioned that the building unit carbazoles are involved in a different way. Thus, two cofacial carbazole units of each nanoring display short C/C distances with the carbazoles of two other surrounding molecules (from 3.31 to 3.49 Å). The two other cofacial carbazoles are also involved in short contacts but with either its own pending ethyl chain or with the ethyl chain of a surrounding molecule (3.44 and 3.46 Å), showing that the pending chains have a role in the packing diagram. Similar conclusions have been drawn with the ethyl chains borne by the bridges of [4]C-diEt-F. This feature could be used in the future to tune the supramolecular arrangement of such nanorings by alkyl chain engineering.

Figure 3 .

 3 Figure 3. Top: Combined absorbance (black) and emission (blue, λexc = 336 nm for the solution and 350 nm for the film) spectra of [4]C-Et-Cbz in THF (solid lines) and in spin-coated film (obtained from a THF solution, 1 mg/mL, dashed lines). Bottom: Representation of the energy levels and the main molecular orbitals involved in the electronic transitions of [4]C-Et-Cbz obtained by TD-DFT B3LYP/6-311+G(d,p), shown with an isovalue of 0.02 [e bohr -3 ] 1/2 . For clarity purposes, only the major contribution of each transition is shown (see SI for details).

  Figure 3 and S34 and S38). The absorption spectra of [4]C-Et-Cbz are similar regardless of the solvent (CHCl3, THF or cyclohexane), with absorption maxima at around 264, 288 and 337 nm

[ 4 ]

 4 C-Et-Cbz is not found for [8]CPP nor [4]C-diEt-F but is observed in Cbz (=292 nm in cyclohexane). Note that the molar absorption coefficient of [4]C-Et-Cbz at 292 nm (=5.7 ×10 4 mol.L -1 .cm -1 in cyclohexane) is almost four times the one of Cbz (=1.6 ×10 4 mol.L -1 .cm -1 in cyclohexane) matching with the involvement of four carbazole units. The fourth band, located around 260 nm, also seems specific to [4]C-Et-Cbz, inherited from Cbz (=255 nm,  = 1.1 ×10 4 mol.L - 10.1002/chem.201901066 Accepted Manuscript 1 .cm -1 ). In summary, the low energy transitions (=340 and 400 nm) are also found in [8]CPP and [4]C-diEt-F (and in nanorings in general) whereas the high energy transitions (=290 and 260 nm) are specific of [4]C-Et-Cbz.

[ 4 ]

 4 C-Et-Cbz and [4]C-diEt-F) leads to an emission spectrum strongly blue shifted (by 46-48 nm) compared to that of non-bridged [8]CPP. As the absorption spectra of [4]C-Et-Cbz and [8]CPP are similar, this induces a

[ 4 ]

 4 C-diEt-F in THF is almost twice (28%) the one of [4]C-Et-Cbz. The fluorescence decay curve of [4]C-Et-Cbz measured in THF (exc=310 nm, Figure

  10.1002/chem.201901066 Accepted Manuscript of [4]C-Et-Cbz is only three times higher than that of [8]CPP (resp. 1.5 ×10 8 s -1 and 5.2×10 7 s -1 ). Thus, this confirms that the increase of the quantum yield induced by the presence of the bridge (from [8]CPP to [4]C-Et-Cbz) is due to a higher transition dipole moment. To conclude, in dilute solution, the incorporation of four nitrogen bridges within the [8]CPP molecular structure significantly shifts the emission maxima to lower wavelengths and increases the quantum yield.

  10.1002/chem.201901066

  10.1002/chem.201901066 Accepted Manuscript material studied in a solution in absence of any electroactive substance (Figure 4, bottom right)

Figure 4 .

 4 Figure 4. Cyclic voltammetry (100 mV/s) of [4]C-Et-Cbz [2×10 -3 M] CH2Cl2/Bu4NPF6 (0.2 M), platinum electrode diameter 1 mm. Top left: one scan 0.25/2.25 V in CH2Cl2; Top right: three scans between 0.25/1 V in CH2Cl2; Bottom left: ten scans 0.25/1.86 V in CH2Cl2; Bottom right: three scans 0.27/1.86 V (modified platinum working electrode).

Figure 5 .

 5 Figure 5. Top: Transfer characteristic in linear regime of OFET with VDS=-10V; Bottom: Output Characteristics: (Left) OFET in digital circuit with two polarization points (On and Off regimes). (Right) OFET in analog circuit with one polarization point.

Finally, in

  order to determine the charge transport properties, [4]C-Et-Cbz has been used as active layer in p-type channel OFETs possessing a bottom-gate bottom-contact (BG-BC)

  10.1002/chem.201901066 Accepted Manuscript low (2.2 V/dec), translating the electrical quality of the nanoring-insulator interface. This can be advantageously used in the future. To go deeper in the understanding of the behaviour of [4]C-Et-Cbz in an OFET, output characteristics have been recorded. Output characteristics constitute important data to

Conclusion

  To conclude, we report in this work a detailed structure-property relationship study of[4]cyclo-N-ethyl-2,7-carbazole [4]C-Et-Cbz and its first incorporation in an OFET. [4]C-Et-Cbz is obtained in a four-steps synthesis with an interesting yield of 22%. The structural and electronic properties have been compared to those of structurally related analogues [4]C-diEt-CF (with carbon bridges) and [8]-CPP (without any bridge) in order to shed light on the impact of the bridging in nanorings. Of particular interest, we have notably shown that [4]C-Et-Cbz

10.1002/chem.201901066 Accepted Manuscript

Acknowledgments

This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 699648 (FRODO Project). This work is supported by the European Union through the European Regional Development Fund (ERDF), the Ministry of Higher Education and Research, the Région Bretagne (ARED DIADEM, FL),

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Accepted Manuscript


Stéphane Desgranges

Orane Lorton

Laura Gui-Levy

Pauline Guillemin

Zarko Celicanin

Jean-Noël Hyacinthe

Romain Breguet

Lindsey A Crowe

Christoph D Becker

Marine Soulie

Jean-Noel Hyacinthe

Marine Soulié

Nicolas Taulier

Christiane Contino-Pépin

Rares Salomir

Micron-sized PFOB

Introduction

High intensity focused ultrasound (HIFU) is a promising non-invasive and non-ionizing treatment for ablation of solid tumours. 1,2 It has FDA approval for the treatment of uterine fibrosis 3 , and is in clinical development to treat several solid malignant tumours including liver, prostate, breast, bladder, kidney and soft tissue sarcoma. [4][5][6][7][8][9][10] However, it possesses several shortcomings such as long treatment duration to fully ablate the tumour 1,11 and, for deep-seated tumours, high ultrasound (US) intensity is required leading to an increase in side effects. In addition, even when US energy is concentrated to the focal point, it can also be deposited along the US beam in front or behind the focus point and cause severe side effects such as skin and bone burning. 8,[12][13][14] Furthermore, given a treatment planning, interpatient variation in the volume and shape of the lesion may be difficult to control and to reproduce. 15 The ablative effects of HIFU are due to the focusing of high energy beams in a small region on the order of the wavelength, i.e. on the millimetre scale. 1 At the focal spot, the temperature can rise by 15 to 50 °C within seconds, resulting in a rapid blood coagulation and inducing cell necrosis. The damage to the tumours involves two synergistic phenomena.

The first is the thermal deposition of energy and is proportional to the coefficient of absorption of the tissue. The second is the inertial cavitation (IC) 1 yielding mechanical damages to cell structure and that can also increase the thermal effect consecutive to the emission of acoustic waves at higher frequency than the incident beam, that is, mode conversion phenomenon. 16 Several approaches have been investigated to amplify the thermal delivery during HIFU and hence to decrease the occurrence of side effects. The first approach was to use microbubbles (MB) filled with perfluorocarbon (PFC) gas that were originally used for ultrasound contrast enhancement. 11,17,18 The MB serve as nuclei for inertial cavitation (IC) and act as enhancers of tissue heating rate as they absorb energy from the sound wave when they oscillate. 16,19 However, as they are ultra-sensitive to US with a very low IC pressure threshold, they may cause unwanted damage along the US beam due to vaporisation of bubbles and/or initiation of IC, producing unwanted side effects such as uncontrollable pre-focal lesion and skin burn. 11,19 Additionally, they exhibit a limited circulation half-life as gases diffuse rapidly in tissue. To overcome this limitation, an alternative approach would be to produce bubbles in-situ without an exogenous agent, however this method it is not satisfactory because of the high IC threshold of tissues, and the results can be highly variable due to the natural heterogeneity of tissues. 20 The use of acoustic droplet vaporisation (ADV) was suggested to be more advantageous to the same purpose. The concept of ADV consists of using phase shift droplets (PSD) filled with a liquid PFC that undergoes a phase change from liquid to gas under the induction of an US wave. 21,22 PFC droplets are able to vaporise, like any volatile liquid, provided a sufficient decrease in pressure below their vapour pressure or an increase in temperature above their boiling point. Under HIFU conditions, at the focal point, the negative pressure peak is sufficient to vaporise droplets into bubbles with a volumetric expansion at least 5 to 6 times the parent droplets. The resulting bubbles can be spatiotemporally controlled 23,24 and, conversely to their unexcited liquid counterpart, possess better echogenic properties. The acoustic negative peak pressure necessary for this vaporisation depends on several factors, such the nature of PFC core, the type of shell and droplet size. These exogeneous droplets that provide bubbles in-situ act as nuclei for in vivo cavitation leading to tissue heating and lesion. 25,26 Moreover, the formation of a bubble cloud would mostly reflect the incident ultrasonic beam and thus protect the far field tissues from US wave effect. 27,28 The backward reflected wave would also contribute to an increase in the pressure amplitude in front of the bubble cloud and hence help further vaporisation. 22 Like microbubbles, droplets are usually constituted of two parts: the core which contains at least one type of PFC and the shell made of a pure surfactant or a mixture of surfactants. For a given droplet composition, the size is an important feature for its vaporisation threshold, as a consequence of the Laplace pressure inside the droplet which is inversely proportional to its radius and to the interfacial pressure between the two liquids.

Therefore, ADV requires more energy than the theoretical condition when a droplet was in contact with air. The larger the droplet, the lower the energy required to vaporise it. 24,29 The choice of PFC core is a key factor. Several groups 30,31 have developed superheated PFC filled droplets, which consist of a PFC with low boiling temperature, usually below human body temperature, that remains in liquid state at 37 °C thanks to either the Laplace pressure and interfacial tension or the metastability of the superheated liquid PFC against homogeneous nucleation. 32 This allows a decrease in the energetic vaporisation threshold and even the use of diagnostic US apparatus for this purpose. Droplets have another major advantage as they possess a longer circulation half-life than their gaseous homologues. 22 There are two kinds of PSD. The first are PFC nanodroplets constituting nanoemulsions, called phase shift nanoemulsions (PSNE). Taking advantage of the enhanced permeability retention (EPR) effect, they can extravasate from the neovasculature and accumulate in the tumour microenvironment. These PSNE are used for imaging and/or therapy. [33][34][35] The second are micron-sized droplets (MSD) which are restrained to the vasculature (endo-vascular). They can be used for enhancing thermal ablation in highly perfused tumours 15,36 and as well for embolotherapy as they generate micro-bubbles able to occlude small capillary vessels. 23,37 In order to reduce the potential side effects ascribed to HIFU therapy when adapting it to highly perfused tumours, we report the use of perfluorooctylbromide (PFOB)-filled MSD to reduce the ultrasound exposure time and energy required for tumour ablation.

Conversely to other reported studies, we used a PFC with a high boiling point (142 °C) in order to gain in droplet stability 26 and allowing a reverse phase shift once the HIFU beam is stopped, in order to avoid cellular and tissue damage as well as blood vessel occlusion. 21 In other terms, high boiling point PFC droplets undergo quasi-reversible interactions with the HIFU beam, which subsequently avoids possible capillary occlusion due to volume expansion, permits recirculation of droplets in the blood stream after exposure to the HIFU and enables accurate spatial control of the thermal effects localized around the focal point.

PFOB, a FDA approved PFC was used as a potential blood substitute because of its oxygen high solubilising ability, inertness and stability. 38,39 It was shown that a formulation of PFOB with lecithin possesses a very low toxicity with a LD 50 in rats of 45 g/kg and a short half-life in the body of about 4 days for 2.5 g/kg administrated. 40 PFC droplets need to be stabilised with amphiphilic molecules such as lipids or fluorinated surfactants. [START_REF] Tadros | Emulsion Formation and Stability[END_REF] The present study uses in-house surfactants called F-TAC which consist of a fluorinated hydrophobic moiety exhibiting a high affinity for PFC droplets and a hydrophilic moiety made of a polyTRIS oligomer [START_REF] Astafyeva | [END_REF] (See Figure 1a). Contrary to commercially available fluorinated surfactants often used in the literature, such as ZONYL (Dupont De Nemours) and CAPSTONE (Chemours), F-TAC surfactants exhibit a good biocompatibility (no hemolytic activity, LD 50 up to 4.5g/kg in rats after i.v. administration), have a ubiquitous distribution in rat after i.v. or per os route, and display a long half-life (30-50h) without any degradation in both plasma and tissues. 43,44 The main goal of the study is the enhancement of HIFU mediated heat deposition by MSD embedded in tissue mimicking material. The influence of the F-TAC chemical structure modification on droplet size and the impact of MSD concentration on the enhancement of HIFU thermal effect was assessed. To do so, the droplets were embedded either in an acoustically absorbent agar-based TMM that mimics the acoustic properties of soft tissue, 45 or in a non-absorbent material made of gelatine gel to gain a better understanding of the mechanism of action of MSD. The velocity of sound in the used gel was assessed, as that gel preparation underwent some substantive change to be compatible with magnetic resonance (MR) imaging and guidance of the HIFU sonication. Given that the depth of US penetration is inversely proportional to the frequency, a 1 MHz frequency used for HIFU sonification was considered to be a good compromise for deep tissue application, for instance in the liver or kidney. The temperature rise was monitored accurately throughout the gel by proton resonance shift frequency (PRSF) MR thermometry, and a diagnostic ultrasound device was used to investigate the attenuation of the backscattered signal from MSD doped gels. 1a).

Experimental (material and methods)

Due to the fluorine-fluorine interaction, the fluorinated part of these amphiphilic molecules exhibits a high affinity for the perfluorocarbon core of the droplets while their polar head ensures the whole water solubility. 43 Their synthesis previously described by Pucci et al. 46 was easily performed in one step by free radical polymerisation, allowing a swift supply of surfactant of high quantity. For a given perfluorocarbon core, the size of the resulting MSD is on one hand correlated to the concentration and chemical structure of the surfactant and, on the other hand, to the level of energy delivered to the solution during the emulsification process. [START_REF] Astafyeva | [END_REF]47 The chemical composition of the liquid core, the surfactant concentration and the process conditions were kept constant and only the impact of the surfactant chemical structure was assessed. (telogen) and azobisisobutyronitrile (AIBN) as a radical initiator. Ten ml of solvent were used per gram of tris(hydroxymethyl) acrylamidomethane (THAM) (C = 0.57 mol/L) and the concentration of AIBN was 0.5 eq of telogen. R 0 is the telogen/monomer molar ratio. The summary of the different polymerisation conditions is listed in Table 1.

Briefly, in a shlenck tube, dry methanol or a mixture of methanol and water (9/1) for the highest DPn, AIBN, THAM and telogen agent were added, the mixture underwent three cycles of freeze, vacuum, thaw and then was heated at 90°C for 4 hours under vigorous stirring until complete disappearance of the monomer. Then the crude product was precipitated twice in diethyl ether and filtrated and dried to recover the expected compound as a white powder with yields ranging from 31.8% to 84.0% (see Table 1). The DPn was assessed by 19 F-NMR as described previously. [START_REF] Astafyeva | [END_REF]

MSD preparation

For our therapeutic purpose, the maximum droplet size in terms of vascular circulation is 6 µm, given the strong requirement to avoid capillary blockage and to allow them to be transpulmonary. 21 In preliminary studies (data no shown) we noticed that employing a high energy process using an ultrasonic device (Bioblock Scientific Vibracell 75043, 13-mm diameter sonotrode) always led to a bimodal population, one in a the nanometric range and one in the micrometric range. Accordingly, the emulsion was prepared using a low energy process using a homogeniser as with a Polytron® system PT 3100 homogenizer from Kinematica (Luzern Switzerland). General procedure: To prepare a 10% volume fraction emulsion, 835 mg of surfactant were dissolved in 58.5 ml of water then 6.5 ml of PFOB were added. The resulting mixture was cooled down with an ice bath and then the resulting emulsion was homogenised three times 15 min at 22500 rpm. Finally, the emulsion was kept at 4°C until use.

Gel preparation

As a proof of concept for the enhancement of HIFU-induced heat deposition by MSD and accurate MR thermometry, MSD were embedded into a tissue mimicking material (TMM).

There are numerous TMM available, among them the most common are agar, urethane rubber, zerdine, silicone polyvinyl alcohol, polyacrylamide and gelatine. [48][49][50] We decided to use the well characterised agar-based gel as it possesses several advantages such as having a sound velocity value close to that of soft tissue, it exhibits almost a linear response of attenuation to frequency and it can be stored for several weeks. 45 Furthermore, this TMM possesses a high melting point of about 80°C and is reusable compared to other gels such as BSA-loaded polyacrylamide. 51

Agar gel (sample #1 and #2)

The composition of the TMM gel 45 was modified in order to be compatible with our experimental setting. The main components of the gel are water, glycerol and agar, the first two compounds mainly contribute to the sound velocity value, while the last one contributes to the stiffness of the gel. The Al 2 O 3 powder, which delivers the attenuation properties of the gel, had to be substituted with SiO 2 because of its interaction with the magnetic field resulting in low MRI signal especially with T 2 * sequences as for PRFS thermometry. The incorporation of SiC , which mimics the backscattering properties along with Al 2 O 3 , was also suppressed as it was not mandatory for our purpose.

General procedure: Proportions of the different ingredients other than water used to reach a constant 290 ml of final gel are provided in mass unit (gram): glycerol = 33.6, BAL = 0.27, agar = 9, Si0 2 (1.5m) = 2.85, Si0 2 (1.5m) = 2.64. Silicon oxide was suspended in 50 ml of degassed water and insonified with a 13 mm sonotrode for 2 min in. The BAL solution, glycerol, the silicon oxide mixture and degassed water (see Table 2) were added to a 400 ml tared beaker. And under mechanical stirring the mixture was heated and then agar was added. The solution was heated at above 90°C for one hour. Then the gel was set to cool down under magnetic stirring and any water lost was compensated with degassed water.

Then when the mixture reached about 40°C; the emulsion (see Table 2), loaded with three drops of methylene blue, was added, homogenised and cool down. The volume fraction of PFOB in TMM was used to describe the MSD concentration (see Table 2).

Gelatine gel (sample #3 and #4)

The purpose of this section was to identify the dominant mechanism producing enhanced acoustic absorption among two hypotheses: 1) the MSD are directly converting the mechanical energy into thermal energy; 2) the MSD act as inelastic scatters producing mode conversion and re-emitting higher frequency than the incident one, with the higher frequencies being absorbed more effectively by the surrounding bulk gel. 52 If the second hypothesis is true, the efficiency of micro-particles should be significantly decreased in a non-absorbent gel. If the first hypothesis is true, their efficiency should be comparable when embedded in an absorbent or non-absorbent bulk gel.

A non-acoustic absorbent gel was prepared with water, gelatine and benzalkonium chloride and its acoustic properties were measured. 9 g of gelatine (brand Vahiné), 285 mg of BAL were added to 276 ml of degassed water. Then the mixture was heated at 45°C for 5-10 min to ensure complete dissolution of the gelatine. The mixture was left to cool down to 25°C and poured in an open glass cylinder, with its base closed by paraffin film. Then 15 ml of emulsion was added and then cooled drawn at 4°C. For the control gel, we used the same protocol, but 285 ml of degassed water was added.

Physical characterization

Particle size

The particle size distribution was assessed using a Mastersizer 2000 laser diffraction particle size analyser (Malvern Instruments, Orsay, France) equipped with Hydro2000S as sample dispersion unit (A) mod using the Mie light scatteringtheory. The refractive indices used were 1.305 for the PFOB and 1.333 for the dispersant (water). Several drops of the emulsion were added with a stirring of 500 rpm to the sample dispersion unit. The Mie theory was used to determine the volume weighted mean diameter D [4,3] and the polydispersity was calculated as d90/d10. d90 is the diameter at which 90% of the sample's volume is comprised of droplets with a diameter less than this value. d10 is the diameter at which 10% of the sample's volume is comprised of droplets with a diameter less than this value. The size distribution histogram is shown in Figure 1b.

Optical microscopy

The optical microscopy was performed on an Olympus BX60 microscope (Olympus, Rungis, France) with 100x magnification (see Figure 1c) and no spectral filter.

Determination of volume fraction

All spectra were recorded on a 400 MHz Bruker Avance II spectrometer with a resonance frequency of 376.53 MHz for 19 F. 19 ppm respectively, thus avoiding problem of keeping a uniform field over a long range. The ratio of integration of the external standard over that of PFOB was plotted against the volume fraction of PFOB to create a calibration curve. All measurements were performed on triplicate samples.

Acoustic velocity measurement

Measurement of sound velocity was performed using the setup schematically described in SI. During a measurement, a burst made of one sinusoidal period was generated by a wave function generator (model 33250A from Agilent, les ulis, France). The burst intensity was amplified 500 times using a RF power amplifier (model A 10-100 from M2S, Argelès sur Mer, France), then went through a duplixer (model RDX-6 from RITEC, Warwick, USA) and eventually reached a transducer. We used three transducers (from Panasonics, Gennevilliers, France) that differ by their resonance frequency; 2.25, 5 and 10 MHz. The burst sinusoidal frequency was chosen to match the transducer central frequency. The transducer was in contact with a gel immersed in water. The short pressure wave produced by the transducer propagated though the gel until it reached the opposed edge of the gel that is in contact with a metallic surface. The wave was reflected back to the transducer and converted into an electrical signal that was sent by the duplixer to a pre-amplifier (PAS-0.1-20 from RITEC), then to a Broadband receiver (BR-640A from RITEC). The signal was visualised on an oscilloscope (model WaveSurfer 424 from Lecroy, Courtaboeuf, France) and recorded after averaging over 400 sweeps. The same measurement was performed after removing the gel where the signal propagates over exactly the same distance in water. The squared signal amplitude was analysed. The temporal position of reflected pulses (determined from its center of gravity) was simply a multiple n times the time 2τ to travel forward and back through the gel. The corresponding forward and back travelling distance, that is twice the gel thickness δ, was determined from the signal measured after removing the gel and using the known ultrasound velocity of water (that is 1480 m/s at a temperature of 20°C). The sound velocity in the gel was then calculated as an average value of sound velocities derived from τ and δ for the n reflected pulses. The mechanical effect of HIFU sonication on the MSD size distribution was investigated in a liquid emulsion using a fixed focal point beam, applied power 135 W, pulse duration 90 ms, duty cycle 90 %, total duration of the sonication paradigm 33 s. To this purpose 3ml of emulsions of MSD stabilized with F 6 TAC 7 or F 8 TAC 7 respectively were inserted in an ultrasound-transparent container centered on the focal point and exposed one, two or three times to the sonication paradigm, separated by 5 minutes intervals. The particle size distribution was measured using the Mastersizer 2000 laser diffraction particle size analyser as described above.

MR thermometry

All measurements were performed using a 3T whole body MR system (Prisma Fit, Siemens, Erlangen, Germany). An 11-cm diameter receive only loop coil was used and placed around the sample. High resolution MR thermometry was performed by Proton Resonance

Frequency Shift (PRFS) thermometry, 53 2.6.4. 19 F MRI of MSD loaded TMM samples 19 F image acquisition of the samples was performed using an RF-spoiled 2D gradient-recalled echo (GRE) pulse sequence in order to confirm the uniform distribution of micro-particles in the gel. A dedicated 19 F quadrature RF-birdcage coil was used, switchable between 1 H and 19 F (Clinical MR solutions, Brookfield, WI). 54 The resonance frequency for 19 F was 115.95

MHz. The coil had internal diameter 4.4 cm and 6 cm length. Due to the small size of the coil, subsamples were cut from the TMM gel native and doped with 0.1% and 0.5% MSD v/v and stacked parallel inside the coil in a miniature water bath, which improved the local magnetic field homogeneity (passive shimming). Main parameters of the gradient echo 19 F sequence were TR = 300 ms, TE = 5.07 ms, NSA = 25, BW = 325Hz/pixel, matrix 96 x 96, FOV = 128 x 128mm, flip angle = 70°, slices per slab = 8, slice thickness = 10 mm, pulse duration = 2 ms, in plane voxel size = 1.67 x 1.33 mm 2 .

Ultrasonography

The acquisition of ultrasound images to investigate the attenuation of the backscattered signal from gel samples was performed using a clinical US system (ACUSON Antares, Siemens Healthcare, Mountain View, CA). The abdominal imaging probe composed of 192 elements operated in harmonic mode at 2.2 MHz. Appropriate near field coupling and far field full absorption of the acoustic field was implemented to avoid beam reflections.

Results and discussion

Characterization of microdroplets

As shown in Table 3, for all surfactants, except for the F 8 TAC 13 and F 8 TAC 17 , MSD possess a size distribution in the micrometric range. For a given hydrophobic tail, the larger the polar head, the smaller the resulting MSD size until reaching a plateau. For both series, the optimal polar head size was found to be around 12 Tris units. Above this value of DPn, the MSD size remains constant for both hydrophobic tails. Furthermore, the size decreased by a factor 6.5 between DPn 7 and 13 for the F 8 series, while for the F 6 series the size decreases only by a factor 2.5 between DPn 7 and 12. On one hand, this trend can be explained by the fact that, during the emulsification process, increasing the polar head size increases the steric hindrance and hence the stability of the MSD until an optimal size is reached. [START_REF] Tadros | Emulsion Formation and Stability[END_REF] On the other hand, once this plateau is achieved (for DPn ≥ 12), the hydrophobic tail seems to play a significant role, as the MSD diameter obtained for F 8 TAC 13 is twice smaller than for F 6 TAC 12 .

This might be either due to an optimal volume ratio between the polar head and the hydrophobic tail of the surfactant and/or to a higher concentration of available surfactant (in the form of free monomer or micelles) in the dispersant phase (i.e. water). In the case of F 8 TAC 13 this leads to a finer emulsion. The surfactant concentration is about 8 (for F 6 TAC 12 )

and 280 (for F 8 TAC 13 ) times over the critical micellar concentration (CMC). It is noteworthy that this difference in droplet size cannot be ascribed to a difference in surface tension between PFOB and water in the presence of each surfactant as they are similar with 12.1 mN m -1 for F 6 TAC 12 and 10.4 mN m -1 for F 8 TAC 13. [START_REF] Astafyeva | [END_REF] Emulsions were stable for several weeks in the refrigerator which is in good agreement with previous work from other groups. 15 Our group has previously shown that the surfactant type F 6 TAC 7 is perfectly biocompatible after i.v.

injection in mice (LD50 above 4.5g/kg). 43 Given that droplets need to be smaller than 6 μm in diameter to avoid capillary thrombosis, and that the larger the droplets diameter the lower the energy required for their vaporisation, 55 MSD made with either F 8 TAC 7 or F 6 TAC 7 surfactant appear to be the best candidates. For the first emulsion the d90 is above 7 µm, while for the second emulsion, d90 was below 6 µm in diameter which makes the latter one a better candidate. However, as

shown by the MSD size distribution in Figure 1.b, the droplets obtained with F 6 TAC 7 displayed a very high polydispersity (d90/d10=4.00) [START_REF] Astafyeva | [END_REF] which was also confirmed by optical microscopy (Figure 1.c).

The method of MSD titration was modified from the one published by Astafyeva et al [START_REF] Astafyeva | [END_REF] in order to obtain a totally homogenous solution containing both PFOB and water. To do so, we used a ternary system made of a mixture of methanol and diethyl ether, the latter being used to ensure complete solubilisation of PFOB in the solution. 56 The concentration of PFOB in the emulsion was thus estimated to be 11.6 ± 0.9 %, which indicated a loss of about 15% of water during the synthesis process.

MR compatibility and acoustics properties

The final gel composition demonstrated perfect MR compatibility in term of local magnetic susceptibility and was shown to be homogeneous at the observation scale of the MR (mmrange), as illustrated in Figure 2 a,b. High resolution GRE proton 3D images also demonstrated that no macroscopic air bubbles were present.

The sound velocity of our TMM was found 1522 ± 5 m/s at 1 MHz, 1521 ± 5 m/s at 2.5 MHz, 1528 ± 5 m/s at 5 MHz and 1532 ± 5 m/s at 10 MHz. These values are close to the speed of sound in soft tissue in vivo (approx. 1540 m/s). Furthermore, usually US devices are calibrated at this speed of sound. 57 The speed of sound of our TMM is a little bit lower than the one found in the original gel from Ramnarine et al. 45 Even if some TMM components were changed, the sound velocity is only proportional to the quantity of water, agar and glycerol and these were used in the same proportion as Ramnarine et al. 58 Furthermore, approximately the same sound velocity is measured for the same TMM but without silica (Data not shown). This discrepancy could be due to the use of different brand of agarose, given that their mechanical properties change according to their molecular weight, 59 and that change in molecular weight affects the gel elasticity with an elasticity decreasing proportionally with the molecular weight.

(

) 1
C ij represents the stiffness coefficient, ρ the mass density and the speed of sound. [START_REF] Laugier | Introduction to the Physics of Ultrasound[END_REF]

Effect of MSD concentration on acoustic properties of the gel (echogenicity)

Four concentrations of MSD using F 6 TAC 7 surfactant were embedded into the TMM to study their impact on the acoustic and echogenic properties. The sound velocity in the different TMM loaded with MSD decreased linearly as a function of their concentration, as PFOB sound velocity is much lower, 623 m/s, than the control TMM, 1522 m/s. 40,[START_REF] Astafyeva | [END_REF] The MSD were not hyper-echogenic in harmonic ultrasound images at 2.5 MHz (see Figure 3) but increasing the MSD concentration induced a significant enhancement of the attenuation of the backscattered acoustic signal as the far field signal become darker as the concentration rises. The backscattered signal was plotted against the depth of the signal source in the image, independently for the four different concentration TMM and fits to an exponential decay function f(x)=exp(-a*x) with the linear attenuation coefficient "a" decreasing linearly with droplets concentration.

MSD interaction with HIFU beam

In absorbent TMM

The 19 F-MR imaging confirmed that the droplets were evenly distributed throughout the gel on the scale of the current resolution (Figure 4).

Only the two lowest MSD concentrations were tested for HIFU thermal enhancement, 0.1 and 0.5 % v/v. These concentrations are more realistic when considering the feasible delivery in living tissue. The additional temperature elevation was approximately 9°C and 15°C for 0.1% and 0.5% concentration of MSD respectively, as illustrated in Figure 2 d-f and Figure 5a, which corresponds to an impressive thermal dose amplification by a factor on the order of 2 9 and 2 15 respectively, according to Sapareto. [START_REF] Sapareto | [END_REF] Table 4 shows the results for the two defined metrics of HIFU enhancing effect in two series of TMM samples, the precision of measurements, the values of the two tailed p-test and the confidence interval (CI). As the p-value was always inferior to 10 -5 in each comparative branch, the reported number of replicates is clearly sufficient and allowed an estimation of the enhanced heating efficacy with 6% precision (second metric). This value is considered sufficient for in vivo application, given the other potential sources of errors in a biological system that largely overweight this uncertainty.

The additional temperature elevation per unit of emitted acoustic energy (first metric) was found 4.30  0.39 °C/kJ in gel series #1 with 0.1% concentration of MSD, 3.45  0.22°C/kJ in gel series #2 with 0.1% concentration of MSD, 7.32  0.57 °C/kJ in gel series #1 with 0.5% concentration of MSD and 5.15  0.28 °C/kJ in gel series #2 with 0.5% concentration of MSD.

The application of the second metric of HIFU enhancing effect yielded an integral enhancement of the thermal energy produced in the MR slice of (3.56  0.44) × 10 -3 in the gel series #1 with 0.1% concentration of MSD, (4.03  0.32) × 10 -3 in the gel series #2 with 0.1% concentration of MSD (6.51  0.72) × 10 -3 in the gel series #1 with 0.5% concentration of MSD, and (7.08  0.40) × 10 -3 in the gel series #2 with 0.5% concentration of MSD. The precision and the confidence interval demonstrated relevant and reproducible measurements.

The relationship between the MSD concentration and HIFU-induced heat generation was not linear as demonstrated by the two metrics. This relationship was also observed with PSNE where increasing the droplet concentration from 0.008 % to 0.020 % result in similar lesion volume in a polyacrylamide gel. 62 Repeated acquisition of MR temperature maps in the plane parallel to the HIFU beam propagation showed no evidence of pre-or post-focal thermal build up during the volumetric HIFU exposure in presence of MSD (Figure 1.c). The heating patterns were localised around the prescribed position of the focal plane and matched the near-elliptical shape predicted by theory (e.g. non-distorted). These findings are very important in the context of the lesion predictability. According to Chen et al, 63 the shape of the lesion was demonstrated to change from a cigar shape to a teardrop shape in the presence of an ultrasound contrast agent around 1 MHz frequency. Lo and Kripfgans 64 found similar results by increasing the amplitude or the number of pulses. In our study, we have demonstrated that the interaction between the HIFU beam and the home-made MSD did not result in the distortion of the lesion shape, within the range of applied power and duty cycle of sonication. This discrepancy might be explained by a different mechanism of interaction of MSD with the acoustic waves. PFC possess a high ability to dissolve gas, especially oxygen, and were reported in literature as oxygen carriers. 39 As postulated by Rapoport et al, 34 during the peak rarefactional pressure, the dissolved gas forms a bubble inside the MSD shell, whereas the PFOB stay in liquid form. These bubbles are capable of undergoing stable cavitation but are less prone to IC which might explain the difference of behaviour compared to other study. 26

Impact of acoustics gel properties on HIFU thermal enhancement by MSD droplets

HIFU sonication yielded a low temperature elevation of only 1.2°C in average in the nonabsorbent gel samples. Comparing the results of heating enhancement by 0.5% MSD in absorbent and non-absorbent gel according to Tables 4 and5 for the second metric and the surfactant F 6 TAC 7 show that the enhancement of the HIFU thermal effect was mainly due to the presence of MSD (90% of the effect) and the intrinsic acoustical absorption properties of the TMM had only a slight impact. The first metric was not used for this comparison as it may be biased by the different heat diffusion coefficient of the gel matrix.

Effect of choice of surfactant on the MSD ultrasound absorption

The best potential candidate surfactant according to Table 3 regarding the average diameter (F 8 TAC 7 and F 6 TAC 7 ) were investigated for comparative MSD effect on the HIFU absorption in non-absorbent gel (Table 5, metric 2). The other surfactants were excluded because of the small size of corresponding MSD. The integral enhancement of the thermal energy deposition in the slice, comparing the 0% and 0.5% concentration of MSD was 6.1 × 10 -3 in non-absorbent gel using F 6 TAC 7 and 4.4 x 10 -3 in non-absorbent gel using F 8 TAC 7 surfactant.

These MSD have comparable ultrasound absorption, but the choice of the surfactant is important for an optimal effect of enhanced HIFU thermal therapy.

Effect of repeated HIFU sonications on MSD

Table 6 showed that after the repetition of HIFU exposure in the non-absorbent gel at the same location, a slight decrease of 5% of thermal deposition per cycle was observed between the first shot and the second shot and 10 % between the first shot and the third shot, this tendency is also illustrated with graphical plots in Figure 5.b. This behaviour confirms that the interaction between the HIFU beam and MSD is mainly a reversible process within the range of sonication parameters used in our study. The measurable loss of heat deposition efficacy between each sonication was observed under static conditions (i.e.

no blood flow) and indicated that the MSD distribution and concentration are marginally evolving, for instance some droplets can coalesce or some of the PFC can be dissolved. 65 One advantage of the MSD stability against repeated HIFU sonication is the reduction of the risk of embolism. 26 Another advantage is the possibility to use respiratory gated sonication, i.e.

delivering temporal fractions of energy periodically and synchronized to tissue motion 66 in order to target the same tissue despite patient breathing.

The repeated exposure of MSD liquid emulsion to HIFU beams yielded a reduction of the average diameter in the range 25% to 75%, depending on the nature of surfactant and on the number of applied cycles of sonication, as shown in Table 7. This result supports the safe use of described endovascular MSD in vivo, as their size decreased upon application of HIFU, without a risk of capillary embolism. Due to the large pool of circulating MSD in the blood, the local denaturation (eg size reduction) of some MSD is not expected to impact the final efficacy as new MSD are continuously supplied to the treated area.

Perspectives

In this proof of concept study, we demonstrated significant enhancement of the HIFU absorption in presence of tailored-made sono-sensitive MSD, however, a parametric study was not performed to determine the influence of the acoustic intensity levels and duration of sonication on the enhancement effect. These investigations are required in order to optimize the HIFU pulse sequence to be applied to the respective MSD.

Future in vivo studies need to be performed to confirm the thermal enhancement produced by the current MSD. As compared to the present in vitro study, there are some different conditions to be considered. Firstly, the fraction of acoustic power transferred to tissue will change as the absorption properties, stiffness and viscosity will be different from our TMM. Secondly, the droplets will be confined to the blood vessels as we target tumours in highly perfused organs (e.g. kidney or liver). Thirdly, we may be not able to reach, in vivo, the droplet concentration added to the gels, however, significant dose reduction is likely to be achieved. According to Figure 5a, the 0.1% MSD gel was heated approximately 2.5 times more than the baseline gel. This ratio largely exceeds the therapeutic need. One should also note in this study that we used not more than 135 acoustic watt. Literature reports 67,68 mention significantly larger acoustic powers in vivo (ie between 300W and 800W).

Unlike phase shift nanoemulsions, 30 when using our micro-droplets, the sonication can start a few minutes after the iv injection, as there is no need to wait some accumulation period. Overall, a HIFU treatment session comprises 10 to 30 minutes of active sonication interval and the MSD are required to be stable during a relatively short period of time.

The reported experiments were performed at ambient temperature to avoid a timeconsuming procedure of stabilizing the TMM temperature at 37 °C inside the MR bore. The temperature can also influence the energy required for ADV and/or IC, knowing that ADV depends both on thermal and acoustic parameters and the latter will foster the physical interaction of HIFU beam with MSD. 65 The pool of MSD interacting with the HIFU beam will be continuously refreshed in vivo due to the blood flow, supporting a higher efficiency.

Overall, the final efficacy in vivo remains to be determined.

Conclusion

As a proof of concept, MSD with a PFOB core were synthesised and introduced into a MRI compatible TMM, in order to enhance the thermal deposition of focused ultrasound. We expect that this effect will allow a decrease in the energy and the time required to perform tumour ablation, and to reduce the risks of HIFU treatment side effects by decreasing the thermal build up in the near and far field.

By varying the chemical structure of an in-house fluorinated surfactant, the size of the MSD could be tuned in the range 0.67 to 4.07 µm. These droplets were embedded in a common agar-based TMM, which mimics the acoustic properties of soft tissue. The gel composition was modified to be MR compatible by substituting the Al 2 O 3 by SiO 2 and the acoustic properties of this TMM new formulation were assessed, yielding a sound velocity very similar to soft tissue.

TMM loaded with various concentrations of MSD did effectively enhanced the heating efficiency around the focal point, potentially reducing treatment time for a given target level of temperature. We noticed that the thermal deposition was not linear with MSD concentration in TMM gels, and that the best specific activity was obtained in vitro at 0.1% concentration. Furthermore, the reiteration of the HIFU burst at the same location only lessened by about 5 % the efficacy of heat deposition between each repetition in static conditions (non-circulating droplets). Moreover, the acoustic properties of the material had little if any influence on the efficiency of the MSD, translated into similar enhancement in both absorbent and non-absorbent gel. Further investigations are required to assess the exact mechanism of acoustic energy conversion into thermal energy, specifically if the droplets undergo phase transition or not. Future studies are planned using ex vivo perfused kidney in order to prove that this effect is transposable to highly perfused organs. The ulterior sonications gradually induce less thermal effect. The blue line corresponds to the first sonication, the red line corresponds to the second sonication and the black line to the third sonication.

2. 6 .

 6 Thermo-acoustic investigation of TMM gels doped with MSD 2.6.1. Focused ultrasound A spherical MR-compatible phased array HIFU transducer (Imasonic, Besançon, France) composed of 256 elements was used for generation of focused ultrasound. The main parameters are frequency range 974 -1049 kHz, focal length 130 mm and aperture 140 mm. The transducer was supplied by a 256-channel beam former (Image Guided Therapy, Pessac, France). The HIFU transducer was placed horizontally on the MR table and emitted vertically. Each gel sample (agarose-based and gelatine-based) was placed in an ultrasound coupling holder filled with degassed water and maintained with a standardised setup using resin moulds (Figure 2 a,b). A standard ultrasonic gel was added on the sample top to avoid interface reflection of the waves. The standardised setup assured reproducible positioning of the sample and a 35 mm identical depth of the focus through the series of experiments.HIFU sonication was performed using the electronic steering of the beam thus describing iteratively a discrete circular pattern of 4 mm diameter composed of 16 points regularly distributed on the circumference. This sonication pattern was chosen in order to average eventual local inhomogeneities of the TMM gel or MSD distribution in the gel, which could exist at infra-millimeter scale. The pattern was covered in 1.65 s and the trajectory was repeated 20 times, yielding a total treatment time of 33 s. The applied acoustic power was 94W and the beam emission duty cycle was set at 70% (sample #1 absorbent gel and #3 nonabsorbent ge) or 90% (sample #2 absorbent and #4 non-absorbent). This is respectively equivalent to 1.4 s or 1.8s cumulated sonication time per ietration locus, corresponding to an effective duty cycle of sonication of droplets (as seen at a given location in the gel) of 4.2% and 5.5%. Sonication planning and hardware control was achieved using Thermoguide™ software (Image Guided Therapy, Bordeaux, France).

  which provides a precise monitoring of temperature evolution at a high frame rate and with a millimetre resolution. To this purpose we used a segmented GRE-EPI sequence with main parameters; TE (echo time) = 10ms, TR (repetition time) = 25ms, flip angle = 8°, BW (bandwidth) = 550Hz/pixel, acquisition matrix 128×128, slice thickness = 5mm, FOV = 128 x 128mm, voxel size = 1x1x5mm 3 , temporal resolution = 1 s., number of averages NSA (number of averages) = 1, phase encoding direction = head-foot (HF), spectroscopic fat saturation. The MSD effect of enhancing the HIFU absorption was measured with two normalized metrics: 1) a differential heating factor was defined as the additional elevation of temperature at the focal point considered at the end point of the sonication interval divided by the total emitted acoustic energy of the sonication [unit °C/kJ], and 2) an integral enhancement of heating was defined as the thermal energy deposited in the MR slice integrated over the voxels heated at least +1°C above baseline at the end point of the sonication interval and divided by the total emitted acoustic energy of the sonication [dimensionless]. The thermal energy was calculated as the product temperature elevation times estimated heat capacity. A total of 38 sonications were analyzed in absorbent or non-absorbent gels doped with MSD and compared with 36 baseline sonications in MSD-free gels. 2.6.3. Treatment planning Positioning of the focal point was prescribed using 3D high resolution images acquired with an isotropic gradient echo sequence with the following parameters: TE = 2.46 ms, TR = 5.36 ms, flip angle = 10°, BW = 390 Hz/pixel, slices per slab = 192, FOV = 256 x 256 mm, slice thickness = 0.8 mm, voxel size = 1.00 x 1.00 x 1.28mm. The focal plane was set at 35 mm depth in the sample in the direction of propagation of the HIFU beam.

FiguresFigure 1 Figure 2 .

 12 Figures

Figure 3 . 1 .

 31 Figure 3. Harmonic ultrasound imaging of loaded MSD gel at 2.2MHz for the four concentrations provided as embedded text. (Left) The fitted function of the backscattered signal corresponding to a decreasing exponential with the linear attenuation coefficient "a" expressed in units mm -1 . Experimental data was taken as the normalized average profile of the US signal intensity in a 150 pixel wide region of interest. (Right) The corresponding native US images.

Figure 4 .

 4 Figure 4. 19F MRI image of sliced gel with different MSD concentration, from left to right : 2%, 0.5%, 1%, 0.1% and without MSD (overlaid frame in the zero-signal area).

Figure 5 .

 5 Figure 5. a) Evolution of temperature at the centre of the sonication trajectory in absorbent gels at different MSD concentration (see legend) during HIFU exposure. b) Impact of HIFU repetitions on temperature rising (non-absorbent gel). The same acoustic parameters were applied after 5 minutes delay at the same location.

  F-NMR spectra were acquired using the inverse-gated decoupling technique. Each spectrum was the result of 256 scans with 131 072 data points using a relaxation delay of 4 s. Peak area was integrated using manufacturer standard software (Topspin, version 3.5pl7, Bruker, Wissembourg, France). A calibration curve was obtained using a mixture of 40 µl of PFOB dissolved in 2.9004 g (4.068 ml) of Et 2 O and a dilution path produced various concentrations. Twenty µl of water were diluted with 600 µl of MeOH, then 600 µl of various concentration Et 2 O/PFOB mixture were added. For the titration, the volume of water was replaced by 20 µl of emulsion and 600 µl Et 2 O were added instead of the mixture Et 2 O/PFOB. Et 2 O was used to solubilise the PFOB and MeOH to have a homogeneous solution. The mixture was homogenised, then 500 µl of this solution were added to the NMR tube, followed by a coaxial capillary filled with a solution of sodium trifluoroacetate (TFA) salt in D 2 O (50mg/ml). The latter was used as external reference and was kept the same for all experiments. The TFA salt was chosen because of the proximity of the signal ascribed to its CF 3 group compared to the CF 3 of the PFOB, at -75.96 and 80.15

Table 1 .

 1 Polymerisation condition of different F-TAC

	Tables
	Surfactant F 8 TAC 7	F 8 TAC 13	F 8 TAC 17	F 6 TAC 7	F 6 TAC 12	F 6 TAC 29
	1/R 0	4		8	12		4	12	20
	Yield	65.2%	84.0%	65.1%	63.4%	81.3%	31.8%
		Control 0.1% 0.5%	1%	2%
	H 2 O *	251.6	248.3 236.6 221.6 191.6
	emulsion	0	3	15	30	60

Table 2 .

 2 Volume of water and emulsion added in the TMM gel series (in mL). This volume includes the 50 ml and 5 ml of water added to SiO 2 and BAL respectively

		Control	0.1%	0.5%	1%	2%
	Volume of H2O (mL)*	251.6	248.3	236.6	221.6	191.6
	Volume of emulsion (mL)	0	3	15	30	60

*

Table 2 .

 2 Volume of water and emulsion added in the TMM gel series (in mL).

	Surfactant	F 8 TAC 7	F 8 TAC 13	F 8 TAC 17	F 6 TAC 7	F 6 TAC 12	F 6 TAC 29
	Size in µm	4.07 ± 0.12 0.62 ± 0.02 0.62 ± 0.09 3.67 ± 0.17 1.48 ±0.22 1.47 ± 0.09
	PDI (d 90 /d 10 )	4.84	3.97	3.18	4.00	2.90	2.00

Table 3 .

 3 Droplet's size and polydispersity according the surfactant structure

			Difference between 0% and 0.1% concentration
	N Metric 1	Precision	P test	95% CI	Metric 2	Precision	P test	95% CI
	#1 F 6 TAC 7 4 #2 F 6 TAC 7 5	(°C/kJ) 4.30 3.45	(°C/kJ) 0.39 0.22	p<10 -5 p<10 -5	3.53-5.07 3.02-3.88	(J/kJ) 3.56 4.03	(J/kJ) 0.44 0.32	p<10 -5 p<10 -5	2.68-4.45 3.39-4.67
			Difference between 0% and 0.5% concentration
	N Metric 1	Precision	P test	95% CI	Metric 2	Precision	P test	95% CI
	#1 F 6 TAC 7 4 #2 F 6 TAC 7 4	(°C/kJ) 7.32 5.15	(°C/kJ) 0.57 0.28	p<10 -5 p<10 -5	6.18-8.47 4.60-5.70	(J/kJ) 6.51 7.08	(J/kJ) 0.72 0.40	p<10 -5 p<10 -5	5.08-7.95 6.27-7.88

Table 4 .

 4 Differential heating factor calculated according to first metrics of MSD absorption effect and integral enhancement of thermal energy absorption by the MSD (second metrics), between 0.0% and 0.1%, and between 0.0% and 0.5% shown for absorbent TMM gel series (sample #1 and #2). N stands for the replicates of sonications.