Transport properties of very overdoped nonsuperconducting Bi2Sr2CuO6+ thin films

H. Raffy,* Z.Z. Li, P. Auban-Senzier Laboratoire de Physique des Solides, CNRS UMR 8502, Bâtiment 510, Université Paris-Saclay, 91405 Orsay, France.

The transport properties, resistance, Hall effect, and low T magnetoresistance for very oxygen overdoped nonsuperconducting Bi2Sr2CuO6+d (Bi2201) thin films are reported. From 20 to 300K, the temperature dependence of the resistance is well described by a law of the form a+bT 4/3 , theoretically predicted to occur in the presence of ferromagnetic fluctuations. In addition, this prediction is reinforced by the analysis of the transverse and the longitudinal low T magnetoresistance. Interestingly, the presence of a weak disorder causing low T electronic localization allows us to evidence very short diffusion lengths, as observed in other systems with ferromagnetic fluctuations.

I. INTRODUCTION.

After a long period during which the studies on cuprates essentially focused on the underdoped and optimally doped regions of their phase diagram (PD), recent studies are considering the overdoped side and particularly the end of the overdoped region. In the underdoped side, it is well known t h a t s u p e r c o n d u c t i v i t y a p p e a r s b y d o p i n g a n antiferromagnetic insulating phase. Increasing the doping, the critical temperature, Tc, undergoes a maximum at optimal doping and decreases in the over doped side to disappear for very over doped (VOD) cuprates, yielding a pure metallic state. This behavior raises a question regarding the reason for which superconductivity disappears with increasing doping or inversely why superconductivity appears with decreasing it. Trying to answer this question constitutes another way for unveiling the mystery of the pairing in the cuprates. This point has been recently considered theoretically [START_REF] Maier | Overdoped end of the cuprate phase diagram[END_REF][START_REF] Li | Superconductor-to-metal transition in overdoped cuprates[END_REF]. Maier et al. [START_REF] Maier | Overdoped end of the cuprate phase diagram[END_REF] attribute the right end of the Tc dome to the combined effect of the decreasing d wave pairing strength, closely coupled to changes in the magnetic fluctuations, and to weak impurity scattering. Although they think reasonable to associate the drop of superconductivity to a weakening of pairing interaction, Li et al. [START_REF] Li | Superconductor-to-metal transition in overdoped cuprates[END_REF] consider that the reduction of t h e s u p e r f l u i d d e n s i t y m a y b e t h e c a u s e o f t h e superconductor-to-metal transition. For these authors, the key ingredient is a combination of d-wave pairing and disorder, leading to a granular superconductor. Experimentally, the loss of superconductivity by doping the cuprates can only be observed in one-layer cuprates, i.e., in La2-xSrxCuO4 [START_REF] Takagi | Systematic Evolution of Temperature-dependent Resistivity in La2-xSrxCuO4[END_REF], Tl2Ba2CuO2 [START_REF] Kubo | Transport and magnetic properties of Tl2Ba2CuO6+d showing a -dependent gradual transition from an 85-K superconductor to a non superconducting metal[END_REF], and Bi2Sr2CuO6 (Bi2201)] [START_REF] Li | Oxygen induced superconducting, metallic or insulating behaviour in as-grown epitaxial Bi2Sr2CuOx thin films[END_REF] considered in this study. In Bi2201, nonsuperconducting metallic samples can be obtained either by cationic substitution Bi/Pb, giving different samples for different cationic substitution, or by oxygen doping of a given thin film [START_REF] Li | Oxygen induced superconducting, metallic or insulating behaviour in as-grown epitaxial Bi2Sr2CuOx thin films[END_REF].The latter technique allows one to observe the evolution of the transport properties on one and the same sample [START_REF] Konstantinovic | Evolution of the resistivity of single layer Bi2Sr1.6La0.4CuOy[END_REF]. The VOD part of the PD is somewhat simpler than the less doped one: the (PG) disappears for a given doping p *( consid ered as a *Corresponding author: helene.raffy@universite-paris-saclay.fr quantum critical point [START_REF] Izaire | Transport signatures of the pseudogap critical point in the cuprate superconductor Bi2Sr2-xLaxCuO6+δ[END_REF], p* being smaller than the doping where superconductivity dies out; scanning tunneling microscopy (STM) experiments have shown [START_REF] Li | Quasiparticle interference and charge order in a heavily overdoped non-superconducting cuprate[END_REF] that charge order and nematicity, reported when superconductivity exists, vanish at high doping when superconductivity has disappeared. At the same time, nanoscale patches of static c h a r g e o r d e r w i t h √ 2 x √ 2 p e r i o d i c i t y are observed. Angle-resolved photoemission spectroscopy (ARPES) experiments on Pb doped-Bi2201 single crystals [9,10.] have shown that the evolution of the Fermi surface (FS) topology with doping exhibits a Lifshitz transition from holelike FS to e-like FS with increasing doping. However, while Ding et al. [START_REF] Ding | Disappearance of superconductivity and a concomitant Lifshitz transition in heavily-overdoped Bi2Sr2CuO6 superconductor revealed by angle-resolved photoemission spectroscopy[END_REF] conclude that this transition coincides with the loss of superconductivity, Valla et al. [START_REF] Valla | Hole-like Fermi surface in the overdoped non-superconducting Bi1.8Pb0.4Sr2CuO6+[END_REF] report that it occurs at higher doping than that where superconductivity disappears. Furthermore, as discussed already in Ref. [START_REF] Castro | Anomalous Fermi Liquid Behavior of Overdoped High-Tc Superconductors[END_REF], the obtained metallic state of VOD does not show the canonical T 2 Fermi liquid behavior. The linear temperature dependence of the resistivity, existing in the whole temperature range at optimal doping, still exists at low temperature with increasing doping within a smaller temperature range. One qualifies this behavior as strange metal.

Another characteristic of the cuprates is the presence of antiferromagnetism which survives in a large part of the PD, from the doped Mott insulator to the overdoped state where the PG disappears [START_REF] Frachet | Hidden magnetism at the pseudogap critical point of a cuprate superconductor[END_REF]. Unexpectedly, it has been predicted theoretically that ferromagnetic fluctuations appear in the nonsuperconducting overdoped (OD) state [START_REF] Kopp | Competing ferromagnetism in high-temperature copper oxide superconductors[END_REF]. Several experiments were conducted to look for ferromagnetic fluctuations in different overdoped (OD) non superconducting cuprates: in OD La2-xSrxCuO4 single crystals by the technique of muon spin relaxation (SR) [14.]; in a OD Bi2201 single crystal by magnetization and muon experiments [START_REF] Kurashima | Development of Ferromagnetic Fluctuations in Heavily Overdoped (Bi,Pb)2Sr2CuO6+d Copper Oxides[END_REF] and also in e-doped La1-xCe2CuO4 thin films by magnetoresistance, magnetic measurements and Kerr effect [START_REF] Wei | Ferromagnetic order beyond the superconducting dome in a cuprate superconductor[END_REF]. These different studies concluded either to the presence of a dilute concentration of static magnetic moments [START_REF] Sonier | Direct search for a ferromagnetic phase in a heavily overdoped nonsuperconducting copper oxide[END_REF] or to the presence of two-dimensional (2D) ferromagnetic fluctuations [START_REF] Kurashima | Development of Ferromagnetic Fluctuations in Heavily Overdoped (Bi,Pb)2Sr2CuO6+d Copper Oxides[END_REF], or to the emergence of itinerant ferromagnetism [START_REF] Wei | Ferromagnetic order beyond the superconducting dome in a cuprate superconductor[END_REF]].

In order to give a complementary characterization of the VOD region of the PD of cuprates, we have studied the transport properties (Hall Effect and low T magnetoresistance) of Bi2201 thin films at high oxygen doping state for which superconductivity is suppressed . The present study complements the study previously published on Bi(La)2201 with Tcmax equal to 30K at optimal doping and Tc= 15K for these maximally oxygen doped Bi(La)2201 samples [START_REF] Konstantinovic | Evolution of the resistivity of single layer Bi2Sr1.6La0.4CuOy[END_REF] due to the presence of La. For VOD thin films presented here, in the absence of superconductivity, a slight increase of the resistance R(T) and of the Hall constant RH (T) appears at the lowest temperatures suggesting a possible presence of disorder. It was shown that the low T magnetoresistance is very anisotropic which will be ascribed to e-e interaction effects and weak electronic localization. Although we could not observe ferromagnetism directly in our transport measurements, magnetoresistance is a probe of the hole dynamics which can disclose a behavior involving spins.

II. EXPERIMENTAL TECHNIQUES

Pure Bi2201 thin films, with a thickness ranging 100-350 nm, were epitaxially grown by a sputtering technique on c-axis oriented SrTiO3 single crystal substrates. , X-ray - diffraction (XRD) studies allowed us to show that these films are single phase and c-axis oriented. Our technique of film synthesis as well as the film characterization were described in our earlier references [START_REF] Li | Oxygen induced superconducting, metallic or insulating behaviour in as-grown epitaxial Bi2Sr2CuOx thin films[END_REF][START_REF] Konstantinovic | Evolution of the resistivity of single layer Bi2Sr1.6La0.4CuOy[END_REF][START_REF] Li | Interplay of doping and structural modulation in superconducting Bi2Sr2-x LaxCuO6+x thin films[END_REF]. We have shown in Ref. [START_REF] Li | Interplay of doping and structural modulation in superconducting Bi2Sr2-x LaxCuO6+x thin films[END_REF] typical XRD characterization results (XRD -2scan and XRD scan in Figs. 4 and5 of Ref. [START_REF] Li | Interplay of doping and structural modulation in superconducting Bi2Sr2-x LaxCuO6+x thin films[END_REF] respectively) as well as a cross section of such Bi2201 films grown on SrTiO3 substrates (in Fig. 1 of Ref. [START_REF] Li | Interplay of doping and structural modulation in superconducting Bi2Sr2-x LaxCuO6+x thin films[END_REF]).

In order to reach by oxygenation the pure metallic phase required for this study, we chose to synthetize thin films with a maximum critical temperature Tcmax around 10 K. Tcmax of Bi2201 thin films is essentially depending on the cationic composition, which was found to be Bi2.1Sr1.85CuOy for the thin films studied here (the same Tc value was reported for thin films prepared by molecular beam epitaxy [START_REF] Osovsky | Anomalous Temperature dependence of the Upper Critical Field In Bi-Sr-Cu-O[END_REF] with comparable cationic composition, superconducting Bi2201 thin films being slightly Sr deficient).

The films have been patterned mechanically into a Hall bar, parallel to the a-crystallographic axis, equipped with six gold contacts for R(T) and Hall measurements. They were overdoped by an oxygen treatment as previously reported [START_REF] Li | Oxygen induced superconducting, metallic or insulating behaviour in as-grown epitaxial Bi2Sr2CuOx thin films[END_REF]. The maximum oxygen doping is obtained by annealing in oxygen plasma.

Transport properties, resistance, Hall effect and magnetoresistance, R(T), RH(T), R(H,T) have been measured

in a Quantum Design physical property measurement system (PPMS) (2K≤T<400K; 0≤H≤9T). One sample was measured down to 40 mK using an adiabatic demagnetization refrigerator adapted to the PPMS setup. The temperature dependence of the Hall coefficient RH(T) has been deduced from the measurements of the magnetoresistance performed successively at 9 and -9T with: RH(T)= (r(T,9T)-r(T,-9T))/2H, a n d r t h e r e s i s ta n c e me a s u r ed o n Ha l l co n t ac t s .

FIG. 1. Temperature dependence of the resistance of thin film

Bi2201 no.1 measured at optimal (OPT) doping (red symbols) and after oxygenation in a VOD state (black symbols). The lines are fits to the linear parts of the R(T) curves: at optimal doping from 50 to 300K (continuous line); in the VOD state, at low T from 65K to 100K (continuous line and inset) and at high T for 250 to 370K (dashed line).

III. RESULTS AND DISCUSSION

A. Resistance vs T

As shown in our previous studies [START_REF] Konstantinovic | Evolution of the resistivity of single layer Bi2Sr1.6La0.4CuOy[END_REF], the increase of oxygen content in a Bi2201 thin film appears clearly in the shape of both R(T) and (see next paragraph) RH(T). Figure 1 displays two typical R(T) curves obtained for Bi2201 no.1 thin film at optimal doping and after plasma oxidation. For the optimally doped state the resistance R(T) is linear in temperature down to the superconducting fluctuations before the superconducting transition (Tc=11.5K). The R(T) curve of the VOD state exhibits lower resistance values at given T, a well-marked increase of the curvature, and no superconducting transition.

Looking for the strange and bad metal behavior of cuprate resistance occurring at low T and high T respectively, one can extract from this latter curve two linear parts (see lines in Fig. 1). The former at low T occurs for 66K≤T≤100K (see inset) with a slope of R(T) equal to 0.149 K and the latter at high T is observed for 250K≤T≤370K with a slope equal to 0.247/K corresponding respectively to slopes of the resistivity equal to 0.805 and 1.334 .cm/K. From the low T slope and half the distance of CuO2 planes c/2=12.33±0.02 Å, we obtain a value of the slope of the resistivity per Cu plane: A=6.5±0.3, comparable to the Planckian limit for Bi2201 [START_REF] Izaire | Transport signatures of the pseudogap critical point in the cuprate superconductor Bi2Sr2-xLaxCuO6+δ[END_REF]: 8±2. Actually, the shape of R(T) shown in Fig. 2 can be well described by a law of the type: a+bT n . One finds n=1.40 for 20K≤T≤300 K and n=1.37 for 20 K≤T≤370 K (see the red line fit in Fig. 2). One may wonder if a linear variation of R(T) replaces the T 4/3 law at higher T. We did not check it above 370K as heating could induce a loss of oxygen and a change of the doping state of the thin film. [START_REF] Kurashima | Development of Ferromagnetic Fluctuations in Heavily Overdoped (Bi,Pb)2Sr2CuO6+d Copper Oxides[END_REF] in nonsuperconducting Bi2201. The fact that R(T) in thin films and single crystal behave the same confirms the quality of the thin films studied. Since the beginning of our studies on BiSrCuO thin films, we have systematically considered the evolution of the exponent n with doping and reported the value n=1.3 for non superconducting VOD Bi2201 thin films [START_REF] Rifi | Propriétés de transport de films de Bi2Sr2CuO6 et de multicouches supraconductrices Bi2Sr2CaCu2O8/Bi2Sr2CuO6 préparées par pulvérisation cathodique[END_REF] as well as for VOD La doped thin films still superconducting (Tcmin = 17K) [START_REF] Konstantinovic | Evolution of the resistivity of single layer Bi2Sr1.6La0.4CuOy[END_REF]. The present paper is an attempt to give an explanation to these values as well as those obtained in our early studies.

As shown in the inset of Fig. 2, an increase of R(T) is observed at low T, below a minimum located between 6 and 10 K for the different Bi2201 films studied with comparable Tcmax values [21]. We attribute this increase to the presence of a weak disorder. We will see in the following that the study of the magnetoresistance in presence of disorder allows us to obtain interesting information on the scattering process present in these films.

In order to observe this resistance increase and to check the absence of superconducting transition at lower temperature, the resistance of Bi2201 no. 2 thin film was measured down to 40 mK with the special equipment mentioned in Sec. II. Below 2K, R(T) increases logarithmically before reaching a broad maximum at 180 mK followed by a slight decrease down to 40 mK (Fig. 3). Applying a magnetic field of H = 800G (the maximum field allowed by our cooling system) R(T, 800 G) is first slightly lower than R(T, 0) in the increasing part of R(T), then below the maximum, R(T, 800 G) becomes higher than R(T, 0) in the decreasing part of R(T).

B. Hall effect

It is well known that the Hall effect is not constant vs T in cuprates and that it does not bring the same information as in classical metal [START_REF] Putzke | Reduced Hall carrier density in the overdoped strange metal regime of cuprate superconductor[END_REF]. The temperature dependence of the Hall coefficient, RH(T), has been measured in a magnetic field of ±9T at optimal doping and at VOD oxygen doping successively on the same film. RH(T) increases from 300 K to a maximum occurring below T=100 K. The comparison of both curves RH(T) obtained for the same film as in Fig. 1, plotted in Fig. 4, shows sthat the carrier concentration is increased by oxygen doping. As, at optimal doping, superconductivity is still present under H=9 T, RH(T) decreases to zero when approaching Tc. In the case of a VOD nonsuperconducting state, RH(T) is still temperature dependent, with a maximum around 100 K as in the optimal doping situation and as it was already reported for all doping states [START_REF] Konstantinovic | Temperature dependence of the Hall effect in single layer and bilayer Bi2Sr2Can-1CunOy thin films at various oxygen doping[END_REF][START_REF] Fruchter | Contribution of disorder to the Hall effect in Bi2Sr2CuO6+[END_REF]. Remarkably enough, a low T increase of the Hall constant is observed for all our nonsuperconducting VOD thin films. It can be seen in Fig. 4 and even more clearly in Fig. 5 for another VOD Bi2201 thin film. It occurs in the T region where R(T) presents an increase attributed to localization and e-e interaction effects. While the low T R(T) increase results both from e localization and e-e interaction, the low T increase of the Hall constant is only ascribed to e-e interaction effect as predicted by the interaction model [START_REF] Altshuler | Magnetoresistance and Hall effect in a disordered twodimensional electron gas[END_REF], and the localization model [START_REF] Fukuyama | Hall effect in two-dimensional Disordered systems[END_REF] As discussed in Ref. [22.] in the conventional transport theory, the Hall number in the low T and high field limit is simply related to the number of charge carriers, nH. It was also shown [START_REF] Putzke | Reduced Hall carrier density in the overdoped strange metal regime of cuprate superconductor[END_REF] that for Bi2201 the field dependence of RH in the normal state is weak (Fig. S3 of Ref. [START_REF] Putzke | Reduced Hall carrier density in the overdoped strange metal regime of cuprate superconductor[END_REF]). From the value of RH measured at our lowest temperature = 2 K under a field H = 9 T, the hole concentration per volume unit nH = 1/eRH can be derived although the small increase of RH due to e-e interaction reduces slightly the value nH. For the sample of Fig. 5, nH is found equal to (6.72±0.05) x 10 21 /cm 3 , while nH = (6.85±0.05) x 10 21 / cm 3 at T= 10 K where RH(T) is minimum. The number of holes per Cu, nCu, is obtained using the volume V of the unit cell of Bi2201 and the number of Cu atoms in the unit cell [START_REF] Ando | Carrier concentration in Bi2Sr2-xLaxCuO6+x single crystals and their relation to Hall coefficient and thermopower[END_REF]. It is found equal to 1.27±0.1, giving a value of the doping per Cu: p = nCu-1= 0.27±0.1.

C. Magnetoresistance R(H, T)

The magnetoresistance (MR) was measured from 2 to 25 K in a perpendicular magnetic field H (transverse MR) and in a magnetic field parallel to the current H (longitudinal MR) for 0 ≤ H ≤ 9T (and also in one case in a transverse field with an angle close to 45° in a plane perpendicular to the film containing the current I.

.

Figure 6 presents a typical result obtained at 3 K in perpendicular and parallel magnetic fields for VOD Bi2201 no. 3 thin film. The longitudinal MR is positive and increases steadily with H. On the contrary, the transverse MR is negative at low magnetic field, becoming positive at high magnetic field. It is also very different from the MR measured when the sample is less doped and still superconducting. In that case the MR is positive, dominated by the suppression of the superconducting fluctuations. These two situations are described in detail in the following paragraphs.

Longitudinal MR

The magnetic field being parallel to the current, one expects the longitudinal magnetoresistance to arise from the coupling of the spins to the magnetic field H. In two dimensions, while the orbital contribution is sensitive only to the magnetic field component normal to the plane, the spin splitting term should be isotropic [START_REF] Lee | Magnetoresistance of weakly disordered electrons[END_REF] (no spin orbit). where R(0) is the value of the resistance in zero field with dR = R(H)-R(0) and BS a characteristic field related to e-e interaction.

If the magnetoresistance was due to the Zeeman effect, as it can be thought with a field parallel to the current, one would expect a parabolic law of the form dR/R(0≈ (H/Bz) 2 with Bz = kB T / g = 0.744 T The values of extracted from the parabolic fits are plotted in the inset of Fig. 7(b). Although Bz (T) is linear, the linear fit does not go through the origin, and its slope (13.2T) is about 20 times higher than that of the Zeeman. field Bz (0.744 T/K). Besides, we do not observe any saturation of the MR with increasing field at the lowest temperatures for which the MR stays linear at the highest field values.

In contrast to the transverse orbital MR (see next paragraph), the longitudinal MR (H//I) could not be analyzed with existing theories. At this point it is interesting to compare these results to experimental ones obtained in previous studies reporting the MR of Pd1-yNiyHx thin films near the paramagnetic-ferromagnetic crossover [START_REF] Raffy | Magnetic properties of Pd1-yNiyHx thin films near the paramagnetic-ferromagnetic crossover[END_REF]. The Pd1-yNiyHx alloys are ferromagnetic for y≥ 2.5 % Ni and x = 0 and become less magnetic and even superconducting when hydrogenated to saturation (x ~ 1). In these films the approach of the ferromagnetic state was monitored by increasing y and decreasing x from the saturated state. It was shown, for paramagnetic states already close to be ferromagnetic (y = 16 at %, x = 1), that the MR is positive and parabolic in longitudinal magnetic field and also in perpendicular magnetic field with smaller MR values, the presence of localization being no longer seen (state very close to a ferromagnetism). Increasing the magnetism by decreasing x (x<1) for a given y, the MR becomes isotropic. In our Bi2201 thin films the localization is still present in the transverse MR, but the transverse MR tends to become positive at high magnetic field. The longitudinal magnetoresistance follows a parabolic variation as described above and the field Bs(T) presents a T linear variation as observed for a characteristic field HN for spin splitting (Fig. 5 of Ref. [START_REF] Raffy | Magnetic properties of Pd1-yNiyHx thin films near the paramagnetic-ferromagnetic crossover[END_REF]). The similar behavior of the longitudinal MR of Bi2201 and Pd1-yNiyHx thin films suggests that the longitudinal magnetoresistance may result from the presence of ferromagnetic fluctuations in VOD Bi2201 thin films. At low T, the parabolic variation of R(H,T) is followed by a linear variation with increasing fields. It would be interesting to measure the MR at higher magnetic fields to see if one still observes a linear variation of R(H) without saturation as reported in perpendicular and parallel magnetic field in Bi2201 single crystals and discussed in [START_REF] Ayres | Incoherent transport across the strange-metal regime of overdoped cuprates[END_REF].

Perpendicular field MR: What is the origin of the negative MR observed at low field?

The low T upturn of R(T) and the negative part of the perpendicular field MR recall the behavior observed in conventional metal thin films in the weak localization regime [START_REF] Bergmann | Inelastic life-time of the conduction electrons in some noble metal films[END_REF][START_REF] Lee | Disordered electronic systems[END_REF]. For instance, in Cu thin films [START_REF] Van Haesendonck | Localization and negative magnetoresistance in thin copper films[END_REF], there is a low T increase of R(T) and a negative transverse MR, attributed at low field to the localization of the carriers in presence of disorder. The application of a perpendicular magnetic field destroys the interference effect of the conduction electrons leading to a negative MR in the absence of spin-orbit effect We have analyzed the orbital part of the MR measured at low T using the expression [START_REF] Hikami | Spin-Orbit interaction and magnetoresistance in the two dimensional random system[END_REF] We have fitted our data with expression (1) between 2 and 20 K. Good fits are obtained for the samples studied. Figure 8 shows a typical result of the fit for the orbital component of the MR at T=3K shown in Fig. 6.

Our analysis of the orbital MR is supported by measurements we made in inclined magnetic field with an angle DD °) with the film plane for which the orbital part of the MR obtained in the same way by subtracting the e-e contribution and plotted vs the perpendicular component of the magnetic field HsinD follows the same law as the MR measured in perpendicular magnetic field: at a given T, the curves representing dRloc(H sinD) vs H sinD and dRloc(H) vs H are well superimposed for H ≤ HsinD.

Figures 8(b) and 8(c) display respectively the temperature dependence of the dephasing magnetic field H(T) and the dephasing length Lfor the same sample. H(T) is increasing with T, almost linearly at low T, and its value is about ten times higher than that observed in ordinary metal films [START_REF] Bergmann | Inelastic life-time of the conduction electrons in some noble metal films[END_REF].

The values of the dephasing lengths in our work are very comparable to those reported by Jing et al. [START_REF] Jing | Anomalous Enhancement of the Electron Dephasing Rate from Magnetoresistance Data in Bi2Sr2CuO6[END_REF] for their non superconducting Bi2201 single crystal. On the other hand, the transport mean free path l, for instance at 5K, is found equal to 4 nm (with kFl = 30 and kF = 7.22.10 7 cm -1 the 2D Fermi vector, deduced from resistivity and Hall constant values).

As indicated in the Introduction, the presence of ferromagnetic fluctuations in VOD cuprates has been predicted theoretically [13. 19] and reported experimentally in VOD Bi2201 single crystals [START_REF] Kurashima | Development of Ferromagnetic Fluctuations in Heavily Overdoped (Bi,Pb)2Sr2CuO6+d Copper Oxides[END_REF]. In that case, it was deduced from magnetization measurements and SR experiments. For thin films, it is difficult to deduce from such experiments valuable information concerning the presence of magnetic fluctuations (too small material quantity, presence of magnetic impurities in the substrate). However, it can be thought that the large values of H(T) obtained for our VOD non superconducting thin films are possibly related to the presence of ferromagnetic fluctuations.

As in the case of the longitudinal MR, we come to this conclusion by considering the experimental results of the preceding work concerning the transverse MR in Pd, PdH [START_REF] Raffy | Comparative study of the inelastic diffusion lengths in weakly localizing Pd and PdHx films[END_REF] and Pd1-yNiyHx (y = 6 at%) [START_REF] Raffy | Localization, magnetism, and superconductivity in two dimensional Pd1-yNiyHx films[END_REF] thin films. Approaching in a controlled manner a ferromagnetic state by decreasing x from 1, the analysis of 2D e localization present in the transverse MR of these films allowed the authors to identify the signature of magnetic fluctuations in these systems. It was shown that the dephasing magnetic fields were larger in pure Pd than in PdH . This effect was related to the tendency for Pd to be closer to a magnetic instability (see Fig. 1 in Ref. [START_REF] Raffy | Comparative study of the inelastic diffusion lengths in weakly localizing Pd and PdHx films[END_REF]) and attributed to the scattering from extended magnetic fluctuations. The study of weak e localization in 2D Pd1-yNiyHx thin film (y = 6 at%, Ref. [START_REF] Raffy | Localization, magnetism, and superconductivity in two dimensional Pd1-yNiyHx films[END_REF])) showed that a strong increase of H in comparison with H in PdH thin films is observed for x = 1 while a decrease of leads a further increase of H (see Fig 5 of Ref. [START_REF] Raffy | Localization, magnetism, and superconductivity in two dimensional Pd1-yNiyHx films[END_REF]). These effects were related to the growth of ferromagnetic fluctuations.

Relying on the results of these experimental studies, we propose to attribute the large values of H (T) in VOD Bi2201 to the presence of ferromagnetic fluctuations.

IV. SUMMARY

The transport properties of Bi2201 thin films in a VOD nonsuperconducting state obtained by plasma oxygenation have been compared to the transport properties of the same samples in an optimally doped state. The temperature dependence of the resistance of VOD thin films follows a law of the form a+bT 4/3 in the range 20-300 K, which was predicted to reveal ferromagnetic fluctuations [START_REF] Kopp | Competing ferromagnetism in high-temperature copper oxide superconductors[END_REF][START_REF] Kurashima | Development of Ferromagnetic Fluctuations in Heavily Overdoped (Bi,Pb)2Sr2CuO6+d Copper Oxides[END_REF]. At low T, the metallic state of these nonsuperconducting cuprates do not present the canonical Fermi liquid behavior. Contrariwise, a low T logarithmic increase of R(T) is observed. Such an increase, also seen under high magnetic field in less doped or optimally doped states of the same samples, is attributed to the presence of a weak disorder in Bi2201 thin films. The Hall constant in the VOD state, still T dependent, with smaller values than for the optimal state, is consistent with high doping. An interesting observation is the low T increase of RH(T) in the same T region where R(T) increases. We attribute the T increase of RH(T) to e-e interaction only, and the resistivity increase to ee interaction and weak electronic localization.

Interestingly, the study of the combined effects of magnetic field and disorder at low T allowed us to probe the importance of excitations existing in this temperature and doping range. The magnetoresistance is anisotropic. The longitudinal magnetoresistance is positive whatever H (0≤ H ≤ 9T) and T (2K ≤T ≤25K). It is attributed to e-e interaction. The analysis of its parabolic law may suggest the presence of ferromagnetic fluctuations. The transverse magnetoresistance of VOD Bi2201, drastically different from the one in the superconducting states, is negative at low field becoming positive at high field. It is supposed to result from a superposition of the isotropic e-e contribution and orbital 2D weak localization. The analysis of the orbital magnetoresistance in terms of 2D weak localization allows us to determine the dephasing fields H(T). The high values of these characteristic fields compared to those reported for classical metallic thin films reveal a large scattering effect which may indicate the presence of ferromagnetic fluctuations. Our conclusions are supported by a comparison of our results with those of previous studies of Pd1y NiyHx [START_REF] Raffy | Magnetic properties of Pd1-yNiyHx thin films near the paramagnetic-ferromagnetic crossover[END_REF][START_REF] Raffy | Localization, magnetism, and superconductivity in two dimensional Pd1-yNiyHx films[END_REF] thin films near the paramagnetic-ferromagnetic cross-over.

More studies would be necessary to understand why superconductivity appears with decreasing doping and how ferromagnetic fluctuations give way to anti-ferromagnetic ones at lower doping when the pseudogap is opened. Spin excitations appears to be present in the different parts of the PD of cuprates and may play a role in their superconductivity [START_REF] Restrepo | Coupled electronic and magnetic excitations in the cuprates and their role in the superconducting transition[END_REF].

FIG. 2 .

 2 FIG. 2. Detailed behavior of the R(T) curve given in Fig.1 for thin film Bi2201 no 1 in the VOD state. The R(T) curve is fitted by a law of the form: R(T) = a+bT n above 20 K, n= 1.4 up to 300 K, and n=1.37 up to 370 K (red lines). The inset visualizes the logarithmic increase of R(T) below 10 K

FIG. 3 .

 3 FIG. 3. R(T) for 40 mK<T<2 K for VOD Bi2201 no. 2 thin film in a semilog plot (blue curve) showing the logarithmic variation of R(T) (red line). The green curve is a plot of R(T, 800G) showing the influence of a perpendicular magnetic field H=800 G. Inset: the same plots in a linear T scale display the maximum of R(T) occurring for T=180 mK, revealing the presence of some superconducting islands.

FIG. 5 .FIG. 6 .

 56 FIG. 4. Temperature dependence of the Hall constant measured under H = 9 T of thin film Bi2201 no 1, in red at optimal (OPT) doping and in blue in the VOD state. At T = 300 K, the Hall numbers are respectively: nH(OPT) = (3.9±0.2)x10 21 /cm 3 and nH(VOD) = (6.5±0.3)x10 21 /cm 3 . Note the slight increase of RH below 10 K in the VOD state. Inset: T dependence of cotg /H.RH (blue points). The red line is the fit of the data with a law of the type: A+BT m (m = 1.73).

FIG. 7 .

 7 FIG. 7.(a) Magnetoresistance of VOD thin film Bi2201 no 3: dR=R(H, T) -R(0, T) measured in a field parallel to the current at various T (2-20 K). ( b) Plot of dR/R(0) vs H 2 // for T ≥ 9 K, where dR is equal to R(H)-R(0), and linear fits giving the values of the characteristic field Bs defined as dR/R(0)=(B/Bs) 2 . The inset shows the T dependence of Bs and the linear fit.

Figure 7 (

 7 Figure 7(a) displays R(H) data obtained on a VOD Bi2201 thin film measured at various T. In a magnetic field parallel to the current, R(H) is alwaysapositive in the domain of temperature study (2 K ≤ T <25 K). The field dependence of R(H) is quadratic at low field: H ≤ 4T for the lowest T values and up to 9 T for T ≥ 10 K [Fig. 7(b)] where the data are fitted by a parabolic law of the form

FIG. 8

 8 FIG. 8 (a) Negative contribution or orbital part of the magnetoresistance of VOD Bi2201 no. 3 thin film at T = 3 K in perpendicular magnetic field fitted by the expression of the MR due to weak localization (see text).The parameters obtained from the fit are: H= (0.7261±0.0035) T and L(3 K) = (15±0.5) nm.(b) Temperature dependence of the characteristic dephasing field Hand (c) of the dephasing length L

  dRloc(H)/RR = -(αe 2 /2 2 ħ)Y[H/ H](1)where Y(x) = ln xx H= ħ/4eDτ is the dephasing magnetic field L= √D is the dephasing length with D the diffusion constant and the dephasing time R is the resistance per square x) is the digamma function showing a quadratic behavior at low x and a ln x behavior at high x values; α is a constant of the order of unity in the absence of spin orbit effect.

ACKNOWLEDGMENTS

H. R. thanks M. Gabay and C. Pasquier at LPS, Université Paris-Saclay, and P. Monceau at CNRS, Institut Néel, Grenoble, for stimulating and very helpful discussions.


Stéphanie Roullet

Norman Luc

Julie Rayes

Jean Solarz

Dante Disharoon

Andrew Ditto

Emily Gahagan

Christa Pawlowski

Thibaud Sefiane

Frédéric Adam

Caterina Casari

Olivier D Christophe

Michael Bruckman

Peter J Lenting

Anirban Sen Gupta

Cécile V Denis
email: cecile.denis@inserm.fraxs262@case.edu

Anirban Sen Gupta

Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in Von Willebrand disease murine models

Key points

• Synthetic platelet nanoparticles improve thrombus formation on collagen using blood from Von Willebrand disease murine models

• Treatment with synthetic platelet nanoparticles reduces blood loss in murine models of Von Willebrand disease

Introduction

Treatment of Von Willebrand disease (VWD) has remained basically unchanged for the past 30 years. [START_REF] Denis | von Willebrand disease: what does the future hold?[END_REF] It rests on 3 pillars: antifibrinolytics, desmopressin, and Von Willebrand factor (VWF) concentrates. However, these different approaches do not consider the heterogeneity of VWD and so far, no type or subtype-specific treatment option has been developed.

In this regard, VWD-type 2B (VWD-2B) could represent an attractive target due to its very specific nature. Indeed, VWD-2B is characterized by «gain-of-function» mutations in the VWF A1 domain, which increase VWF binding to its platelet receptor, glycoprotein (GP) Ibα.

Consequently, it is the only VWD subtype associated with a platelet phenotype including the presence of circulating platelet aggregates, giant platelets, a fluctuating thrombocytopenia, and a platelet function defect. [START_REF] Casari | von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin alphaIIbbeta3[END_REF][START_REF] Federici | Clinical and molecular predictors of thrombocytopenia and risk of bleeding in patients with von Willebrand disease type 2B: a cohort study of 67 patients[END_REF][START_REF] Kauskot | LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B[END_REF] These distinctive features raise the question of whether correcting the platelet defect could improve hemostasis in VWD-2B. Thrombocytopenia can be addressed with platelet transfusions [START_REF] Kruse-Jarres | How I treat type 2B von Willebrand disease[END_REF] or even with thrombopoietin receptor agonists 6,7 but these approaches do not solve the thrombocytopathy since the mutant VWF will still bind spontaneously to platelet GPIbα. Strategies that can bypass the VWF-GPIbα axis while supporting platelet adhesion and aggregation may offer new possibilities to treat VWD-2B. In this regard, platelet-inspired synthetic hemostatic nanoparticles represent a very attractive alternative. These 'synthetic platelet' (SP) nanoparticles are made of a phospholipid-based liposomal template that is surface-decorated with three different peptides: a fibrinogen mimetic peptide (FMP) that can bind to the active form of platelet integrin aIIbb3, a collagenbinding peptide (CBP) that can bind to exposed subendothelial fibrillar collagen, and a VWFbinding peptide (VBP), derived from the C2 domain of Factor VIII that can bind to the D'-D3 domain of VWF. [START_REF] Haji-Valizadeh | A factor VIII-derived peptide enables von Willebrand factor (VWF)-binding of artificial platelet nanoconstructs without interfering with VWF-adhesion of natural platelets[END_REF][START_REF] Ravikumar | Mimicking adhesive functionalities of blood platelets using ligand-decorated liposomes[END_REF][START_REF] Ravikumar | Peptide-decorated liposomes promote arrest and aggregation of activated platelets under flow on vascular injury relevant protein surfaces in vitro[END_REF] SP have been shown to collaborate with residual platelets to restore hemostatic efficacy in tail-clip model in severely thrombocytopenic mice. [START_REF] Luc | Bioinspired artificial platelets: past, present and future[END_REF][START_REF] Sekhon | Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of bleeding[END_REF][START_REF] Shukla | In vitro characterization of SynthoPlate (synthetic platelet) technology and its in vivo evaluation in severely thrombocytopenic mice[END_REF] Their hemostatic efficacy was also demonstrated in several animal models of traumatic injury, including a murine model of hepatic laceration, [START_REF] Dyer | Intravenous administration of synthetic platelets (SynthoPlate) in a mouse liver injury model of uncontrolled hemorrhage improves hemostasis[END_REF] a porcine model of traumatic arterial hemorrhage [START_REF] Hickman | Intravenous synthetic platelet (SynthoPlate) nanoconstructs reduce bleeding and improve 'golden hour' survival in a porcine model of traumatic arterial hemorrhage[END_REF] and a rodent model of traumatic liver resection injury. [START_REF] Sekhon | Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of bleeding[END_REF] Due to their promising hemostatic performance efficiency in these models and their unique mode of action towards VWF, i.e.

binding to the D'-D3 region and not to the A1-domain like platelets, we hypothesized that SP could be used to improve hemostasis in VWD-2B. Furthermore, since SP maintains the ability to bind to collagen irrespective of VWF presence 8 and still co-aggregate with active platelets to reduce bleeding, we postulated that they might also provide hemostatic support in a situation where native platelet adhesion to VWF is lacking due to the absence of VWF. Therefore, we also tested the hemostatic capability of SP in a VWF-knockout (KO) murine model.

Methods

Additional description of the experimental procedures can be found in the Supplementary Material (available on the Blood website).

Animals and ethics statement

VWD-2B mice harboring the p.V1316M mutation in murine VWF and VWF-deficient mice (VWF-KO) have been described previously. [START_REF] Adam | A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation[END_REF][START_REF] Denis | A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis[END_REF] Male and female mice were used throughout the study (8-24 weeks old). This project was approved by the ethical committee CEEA26 (number APAFIS#27290-2020091714275854 v1).

Particle Manufacturing Methods

VBP and CBP were conjugated to DSPE-PEG2K-Mal via thiol-maleimide coupling to form DSPE-PEG2K-VBP and DSPE-PEG2K-CBP and FMP was conjugated to DSPE-PEG2K-azide via coppercatalyzed alkyne-azide cycloaddition (CuCAAC) or 'click chemistry' to form DSPE-PEG2K-FMP.

All lipid-peptide conjugates were purified by dialysis and characterized by MALDI-TOF mass spectrometry. For manufacturing liposome-based SP nanoparticles surface-decorated with all three peptides, DSPE-PEG-VBP and DSPE-PEG-CBP were combined at 1 mol% each with DSPE-PEG-FMP (15 mol%), DSPC (40 mol%), cholesterol (40 mol%), DSPE-mPEG1K (2 mol%), and DHPE-RhB (1 mol%) in 1:1 chloroform:methanol, subjected to solvent evaporation under reduced pressure to form thin lipid films, rehydrated with 0.9% NaCl, then subjected to 10 freeze-thaw cycles using alternating exposure to liquid nitrogen and 60°C water bath, and finally extruded five times through a 200 nm pore size polycarbonate filter (thin film rehydration and extrusion method) (Fig. 1A). [START_REF] Zhang | Thin-Film Hydration Followed by Extrusion Method for Liposome Preparation[END_REF] For nanoparticles with two, one, or no peptides, the specific lipid-peptide conjugates were replaced with an equivalent mol% of DSPE-mPEG2000, and subjected to the same process of liposomal nanoparticle fabrication as described above. Prior to use, nanoparticle size was characterized by dynamic light scattering (Fig. 1B) and cryo-transmission electron microscopy (Fig. 1C).

BioFlux microfluidic assay to study effect of SP on human platelets in absence of VWF

Human platelet suspensions in buffer, containing 200,000 platelets per μL (Plt-200k), 50,000 platelets per μL (Plt-50K) or 20,000 platelets per μL (Plt-20k) were perfused on collagen-coated channels at 60 dyn/cm 2 for 10 minutes and the green fluorescence (calcein) for surfaceadhered and aggregated platelets was imaged in real time. Next, rhodamine-B labeled (red fluorescence) control nanoparticles (CP) or SP nanoparticles at a particle:platelet ratio of 1000:1 were added to Plt-50K and Plt-20K conditions, to evaluate the ability of CP vs SP in rescuing platelet recruitment on the collagen-coated surface.

Total thrombus formation analysis system (T-TAS®) perfusions

The T-TAS® Plus (Zacros Fujimori Kogyo, Tokyo, Japan) is an automated microchip-based flow chamber system developed for assessment of platelet thrombus formation under flow conditions. [START_REF] Hosokawa | A microchip flow-chamber system for quantitative assessment of the platelet thrombus formation process[END_REF][START_REF] Hosokawa | A novel automated microchip flow-chamber system to quantitatively evaluate thrombus formation and antithrombotic agents under blood flow conditions[END_REF][START_REF] Sueta | A novel quantitative assessment of whole blood thrombogenicity in patients treated with a non-vitamin K oral anticoagulant[END_REF] Murine blood was collected under general anesthesia (isoflurane) by retroorbital puncture within a non-heparinized glass capillary, in one-tenth volume trisodium citrate (13.8 mM final concentration). Blood was half-diluted in citrate solution (3.2% trisodium citrate solution/ 0.9% NaCl at the ratio of 1:9) and complemented with undecorated (control) nanoparticles or SP at a ratio of 50 particles per platelet. T-TAS® Plus perfusions were performed on AR-chips coated with collagen and tissue factor at a shear rate of 600 s -1 for up to 30 min. Area under the curve (AUC), representative of the increase in pressure which relates to thrombus formation, was calculated or extrapolated at 30 min.

Parallel plate flow chamber

Murine blood half-diluted in Tyrode's buffer was labeled with rhodamine 6G and complemented with control nanoparticles (CP) or SP at a ratio of 50 particles per platelet.

Thrombus formation was evaluated after perfusion assay on a fibrillar collagen matrix under arterial shear conditions (shear rate of 1500 s -1 ). 22

Tail-clip bleeding assay

After general anesthesia with ketamine and xylazine, mice received different formulations of particles via intravenous injection (2 mg/kg, in a final volume of 100 µL NaCl 0.9%, leading to a platelet: particle ratio between 1:5 to 1:10). Ten minutes later, 3 mm of the distal tip of the tail was amputated. Blood collection and blood loss measurement were performed as previously described. [START_REF] Ferriere | A hemophilia A mouse model for the in vivo assessment of emicizumab function[END_REF] Of note, bleeding was assessed over 30 min for VWD-2B and 20 min for VWF-KO mice.

Immunofluorescence imaging of amputated tails

At the end of the tail-clip bleeding assay, 1 cm of the distal tip of tails from VWF wild-type (WT) mice or VWF-KO mice injected with SP was amputated and fixed in 4% paraformaldehyde (PFA) for 24 h. Sections were stained for platelets using an anti-mouse CD41 antibody and for fibrin. Analysis was done in a selected region of interest which was defined inside the vessel.

Sections were imaged using epifluorescent microscopy and analyzed using ImageJ software.

Scanning electron microscopy observation of clot morphology after tail vein transection

Untreated VWF-KO mice or VWF-KO mice injected with SP underwent a tail vein transection procedure as previously described. [START_REF] Johansen | Development of a tail vein transection bleeding model in fully anaesthetized haemophilia A micecharacterization of two novel FVIII molecules[END_REF] After 10 min, the tail was immersed in 4% PFA/1% glutaraldehyde and cut. The sample was then immediately immersed in 10 ml 4% PFA/1% glutaraldehyde and fixed overnight at 4°C. Then, a 1 cm tail section was cut, centered on injury.

A "Nanosuit" was used to protect the samples from electron beam and hard drying [START_REF] Takaku | A modified 'NanoSuit(R)' preserves wet samples in high vacuum: direct observations on cells and tissues in field-emission scanning electron microscopy[END_REF] The tails were dipped three times during one minute in the nanosuit and then pre-irradiated by a sputtering device without the metal emitter. Finally, silver metallization (setting: 10 nm of silver: 120 seconds at 5 cm and 30 mA) of the complete tail section was performed, and the samples were observed with scanning electron microscopy at different magnifications.

Statistical analysis

Data are presented as mean±standard deviation (SD) unless indicated otherwise. Mann-Whitney test was performed for two-groups comparison. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was performed when comparing multiple groups. P <0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 7 software (La Jolla, CA, USA).

Results

SP enhances platelet surface-coverage in BioFlux microfluidics in VWF-deficient condition

Ex-vivo addition of SP to VWD blood improves T-TAS® perfusions' parameters

In the T-TAS® perfusion system, the AUC is an integrated parameter, representative of the pressure generated by platelet adhesion and thrombus formation inside a collagen-and tissue factor-coated chip. By analyzing changes in pressure as a function of time, Fig. 2A left panel clearly shows that SP addition to murine VWD-2B blood led to a significant increase in AUC (1308 with a 95%CI 1278-1329) compared to untreated VWD-2B blood (710, 95%CI 687-734, P<0.0001) or to undecorated/CP-supplemented blood (733, 95%CI 717-748, P<0.0001). For murine VWF-KO blood, we also measured significantly increased AUC when blood was supplemented with SP (988, 95%CI 954-1022) compared to untreated blood (422, 95%CI 407-436, P<0.0001) or to CP-treated blood (558, 95%CI 541-575, P<0.0001) (Fig. 2A, right panel).

For SP-treated VWD-2B or SP-treated VWF-KO blood, the AUC remained lower than for wildtype blood (1916, 95%CI 1900-1933), indicating that thrombus formation was not completely rescued.

Altogether, the T-TAS® perfusion assays show a significant improvement of platelet adhesion when SP were added to VWD blood.

Ex-vivo addition of SP to VWD blood significantly improved thrombus formation in a parallel plate flow chamber

To build on the encouraging results obtained with the T-TAS® system, additional blood perfusion studies were performed using a more "classical" collagen-coated parallel plate flow chamber. Addition of SP to VWD-2B blood significantly increased thrombus formation compared to both untreated blood or blood supplemented with CP (174±59 vs 58±9, P=0.016 and 174±34 vs 55±2, P=0.015, respectively) (Fig. 2B, upper panels) as measured by the mean fluorescent intensity, a surrogate marker for thrombus size (Fig. 2C, left panel).

Addition of SP to VWF-KO blood also led to a strong increase in thrombus formation compared to untreated blood (523±213 vs 28±14, P=0.002) and blood supplemented with CP (523±213 vs 31±2, P=0.003), (Fig. 2B, bottom panels and Fig. 2C, left panel). Interestingly thrombi formed in VWF-KO blood supplemented with SP appeared bigger than those formed in VWD-2B supplemented with SP.

In terms of percentage platelet coverage, SP addition to VWF-KO blood led to an increase in platelet adhesion (Fig. 2C, right panel) with 20±2% covered area compared to 5.2±1.9% with untreated blood and 5.3±2% with CP. The results are less clear with VWD-2B blood where in terms of percentage covered area, an increase was seen only in the presence of CP (24±3.4%) compared to untreated blood (13.4±3.7%) or to SP-supplemented blood (13.4±0.2%).

However, despite this increased surface coverage seen with control particles, no thrombus formation was observed.

Compared to wild-type conditions, only thrombus size obtained in SP-treated VWF-KO blood was normalized. All other parameters remained below wild-type read-outs.

Co-localization of platelets with SP can clearly been seen when both entities were labeled with distinct fluorochromes in thrombi formed using SP-treated VWF-KO blood (Fig. S3).

SP significantly reduced bleeding in VWD mice

We next assessed the bleeding phenotype of VWD-2B and VWF-KO mice treated with SP. As expected, untreated VWD-2B mice lost large amounts of blood compared to WT mice (938±161 vs 18±17 µL, P<0.0001; Fig. 3A). Treatment with CP did not improve hemostasis in these mice (966±192 vs 938±161 µL, P=0.98). In contrast, addition of SP significantly reduced blood loss by an average of 35% (631±199 vs 966±192 µL with control particles, P<0.001 and 631±199 vs 938±161 µL without treatment, P=0.001). Nevertheless, complete correction was not obtained (631±199 vs 18±17 µL in WT mice, P<0.0001) (Fig. 3A). Of note, SP did not affect the multimeric profile of VWD-2B mice (Fig. S4).

For VWF-KO mice, untreated mice as well as CP treated-mice bled profusely compared to WT mice (561±207 µL and 539±194 µL vs 19±17 µL) (Fig. 3B). SP treatment of VWF-KO mice demonstrated an important efficacy in reducing bleeding (183±167 µL vs 561±207 µL in untreated mice, P=0.01). Strikingly, blood loss after SP treatment was no longer statistically different than blood loss in WT mice (183±167 µL vs 19±17 µL, P=0.15). SP thus appeared more efficient in VWF-KO mice compared to VWD-2B mice, probably due to the platelet function defect described in the latter model.

Mechanisms underlying hemostatic efficacy of SP in VWD

In order to investigate whether the three different peptides were equally important in the hemostatic efficiency of SP in VWD murine models, we tested different combinations of particles harboring only one peptide or combinations of two peptides (Fig. 4).

In VWD-2B, particles decorated with any of the single peptides were not efficient in reducing blood loss (CBP: 761±287 µL, FMP: 1072±174 µL and VBP: 856±125 µL vs 966±192 µL with CP, P=0.08, 0.71 and 0.81 respectively). Second, when using particles decorated with combinations of two peptides, only 'CBP + VBP' and 'CBP + FMP' combinations were as efficient as SP in correcting blood loss (CBP+VBP: 587±255 µL and CBP+FMP: 568±173 µL vs 631±199 µl with SP; P=0.99 and =0.97 respectively). In contrast, particles decorated with the 'VBP + FMP' combination did not show any efficacy (929±156 µL vs 966±192 µL with CP, P=0.99) (Fig. 4A).

In VWF-KO mice, similar to VWD-2B mice, particles decorated with single peptides were not efficient in reducing blood loss with even a slight increase with FMP (CBP: 636±239 µL, FMP: 859±161 µL, VBP: 618±200 µL vs 539±194 µL with CP, P=0.16, 0.0016 and 0.82 respectively).

Surprisingly, the combination of CBP+FMP was less efficient in reducing blood loss than SP (530±100 µL vs 183±167 µL, P=0.0001), pointing to an unexpected effect of the VBP (Fig. 4B), likely due to the phospholipid binding capacity of this factor-VIII derived peptide. However, particles decorated with VBP only did not show any hemostatic efficacy. Complete statistical analysis is provided in Supplementary Table 1.

In conclusion, CBP appears to be the most important peptide in the SP but is not sufficient by itself to support hemostasis in VWD-2B mice while all three peptides are necessary in VWF-KO mice.

SP colocalizes with platelets and fibrin in the clot observed after tail-clip

We next sought to image the clots observed after SP treatment in the tail clip experiment.

Here, we only imaged clots from the treated VWF-KO mice. SP, labeled with Rhodamine B could be seen as colocalizing with platelets and fibrin in the clots formed in VWF-KO mice after SP treatment (Fig. 5A). A large clot, occluding the vessel from side to side could clearly be seen in SP-treated VWF-KO mice. Although this experiment is mostly of a qualitative nature, our attempt to quantify these data suggested that both the area and fluorescence intensity of the platelets and fibrin signals were similar to those obtained after imaging of a clot formed in WT mice (Fig. 5B).

SP leads to substantial hemostatic clot as visible by scanning electron microscopy

To extend our observation of the SP-induced clot formation in VWF-KO mice, we collected the clot obtained after lateral tail vein transection in untreated and SP-treated mice. On the upper panels of Fig. 5C, the sectioned hair indicates the area where the cut was performed (shown by the black arrows). In the untreated VWF-KO mouse, a thin thrombus could be spotted as covering the scar, thrombus formed of individualized red blood as visible at higher magnification. Upon SP treatment (Fig. 5C, right panels), a larger thrombus was visible, covering the entire scar section with red blood cells aggregated together in the clot.

Discussion

Platelet-mimicking nanoparticles are currently under pre-clinical development to mitigate bleeding in various hemostatic dysfunction settings spanning thrombocytopenia, coagulopathy and trauma. 11-15 However, they have not yet been tested in congenital bleeding disorders linked to platelet and VWF defects. Considering our team's current interest in developing tailored therapeutic options for different VWD types/subtypes, we hypothesized that, by circumventing the VWF-GPIbα axis, SP could represent an attractive option for VWD type 2B. In vitro, using a Bioflux microfluidic system, we first showed the capacity of SP to rescue human platelet recruitment in thrombocytopenic conditions and in VWF-deficient conditions, serving as a basis for testing their efficacy in our VWD-2B and VWF-KO mouse models. Next, using VWD-2B or VWF-KO murine blood, we confirmed that SP indeed significantly improved thrombus formation in perfusion assays both in a regular parallel plate perfusion chamber (Maastricht flow chamber), [START_REF] Provenzale | Whole Blood Based Multiparameter Assessment of Thrombus Formation in Standard Microfluidic Devices to Proxy In Vivo Haemostasis and Thrombosis[END_REF] as well as in the T-TAS® assay.

Building on these in vitro results, and when injected in vivo, SP were able to reduce bleeding in VWD-2B mice and to achieve near-complete correction of hemostasis in VWF-KO mice.

These results raise a number of questions: firstly, regarding why SP are functional in VWF-KO mice since platelets are fully functional in these mice, and second, why SP are more efficient in the VWF-KO compared to VWD-2B mice? SP have been shown to collaborate with platelets [START_REF] Ravikumar | Peptide-decorated liposomes promote arrest and aggregation of activated platelets under flow on vascular injury relevant protein surfaces in vitro[END_REF][START_REF] Luc | Bioinspired artificial platelets: past, present and future[END_REF][START_REF] Sekhon | Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of bleeding[END_REF][START_REF] Shukla | In vitro characterization of SynthoPlate (synthetic platelet) technology and its in vivo evaluation in severely thrombocytopenic mice[END_REF] and in the absence of platelet-VWF interaction in VWF-KO mice, we can rationalize that the adhesion of SP to vascular collagen creates a new landing platform for native platelets to adhere to. Platelets would therefore be recruited to sites of vascular injury via SP, a hypothesis supported by the co-localization of SP with platelets and fibrin following tail-clip injury. As to why SP display higher efficiency in VWF-KO mice compared to VWD-2B animals, it should be emphasized that the latter model is characterized by thrombocytopenia/thrombocytopathy. Platelet counts in VWD-2B mice are reduced by 50% compared to VWF-KO. [START_REF] Adam | A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation[END_REF] Although this decrease is not very profound, the inability of these VWD-2B platelets to fully activate 27 may also explain why less coagulation will take place on their surface, limiting fibrin formation and ultimately, hampering bleeding correction.

To investigate mechanisms by which SP contributed to haemostasis in VWD mice, we tested several types of particles decorated with different peptide combinations. In VWD-2B mice, only particles decorated with combination of 'CBP + VBP' and 'CBP + FMP' proved as efficient as SP to reduce bleeding, highlighting the pivotal role of collagen in injury site-specific particle anchoring and haemostatic action. However, the CBP alone was not efficient, suggesting that at least, another platelet-mimicking characteristic was necessary for particle efficiency.

Nevertheless, restoring full haemostasis in VWD-2B remains difficult since both defects in VWF and in platelets need to be overcome. [START_REF] Rayes | Mutation and ADAMTS13-dependent modulation of disease severity in a mouse model for von Willebrand disease type 2B[END_REF] New generations of platelet-inspired nanoparticles are currently under development: platelet-mimicking procoagulants nanoparticles, with a plasmin-triggered exposure of phosphatidylserine at the site of injury, [START_REF] Sekhon | Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of bleeding[END_REF] or the release of thrombin at the site of injury. [START_REF] Girish | Platelet-Inspired Intravenous Nanomedicine for Injury-Targeted Direct Delivery of Thrombin to Augment Hemostasis in Coagulopathies[END_REF] Increasing site-specific coagulation outputs with such particles might be key to improve their potential use in VWD-2B. It should also be emphasized that our VWD-2B mouse model carries the p.V1316M variant, one of the most severe VWD-2B causing mutation. Milder forms of VWD-2B may potentially be more responsive to SP treatment.

In VWF-KO mice, the result that the combination of 'CBP+FMP' was less efficient than SP to correct the haemostatic defect can appear very surprising at first glance. Indeed, VBP, the missing peptide in these dual-decorated particles, should be of no use in the absence of VWF.

However, this VBP is derived from the FVIII C2 domain and binds not only to the VWF D'-D3 region but also to phospholipids. [START_REF] Haji-Valizadeh | A factor VIII-derived peptide enables von Willebrand factor (VWF)-binding of artificial platelet nanoconstructs without interfering with VWF-adhesion of natural platelets[END_REF] It seems conceivable therefore that even in the absence of VWF, this peptide contributes to the hemostatic response by re-enforcing the interactions between SP and activated platelets. However, particles decorated with the VBP peptide alone have no hemostatic efficiency by themselves. Although theoretically, this peptide could compete with FVIII for VWF binding in the VWD-2B mice, this is unlikely to occur given that its concentration (≤500 nM) is lower than the IC50 (6 µM). [START_REF] Saenko | A role for the C2 domain of factor VIII in binding to von Willebrand factor[END_REF] Imaging of the clots in the presence of SP was carried out only in VWF-KO mice for technical reasons. Indeed, imaging clots of bleeding mice, such as was the case for VWD-2B mice, proved unsuccessful. The clots obtained in VWF-KO mice treated with SP appeared very similar to those obtained in wild-type mice as visible by fluorescent analysis. The overlap between platelets and fibrin in SP-treated mice suggests that by recruiting platelets, SP contribute not only directly to the formation of the platelet-enriched thrombus but also indirectly to the availability of platelet-exposed procoagulant phospholipids on which coagulation can occur.

Scanning electron microscopy confirmed that SP led to a more compact and solid thrombus than those obtained in untreated VWF-KO mice.

So where does such an approach fit as a potential treatment for VWD? Originally, the study was intended to test a new therapeutic for VWD-2B but, although promising, the SP particle design does not appear to be optimally efficient in this VWD subtype. However, this technology has the advantage that it can be further tailored for augmenting coagulation by incorporating additional pro-hemostatic molecules on the particle surface or in the particle core, mimicking platelet's procoagulant and secretory functions. Therefore, the present study should be seen as a first proof-of-concept that platelet-mimicking particles can be used for VWD-2B patients. For severe VWD (type 3), SP particles have proven very efficient. Due to their unique characteristics, they can thus be considered as a potential new therapeutic approach for severe VWD. Indeed, their manufacturing process and stability, i.e. lyophilizability, no requirement for cold storage and >6 months shelf-life, may render them very attractive compared to VWF concentrates. Furthermore, from a pharmacokinetic point of view, SP have a clear advantage over VWF concentrates. In mice, 2 hours after injection, only about 10% of VWF can still be detected. [START_REF] Lenting | An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation[END_REF] In contrast, 2 hours after injection in mice, only 15% of SPs have been cleared from the circulation, [START_REF] Shukla | In vitro characterization of SynthoPlate (synthetic platelet) technology and its in vivo evaluation in severely thrombocytopenic mice[END_REF] and recent studies with procoagulant platelet-mimicking particles have reported a half-life of 12 hours. 12 SP thus display a much longer circulatory half-life than VWF. The use of SP could also be considered in severe VWD patients who have developed allo-antibodies, precluding the use of VWF concentrates.

Altogether, there is clearly room for such a technology in a VWD setting and our results warrant further investigation of platelet-inspired nanoparticles as a novel strategy for the hemostatic management of VWD.

Fig. 1D shows

  Fig. 1D shows representative fluorescence images of platelet surface-coverage in Plt-200k vs Plt-20k vs 'Plt-20k + CP' vs 'Plt-20k + SP' conditions on collagen-coated BioFlux microfluidic channels in the absence of any plasma VWF. As evident from the images, the surface coverage was drastically reduced in Plt-20K condition compared to Plt-200K condition. Adding CP in Plt-20K condition did not rescue or improve platelet surface-coverage. In contrast, adding SP in Plt-20K condition substantially improved platelet surface-coverage, and a high co-localization of red SP nanoparticles with green platelets was observed (appearing yellow in the merged image), suggesting that SP nanoparticles were able to rapidly bind to the exposed collagen even in the absence of VWF and enable concomitant binding of active platelets onto them to render enhanced recruitment and coverage of platelets on the surface. Fig. 1E shows quantitative analysis of the results from these studies (n = 5 per condition), confirming that surface-coverage of platelets on collagen-coated microchannels is significantly reduced (P<0.0001) in Plt-20K condition (1.2±0.8%) compared to Plt-200K condition (14.5±3.2%), and addition of SP significantly rescued (7.0±2.2%, P=0.0014) this surface-coverage compared to addition of CP (1.4±0.9%). Surface-averaged platelet fluorescence intensity (green fluorescence, arbitrary units) from the same conditions, normalized to Plt-200K condition, are shown in Fig. S1. Supplementary movies A, B, C and D show representative microfluidic imaging of Plt-200K, Plt-20K, Plt-20K + CP and Plt-20K + SP conditions. Additionally, Fig. S2 shows representative fluorescence images and quantitative analysis data for SP treatment of Plt-50K condition, once again indicating the ability of SP to rescue platelet coverage on collagen-coated microfluidic surface. Altogether, these results indicate that SP nanoparticles can potentially render improved hemostatic responses in VWF-deficient and platelet-depleted conditions, as long as the concomitant collagen-binding (via CBP) and integrin αIIbβ3-binding (via FMP) properties of SP are able to enhance the recruitment of active platelets on collagenexposing surface.

Figure 1 .

 1 Figure 1. Physical and functional characterization of the synthetic platelet (SP) nanoparticles A) Schematic of the manufacturing process for synthetic platelet (SP) nanoparticle with lipids and lipid-peptide conjugates using the thin film rehydration followed by extrusion methodology. B) Dynamic Light Scattering (DLS) characterization and C) Cryo-transmission electron microscopy (Cryo-TEM) imaging of SP nanoparticles indicates a diameter of approximately 150-200 nm. D-E) Representative fluorescence microscopy images and quantitative analysis of imaging data from BioFlux microfluidics studies with platelet suspensions indicate that depletion of platelets from 200,000 per µL (Plt-200K) to 20,000 per µL (Plt-20K) results in drastic reduction of platelet coverage of collagen-coated microfluidic channel surface. Treatment of Plt-20K with control nanoparticles (CP) does not rescue this while treatment of Plt-20K with SP nanoparticles significantly rescues platelet recruitment and coverage on the collagen-coated channel surface (colocalization of Rhodamine B-labeled red fluorescent SP with calceinstained green fluorescent platelets appear yellow). The particle/platelet ratio was 1000:1. Means ± SD are represented. Statistical analysis was performed using a one-way Anova with Tukey's correction. ns: non-significant, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Figure 2 .

 2 Figure 2. In vitro effect of SP nanoparticles added to VWD murine blood in perfusion assays A) Synthetic platelets or control nanoparticles were added to VWD-2B blood (left panel) or to VWF-KO blood (right panel) at a ratio of 50 particles per platelet. Particle-supplemented blood or unsupplemented blood (untreated) was perfused over AR chips in the T-TAS® Plus system. Graphs represent changes in pressure (a measure for thrombus formation) as a function of time. The colored area represents the standard error of the mean (SEM) for each condition. Statistical analysis was performed using a one-way Anova with Tukey's correction. B-C) Synthetic platelets or control nanoparticles were added to VWD-2B murine blood or to VWF-KO murine blood at a ratio of 50 particles per platelet. Particle-supplemented blood or unsupplemented blood (untreated) was perfused over collagen in a parallel flow chamber at 1500 s -1 . untreated Representative images of thrombus formation are shown in B while percentage platelet coverage, representing adhesion or mean fluorescence intensity (MFI) as

Figure 3 .

 3 Figure 3. In vivo effect of SP nanoparticles in VWD-2B and VWF-KO murine models Mice were injected or not with control nanoparticles or synthetic platelet nanoparticles (2 mg/kg). Bleeding was measured for 30 min (VWD-2B) (A) or 20 min (VWF-KO) (B) after amputation of 3 mm of the tail tip. Blood was collected in warm saline and quantified using a hemoglobin calibration curve. Each dot represents an individual mouse. Data are presented as mean ± SD. Statistical analysis was performed using a one-way Anova with Dunnett's correction. ns: not significant, * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001. WT: wild-type.

Figure 4 .

 4 Figure 4. In vivo effect of various peptide-decorated particles in VWD-2B and VWF-KO murine models Mice were injected with control particles, synthetic platelets nanoparticles or particles decorated with various combinations of peptides (2 mg/kg). Bleeding was measured for 30 min (VWD-2B) (A) or 20 min (VWF-KO) (B) after amputation of 3 mm of the tail tip. Blood was collected in warm saline and quantified using a hemoglobin calibration curve. Each dot represents an individual mouse. Data are presented as mean ± SD. Statistical analysis was performed using a one-way Anova with Dunnett's correction. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Synthetic platelets nanoparticles (3 peptides) are represented with the even grey bar while particles decorated with dual peptides appear in diagonally-striped bars and singledecorated particles appear in horizontally-striped bars. CBP: collagen binding peptide; FMP: fibrinogen mimetic peptide; VBP: Von Willebrand factor binding peptide.

Figure 5 .

 5 Figure 5. Imaging of the clots obtained after synthetic platelets (SP) treatment of VWF-KO mice A) Representative images of the fluorescent staining of clots recovered after amputation of the tail tip of VWF WT mice and VWF-KO mice injected with SP (2mg/kg). Dashes represent the vessel's edges. Rhodamine-labeled SP are visible in red, fibrin/fibrinogen is visible in magenta and CD41-labeled platelets appear in green.

Acknowledgements

The work was supported by INSERM (Institut National de la Santé et de la Recherche Médicale) and by the Agence Nationale de la Recherche (ANR-21-CE14-0076-01 to CVD). JR holds a British Heart Foundation (BHF) Intermediate Fellowship (FS/IBSRF/20/25039). ASG is supported by NIH R01 HL121212. B) Quantification of the platelets (CD41+) and fibrin area and fluorescence intensity in WT (n=3) and VWF-KO mice injected with SP (n=3). Data are presented as mean ± SD. Statistical analysis was performed using a Mann-Whitney test. C) Scanning electron microscopy of the clot recovered after tail vein transection in untreated VWF-KO mouse and SP-treated VWF-KO mouse. On the upper images, black arrows indicate the transection area recognizable by the hair that appear sectioned and the blue arrows point to the clot. On the bottom left image, red arrows show individualized red blood cells. On the bottom right image, blue arrow shows the clot characterized by aggregated red blood cells and red arrows show individualized red blood cells. Two mice per group were imaged and representative images are shown.

are available upon reasonable request to the corresponding author

Authorship Contributions

SR, NL, JR, JS, DD, TS, FA, CCC, ODC, PJL and CVD performed experiments and analyzed the data. AD, EG, CP, MB and ASG provided essential reagents. JR, PJL, ASG and CVD designed the study. SR, CVD and ASG wrote the manuscript. All authors contributed to the final editing of the manuscript.

Disclosure of Conflicts of Interest

AD, EG, CP and MB are employees of Haima Therapeutics LLC. ASG is co-founder and chief scientific advisor for Haima Therapeutics.