L Somaglino

L Mousnier

A Giron

W Urbach

N Tsapis

N Taulier

In vitro evaluation of polymeric nanoparticles with a fluorine core for drug delivery triggered by focused ultrasound

Keywords: ultrasound, drug delivery, nanoparticles, PFOB, cavitation

Polymeric nanoparticles are being intensively investigated as drug carriers. Their efficiency could be enhanced if the drug release can be triggered using an external stimulus such as ultrasound. This approach is possible using current commercial apparatus that combine focused ultrasound with MRI to perform ultrasonic surgery. In this approach, nanoparticles made of a perfluoro-octyl bromide core and a thick polymeric (PLGA-PEG) shell may represent suitable drug carriers. Indeed, their perfluorocarbon core are detectable by 19 F MRI, while their polymeric shell can encapsulate drugs. However, their applicability in ultrasound-triggered drug delivery remains to be proven. To do so, we used Nile red as a model drug and we measured its release from the polymeric shell by spectrofluorometry. In the absence of ultrasound, only a small amount of Nile red release was measured (<5%). Insonations were performed in a controlled environment using a 1.1 MHz transducer emitting tone bursts for a few minutes, whereas a focused broadband hydrophone was used to detect the occurrence of cavitation. In the absence of detectable inertial cavitation, less than 5% of Nile red was released. In the presence of detectable inertial cavitation, Nile red release was ranging from 10 to 100%, depending of the duty cycle, acoustic pressure, and tank temperature (25 or 37℃). Highest releases were obtained only for duty cycles of 25% at 37℃ and 50% at 25℃ and for a peak-to-peak acoustic pressure above 12.7 MPa. Electron microscopy and light scattering measurements showed a slight modification in the nanoparticle morphology only at high release contents. The occurrence of strong inertial cavitation is thus a prerequisite to induce drug release for these nanoparticles. Since strong inertial cavitation can lead to many unwanted biological effects, these nanoparticles may not be suitable for a therapeutic application using ultrasound-triggered drug delivery.

Introduction

Although nanomedicines have exhibited promising preclinical efficacy to treat cancer, only a few have reached the market until now. [START_REF] Fattal | Nanomedicine technology: current achievements and new trends[END_REF] Among clinically approved nanomedicines, one can cite PEGylated liposomal doxorubicin (Doxil / Caelyx ). It did not improve the overall survival in breast cancer but it is a major evolution in the treatment of Kaposi sarcoma. [2,[START_REF] Stewart | Randomized comparative trial of pegylated liposomal doxorubicin versus bleomycin and vincristine in the treatment of aids-related 650 kaposi's sarcoma. international pegylated liposomal doxorubicin study group[END_REF] Many nanomedicines that have been developed for cancer treatments rely on the so-called enhanced permeation and retention effect (EPR), which facilitates the escape of nanomedicines through leaky solid tumor vasculature and retention due to reduced lymphatic drainage. [START_REF] Maeda | The enhanced permeability and retention (epr) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting[END_REF][START_REF] Maeda | The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo[END_REF]6] Thanks to the EPR effect, tumor passive targeting via nanomedicine can be achieved and the drug concentration in the tumor is enhanced compared to a free drug, leading to an enhanced efficiency. [START_REF] Drummond | Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors[END_REF] But the proportion of nanomedicines usually reaching the tumor site is below 10% of the injected dose. [START_REF] Diou | Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19 FMRI[END_REF] In addition, if tumor models implanted in mice almost systematically exhibit an EPR effect, this is far to be the case for patient 20 tumors. [START_REF] Crommelin | Towards more effective advanced drug delivery systems[END_REF] To overcome the unreliability of the presence of the EPR effect in patients, stimulus-sensitive systems have been developed. [START_REF] Mura | Stimuli-responsive nanocarriers for drug delivery[END_REF] The local application of the stimulus allows to trigger the drug release in the vicinity of the solid tumor and therefore increase drug concentration 25 therein. Stimuli can be exogenous [START_REF] Yao | External-stimuli responsive systems for cancer theranostic[END_REF] such as magnetic field, temperature, ultrasound, light or electric pulses; or endogenous [START_REF] Ganta | A review of 685 stimuli-responsive nanocarriers for drug and gene delivery[END_REF] such as intracellular enzymes, pH or redox gradients. Ultrasound represents an attractive technique as it is widely available in hospitals through echographs [START_REF] Pisani | Polymeric nano/microcapsules of liquid perfluorocarbons for ultrasonic imaging: Physical characterization[END_REF] and is easily manipulated. [START_REF] Boissenot | Ultrasoundtriggered drug delivery for cancer treatment using drug de-690 livery systems: From theoretical considerations to practical applications[END_REF] In addition, commercial apparatus using the MRI-guided high intensity focused ultrasound (MRI-g-HIFU) technique are currently used in clinics for the treatment of fibroids, [START_REF] Clark | Reproductive impact of mri-guided focused ultrasound surgery 695 for fibroids: a systematic review of the evidence[END_REF] and in ongoing clinical trials for the treatment of breast, liver, prostate, 35 and brain cancer. [START_REF] Jolesz | Mri-guided focused ultrasound surgery[END_REF] Most carriers under development for ultrasound triggered delivery are liposomes encapsulating a cytotoxic drug, mostly doxorubicin [START_REF] Dromi | Pulsed-high intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and 705 antitumor effect[END_REF][START_REF] Pitt | Preliminary Results of Combining Low Frequency Low Intensity Ultrasound and Liposomal Drug De-710 livery to Treat Tumors in Rats[END_REF][START_REF] Eggen | Ultrasound improves the uptake and distribution of liposomal doxorubicin in prostate cancer xenografts, 715[END_REF][START_REF] Mestas | Therapeutic efficacy of the combi-720 nation of doxorubicin-loaded liposomes with inertial cavitation generated by confocal ultrasound in AT2 Dunning rat tumour model[END_REF] as liposomal doxorubicin formulations have already been approved by several major drug agencies, including Federal Drug Agency (FDA) and European Medicines Agency (EMA), and are currently commercialized (Myocet and Doxil /Caelyx ). For example, Thermodox , a thermosensitive liposomal doxorubicin is currently under clinical trials. [START_REF] Poon | Lyso-thermosensitive liposomal dox-725 orubicin: an adjuvant to increase the cure rate of radiofrequency ablation in liver cancer[END_REF][START_REF] Boissenot | Ultrasoundtriggered drug delivery for cancer treatment using drug de-690 livery systems: From theoretical considerations to practical applications[END_REF] Ultrasound triggered delivery is either arising from an increase of temperature when using thermosensitive liposomes, [START_REF] Escoffre | Focused ultrasound mediated drug delivery from temperature-sensitive 730 liposomes: in-vitro characterization and validation[END_REF] or due to cavitation for non-thermosensitive liposomes. [START_REF] Afadzi | 735 Effect of ultrasound parameters on the release of liposomal calcein[END_REF] However, the combination of temperature and cavitation enhances drug delivery for thermosensitive liposomes. [START_REF] Dromi | Pulsed-high intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and 705 antitumor effect[END_REF] Polymer systems were also considered such as polymeric micelles, [START_REF] Husseini | Factors affecting acoustically triggered release 740 of drugs from polymeric micelles[END_REF][START_REF] Marin | Drug delivery in pluronic micelles: effect of high-frequency ultrasound on drug release from micelles 745 and intracellular uptake[END_REF] for which drug delivery is triggered by cavitation, and polymeric nanoparticles. [START_REF] Boissenot | Paclitaxel-loaded {PEGy-lated} nanocapsules of perfluorooctyl bromide as theranostic 750 agents[END_REF][START_REF] Boissenot | Ultrasoundinduced mild hyperthermia improves the anticancer efficacy 755 of both taxol® and paclitaxel-loaded nanocapsules[END_REF][START_REF] Luo | On-demand drug release from 760 dual-targeting small nanoparticles triggered by high-intensity focused ultrasound enhanced glioblastoma-targeting therapy[END_REF] For the latter, nanoparticles made of a perfluorooctyl bromide (PFOB) core and a PLGA polymer shell coated with polyethylene glycol (PEG) [START_REF] Diou | Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19 FMRI[END_REF] represent potential candidates to be used as theranostic agents [START_REF] Boissenot | Paclitaxel-loaded {PEGy-lated} nanocapsules of perfluorooctyl bromide as theranostic 750 agents[END_REF][START_REF] Boissenot | Ultrasoundinduced mild hyperthermia improves the anticancer efficacy 755 of both taxol® and paclitaxel-loaded nanocapsules[END_REF] in combination with MRI-g-HIFU technique. Indeed, these nanoparticles can be used as contrast agents for 19 F MRI [START_REF] Pisani | Perfluorooctyl Bromide Polymeric Capsules as Dual Contrast Agents for Ultrasonography and Magnetic Resonance Imaging[END_REF][START_REF] Diou | Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19 FMRI[END_REF] while encapsulating a cytotoxic drug. Boissenot et al. [START_REF] Boissenot | Ultrasoundinduced mild hyperthermia improves the anticancer efficacy 755 of both taxol® and paclitaxel-loaded nanocapsules[END_REF] studied the drug delivery triggered by ultrasound from these systems and observed only a small drug delivery with no vaporization of the PFOB core. Luo et al. [START_REF] Luo | On-demand drug release from 760 dual-targeting small nanoparticles triggered by high-intensity focused ultrasound enhanced glioblastoma-targeting therapy[END_REF] used similar nanoparticles to successfully deliver drugs into glioblastoma cells implanted in mice using high intensity focused ultrasound (HIFU). In their case, a rapid and important release of doxorubicin is observed which, according to the authors, is induced by a vaporization of the PFOB liquid. In order to obtain a better understanding of the ultrasound triggered release from these types of nanoparticle, we have studied by spectrofluorometry the release of encapsulated Nile red using focused ultrasound for various ultrasonic properties. Nile red was chosen because it possesses an hydrophobicity similar to hydrophobic anticancer and non-fluorescent drugs such as paclitaxel or docetaxel, while exhibiting the advantage to be fluorescent only in hydrophobic environments as well as being less cytotoxic than drugs. We showed that it is indeed possible to release a large quantity of Nile red in a short time but only for specific ultrasonic parameters.

Experimental

Materials

Methylene chloride RPE-ACS 99.5% was provided by Carlo-Erba Reactifs (France). Sodium cholate (SC) and Nile red were purchased from Sigma-Aldrich (France). Poly (D,L-lactide-co-glycolide) -poly(ethylene glycol) (PLGA -PEG) Resomer RGP d 50105 (PLGA Mn = 45000 g/mol and 10 wt% of PEG Mn = 5000 g/mol, intrinsic viscosity 0.72 dl/g) were obtained from Boehringer-Ingelheim (Germany). Perfluorooctyl bromide (PFOB) was purchased from Fluorochem (United Kingdom). Water was purified using either a RIOS/Milli-Q system (Millipore, France) or a PURELAB Option-Q unit (ELGA LabWater).

Nanoparticle preparation

The formulation of nanoparticles was prepared using 95 an emulsion-evaporation method where the emulsion is a single oil (methylene chloride) in water emulsion where PFOB, PLGA-PEG and Nile red are all miscible in methylene chloride. [START_REF] Diou | Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19 FMRI[END_REF] The evaporation of methylene chloride leads to the formation of PLGA-PEG shell surrounding 100 a PFOB core and solubilizing Nile red. [START_REF] Pisani | Surfactant dependent morphology of polymeric capsules of perfluorooctyl bromide: Influence of polymer adsorption at the dichloromethane-water interface[END_REF] Specifically, 100 mg of PLGA-PEG was first dissolved into 3.9 ml of methylene chloride before adding 100 µl of a solution containing 60 µg/ml of Nile red in methylene chloride. After complete dissolution of the polymer, 60 µl of PFOB was 105 added dropwise to the solution until complete miscibility. This organic phase was then emulsified into 20 ml of 1.5% sodium cholate (w/v) aqueous solution using a vortex for 1 min, followed by sonication at 30% of the maximum power for 1 min over ice using a vibrating metallic tip 110 (Digital Sonifier 250, Branson). Methylene chloride was then evaporated under magnetic stirring at 300 rpm for about 3 h in a thermostated bath at 20℃. After evaporation, nanoparticles were filtered through a 0.45 µm PVDF membrane and then washed by an ultracentrifugation cy-115 cle of 1 h at 27,440 g and 4℃. The supernatant was discarded and the final pellet of nanoparticles was resuspended into fresh water. To determine the final concentration of nanoparticles in the suspension, small known volumes of the suspension were freeze-dried for 24 h us-120 ing Alpha 1-2LD Plus apparatus (Christ), and weighed.

In our experiments, all suspensions were prepared so that the nanoparticle concentration was equal to 0.25% (w/v).

Liposomes

We used Caelyx, a liposomal doxorubicin formulation 125 made of 2 mg/ml of doxorubicin•HCl encapsulated inside liposomes. The liposome bilayer is made of N-(Methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanola-mine sodium salt (MPEG-DSPE), fully hydrogenated soy phosphatidylcholine (HSPC), and choles-130 terol. The liposomal formulation was suspended in a 10 mM histidine buffer (pH 6.5) containing 300 mM of sucrose, giving a osmolality of 346 mOs/Kg, in order to equilibrate the osmolality between the medium inside and outside of liposomes. In our measurements, the initial liposomal sus-135 pension was diluted 500 times in the same histidine buffer containing sucrose.

Fluorescence

Fluorescence spectra were acquired using a spectrofluorometer (model FP8300 from Jasco). Fluorescence in-140 tensities were measured for suspensions containing either nanoparticles or liposomes.

For liposomes, the fluorescence signal coming from encapsulated doxorubicin is quenched due to the high concentration of doxorubicin into the liposome. An increase in fluorescence intensity is expected upon the release of doxorubicin and the percentage of released doxorubicin was determined using:

R dox = I -I init I 100% -I init × 100 (1)
where I init , I, and I 100% are respectively the fluorescence intensities of the liposome suspension before insonation, 20 min after HIFU exposure, and after the addition of triton X100 (at a concentration of 20 µl/ml) which leads to the release of all doxorubicin. Fluorescence intensities were measured at an emission wavelength of λ em = 558 nm using an excitation wavelength of λ ex = 475 nm. The 20 min delay before fluorescence measurements was chosen to al-150 low the samples, previously insonified at 37℃, to cool down to ambient temperature at which fluorescence spectra were performed.

For nanoparticle suspensions, the release of encapsulated Nile red is accompanied by a decrease in fluorescence intensity as Nile red looses its fluorescence property in water. Thus, fluorescence intensity provides this time the quantity of Nile red remaining encapsulated. Note that the concentration of Nile red inside the polymeric shell is low enough to avoid fluorescence quenching. A 100% release of Nile red corresponds to nanoparticles devoid of Nile red. Consequently, the percentage of released Nile red, R red , was calculated using:

Ultrasonic setup

The ultrasonic setup is schematically described in Fig. 1. Acoustic waves were generated by a focused transducer (Model H-101-G from Sonic Concepts), which input electrical signal came from a RF amplifier (model 150A100C from AR france) connected to the output of a waveform generator (model 33220A from Agilent). The acoustic wave propagated into a closed thermostated tank (at either T tank = 25 or 37 ± 0.2℃) containing highly degassed water, thanks to a degassing machine (Model WDS-105 from Sonic Concepts) connected to the tank. A medical balloon (purchased from Nordson Medical), whose polyethylene terephthalate wall was 35 µm thick, was placed at the focus of the transducer so that the focal region (1.5 mm in diameter and 8 mm in length) lied inside the balloon. The balloon was filled with 2.5 ml of the solution to be studied and exhibited a tiny air/solution interface area (≈ 1.3 -2.3 mm 2 ) at both ends. An ultrasonic absorber covered the lid of the tank as well as all its walls, except the 180 one supporting the transducer, to avoid ultrasonic wave reflections inside the tank. Inertial cavitation occurring into the balloon solution was monitored using a focused broadband hydrophone (model Y-107 from Sonics Concepts). Tone bursts at 1.1 MHz with a 200 Hz pulse repetition 185 frequency were used to sonicate samples. Typical sonication durations were 3 min for nanoparticle suspensions and 1 min 30 s for liposome suspensions, while duty cycle (from 5 to 50%) and acoustic pressure (up to 15.6 MPa) were varied to study the release behavior of both suspen-190 sions. A directional power sensor (model NAP-Z8 from Rohde & Schwarz), connected to a power reflection meter (Model NRT from Rohde & Schwarz), measured the average electrical power that was converted to an average acoustic power by the transducer. Finally, the acoustic 195 peak-to-peak pressure P pkpk at the transducer focus and the intensity I spta (that is the spatial peak intensity averaged over the period of exposure at the focal point which surface section is 0.0177 cm 2 ) were derived from the average acoustic power. Absorption due to the balloon wall 200 was neglected in those calculations as the balloon wall was very thin.

Finally, two temperature probes were used: a first one to measure T tank , the temperature of the degassed water inside the tank, and a second one to record T balloon , the 205 temperature at the balloon outside surface. Note that the use of a hydrophone or a temperature probe inside the medical balloon was avoided as they will become the source of cavitation nuclei and bias the measurements.

Size measurements 210

Average hydrodynamic diameter (D) and polydispersity index (P DI) of nanoparticles were measured by dynamic light scattering at a scattering angle of 173°(using a Nano ZS from Malvern Instruments). The nanoparticle suspensions were diluted in water and measured in triplicate at 25℃. Zeta potential measurements were determined with the same equipment, after a dilution in 1 mM NaCl at 25℃.

TEM measurements

Nanoparticles were observed by transmission electron microscopy (TEM) using a JEOL 1400 (Jeol Ltd, Japan) operating at 80 kV fitted with a SC1000 Orius camera (Gatan Inc, US). Copper grids coated with a formvar film were deposited on a 20 µl drop of nanoparticle suspension (0.05% (w/v) concentration) for 1 min and then on a 20 µl drop of phosphotungstic acid 2% solution for negative staining. The excess of liquid was then blotted off the grids using filter paper. Grids were observed the same day of preparation.

Lactic acid quantification

After the incubation or insonation of samples at 25 and 37℃, the amount of lactic acid was measured as previously detailed [START_REF] Pisani | Polymeric nano/microcapsules of liquid perfluorocarbons for ultrasonic imaging: Physical characterization[END_REF] in the supernatant of the centrifuged nanoparticle suspensions.

Statistics

All statistical analyses were performed using the JMP software (SAS Institute Inc., Cary, NC, USA). The statistical level of significance was set to p = 0.05. Scores were reported as means and standard deviation. Comparison of groups were performed with ANOVA, Chi-square tests or non-parametric Wilkoxon tests on ranks when necessary. Linear model was used to study relation between release rate or energy and experimental conditions. Logistic regression and ROC curves helped in defining the best threshold to separate high release rate (> 0.30) from moderate release rate (< 0.30) using AUC (Area Under Curve) and accuracy (proportion of cases correctly predicted).

Results

HIFU-triggered release of Nile red encapsulated in nanoparticles

The preparation of nanoparticles followed a reliable and proven protocol that ensured that all nanoparticles are composed of a PFOB liquid core and a PLGA-PEG shell encapsulating Nile red. [START_REF] Diou | Pegylated nanocapsules of perfluorooctyl bromide: mechanism of formation, influence of polymer concentration on morphology, and mechanical properties[END_REF][START_REF] Boissenot | Paclitaxel-loaded {PEGy-lated} nanocapsules of perfluorooctyl bromide as theranostic 750 agents[END_REF] The properties of these nanoparticles are also well defined, [START_REF] Diou | Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19 FMRI[END_REF][START_REF] Astafyeva | Properties of theranostic nanoparticles determined in suspension by ultrasonic spectroscopy[END_REF] in particular their mean diameter is approximately equal to 140 nm while their polymeric shell is about 30 nm thick. [START_REF] Diou | Pegylated nanocapsules of perfluorooctyl bromide: mechanism of formation, influence of polymer concentration on morphology, and mechanical properties[END_REF] Insonation was performed for 3 min at a frequency of 1.1 MHz and a pulse repetition frequency of 200 Hz on suspensions (at a nanoparticle volume fraction of 0.25%) filling the medical balloon located inside the thermostated tank. Fluorescence intensities of the sample were recorded before and after insonation and were used to derive the amount, in percent, of released Nile red R red . The values of R red are plotted in Fig. 2 as a function of the total averaged acous-265 tic energy applied at the transducer focus, [START_REF] Carnes | In utero measurements of ultrasonically induced fetal mouse temperature increases, Ultrasound[END_REF] calculated by taking the spatial peak temporal average intensity I spta times the time of insonation (i.e. τ = 3 min). I spta is often considered as an indicator of the ultrasonic thermal deposition. [START_REF] Carnes | In utero measurements of ultrasonically induced fetal mouse temperature increases, Ultrasound[END_REF][START_REF] King | Quantification of temperature rise within the lens of the porcine eye caused by ultrasound insonation[END_REF] R red values are also plotted in Fig. 3 270 as a function of P pkpk , which is the acoustic parameter, in our experiments, that will lead to cavitation and modulate cavitation strength. In our conditions, the threshold for inertial cavitation occurrence was about 6.4 MPa, at which the focused hydrophone started to detect a charac-275 teristic signal made of a broadband background noise and harmonics. For some experiments, a temperature probe was kept in contact with the outside surface of the medical balloon during insonation, which provides T balloon . In , , and ).In these measurements, the duty cycle was set either at 0 ( ), 5% ( ), 10% ( ), 25% ( and ) or 50% ( ). Vertical dashed lines are guides for eyes to indicate critical values of Ispta × τ (see text): the black, red and blue lines are at 79, 380 and 700 J/cm 2 . Note that while Ispta is expressed in W/cm 2 , the unit of Ispta × τ is J/cm 2 . The latter allows to make comparison between measurements made at different insonation time τ . Fig. 2 and3, the experimental points exhibiting the same 280 symbol and color correspond to similar conditions of insonation (identical DC and τ ) and temperature (identical T tank ). Disparities can be observed due to differences in the nanoparticle formulations (e.g. in diameter and shell thickness) used in our experiments. Indeed, the behav-285 ior of the released points in Fig. 2 and 3 from the same batch and performed at different days exhibit a better reproducibility than when comparing released points from different batches. In Table 1, the temperature values at the balloon surface were averaged over several measure-290 ments, and the corresponding estimated P pkpk range was given.

First measurements were conducted with a water tank thermostated at T tank = 37℃. The results are shown as red symbols in Fig. 2 and3 where the duty cycle (DC) 295 was set at either 5 ( ), 10 ( ) or 25% ( ). A weak release (< 5%) of Nile red was measured below a total average intensity of I spta × τ = 79 kJ/cm 2 (I spta ≈ 0.44 kW/cm 2 ) in Fig. 2 or below a peak-to-peak pressure of P pkpk = 6.4 MPa in Fig. 3. Above these values, the Nile red release first increased at approximately 20%. Then, for a duty cycle of 25%, the values could further increase up to 100% for values of I spta ×τ and P pkpk that have been determined to be respectively 380 kJ/cm 2 (I spta ≈ 1.5 kW/cm 2 , AUC= 0.85 and accuracy= 0.76) and 13 MPa (AUC= 0.86 and accuracy= 0.83). For DC = 25%, the probe measured a rise in temperature from 37℃ to 37.7, 44.3 and 45.7℃ on the balloon surface during insonation when P pkpk was equal to 6.2, 11.4-11.6 and 13.4-14.3 MPa, respectively (see Table 1). Whereas for DC = 5% a maximum temperature of 41.3℃ was measured at the balloon surface when P pkpk = 13.1 -13.9 MPa. Similar experiments were per- formed with a water tank set at 25℃. Since duty cycles of 5 and 10% were previously inducing only a small Nile red release (< 20%), only a DC of 25% was considered while keeping the other ultrasonic parameters the same as previously. The use of a 25% duty cycle did not lead to a high Nile red release (i.e. > 35%) when T tank = 25℃ contrary to measurements made at T tank = 37℃, as shown in Fig. 2 and3, and this difference is significant (p < 0.005). 320 However, we succeeded at achieving a higher release (i.e. > 35%) by using a higher duty cycle of 50% but without reaching a 100% release. For DC = 50%, the best discriminating total average energy for having a high release rate is around 700 (I spta ≈ 3.9 kW/cm 2 , AUC = 0.94 and accuracy = 0.88) while the best discriminating pressure is around 14 MPa (AUC = 0.79 and accuracy = 0.88). The temperature at the balloon surface was increasing from 25℃, before insonation, to 30.8 and 33.1℃ at a duty cycle of 25% when P pkpk was equal to 10.7 and 13.6-15.3 MPa, 330 respectively and to 33.4 and 36.2℃ at a duty cycle of 50% when P pkpk was equal to 13.2 and 14.1-15.0 MPa, respectively.

Overall, when considering I spta × τ as a linear model of the variables DC and T tank (R 2 = 0.79), there is a signif-335 icant influence of both duty cycle (p < 0.0001) and tank temperature (p = 0.01) to I spta × τ , but with no significant interaction between them. This is also true when considering the release rate R red as a linear model of DC and T tank (R 2 = 0.25), with a significant influence of both 340 DC (p < 0.005) and T tank (p < 0.0001) but no significant interaction between them.

The insonation time of 3 min was chosen so that it was possible to reach 100% release of Nile red when using optimal parameters at 37℃. When decreasing the insonation 345 times, while keeping a constant duty cycle of 25% and P pkpk ≈ 13.4 -14.1 MPa, the amount of released Nile red was proportionally reduced as shown in Fig. 4. During the insonation, the temperature at the balloon surface was equal to 43.1, 46.6, and 45.7 ± 1.5℃ at 1, 2 and 3 min 350 (see Table 1). In the absence of insonation, nanoparticle suspensions kept 3 to 7 min at 37℃ exhibited only a slight release of Nile red with R red ≈ 2.7%.

Furthermore, we used TEM to monitor any change in nanoparticles morphologies induced by insonation at 355 T tank = 25℃ and DC = 50%, then at 37℃ and 25%, by comparing non-insonified suspensions with samples insonified at low and high values of P pkpk , as shown in Fig. 5. At both temperatures, we observed a few damaged nanoparticles after insonation only for high values of P pkpk 360 (≈ 14.5 MPa). We also measured in Fig. 6 the mean diameter of the nanoparticles before and after insonation, at 25 and 37℃. It did not vary significantively between the various samples, even is the mean diameter for samples insonified at P pkpk ≈ 14.5 MPa, is slightly higher than those 365 observed without insonation or at P pkpk ≈ 11.3 MPa. Finally, the amount of lactic acid was measured to 0.22 ± 0.01% and 0.17 ± 0.01% for samples incubated respectively at T tank = 25 and 37℃. At 37℃, this amount remained constant at 0.17 ± 0.01% for samples insonified at DC = 25% and at P pkpk = 11.3 or 14.3 MPa. Whereas at 25℃ and DC = 50%, the amount of lactic acid was measured to 0.21 ± 0.04 and 0.29 ± 0.08, respectively when P pkpk = 11.3 and 14.6 MPa.

HIFU-triggered release of doxorubicin encapsulated inside

liposomes

We performed similar experiments using Caelyx, a liposomal form of doxorubicin•HCl. The mean diameter of Caelyx liposomes were measured by DLS and was equal 170 ± 20 nm in good agreement with the supplier speci-380 fications (mean size ranging from 160 to 180 nm) with a polydispersity index lower than 0.08. The initial liposomal suspension was diluted 500 times (to be in the same conditions as in the study of Somaglino et al. [START_REF] Somaglino | Validation of an acoustic cavitation dose with hydroxyl radical production generated by inertial cavitation in pulsed mode: Application to in vitro drug release from liposomes[END_REF]) before being introduced into the balloon of the experimental setup.

385

The solution was then insonified with the exactly same ultrasonic parameters as for polymeric nanoparticles except we only used a duty cycle of 25% and the time of insonation was set to 1 min 30 s. Indeed, when using a longer time of 3 min as was done previously we systemat-390 ically reached a 100% doxorubicin release. A shorter time allowed us to vary the quantity of released doxorubicin as a function of P pkpk or I spta × τ . Insonation was performed with a water tank set at either T tank = 25 or 37℃ with a P pkpk value varying from approximatively 8.5 to 395 14.1 Mpa, for which inertial cavitation was always detected by the hydrophone as it has already been shown that the doxorubicin release can be triggered by ultrasound only in presence of inertial cavitation [START_REF] Somaglino | Validation of an acoustic cavitation dose with hydroxyl radical production generated by inertial cavitation in pulsed mode: Application to in vitro drug release from liposomes[END_REF][START_REF] Dromi | Pulsed-high intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and 705 antitumor effect[END_REF]. The amount of released doxorubicin from liposomes, R dox , was recorded 400 by fluorescence spectroscopy and the values are reported in Fig. 2 and3. For both temperatures, we measured a continuous increase in doxorubicin release as I spta × τ or P pkpk was increasing, reaching a maximum release at about 212 kJ/cm 2 and 12.7 MPa at 25℃, and at higher values 405 (extrapolated to approximately 283 kJ/cm 2 and 15.6 MPa) at 37℃ (see Fig. 2 and3). The release rate was significantly different (p < 0.005) between doxorubicin-loaded liposomes and Nile red-loaded nanoparticles experiments at both 25℃ and 37℃.

Discussion

Ultrasound are mechanical waves whose compression and depression induce a mechanical stress, they are also carrying energy that is dissipated through the medium in which ultrasound propagates, thus locally rising the temperature. When the acoustic pressure is high enough, cavitation occurs which further increases the local temperature and mechanical stress as well as creates radicals. In our case, cavitation started to be detected by the focused hydrophone when the peak-to-peak pressure P pkpk was larger than 6.4 MPa. Published investigations showed that the ultrasound triggered doxorubicin release from the non-thermosensitive liposomal formulation caelyx is only due to inertial cavitation [START_REF] Somaglino | Validation of an acoustic cavitation dose with hydroxyl radical production generated by inertial cavitation in pulsed mode: Application to in vitro drug release from liposomes[END_REF][START_REF] Dromi | Pulsed-high intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and 705 antitumor effect[END_REF]. Literature data [START_REF] Schroeder | Controlling Liposomal Drug Release with Low Frequency Ultrasound: Mechanism and Feasibility[END_REF][START_REF] Hansen | Ultrasonic drug release: impact on liposomal doxorubicin in collagens gels[END_REF][START_REF] Afadzi | 735 Effect of ultrasound parameters on the release of liposomal calcein[END_REF][START_REF] Escoffre | Focused ultrasound mediated drug delivery from temperature-sensitive 730 liposomes: in-vitro characterization and validation[END_REF] show a linear behavior for the release from non-thermosensitive liposomal formulation as a function of acoustic pressure (or mechanical index), that starts at the critical pressure, P cav , at which cavitation appears with a temperature dependant slope α(T ): i.e. R dox (P ) = α(T )(P pkpk -P cav ). Here, the temperature dependence of α is not related to a gel-to-liquid transition (as the lipid membrane remains in a gel phase in these liposomal formulations) but to a change in water temperature-dependent properties [START_REF] Afadzi | 735 Effect of ultrasound parameters on the release of liposomal calcein[END_REF][START_REF] Apfel | Gauging the likelihood of cavitation from short-pulse, low-duty cycle diagnostic ultrasound[END_REF][START_REF] Bel | Sonolysis of ciprofloxacin in aqueous solution: Influence of operational parameters[END_REF]: in particular in the amount of gas dissolved in water (involved in nuclei appearance), in the surface tension at water/gas interface (involved in bubble stability), and in the vapor pressure, density and viscosity of water (involved in bubble volume oscillation and implosion). Moreover, Somaglino et al. [START_REF] Somaglino | Validation of an acoustic cavitation dose with hydroxyl radical production generated by inertial cavitation in pulsed mode: Application to in vitro drug release from liposomes[END_REF] showed that the percentage of doxorubicin delivered from the liposomal formulation caelyx is proportional to the inertial cavitation dose. In our experiments, we also observed a linear behavior for the release of doxorubicin from caelyx as a function of P pkpk . This indicates that when the value P pkpk increases the inertial cavitation dose is also increasing, resulting in a higher doxorubicin release. However, this linear behavior is not observed for Nile red release from polymeric nanoparticles, suggesting a release mechanism that is not continuously proportional to the inertial cavitation dose. Nile red release occurs only for specific ultrasound properties, as below critical values of P pkpk or I spta × τ only a low Nile red release was observed. Indeed, when considering Fig. 3, where R red is plotted as a function of P pkpk , the curves obtained at 25 and 37℃ follow the same behavior. A high Nile red release (R nano > 30%) is measured only for P pkpk > 13 MPa. A moderate release 5% < R nano < 30% is observed for 6.4 < P pkpk < 13 MPa, knowing that inertial cavitation occurs above 6.4 MPa in our setup, while only a low release is measured (R nano < 5%) in the absence of inertial cavitation (i.e. when P pkpk < 6.4 MPa), similar to what is measured for non-insonified solutions. However, the above behavior is only observed for a duty cycle equals to 25% at 37℃ and 50% at 25℃. Indeed, a strong release (> 30%) was never measured for P pkpk > 13 MPa when the duty cycle was 5% or 10% at 37℃ or 25% at 25℃. Meanwhile, the temperature measured at the balloon surface increases when the values of duty cycle and P pkpk are increasing. For the same P pkpk range (around 14.1 MPa), a similar temperature elevation is measured at 25% DC: 470 +8.7℃ at 37℃ and +8.1 ℃ at 25℃, while the temperature elevation is +11.2℃ for a duty cycle of 50% at 25℃. At 5% DC and 37℃, the temperature elevation was about 4.1℃, twice less than for DC = 25%. Alternatively, for the same value of duty cycle (i.e 25%) the temperature T balloon 475 decreases by about 2.3℃ when P pkpk decreases from 13.5-15.3 to 10.7 MPa when T tank = 25℃ (see Table 1). It should be noted that these temperature elevations can be higher at the focal point, inside the balloon. Given the definition of I spta , its value increases for either increasing 480 the value of duty cycle or acoustic pressure when all other ultrasonic parameters are kept constant. Since the local temperature increases when DC increases, I spta effectively represents in our case a marker of the quantity of heat deposited from ultrasound at the focal zone. Consequently, 485 the reason why the critical value of I spta × τ is larger at 25℃ than at 37℃ in Fig. 2 diffusion) always remains below 5% after 3 to 7 min of incubation (data points at 0 in Fig. 2 and3), in agreement with other studies on similar polymer. [START_REF] Elkharraz | Paclitaxel-loaded microparticles and implants for the treatment of brain cancer: Preparation and physicochemical characterization[END_REF] Note that in the latter case, the decrease in Nile red fluorescence intensity may partially originate from photobleaching. Moreover, 515 the reason why a temperature rise is needed in addition to sufficient inertial cavitation cannot be explained by the glass transition temperature, T g , of the polymeric shell since for the diblock polymer PLGA-PEG this value is equal to 27℃, a value obtained by differential scanning 520 calorimetry. [START_REF] Diou | Pegylated nanocapsules of perfluorooctyl bromide: mechanism of formation, influence of polymer concentration on morphology, and mechanical properties[END_REF] This value is smaller than that of PLGA (around 40℃). It is also worth noting that T g rapidly decreases due to polymer hydrolysis. For instance, the value of T g for PLGA nanoparticles goes below 20℃ after several days of degradation. [START_REF] Elkharraz | Paclitaxel-loaded microparticles and implants for the treatment of brain cancer: Preparation and physicochemical characterization[END_REF] The fact that cavitation is required to induce a drug delivery from polymeric nanoparticles is consistent with the literature. For instance, Kost et al. [START_REF] Kost | Ultrasound-enhanced polymer degradation and release of incorporated substances[END_REF] studied the ultrasound-triggered release of p-Nitroaniline encapsulated into various polymers, using an ultrasonic bath at 75 kHz.

The authors observed that the release of p-Nitroaniline was drastically reduced when cavitation was minimized. Luo et al. [START_REF] Luo | On-demand drug release from 760 dual-targeting small nanoparticles triggered by high-intensity focused ultrasound enhanced glioblastoma-targeting therapy[END_REF] studied the ultrasound-triggered delivery of doxorubicin encapsulated inside PLGA-PEG nanoparticles with a diameter of 40 nm comprising a small amount of PFOB.

The authors used their ultrasonic device at a power of 8.5-10.5 W but they did not provide pressure values nor the frequency used. They achieved in vitro an important ultrasound-triggered release (50-80%) that is accompanied, after insonation, with a large increase in particle size (up to 4 times), a decrease in zeta potential, and a change in droplet morphology that suggests the occurence of cavitation induced by PFOB vaporisation. However, Boissenot et al. [START_REF] Boissenot | Ultrasoundinduced mild hyperthermia improves the anticancer efficacy 755 of both taxol® and paclitaxel-loaded nanocapsules[END_REF] could not trigger a significant release of paclitaxel encapsulated in the same nanoparticles used in our study, although cavitation occured during insonation. The authors used a maximum peak negative pressures of 7 MPa with a duty cycle of 20% (at 37℃), which values may not be sufficient to obtain a high release in regard to our results. That may also be the case for Abed et al. [START_REF] Abed | Effects of ultrasound irradiation on the release profile of 5-fluorouracil from magnetic polylactic co-glycolic acid nanocapsules[END_REF] who could never induce a release of 5-fluorouracil larger than 6% at 37℃ (measured just after insonation) when encapsulated into nanoparticles made of a PLGA shell and encapsulating a small magnetite core. Indeed, the authors used small ultrasonic intensities (< 1 W/cm 2 ) at a frequency of 3 MHz.

In our experiments, after high pressure insonation, we observed no significant change in particle size and no dramatic structural modifications of the nanoparticles as those measured by Luo et al. after PFOB vaporization, and our acoustic spectra exhibit no evidence of PFOB vaporization. The difference may come from the nanoparticles size and from the fact that our nanoparticles contain a well defined PFOB core representing approximately 30% of the nanoparticle volume whereas for the nanoparticles of Luo et al., the quantity of PFOB represents only 1% of the droplet weight and may be distributed in several locations into the nanoparticle. PFOB is known to solubilize important quantity of oxygen (20 times more than water) [START_REF] Lowe | Perfluorochemicals: their applications and benefits to cell culture[END_REF] which can favor PFOB vaporization. But in our case, the presence of a thick polymer shell strongly acts against an expansion of the PFOB volume. However, it has been shown that the quantity of oxygen solubilized in perfluorocarbons varies with temperature and it increases in the case of PFOB [START_REF] Johnson | In vitro comparison of dodecafluoropentane (ddfp), perfluorodecalin (pfd), and perfluoroctylbromide (pfob) in the facilitation of oxygen exchange[END_REF] while it decreases for water when the temperature rises. This variation should induce a migration of oxygens from water to PFOB, through the polymer, thus enriching the polymer with oxygen. Considering this fact, we hypothesize for the nanoparticles studied here that when P pkpk is large enough, the increase in R red from 3 to 20% observed for P pkpk < 13 MPa is due to a degradation of the polymer shell surface induced by the cavitation occuring in the aqueous solvent surrounding the nanoparticles. While for P pkpk > 13 MPa, cavitation may also occur inside the polymer shell if the temperature is large 585 enough to enrich the shell with oxygen due to its transfer from water to PFOB. This internal degradation should allow a larger diffusion of Nile red out of the polymer. This hypothesis offers the advantage to take into account the fact that both a high acoustic pressure and sufficient tem-590 perature elevation are required to induce a high Nile red release, while either a high acoustic pressure or a temperature elevation are not sufficient alone to induce this high release. However, more investigations are needed to validate this hypothesis. Future investigations should focus 595 on the monitoring of oxygen inside the nanoparticles as a function of temperature. 19 F MRI could be used for such studies although the required acquisition time will prevent a dynamic monitoring of oxygenation. [START_REF] Lorton | Molecular oxygen loading in candidate theranostic droplets stabilized with biocompatible fluori-nated surfactants: Particle size effect and application to in situ 19f mri mapping of oxygen partial pressure[END_REF] Finally, considering the required values of pressure and 600 duty cycle as well as the fact that strong inertial cavitation is needed to induce Nile red release, these nanoparticles are not suitable for therapeutic applications using ultrasoundtriggered drug delivery.

Conclusion 605

We have investigated the ultrasound triggered release of Nile red encapsulated into nanoparticles made of a PLGA-PEG shell surrounding a PFOB core. We could induce an important release (>30%) of Nile red only if the pressure and temperature were high enough (i.e. P pkpk > 13 MPa and T > 45℃). Otherwise, moderate release is obtained but only in the presence of inertial cavitation. Thus, these ultrasonic parameters make these nanoparticles unsuitable for therapies using ultrasound-triggered drug release.

acknowledgement 615

This work is supported by public grant ANR-10-NANO-06 overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program and by the european program EuroNanoMed II (project SonoTherag). The present work has benefited from the facilities and ex-620 pertise of the Electron Micoscopy facilities of Imagerie-Gif (http://www.i2bc.paris-saclay.fr/spip.php?article282) with the precious help of C. Boulogne. This core facility is member of the Infrastructures en Biologie Santé et Agronomie (IBiSA), and is supported by the French mal doxorubicin plus docetaxel significantly improves time to progression without additive cardiotoxicity compared with docetaxel monotherapy in patients with advanced breast cancer previously treated with neoadjuvant-adjuvant anthracycline therapy: Results from a randomized phase iii study, J. Clin. Ondoi:10.1016/j.addr.2012.10.002.

[6] H. Maeda, T. Sawa, T. Konno, Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS, J. Contr. Release 74 (1-3) (2001) 47-61. doi:

R

  red = 1 -I -I void I init -I void × 100 (2) where I void , I init , and I are respectively the fluorescence intensities of a suspension containing nanoparticles with-155 out any Nile red, of a suspension of nanoparticles containing Nile red at the beginning of the experiment, and at the end of experiment (i.e. 20 min after insonation or temperature incubation). Fluorescent intensities were measured at an emission wavelength of λ em = 575 nm and an exci-160 tation wavelength of λ ex = 530 nm.

Figure 1 :

 1 Figure 1: Scheme of the ultrasonic setup used to perform ultrasoundtrigger drug release experiments.

Figure 2 :

 2 Figure 2: Percentage of released doxorubicin R dox from liposomal doxorubicin at 25℃ ( ) and 37℃ ( ). Percentage of released Nile red from nanoparticles at 25℃ ( and ) and at 37℃ ( ,, , and).In these measurements, the duty cycle was set either at 0 ( ), 5% ( ), 10% ( ), 25% ( and ) or 50% ( ). Vertical dashed lines are guides for eyes to indicate critical values of Ispta × τ (see text): the black, red and blue lines are at 79, 380 and 700 J/cm 2 . Note that while Ispta is expressed in W/cm 2 , the unit of Ispta × τ is J/cm 2 . The latter allows to make comparison between measurements made at different insonation time τ .

Figure 3 :

 3 Figure 3: Percentage of released doxorubicin R dox from liposomal doxorubicin at 25℃ ( ) and 37℃ ( ).Percentage of released Nile red from nanoparticles at 25℃ ( and ) and at 37℃ ( , , , and ).In these measurements the duty cycle was set either at 0 ( ), 5% ( ), 10% ( ), 25% ( and ) or 50% ( ). Vertical dashed lines are guides for eyes to indicate critical values of P pkpk (see text): the black, red and blue lines are at 6.4, 13 and 14 MPa.The dotted red and blue lines indicate the linear behavior expected for liposomal doxorubicin (starting around 6.4 MPa, at which strong inertial cavitation starts to occur).

Figure 4 :

 4 Figure 4: Variation in the percentage of released Nile red from nanoparticles using a constant value for Ispta but with a sonication time equals to 1 ( ), 2 ( ) or 3 min ( ). Measurements were performed at 37℃ with a 25% duty cycle and a P pkpk in the range of 13.4-14.1 MPa.

Figure 5 :

 5 Figure 5: TEM pictures of nanoparticle solutions kept at 37℃ (top row) without insonation (top left), insonified with a duty cycle of 25% at low (top middle) and high (top right) P pkpk (resp. Ispta ×τ ) equal to 11.3 (resp. 279) and 14.3 MPa (resp. 434 kJ/cm 2 ), respectively. We respectively observed a 2.7, 22.1, and 88% release of Nile red. In the bottom row, TEM pictures are for nanoparticle solutions kept at 25℃ without insonation (bottom left), insonified with a duty cycle of 50% at low (bottom middle) and high (bottom right) P pkpk (resp. Ispta ×τ ) equal to 11.3 (resp. 566) and 14.6 MPa (resp. 912 kJ/cm 2 ), respectively. We respectively observed a 2.4, 13.2, and 76.4% release of Nile red. The scale bar is valid for all pictures. The red arrows indicate examples of damaged nanoparticles.

Figure 6 :

 6 Figure 6: Diameter of polymeric nanoparticles were measured by DLS when they were not insonified (Reference), after an insonation with a medium P pkpk (≈ 11.3 MPa) or a high P pkpk (≈ 14.5 MPa), at 25℃ with a 50 % duty cycle (blue bars) and 37℃ with a 25% duty cycle (red bars).

  410

Table 1 :

 1 The table displays the mean value of T balloon measured during insonation at the balloon outside surface, for various tank temperatures T tank , duty cycles DC, insonation times τ , and peak pressures P pkpk .

	T tank DC τ	P pkpk	T balloon
	(℃)	(%) (min) (MPa)	(±1.5℃)
	37	5	3	13.1-13.7 41.3
		25	1	14.0-14.1 43.1
			2	13.7-15.3 46.6
			3	6.1	37.7
				11.4-11.5 44.3
				13.4-14.3 45.7
	25	25	3	10.7	30.8
				13.5-15.3 33.1
			10	13.8	32.4
		50	3	13.1	33.4
				14.0-14.9 36.2

  may come from the fact that more heat needs to be locally delivered by ultrasound at 25℃ than at 37℃ to induce a strong Nile red release.Overall, our data suggest that the release of Nile red 490 is triggered by a synergistic effect of temperature elevation and cavitation strength. Indeed, on one hand a high cavitation dose (that increases with P pkpk ) cannot alone induce a high delivery rate if the temperature at the focal region is not high enough. This is illustrated by the data

	495
		performed above 13 MPa. A duty cycle of 25% can lead
		to 100% of Nile red release at T tank = 37℃, while increas-
		ing the local temperature by 8.1℃ to reach 45.7℃. But
		at T tank = 25℃, this value of duty cycle only promotes
	500	less than 30% of Nile red release for a similar temperature
		elevation of 8.7℃, which results in a temperature on the
		balloon surface of 33.1℃. Even an increase of duty cycle
		to 50% does not allow to reach 100% release, while the lo-
		cal temperature being 36.2℃. On the other hand, the sole
	505	temperature elevation cannot be held responsible for the
		observed high Nile red release, the application of a high
		dose of cavitation is required. Indeed, at 37℃ and in the
		absence of insonation, no change in the nanoparticle mor-
		phologies is observed and the passive release of Nile red
	510	(due to polymer shell hydrolysis combined with Nile red

national Research Agency under Investments for the Future programs "France-BioImaging", and the Labex "Saclay Plant Science" (ANR-10-INSB-04-01 and ANR-11-IDEX-0003-02, respectively). Institut Galien Paris-Saclay is a member of the Laboratory of Excellence LERMIT sup-630 ported by a grant from ANR (ANR-10-LABX-33). The authors also thank Domingues Gonçalves for building the ultrasonic tank used in this study.


Matthias Macé

Camille Richeval

Patrice Gérard

Sylvie Duchesne

Catherine Cannet

Irina Boyarskikh

Annie Gérault

Vincent Zvénigorosky

Darya Nikolaeva

Charles Stépanoff

Liubomira Romanova

Charles Stepanoff

Delphine Allorge

Michele Debrenne

Bertrand Ludes

Anatoly Alexeev

Jean-Michel Gaulier

Eric Crubézy

Impact of socio-economic traditions on current tobacco and tea addictions (Siberia 17 th to 20 th century)

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Understanding the historical foundations of contemporary addictions is challenging because historical data do not always reflect biological data and the latter are scarce. We had the opportunity to excavate more than 150 frozen graves in East Siberia (Yakutia) dating from the 17th to the 19th century (Figure 1). Some revealed smoking material (Figure 2), another the most northerly English teapot ever found in an archaeological excavation (Figure 3). We were able to find metabolites of tea and tobacco in the hair of 47 subjects. These analyzes, confronted with historical, economic and ethnological studies, reflect the way in which green tea, and later black tea and tobacco spread in Siberia and how these substances competed with native herbal teas. Passive smoking in young people and comorbidities can be described. Certain dissemination processes among these indigenous populations, either through the elites and heavy consumers or through certain distribution channels, are the same as those which today favor addictions. More curiously still, certain specificities which developed with the first consumers of the 18th century are still part of the epidemiological characteristics of modern Yakutia. This study supports public health policies based on intervening on economic circuits or modes to curb certain addictions, as it shows us that the origin of these addictions is sometimes older and more complex than one might suppose.

Material and Methods

More than 150 bodies found in frozen tombs four geographical regions (31,000 km²) of the Sakha Republic (Yakutia) were studied. They were classified into five chronological periods: before 1700, 1700-1750, 1750-1800, 1800-1850 and after 1850 AD. The first contacts of the autochthonous population with the Russians date back to 1620 AD but there was almost no trace of these contacts in material culture before 1689/1700AD. Assimilation in the Russian Orthodox culture was fully accomplished after 1800 AD.

We categorized and analyzed the characteristics of these ancient subjects and studied their familial relationships, social status and diet (SD1). Among these, 47 had well-preserved hair without contact with sediment. Hair samples were collected and stored during the autopsy of the subjects following procedures ruling out the possibility of outside contamination. They were decontaminated in the laboratory. We analyzed proximal hair segments (2 to 3 cm length) close to the skull corresponding to the last few weeks of life. Levels for the main compounds related to tea and tobacco consumption were analyzed in hair sampled from 47 frozen bodies, dated from the early contact with Europeans (1620 AD) to the assimilation of native people into Russian society (end of the 19 th century). In one case, remains of tea leaves were found in a teapot associated with a woman and analyzed. We were able to look for traces of pulmonary anthracosis in 14 subjects and pulmonary histological investigations for potential comorbidities were also carried out. In 7 out of these 14 cases we were able to analyze both hair and lungs, and for one woman a breast concomitantly, to establish whether the breastfeeding woman had high nicotine use (SD2).

Previous studies investigated nicotine and caffeine in hair [1,2] and used LC-HRMS and LC-MS/MS tools for toxicological investigations in hair [START_REF] Dulaurent | Simultaneous determination of Δ9tetrahydrocannabinol, cannabidiol, cannabinol and 11-nor-Δ9-tetrahydrocannabinol-9carboxylic acid in hair using liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Maublanc | Identification and quantification of 35 psychotropic drugs and metabolites in hair by LC-MS/MS: application in forensic toxicology[END_REF]. We combined these two techniques according to previously published methods [START_REF] Boumrah | In vitro characterization of potential CYP-and UGT-derived metabolites of the psychoactive drug 25B-NBOMe using LChigh resolution MS[END_REF][START_REF] Kintz | Detection of the designer benzodiazepine metizolam in urine and preliminary data on its metabolism[END_REF] for screening drugs and toxics compounds, psychoactive drugs including natural alkaloids, and tea methylxanthines (caffeine, theobromine, theophylline) and tobacco alkaloids (nicotine) and metabolite (cotinine) (SD3).

Another large toxicological screening for psychoactive drugs complied with international recommendations [7,8].

Statistical analyses

Principal Component Analysis was performed over the levels of the five combined analytes and outliers defined qualitatively (SD4). The number of tea metabolites detected in hair varied from zero to three (theophylline, theobromine, caffeine) depending on the individual. Multivariate logistic regressions were used to assess a putative shift in the use of tobacco and tea over time (before and after 1800 AD, see SD4 for details), adjusting for gender, age, social status and presence of pipe. Spearman's correlation coefficient between nicotine and cotinine concentrations was computed within each age class (0-15, 15-30, 30-50 & over 50 years old). Co-correlation between age classes was assessed between the age class of the children (0-15 y/o) and each of the other age classes, including Zou's confidence intervals [9]. Computations were performed using the Cocor R package [START_REF] Diedenhofen | Cocor: A comprehensive solution for the statistical comparison of correlations[END_REF].

We assessed the association between xenobiotic concentrations and geography using several permutation-based methods (SD4). We then assessed the correlation (Spearman's rank test) between analyte concentrations and distances from the main trading posts and/or fairs in the east through which tea and/or tobacco was likely to transit as well as the known entry areas through which it was likely to arrive (SD9). Strength of association was tested using a permutation over individual concentrations. Given the significance of the association between the levels of cotinine and/or nicotine with the pipes (SD5), we looked for the same associations, on the totality of the pipes (for a majority of subjects the hair was not preserved), before and after 1800 AD. The PCA first axis values computed as described above were interpolated over a rhomboid area comprised between the four geographical regions using spline interpolation and triangulation based on linear interpolation as implemented in the akima R package [11]. Maps were produced using the marmap R package and the NOAA bathymetrics and global relief data (resolution of 5 minutes) and then drawn following an orthographic projection for 100° longitude, 60° latitude and 0 m elevation.

Results

Hair analyses showed a LOD of 0.01 ng/mg and LLOQ of 0.02 ng/mg for nicotine and cotinine; a LOD of 0.05 ng/mg and LLOQ of 0.01 ng/mg for caffeine, theobromine and theophylline (Erreur ! Source du renvoi introuvable.Erreur ! Source du renvoi introuvable.). It is of note that no other natural alkaloid compound or related metabolite (e.g. morphine, codeine, atropine, scopolamine, …) was detected in the 47 analyzed hair samples (SD6). The last dichloromethane bath used for the hair decontamination step tested negative for all compounds and for all samples. Theobromine (n=31) was more often detected (including in one 6 to 9-month-old child) than caffeine (n=29), which was itself more often detected than theophylline (n=19). After 1800 AD, at least one substance was detected in all subjects, and the three substances were detected in 8/15 subjects (Figure 4). The subject presenting the highest concentration was a woman buried with a teapot (SD7 and SD11). Before 1800 AD, no substance was detected in 8 hair samples and the three substances were detected in only 6/32 samples. The most frequent case (9/32) was that of subjects with hair samples containing both theobromine and caffeine, essentially women (8 females out of 9 individuals: p=0.0182) and the only male with the two substances dated from the period prior to 1700 AD (SD8). We found a sub-significant association between the status of shaman and theobromine concentrations (SD7).

Cotinine (n=47, 100%) was more often detected than nicotine (n=43, 91.4%). The two subjects with the lowest hair cotinine concentration were a child aged 6 to 12 months and a child aged 6 to 9 months. Cotinine was sometimes found in some adults especially those dated before 1700 AD.

The ten subjects buried with a pipe had traces and/or significant hair concentrations of cotinine and/or nicotine, one had no trace of nicotine and traces of cotinine. For graves with pipes, before 1800 AD we found a sub-significant association between the presence of pipe and the distance to Yakutsk or the distance to the south-west of the studied area (SD9). The nicotine/cotinine correlation increased with age and the correlation was significantly different between the young (0-15 years old) and the oldest quartile (over 50 years old) -SD7 -. An older woman from the end of the 18 th century was the only subject with a level of cotinine associated with the highest level of nicotine; anthracosis pigments found in her lungs can indicate death by -SD2-.

Eighty-two percent (39/47) of the subjects had traces of tea and tobacco in hair samples. The highest users of the two substances were from the 19 th century, and heavy tea users were only found in the 19 th century. Moderate tea users in the 19 th century were also tobacco users. There were families of heavy smokers and non-smokers, and in one family of heavy smokers one of the women was breastfeeding at the time of her death. In the case of caffeine and cotinine, we were able to show a negative correlation between the concentration of these substances and the distance from sampling location to the three main trading posts and/or fairs in the east through which the tea was likely to have transited (SD9). The highest correlation was with the northeast, which represents the point of entry of the trade route by which tea was brought at this period from the Pacific harbor of Okhotsk into Yakutia (p-value = 0.0112). The robustness of this association was challenged by permuting 10% of the metabolic concentration values among individuals (10,000 replicates). The association was confirmed with probability of observing rs above 0.5 above 0.9 for Zachiversk harbor. This is consistent with a NE-SW gradient in caffeine concentration after 1800 AD (Figure 5).

Discussion

The use of tobacco products is the deadliest addiction worldwide and a positive association between smoking and caffeine intake is found in some populations, although the preference for the type of caffeinated drink -particularly tea versus coffee -varies greatly among countries [START_REF] Treur | Associations between smoking and caffeine consumption in two European cohorts[END_REF]. Despite prevention campaign efforts to reduce the public health burden of tobacco, prevention has not benefitted all strata of various societies [START_REF] Leventhal | The Sociopharmacology of Tobacco Addiction: Implications for Understanding Health Disparities[END_REF], because addictions involve the interplay of pharmacology, genetics, and social factors together with environmental factors, including fashion and identity [START_REF] Ferland | Mémoires tabagiques. L'usage du tabac, du XV siècle à nos jours[END_REF]. Although multilevel transdisciplinary models are needed to comprehensively understand sources of tobacco-related health disparities (TRHD), the incorporation of historical context into TRHD research remains infrequent. Yakutia (north eastern Siberia) offers a unique opportunity for such research. Our study is the first to analyze such a large number of hair samples collected in the minutes or hours following the excavation of frozen bodies. Considering that the growth rate of human scalp hair ranges from 0.7 to 1.4 cm/month with a mean of about 1 cm/month [START_REF] Pecoraro | Measurements of Hair Growth Under Physiological Conditions[END_REF] we analyzed samples of hair corresponding to approximately two to three months' growth before the subject's death, much less for the children for whom the hair was not very long. Xenobiotics are well preserved in hair [START_REF] Ashford | Measuring prenatal secondhand smoke exposure in mother-baby couplets[END_REF], we were able to determine strong associations between hair concentrations of some substances and cultural factors even if exposure to second-hand smoke during the lifetime of the patients cannot be excluded [START_REF] Musshoff | Ethyl glucuronide findings in hair samples from the mummies of the Capuchin Catacombs of Palermo[END_REF] (SD10). The Yakuts buried their dead with items representative of their lives and/or occupations. Some strong empirical associations were found: the teapot was associated with the highest tea consumer, the heaviest smoker presented pulmonary emphysema, and the presence of a pipe was associated with smoking in 9 out of 10 cases. Cotinine/nicotine correlation increased with age, probably reflecting passive smoking in young people.

Tea contains some of the 3-methylxanthines (caffeine, theophylline and theobromine), but its chemical composition is significantly affected by tea processing, leaf maturation, botanical varieties, geographical origin, agricultural practice [START_REF] De Mejia | Bioactive components of tea: Cancer, inflammation and behavior[END_REF] and the way to prepare it [START_REF] Hicks | Tea preparation and its influence on methylxanthine concentration[END_REF]. The influence of physiological variations from one subject to another on the incorporation of metabolites in hair is, to our knowledge, unknown. However, while all three 3-methylxanthines are present in most black teas, theophylline is often absent in green teas [START_REF] Bi | Investigation of free amino acid, total phenolics, antioxidant activity and purine alkaloids to assess the health properties of non-Camellia tea[END_REF] and in some black teas [START_REF] Fernández | The Use of Catechins and Purine Alkaloids as Descriptors for the Differentiation of Tea Beverages[END_REF]. In our sample, women seem to have consumed green tea from the end of the 17th century. Black tea consumption, which appeared punctually before 1750 AD, became widespread in the 19th century, consistent with historical data [START_REF] Yoder | Tea time in Romanov Russia: acultural history, 1616-1917[END_REF]. Ivan tea, a fermented herbal tea from Chamerion latifolium and Chamerion angustifolium which was drunk at that time, contains only theobromine in quite large quantities [START_REF] Pakhomova | Definition of theobromine in the Russian Federation fermented herbal tea[in russian[END_REF]. This native herbal tea was consumed by five subjects of various ages from the 17th and 18th centuries; in the 19th century it was only detected in three subjects, two of whom were less than one year old, suggesting that at that time it could have been still used for medicinal purposes. It cannot be ruled out that other herbal teas may have been consumed. (SD11).

All periods combined, we found a few heavy tobacco consumers, men and women, amongst light or passive consumers, but tobacco-related co-morbidity was only recorded after 1750 AD (in a woman) at a time when a large part of the population, even older children, became addicted according to travelers' accounts [START_REF]Olyksandrovič. Ugolok dalekogo severa [A little corner in the Far North][END_REF][START_REF] Georgi | The description of all the peoples of the Russian Empire[END_REF]. Heavy tea users were only detected in the 19 th century and the heaviest users of the two substances dated from this century. In the 19th century, while the Yakut population assimilated into the Russian orthodox way of life, several contemporary trends were detected: all subjects seemed to become, at least, light consumers of tea, and moderate tea users were also tobacco users. The average tobacco-related xenobiotic hair concentration was higher in women than in men before 1800 AD (Figure 4) and this peculiarity is still found today in the Sakha republic and the current population of the Republic currently exhibits generalized tea drinking habits and a higher smoking incidence than any other part of the Russian Federation [START_REF] Egorova | Evaluation of the influence of sociohygienic factors on the formation of diseases of the circulatory system in people of working age in the Republic of Sakha (Yakutia)[in russian][END_REF][START_REF] Gambaryan | Epidemiology Of Chronic Respiratory Disease In Different Climatic And Geographic Regions Of Russia[END_REF]. As far as the further away from Verkhoyansk (in the north), the lower the nicotine concentration in hair. This probably established the current feature of the prevalence of smokers in the north [START_REF] Egorova | Evaluation of the influence of sociohygienic factors on the formation of diseases of the circulatory system in people of working age in the Republic of Sakha (Yakutia)[in russian][END_REF].

The generalization of tea and tobacco is linked to economic circuits. In our study area, tobacco use spread faster than tea use, probably because tobacco is intrinsically more addictive than tea, and because of the absence of a previous tradition in the use of these substances. A variety of herbal teas -specially Ivan tea-have been used by the Yakuts for centuries. Herbal teas were widely consumed as beverages and medicines for disease prevention and treatment but also for shamanic reasons as attested by the theobromine levels in female shamans. The use of green tea by women in the 18 th century also support this hypothesis, and the generalization of the use of tea follows the development of tuberculosis [START_REF] Dabernat | Tuberculosis epidemiology and selection in an autochthonous Siberian population from the 16 th -19 th century[END_REF] for which green tea was used as a treatment, perhaps with some results [START_REF] Chen | Impact of tea drinking upon tuberculosis: a neglected issue[END_REF]. In the 19 th century, tea had made the transition from a beverage used in medicine and spirituality-based socialization, to being used by elites with the tea ceremony led by women of fashion and means. The ceremony dictating that tea must be brewed and drunk using specialized equipment (Erreur ! Source du renvoi introuvable.) soon became established as a more generalized practice. For tobacco there were no previous traditions; socio-economic and family contacts seem to have played a decisive role very early with this substance, which is more addictive than tea.

The earliest subject (#4) presented only a few traces of cotinine and nicotine in hair, perhaps reflecting passive exposure due to contact with Europeans as some of his artefacts demonstrate. Another man (#19) with slightly higher concentrations of these substances died at a time when a 1680 report from the European mercenaries underlined that they traded tobacco for furs with autochthones [START_REF] Safronov | Russian trade and merchandise in Northeast Asia in the seventeenthmid-nineteenth century[END_REF]. Less than a century after contact, proximity to the major tobacco import trading post of Kiakhta was a determining factor in consumption as were direct relationships of individuals with this spot. The development of economic circuits affected consumption, which largely flouted laws -as early as 1634 AD, smoking was prohibited in Russia on punishment of death (1634) and it remained so for autochthonous people until 1822 AD. The development of smoking was linked to the economic circuits in the 18 th century, while in the 19 th century tobacco consumption became generalized and economic circuits were detectable only for tea which was imported via the Pacific harbors. Between 1700 AD and 1800 AD two men of the elite (#29 and #30) with a Mongolian haircut and a southern fashion for their clothes were heavy consumers of tobacco. One of them was found with a beautiful mammoth ivory pipe. The manufacturing of highly attractive smoking accessories was instrumental in the dissemination of tobacco (SD 5). The custom then was to pass the pipe from one person to the other to smoke thus encouraging family and matrilineal transmission. These men probably travelled to a trading post on the Mongolian border in contact with China where tobacco, tea and furs were traded. Two other contemporary heavy smokers, females in this case (#12 and #26), were related to one of them and one was breastfeeding when she died. The mother of #29 and #30, who was not supposed to be living under the same roof as them when she died, was not a heavy tobacco user and drank a different tea than her daughter. Similar family habits were also observed in the 19 th century.

Conclusions

After the first contact, teas were widely consumed as beverages and medicines but also for shamanic reasons; for tobacco, socio-economic factors, fashion and family contacts seem to have played a decisive role very early on. It seems therefore that behavioral evolution governed the process of integration of tobacco and tea into the Yakut culture, and in so doing, led to the continuity of use of these substances over a long period of time. Some epidemiological characteristics of present-day Yakutia find their origins in the diffusion phenomena of the 18th and 19th century. Analyzing the respective contributions of social and economic processes in the use of these substances opens avenues of investigation for today's public health.

Compliance with Ethical Standards

Funding: This work was supported by the program of the France-Russia Associated International Laboratory (LIA COSIE number 1029), associating the North-Eastern Federal University (Yakutsk, Sakha Republic), the State Medical University of Krasnoyarsk, the Russian Foundation for Fundamental Research (Moscow, Russia), the University of Paul Sabatier Toulouse III, the University of Strasbourg I (France) and the National Centre for Scientific Research (Paris, France). Funding for excavations was provided by the French Polar Institute Paul Emile Victor, the French Archaeological Mission in Oriental Siberia (Ministry of Foreign and European Affairs, France), the North-Eastern Federal University (Yakutsk, Sakha Republic).

Ethical approval: this article does not contain any studies with human participants or animals that were performed by any of the authors.

Informed consent: not applicable