David Brasse
email: david.brasse@iphc.cnrs.fr

Hélène Burckel

Patrice Marchand

Marc Rousseau

Ali Ouadi

Marie Vanstalle

Christian Finck

Patrice Laquerriere

Frederic Boisson

Brasse David

Burckel Hélène

Marchand Patrice

Rousseau Marc

Ouadi Ali

Vanstalle Marie

Finck Christian

Laquerriere Patrice

Boisson Frédéric

]-FLT PET Tracers in the Evaluation of the Preclinical Proton Therapy Response in Hepatocellular Carcinoma

Keywords: PET, Proton therapy, Preclinical studies, Longitudinal study, Hepatocellular carcinoma

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Introduction

Hepatocellular carcinoma (HCC) is a type of tumor that requires a specific irradiation schedule to control its development. Radiotherapy has emerged as an alternative for patients with HCC when other curative modalities are not suitable [START_REF] Ohri | Radiotherapy for Hepatocellular Carcinoma: New Indications and Directions for Future Study[END_REF]. In addition, various reports have described favorable clinical outcomes of proton therapy compared to traditional approaches such as Xray therapy [START_REF] Yoo | Proton therapy for hepatocellular carcinoma: Current knowledges and future perspectives[END_REF].

Proton therapy centers are recruiting patients to highlight the efficacy of proton-beam therapy against advanced diseases such as HCC [START_REF] Klein | Hepatocellular carcinoma radiation therapy: review of evidence and future opportunities[END_REF][START_REF] Dionisi | Is there a role for proton therapy in the treatment of hepatocellular carcinoma? A systematic review[END_REF]. Although proton therapy has been demonstrated to be effective in the treatment of HCC, Eggert and Greten highlighted its limited availability as the major disadvantage of this radiation modality [START_REF] Eggert | Current Standard and Future Perspectives in Non-Surgical Therapy for Hepatocellular Carcinoma[END_REF], probably due to infrastructure costs and to the fact that indications are still evolving and being investigated [START_REF] Yeung | Proton beam therapy for hepatocellular carcinoma[END_REF].

Positron emission tomography (PET) functional imaging modality combined with computerized tomography (PET/CT) has found a well-established role before and during the radiotherapy planning process, mainly when associated with the tracer 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]-FDG), such as in head-and-neck [START_REF] Grégoire | Molecular Imaging-Guided Radiotherapy for the Treatment of Head-and-Neck Squamous Cell Carcinoma: Does it Fulfill the Promises?[END_REF], hepatobiliary [START_REF] Bajpai | Image-guided Treatment in the Hepatobiliary System: Role of Imaging in Treatment Planning and Posttreatment Evaluation[END_REF], esophageal [START_REF] Huang | FDG PET using SUVmax for preoperative T-staging of esophageal squamous cell carcinoma with and without neoadjuvant chemoradiotherapy[END_REF] and lung malignancies [START_REF] Cremonesi | Role of interim 18F-FDG-PET/CT for the early prediction of clinical outcomes of Non-Small Cell Lung Cancer (NSCLC) during radiotherapy or chemo-radiotherapy. A systematic review[END_REF]. PET imaging has also found a place in the evaluation of radiotherapy response [START_REF] Rafat | Imaging radiation response in tumor and normal tissue[END_REF][START_REF] Cliffe | Radiotherapy response evaluation using FDG PET-CT-established and emerging applications[END_REF][START_REF] Decazes | How to use PET/CT in the evaluation of response to radiotherapy[END_REF] and the long-term evaluation of the effect of proton treatment [START_REF] Ryttlefors | Long-term evaluation of the effect of hypofractionated high-energy proton treatment of benign meningiomas by means of (11)C-Lmethionine positron emission tomography[END_REF].

[ 18 F]-FDG provides a basic level of information on the tumor metabolism and can also be useful to follow the side effects of irradiation in healthy tissue due to its uptake in inflammatory tissues [START_REF] Van Elmpt | Response assessment using 18F-FDG PET early in the course of radiotherapy correlates with survival in advanced-stage non-small cell lung cancer[END_REF][START_REF] Pommier | Impact of (18)F-FDG PET on treatment strategy and 3D radiotherapy planning in non-small cell lung cancer: A prospective multicenter study[END_REF]. 3′-[ 18 F]fluoro-3′-deoxythymidine ([ 18 F]-FLT), a fluorinated analog of thymidine, was developed to investigate cellular proliferation in cancer. Its uptake in cells depends on thymidine kinase (TK-1) activity and therefore on the mitotic activity of the cells, based on the thymidine salvage pathway.

Clinical trials involving [ 18 F]-FLT PET have also demonstrated its potential as an early predictor of outcome after radiotherapy or chemotherapy [START_REF] Hoeben | 18F-FLT PET during radiotherapy or chemoradiotherapy in head and neck squamous cell carcinoma is an early predictor of outcome[END_REF] and have been suggested to monitor the proliferative activity of HCC [START_REF] Eckel | Imaging of proliferation in hepatocellular carcinoma with the in vivo marker 18F-fluorothymidine[END_REF]. It has also been proposed to assess the early response to radiation therapy due to its non-specific radiation-induced inflammation, in contrast to [ 18 F]-FDG [START_REF] Lin | Predictive Value of Early-Stage Uptake of 3'-Deoxy-3'-18F-Fluorothymidine in Cancer Cells Treated with Charged Particle Irradiation[END_REF].

To effectively appreciate and promote the clinical potential benefits of proton therapy, it is necessary to understand the radiobiological properties involved. Although several biological questions have been investigated in vitro, mainly related to cell survival and proliferation, there are still ongoing studies and a need for the development of proton irradiation platforms dedicated to preclinical investigations of both in vitro experiments and in vivo small animal models [START_REF] Ford | An image-guided precision proton radiation platform for preclinical in vivo research[END_REF][START_REF] Almeida | Exploring the feasibility of a clinical proton beam with an adaptive aperture for pre-clinical research[END_REF][START_REF] Greubel | Scanning irradiation device for mice in vivo with pulsed and continuous proton beams[END_REF][START_REF] Meyer | Biological and dosimetric characterisation of spatially fractionated proton minibeams[END_REF][START_REF] Vanstalle | Investigation of Optimal Physical Parameters for Precise Proton Irradiation of Orthotopic Tumors in Small Animals[END_REF].

In the present study, we used a preclinical proton therapy platform based on a 25 MeV cyclotron. SK-HEP-1 cells mimicking human HCC were injected into mice to monitor the efficacy of the treatment using preclinical PET imaging. These cells have widely served as a model of HCC since their establishment in 1971. Despite a recent study demonstrating that SK-HEP-1 should be considered an immortal human liver sinusoidal endothelial cell line instead of an HCC cell line [START_REF] Tai | SK-Hep1: not hepatocellular carcinoma cells but a cell model for liver sinusoidal endothelial cells[END_REF], its aggressive migration [START_REF] Eun | Hepatoma SK Hep-1 cells exhibit characteristics of oncogenic mesenchymal stem cells with highly metastatic capacity[END_REF] combined with its radioresistance made it a good candidate for the current study [START_REF] Altmeyer | Pharmacological enhancement of autophagy induced in a hepatocellular carcinoma cell line by high-LET radiation[END_REF].

The main goal of the present study was to compare the [ 18 F]-FDG and [ 18 F]-FLT PET imaging radiolabeled molecules for the longitudinal follow-up of small animal proton therapy studies in HCC to highlight their potential interests to monitor the treatment depending on the irradiation scheme.

Materials and Methods

Cell line and culture conditions

The HCC cell line SK-HEP-1 was obtained from the American Type Culture Collection (HBT-52, ATCC, USA). Cells were grown in Dulbecco's modified Eagle's medium (Pan Biotech, Dutscher, France) supplemented with 10% fetal bovine serum, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid buffer, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 100 U/mL penicillin and 100 µL/ml streptomycin. Cells were maintained in an incubator at 37 °C in a humidified atmosphere.

Animal model

Six-week-old, athymic, female NMRI-Foxn1 nu/nu mice were purchased from Janvier Laboratories (Saint Berthevin, France) and housed under pathogen-free conditions.

Animal procedures were performed in accordance with experimental protocols and guidelines for the care and use of animals and approved by the Strasbourg ethics committee for animal experimentation and the French ministry in charge of research (CREMEAS, Strasbourg, France, Apafis #261).

One week after the mice arrived, SK-HEP-1 cells (10×10 6 ) were injected subcutaneously into the left hindlimb of mice in 50 µL of phosphate-buffered saline. The animal weight and tumor diameter were recorded three times a week during the experiment and followed up to detect any weight loss or change. The small and long axes of the tumor were measured using calipers, and the mean value was recorded as the diameter. Tumor growth occurred over a two-week period, during which a typical diameter of 3-10 mm was observed. Seven days post-cell injection (PCI), 90% of the tumor had reached a suitable diameter (< 6 mm in depth) adapted to the 25 MeV proton beam. The mice were then randomized into several groups of 10 mice to undergo PET exams and immunohistochemical analysis.

Molecule synthesis

Pharmaceutical-grade [ 18 F]-FDG was purchased from IBA (France). [ 18 F]-FLT was synthesized using known radiolabeling procedures on automated radiosynthesizers with a specific activity ranging from 35 to 72 GBq/µmol [START_REF] Marchand | Automated and efficient radiosynthesis of [(18)F]FLT using a low amount of precursor[END_REF] (Raytest Synchrom Evo III or Trasis AllInOne). The synthesis was controlled by high performance liquid chromatography (HPLC) (purity >95%) and, if necessary, gamma-thin-layer chromatography (mini-Gita, Raytest Germany). The stability over 6 h was assessed by HPLC, and no degradation was observed. Radiotracers were formulated in 0.9% sterile NaCl (veterinary grade, B. BRAUN) for i.v. injections.

Irradiation setup

The 25 MeV proton beam was extracted from the cyclotron (TR24, ACSI, Canada) and shaped towards the tumor through a vacuum pipe. Before reaching the irradiation area, a homogeneous broad beam was obtained with an Al-scattering foil (200 µm thick) set behind a 5 mm Al collimator placed 240 cm upstream. The spread-out Bragg peak (SOBP) was achieved using an energy degrader consisting of 20 Al disks of increasing thickness ranging from 147 to 2761 𝜇m, leading to linear energy transfer variation and SOBP delivery [START_REF] Vanstalle | Analytical dose modeling for preclinical proton irradiation of millimetric targets[END_REF]. An additional 10 mm collimator was located after the degrader to finalize the beam shaping (Fig. 1). The dose delivered to the tumor was set up both by the beam intensity and the irradiation duration, controlled with a beam kicker located between the source and the entrance of the cyclotron cavity, allowing fast beam shutoff. More information on the preclinical proton beamline and the associated dosimetry can be found in [START_REF] Constanzo | Dosimetry and characterization of a 25-MeV proton beam line for preclinical radiobiology research[END_REF]. During the whole irradiation procedure, each mouse was anaesthetized using IP injection of ketamine (65 mg/kg)/xylazine (10 mg/kg). The mouse body temperature was maintained at 36 ± 1 °C using a vivarium heating lamp.

Description of the experimental protocol

For the evaluation of both molecules, the experimental protocol consisted of eight groups of ten mice. Each group represented a particular dose/radiotracer condition. The proton singlefraction doses were 5, 10 and 20 Gy, with a dose rate of 10 Gy/min, plus a control group with no dose delivered for each molecule. The delivered doses were chosen to range from low to high dose and according to previous preclinical results obtained with external beam irradiation [START_REF] Lin | Predictive Value of Early-Stage Uptake of 3'-Deoxy-3'-18F-Fluorothymidine in Cancer Cells Treated with Charged Particle Irradiation[END_REF][START_REF] Wilson | Glucose metabolism gene expression patterns and tumor uptake of 18 F-fluorodeoxyglucose after radiation treatment[END_REF]. For each irradiated mouse, the dose was uniformly distributed in a cylindrical volume of 10 mm diameter and 5 mm depth, centered in the tumor. A reference time point was defined as the day before the irradiation procedure, seven days PCI, at which a reference PET exam was acquired for each mouse. Additional PET exams were then performed on each mouse every three days up to 16 days after irradiation.

Small animal PET imaging protocol

Animals were scanned with a dedicated preclinical PET (IRIS PET, Inviscan). The injected activities for both radiolabeled molecules were 10 MBq via the tail vein. Static image acquisitions were performed 45 minutes ([ 18 F]-FDG groups) and 90 minutes ([ 18 F]-FLT groups) after injection. The time between the radiotracer injection and the different PET exams was investigated and was chosen to optimize the tumor-to-background uptake ratio and according to the literature. To optimize [ 18 F]-FDG tumor uptake, the mice were fasted overnight and kept warm and anesthetized during the 45-minute biodistribution [START_REF] Fueger | Impact of animal handling on the results of 18F-FDG PET studies in mice[END_REF]. During the entire exam, each mouse was maintained under isoflurane anesthesia (2%, MINERVE company).

Two photons detected within a 5 ns coincidence timing window and with an energy ranging from 250 to 750 keV were defined as a coincidence. For each PET exam, the coincidences were acquired for 10 minutes. Data were reconstructed into a 201x201x120 volume using the iterative 3D ordered-subset expectation-maximization algorithm. The resulting voxel size was equal to 0.42 mm in the transverse plane while the slice thickness was equal to 0.855 mm. PET data were fully corrected for normalization, random coincidences, radioactive decay and dead time during the reconstruction process. No attenuation and scatter corrections were applied.

PET Data analysis

PET data analyses were performed using the AMIDE software package [START_REF] Loening | AMIDE: a free software tool for multimodality medical image analysis[END_REF]. A focal nonphysiologic increase in radiotracer uptake in the area of tumor development was considered positive PET. An ellipsoid was manually drawn on the tumor, large enough to account for the entire radiotracer uptake of the tumor. The standardized uptake value (SUV) was calculated for all voxels and the resulting highest value was defined as the SUVmax. Inside the volume, a region of interest (ROI) was defined by all the voxels with a value greater than 40% of the SUVmax value. The SUV mean value (SUVmean) inside the ROI was calculated, as was the functional volume, defined as the sum of all the voxels in the ROI. Activity was also calculated as the product between the mean voxel value calculated inside the ROI (Bq/mL) and the functional volume (mL). The functional volume and the functional activity are equivalent to the metabolic tumor volume and the total lesion glycolysis specifically defined for [ 18 F]-FDG.

To investigate the behavior of the different calculated figures of merit after irradiation, all the calculated values were divided by the values obtained at the reference time point, seven days PCI.

Immunohistochemical procedure

Three groups of 40 mice each were involved in the immunohistochemical procedure. Each group represented a time point and consisted in 10 mice per irradiation condition (control, 5

Gy, 10 Gy and 20 Gy). Mice were sacrificed at accurate times before and after irradiation (-1, +3 and +16 days). Tumor samples were removed and fixed in buffered formalin for at least 24 h, dehydrated and paraffin embedded. Standard 5 µm sections were sliced, and histological staining was carried out with hematoxylin-eosin to analyze the mode of organization of the cells, the size and shape of the tumor and the degree of tissue damage. The expression of the Ki-67 antigen was detected by immunohistochemistry for use as a marker of tumor proliferation. This antigen was detected using the mouse monoclonal antibody against Ki-67 (clone MIB1, DAKO M7240, 1/50 e dilution). The quantitative evaluation was performed by counting with ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] after observing tissue sections at high magnification (x40) and determining the number of positive tumor cells relative to the total number of tumor cells present on the same field of microscopic observation. The results were evaluated by counting the number of nuclei positive for immunostaining out of a total of 1000 tumor cells (10 to 20 observation fields).

Statistical analysis

We compared each variable between the groups using the Mann-Whitney rank test function.

Statistical calculation was performed using the Python programming platform associated with the Pandas and SciPy libraries [START_REF] Mckinney | Data Structures for Statistical Computing in Python[END_REF][START_REF] Virtanen | SciPy 1.0: fundamental algorithms for scientific computing in Python[END_REF]. The level of significance was p < 0.05. Descriptive statistics and box plots were mainly used for data analysis.

Results

Visual analysis of reconstructed images demonstrated a better tumor-to-surrounding background uptake ratio for [ 18 F]-FLT compared to [ 18 F]-FDG leading to a better delineation for VOI placement (Fig. 2). The SUVmean, the functional volume and the resulting activity were calculated and normalized to evaluate the [ 18 F]-FDG and the [ 18 F]-FLT uptakes under control and irradiation conditions.

In vivo characterization of [ 18 F]-FDG uptake

The results obtained for the control condition are shown in in Figures 3a-c. Between 7 and 11 days PCI, a first phase could be defined as a tumor expansion period, when a decrease in the normalized SUVmean (p=3e-4) and an increase in the normalized functional volume (p=3e-4) were observed, resulting in stable normalized activity values (p=3e-1). In the second phase, a decrease in the normalized functional volume up to 17 days PCI (p=4.8e-2) was noted. The increase in the normalized SUVmean in the same period was also statistically significant (p=1e-2). For the last phase, starting between 14 and 17 days PCI, a decrease in the normalized SUVmean (p=2e-2) and an increase in the normalized functional volume were highlighted, indicating tumor recovery (p=1e-3).

The results for [ 18 F]-FDG PET imaging after proton irradiation are given in Figure 3d-f, where only the normalized activity is presented. While a decrease in the normalized activity after 10 (p=2e-4) and 20 Gy irradiation (p=1e-2) over the examination period was observed, the normalized activity decreased at the beginning of the experimental protocol (p=3e-2 between 3 and 6 days after irradiation) and remained stable after 9 days postirradiation in the 5 Gy group.

The difference between 5 Gy and the other irradiation doses was amplified after 9 days. No significant differences were observed between the control group and the tumors irradiated with 5 Gy.

In vivo characterization of [ 18 F]-FLT uptake

The results obtained for the control condition are shown in Figure 4a-c. Between the 7 th and the 14 th day PCI, a growth phase was characterized by an increase in both the normalized SUVmean (p=4e-2) and the normalized functional volume (p=4e-3), resulting in an increase in the normalized activity (2.63 ± 1.5, p=2e-4). Despite a visual increase of the SUVmean between the 11th and the 14th day PCI, this obtained difference was not statistically significant (p=6e-2). The behavior of the 3 figures of merit was stable after day 14. F]-FLT tumor uptake after 5, 10 and 20 Gy proton irradiation. Three days after irradiation, an increase in normalized [ 18 F]-FLT activity in the 5 Gy group was observed (p=1e-4), while no change was seen in the 10 Gy group (p=4e-1), and a decrease was seen in the 20 Gy group (p=2e-2).

Figures 4d-f present the normalized activities of [

After these 3 days, the normalized [ 18 F]-FLT activities remained constant, with no significant difference in any proton-dose group except a significant decrease after 20 Gy the last day of the experiment (p=4.7e-2 between 6 and 16 days after irradiation).

A difference between the normalized [ 18 F]-FLT activity in the 5 and 20 Gy groups three days after irradiation was observed (p=2e-3). A significant difference was observed between the mice irradiated with 10 or 20 Gy and the control mice from six days after irradiation (p=2e-3) until the end of the experimental protocol (p=3e-3).

Conversely, no significant difference was observed between the data obtained in the 5 Gy group and the control group except at 9 days after irradiation, where the calculated median activity in the 5 Gy group (1.9) was significantly lower than the median in the control mice (2.5) (p=4.6e-2).

Comparison of both molecules at an early stage after irradiation

The interpretation of [ 18 F]-FDG uptake in the early days following irradiation was difficult because [ 18 F]-FDG responds to inflammatory changes. Three days after irradiation, the normalized SUVmax calculated after [ 18 F]-FDG injection increased by a factor proportional to the irradiation dose (Figure 5a). The p-values were 4.8e-2 for the control group compared to the 5 Gy group, 3.6e-2 for 5 Gy compared to 10 Gy and 2.8e-2 for 10 Gy compared to 20 Gy.

We observed a decrease of the normalized functional volume when the irradiation dose increased leading to a stable normalized activity (data not shown).

The observed behavior of [ 18 F]-FDG uptake at the early stage was counterbalanced by the decrease in [ 18 F]-FLT uptake as the proton dose increased (Figure 5b). Going from 5 to 10 Gy, the median of the normalized SUVmax fell by 21% (p=1.1e-2), and the drop was 36% when 5

Gy was compared to 20 Gy (p=1.2e-3). We also observed a decrease of the normalized functional volume leading to a decrease of the normalized activity when the irradiation dose increase (data not shown).

Evaluation of the Ki-67 index

Variations in the expression of the Ki-67 index as a proliferation marker are presented in Figure 6. Nonirradiated SK-HEP-1 tumors showed an important Ki-67 proliferation index between 65 and 75%, regardless of the day of collection. Three days after irradiation, irradiated tumors presented a significant decrease in their Ki-67 indexes of approximately 40%, irrespective of the irradiation dose (p=1e-4). Then, between 3 and 16 days postirradiation, the Ki-67 index of the tumors irradiated with 5 Gy remained stable, while a decrease in Ki-67 was observed in the 10 and 20 Gy groups. Sixteen days postirradiation, Ki-67 varied from 42% after 5 Gy to 26% after 10 Gy to 10% after 20 Gy irradiation. At the end of the experimental protocol, a significant dose-dependent decrease was observed in the Ki-67 of tumor cells between the 5 and 20 Gy groups (p=1e-4).

Discussion

In this work, we used static PET imaging to evaluate the behavior of metabolism and cell proliferation in tumors after preclinical proton therapy procedures. The main objective was to compare the [ 18 F]-FDG and [ 18 F]-FLT radiolabeled molecules to monitor the treatment in an HCC context.

The results obtained for each dose/radiotracer condition showed important variations per analyzed day. This variation is due not only to statistical fluctuations (N=10 per condition) but also to factors related to the imaging technique itself, the quantification approach and other animal-specific parameters. In our study, we tried to reduce these factors by optimizing the experimental conditions [START_REF] Kuntner | Quantitative preclinical PET imaging: opportunities and challenges[END_REF] and by normalizing each investigated figure of merit to a reference value measured before the irradiation. To overcome some limitations of PET SUV measurements, we also measured the functional volume to derive an activity value representative of the overall metabolism and the cell proliferation behavior at the tumor level.

The single-fraction procedure used in our study is not fully representative of the irradiation protocol used for human therapy, where radiation is usually administered to patients in multiple fractions. However, clinical studies on liver demonstrated high rates of local tumor control using high-dose hypofractionated stereotactic body radiation therapy [START_REF] Schaub | Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma: Current Trends and Controversies[END_REF]. Therefore, our protocol mimics the effect of a high dose delivered on a mouse model of HCC. Additional experiments have to be addressed before extending our conclusions to patients.

[ 18 F]-FDG uptake showed a linear dose-dependent increase in the first days after treatment (37%, p<0.05), while [ 18 F]-FLT uptake decreased in a dose-dependent manner (e.g 21% for 5

Gy compared to 10 Gy, p=1.1e-2). We made the assumption that the increase of [ 18 F]-FDG uptake in the first days following the irradiation was due to potential inflammatory changes.

However, previous study showed that in vitro radiation of tumor cells using X-rays could result in an increase of [ 18 F]-FDG uptake without presenting inflammatory cells [START_REF] Higashi | In vitro assessment of 2-fluoro-2-deoxy-Dglucose, L-methionine and thymidine as agents to monitor the early response of a human adenocarcinoma cell line to radiotherapy[END_REF]. Further investigation on proton associated with SK-HEP-1 cells should be conducted to clarify this specific point.

At the later time point, [ 18 F]-FDG normalized activity showed an 85% decrease (p<0.01) for both 10 and 20 Gy doses and no variation for 5 Gy. Conversely, a significant 61% (p=0.002) increase was observed for [ 18 F]-FLT normalized activity at 5 Gy and no variation for higher doses. The current results obtained with [ 18 F]-FLT are consistent with those already published by Lin et al, demonstrating the value of using [ 18 F]-FLT to monitor the early response of cell proliferation to proton therapy in a colon carcinoma cell line [START_REF] Lin | Predictive Value of Early-Stage Uptake of 3'-Deoxy-3'-18F-Fluorothymidine in Cancer Cells Treated with Charged Particle Irradiation[END_REF]. In the current study, we observed the same behavior of [ 18 F]-FLT activity and proliferation activity (Ki-67) two weeks after irradiation. Previous work also demonstrated a significant correlation of [ 18 F]-FLT with the proliferating fraction of HCC as assessed by Ki-67 immunohistochemistry [START_REF] Eckel | Imaging of proliferation in hepatocellular carcinoma with the in vivo marker 18F-fluorothymidine[END_REF][START_REF] Chalkidou | Correlation between Ki-67 immunohistochemistry and 18F-fluorothymidine uptake in patients with cancer: A systematic review and meta-analysis[END_REF].

Considering that tumor cell proliferation is a mechanism of therapy resistance, the use of [ 18 F]-FLT can provide an effective decision tool for treatment management.

The literature on the use of [ 18 F]-FLT in the clinical routine reveals high background signals in the liver, which can be a potential drawback of using this molecule in HCC [START_REF] Tehrani | PET imaging of proliferation with pyrimidines[END_REF]. Despite this physiologic retention in the liver, Eckel et al [START_REF] Eckel | Imaging of proliferation in hepatocellular carcinoma with the in vivo marker 18F-fluorothymidine[END_REF] reported a median 20% higher focal uptake by malignant tumors than the surrounding liver, leading to a 69% sensitivity for the detection of HCC. To optimize the tumor-to-liver contrast, Sharma et al proposed in a pilot study to classified each voxel according to their time activity curve [START_REF] Sharma | Monitoring Response to Transarterial Chemoembolization in Hepatocellular Carcinoma Using 18F-Fluorothymidine PET[END_REF]. However, they showed that the high and variable SUV limited the improvement of tumor visualization. During the last decade, several approaches have been proposed to improve the sensitivity of PET/CT in HCC diagnosis [START_REF] Ronot | Functional imaging in liver tumours[END_REF][START_REF] Lu | Positron-emission tomography for hepatocellular carcinoma: Current status and future prospects[END_REF]. However, according to the European Association for the Study of the liver, there is no single radiolabeled molecule recommended for either diagnosis or response to therapy procedures in HCC [START_REF] Liver | Electronic address: easloffice@easloffice.eu[END_REF]. The development of new probes and new procedures should not only improve the diagnosis but also the therapy response assessment.

Conclusions

We showed that the use of the [ 18 F]-FDG and [ 18 F]-FLT radiolabeled molecules can provide useful and complementary information for longitudinal follow-up of small animal proton therapy studies in the context of HCC. Our conclusions were drawn from the results obtained from HCC cell line SK-HEP-1. [ 18 F]-FDG, a reference molecule for PET imaging, enables a treatment monitoring several days / weeks postirradiation. On the other hand, [ 18 F]-FLT could represent a good candidate to monitor the treatment few days postirradiation, in the context of hypo-fractioned and close irradiations planning. This opens new perspectives in terms of treatment efficacy verification depending on the irradiation scheme.

Fig. 1

 1 Fig. 1 Picture of the horizontal beam line, including the rotating wheel used to degrade the

Fig. 2

 2 Fig. 2 Coronal sections of mice scan after (a) [18F]-FDG injection and (b) [18F]-FLT injection

Fig. 3

 3 Fig. 3 Results of [ 18 F]-FDG uptake. Mice (N=10) were imaged 7 days PCI, treated on day 8,

Fig. 4

 4 Fig.[START_REF] Dionisi | Is there a role for proton therapy in the treatment of hepatocellular carcinoma? A systematic review[END_REF] Results of [ 18 F]-FLT uptake. Mice (N=10) were imaged 7 days PCI, treated on day 8,

Fig. 5

 5 Fig. 5 (a) Normalized SUV max of [ 18 F]-FDG uptake and (b) [ 18 F]-FLT uptake in the different

Fig. 6

 6 Fig. 6 Immunohistochemical evaluation of the Ki-67 cell proliferation index in SK-HEP-1

Figure 1

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Figure 2

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Figure

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3

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Figure 4 25 Figure 5

 255 Figure 4

Figure 6

 6 Figure 6

  18

Acknowledgments

This work was partly supported by ITMO Cancer AVIESAN (Alliance Nationale pour les Sciences de la Vie et de la Santé, National Alliance for Life Sciences and Health) within the framework of the Cancer Plan and by IN2P3. The authors wish to thank Bruno Jessel, Lionel Thomas, Jacky Schuler, Michel Pellicioli, Julie Constanzo and Cédric Mathieu for their help in performing all the experiments. We also thank all the members of the mechanical workshop involved in the development of the beam line.

Author contributions

All authors discussed and conceived the generic concept and reviewed the manuscript. D.B.

Conflict of Interest

The authors declare that they have no conflict of interest.

Clinical Practice Guidelines: Management of hepatocellular carcinoma. J Hepatol 69:182-236.


L Lengaigne

W Gauthier-Manuel

S Daniau

J M Triscone

Hauden

E Courjon

N Bodin

G Lengaigne

L Gauthier Manuel

W Daniau

S Ballandras

P Paruch

J Hauden

Fabrication of periodically polled domains transducers on liNbO3

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

INTRODUCTION

The possibility to manufacture elastic wave-guide based on a thin ferroelectrics film deposited on a single crystal substrate and periodically poled to enable the excitation of surface acoustic wave has been demonstrated recently [1,[START_REF] Sarin Kumar | A High Frequency Surface Acoustic Wave Device Based on Thin Film Piezoelectric Inter-digital Transducers[END_REF].

The so-called piezoelectric inter-digital transducer (PIT) simply consists in two electrically conductive media embracing a poled ferroelectrics layer. It has been successfully implemented and tested for the excitation of elliptically polarized waves in the frequency range 1-3 GHz [START_REF] Sarin Kumar | A High Frequency Surface Acoustic Wave Device Based on Thin Film Piezoelectric Inter-digital Transducers[END_REF].

In this work, the possibility to develop PITs on LiNb03 is investigated, since this material is classically identified as well-adapted for very high frequency applications. The first experiments have been performed on 500µm thick 3" wafers of optical quality Z-cut answering severe specification on total thickness variation and side parallelism. The fabrication of PITs on such wafers allows for the excitation of symmetrical Lamb modes with an operating frequency twice higher than those obtained using standard inter-digital transducers. A photo-resist pattern is defined on one side of the wafer, enabling the implementation of a poling process using a liquid electrode. A poling sequence [START_REF] Myers | Quasi-phase-matched optical parametric oscillatorJOSA B[END_REF] III.

EXPERIMENT AL RESULTS

In this section, we present the electrical measurements obtained using a Suss Microtec RF probing bench using the We have no explanation to propose for that result, since all the transducers were processed at the same time. Vi. g

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(a) is not rigorously equal to 50% (for instance 40% Z+ I 60%

Q "••• ••••••• -• •••••---
Z-within the period), the longitudinal bulk wave and its odd harmonics (see the contribution at 21 MHz on fig. 5a) can be weakly excited. This was simulated for the lOOµm period device and is reported in fig. 6.

  Figure 1. Typical geometry of piezoelectric interdigited transducer (PIT) based on fe rroelectric poling As mentioned in introduction, the poling process can be rather easily applied to PZT for which the coercitive field is small enough to allow for an efficient control of the domain polarity. In the case of lithium niobate, this situation is quite different because of the large value of its coercitive field (22 MV.m-1 compared to 2.5 MV.m-1 max. for PZT). As a consequence, the development of a dedicated poling bench was required to control the poling of thick (500µm) Z-cut LiNb03 plates. This is detailed in ref. [3]. Consequently, only a short recall of the bench principle is reported below. The poling bench mainly consists in a high voltage amplifier used to submit the LiNb03 wafer to an electric field strong enough to invert the native poling of the plate. To achieve such an operation, one needs the use of optical grade Z-cut LiNb03 plates. Wafers are cut in the same boule to well control the poling condition. A photoresist mask is achieved atop one wafer surface, which will control the poling location. An lithium chloride electrolyte is used to drive the poling high voltage atop the wafer surfaces. A dynamic poling sequence is then imposed to the wafer, progressively reaching the coercitive field of lithium niobate. An evidence of succesfull poling is obtained by measuring the current of the whole electrical system. Once evidence of transient current measured, the device is considered to be poled. Following this sequence, and providing no short circuit occurs, an almost perfect poling can be achieved. Figure 2 shows a principle scheme of the poling bench.

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Scheme of the ferroelectric plate poling bench

  so-called Z-probes connected to a Rhode & Schwarz ZVRC network analyzer. The electrical admittances of the test devices have been measured and are compared to the harmonic admittance of the corresponding configuration. These results are reported in fig. 5(a,b,c) for the three considered configurations. In fig.Sc, we have reported the response of an un-poled device, as only the bulk wave signature and its odd harmonics appears on the experiments.

4

 4

Figure 5 .

 5 Figure 5. Comparison between theory and experiment for the three test vehicles (a) periodp=IOOµm (b) p=200µm (c) p=400µm (poling failed) We have related the contribution on the experimental curves near 7 MHz of fig 5a&b to parasitic bulk waves due to the actual volume fraction of positively and negatively poled domains of the period. In the case the volume fraction

Figure 6 .

 6 Figure 6. Infl uence of a volume fraction defect on the transducer response ( 40/60% instead of 50% as expected) Thanks to the very good agreement between theory and experiments on fig 5a&b, one can easily identify the shape of the mode of the different Lamb-wave contributions, allowing to select one of those for its particular properties and polarization. Fig.7 (a,b,c) shows some examples of modes excited in the plate for a 200µm period transducer. It can be seen on these graphs that elliptically polarized modes as well as almost purely longitudinal waves can be excited in the plate thanks to our PIT.

Figure 7 .

 7 Figure 7. Shape of given modes of the IOOµm period transducer (fi g. Sb) (a) fundamental at 19 MHz (b) mode#6 at 28 MHz (c) mode#8 at 34 MHzThe last experimental work that has been achieved for this paper is to test the robustness of the polarization to lapping and polishing techniques. These operations are required in order to fabricate LiNb03/Silicon wave-guides, as expected as a future issue of our work. We consequently have lapped down the plate to a thickness of 250µm and then achieved new admittance measurements. Again, the very nice theory/experiment agreement is met as shown in fig.8, proving that the poling is strong enough to persist even after such treatments.

Figure 8 .

 8 Figure 8. Theory/experiment assessement after lapping the plate down to 250µm, 200 µm period PIT

ACKNOWLEDGMENT This work has been achieved in the Dominos program framework, funded by the European Community as the InterReg project DOMINOS.


Céline Hubert

Cyril Chomette

Anthony Désert

Alexandra Madeira

Adeline Perro

Ileana Florea

Dris Ihiawakrim

Ovidiu Ersen

Anna Lombardi

Etienne Pertreux

Fabien Vialla

Paolo Maioli

Aurélien Crut

Natalia Del Fatti

Fabrice Vallée

Jérôme Majimel

Serge Ravaine
email: serge.ravaine@crpp.cnrs.fr

Etienne Duguet
email: etienne.duguet@icmcb.cnrs.fr

Mona Tréguer- Delapierre

Versatile template-directed synthesis of gold

Introduction

Due to their fascinating tunable localized surface plasmon resonance (LSPR) properties, [1][2][3] and their growing potential for a wide range of applications including light harvesting 4 ultrasensitive detection 5 and metamaterials, 6 the fabrication of plasmonic nanostructures with a well-controlled shape and morphology has attracted considerable research effort in recent decades. [7][8][9] Among all the investigated approaches, a huge number are based on the use of a template, 10 with the global aim of obtaining nanostructured architectures unattainable through other means. Organized films of colloidal particles deposited onto a substrate have been extensively used as surface mask templates to create a large panel of plasmonic nanostructures, [11][12][13] while porous membranes are routinely used to guide the synthesis of mostly one-dimensional plasmonic nanomaterials. 14,15 A third category of templated syntheses refer to processes occurring in solution. 16 The template can serve either as a platform upon which the material is sculpted through its partial dissolution 17 or as a scaffold to which the growing material conforms. 18 These subtractive and additive processes can also be combined. One of the most straightforward examples of the use of templates in solution is the synthesis of hollow metal nanostructures. These are of special interest because they have higher surface-to-volume ratios compared to their solid counterparts, making them good candidates for catalysis [19][20][21] and trace detection. [22][23][24] Moreover, their LSPR absorption band lies in the near-infrared domain when their thickness is sufficiently small, [25][26][27] which makes them useful for in vivo imaging [28][29][30][31][32][33] or photothermal therapy. 34,35 To date, the large majority of metallic nanocage synthesis routes are based on a galvanic replacement reaction wherein the template is oxidized and dissolved, while metal ions are reduced and plated onto the surface of the template. Following the pioneering work of Xia and coworkers who synthesized hollow gold nanostructures from silver nanospheres, 36 a lot of metal nanostructures with hollow interiors such as nanoframes, nanocages and nanoboxes with various morphologies were synthesized from templates. [37][38][39][40][41][42][43][44][45][46][47] Since a complete removal of the less stable metal is hardly obtained, most of the as-prepared hollow nanostructures consist of alloy shells. To complete the dealloying process, the addition of an etchant, e.g. Fe(NO3)3 48 or O2, 49 has been proposed by several groups to yield hollow nanostructures with better controlled wall thicknesses and porosities. Nanostructures with greatly increased complexity such as nanorattles 50,51 or multi-walled nanoshells [52][53][54] were also obtained by coupling galvanic replacement with other physical/chemical processes such as co-reduction, sequentially deposited templates, or the Kirkendall effect. These combined routes offer high precision but limited control over the composition and the scale-up.

To tackle these restrictions, we envisioned a new paradigm based on sacrificial templates made of two phases with different surface energies: only one phase shows affinity for the coating material in order to achieve a discontinuous shell and therefore simultaneously carve the walls and the windows of the cage. Therefore, the cage structure may be fully programmed through the control of the biphasic template. Recently, we implemented and validated this original pathway to fabricate titania nanocages, but titania growth was not as regioselective as desired and complete template etching was not achieved. 55 The group of Stein developed a similar concept for an ensemble of particles (3D close-packed colloidal crystals) but not for individual ones. 56 Thus, the creation of cages from our approach offers numerous advantages due to the very low polydispersity of the templates, their high symmetry and geometries, their specific customization and their production at the gram scale or more. Also, our process allows to fill the nanocages with a guest particle of same or different nature.

Here, with complete success, gold nanocages are grown using highly symmetrical silica/polystyrene (PS) sacrificial templates. These templates are multipod clusters made of a core silica sphere surrounded by a controlled number of PS latex spherical satellites. Inspired by the pioneering works of Halas and coworkers [57][58][59][60] alongside more recent publications, [61][62][63][64] we took advantage of the regioselective attachment of gold nanoseeds onto the accessible surface of the silica cores of binary clusters and their subsequent growth. Because of the lack of affinity between gold and the surface of the PS satellites, gold was selectively reduced onto the seeds, creating the cage structure in the interstitial gaps between PS satellites. Electron microscopy reveals the well-defined shape of the gold nanocages upon dissolution of the binary templates, demonstrating that the number and positions of the windows directly correlate to the number and positions of the PS nodules. The optical properties of the as-prepared hollow nanostructures were extracted at the single particle level by spatial modulation spectroscopy. Experimental data are compared to the results of numerical simulations. Furthermore, we demonstrate that this approach can be used to fill the nanocages with a guest magnetic nanoparticles, such as iron oxide.

Results and discussion

Fig. 1 schematically illustrates the multi-step process employed in the synthesis of the gold nanocages with twelve regularly-spaced and calibrated windows. We start from silica/PS dodecapods which may be described as icosahedral clusters of PS spheres attached to a central silica sphere of similar diameter. We then overgrow the core and functionalize the silica in the void space between the PS satellites via surface amination using N-[3-(trimethoxysilyl)-propyl]ethylenediamine (EDPS). The anchored amine moieties enable a regioselective coating by gold. The regioselective gold coating is obtained in two steps: the adsorption of tiny gold nanoparticles 65 on the freshly aminated silica surface 62 and their subsequent regrowth using a reducing agent (formaldehyde) and a gold plating solution (tetrachloroauric acid/potassium carbonate). 63 Polyvinylpyrrolidone is added to the growth medium to prevent colloidal aggregation. Finally, by selective dissolution/etching of the silica/PS template, we release the nanocage. It presents the high symmetry of a hollow dodecahedron whose thirty edges are made of gold and slightly curved, and whose twelve faces are drilled with circular windows. We have previously reported the fabrication of the silica/PS dodecapod-like precursors at the gram scale by a seedgrowth emulsion polymerization of styrene in the presence of 85 nm silica spheres. 63 They are readily obtained with a morphological purity of ∼75%. The nature and proportion of the side-products, i.e. essentially clusters with a lower number of PS satellites or made of several silica cores, are described in Table S1 (ESI †).

Due to its excellent spatial resolution, transmission electron microscopy (TEM) is well adapted for verifying the success of the different synthesis stages and analyzing the final morphology of the nanocages (Fig. 2). TEM images confirm the window morphology, their symmetrical arrangement and the hollow interior of the nanoparticles. The inner diameter, which corresponds to the value of the silica diameter after regrowth, is 125 nm. The windows are large and regularly distributed across the surface. Electron tomography reconstructs three-dimensional images of the nanoparticles, precisely measuring gold edges 20 nm in thickness and windows of 48 nm in diameter (Fig. 2 and Fig. S1, ESI †). The gold shell is rather rough since the Au deposits consist of agglomerates of gold nanoparticles. The morphological yield of the nanocages is similar to that of the silica/PS dodecapod-like templates (∼75%), which shows that our synthesis pathway is very efficient at preserving the shape of the sacrificial template. The nanocages can have tunable dimensions, including the gold wall thickness and pore diameters. They can be adjusted by varying the total amount of gold or silica deposited during the multi-step process.

The simplicity and versatility of our approach was further evidenced by extending the strategy to the fabrication of nano-cages with 2, 3, 4 and 6 windows from bipod, tripod, tetrapod and hexapod silica/PS templates, respectively (Table S1 and Fig. S2-S5, ESI †). Gold maps obtained by scanning TEM coupled with energy-dispersive X-ray spectroscopy (STEM-EDX) of Fig. 3 show the morphologies of the as-obtained nanocages. They evidence that the high symmetry of the template is systematically transferred to the nanocages, especially the relative position of the windows, i.e. in line, triangular, tetrahedral and octahedral spatial windows. Similarly to the dodecapods, these nanocages can be produced on a large scale, as the templates are readily fabricated in gram quantities and with morphology purity from 66% to 91% (Table S1, ESI †). Detailed investigations of the optical responses of two types of synthesized nanocages, presenting four and twelve windows, performed in different environments (ensembles of nanoparticles in solution, or individual nanoparticles deposited on transparent and absorbing substrates) are presented in Fig. 4 and5.

Fig. 5 Optical properties of nanocages with twelve windows. (a) Absorbance spectrum of the synthesized nanocage dispersion. (b) Extinction spectra of two individual nanocages deposited on a thin carbon membrane, measured using SMS. (c) Dependence of their extinction cross-sections on the polarization direction of the incident light wave, measured by SMS at λ ≈500 nm wavelength (squares) and sinusoidal fits (lines). The TEM image of one of the investigated nanocages (red lines in panels (b and c)) is shown on top (scale bar:

100 nm).

Average information on the optical properties of the produced gold nanocages can be obtained by measuring the absorbance of the synthesized solutions (proportional to the extinction of the nanocages according to Beer-Lambert law). The absorbance spectrum is obtained from nanocage dispersions having four windows, shown in Fig. 4a. It does not display any clear resonance in the 700-1050 nm wavelength range, and weakly increases with wavelength. However, this optical response does not necessarily reflect that of the individual nanocages present in the investigated solution, as absorbance measurements are affected by the nanoparticle polydispersity (which results in the broadening -or even complete obscuration -of optical resonances). These drawbacks can all be avoided by using spatial modulation spectroscopy (SMS), a single-particle technique based on the modulation of a nanoparticle position in a strongly focused laser beam which allows the quantitative determination of its extinction cross-section as a function of light wavelength and polarization. 2,66,67 P a g e | 6

The extinction spectra measured for three individual gold nanocages with four windows deposited on a thin silica membrane are shown in Fig. 4b. These spectra differ considerably from the absorbance spectrum of the nanocage dispersion (Fig. 4a), highlighting that using a single-particle approach is indeed crucial for an accurate optical characterization of this sample (more generally, single particle approaches are especially useful in the case of colloidal solutions composed of nano-objects presenting a large dispersion in composition and morphology and/or when strong interactions between the nano-objects present in the solution modify their optical properties). Singleparticle spectra are characterized by marked LSPRs centered at about 900 nm wavelength. The transmission electron microscopy images of the optically investigated nano-objects (one of them being shown in Fig. 4) confirm their four windows. The variation of the resonance position for the three investigated nanocages (from 890 to 950 nm) shows that the large width of the ensemble spectrum results at least partly from the morphological dispersion of the synthesized nano-objects. One could also a priori ascribe this LSPR variation to a different nanocage orientation on the underlying substrate, but the theoretical simulations presented below allow this hypothesis to be ruled out.

SMS also probes the effect of light polarization direction on nanoparticle optical extinction. Fig. 4c presents such measurements on the four nanocage windows. The extinction spectra show sinusoidal oscillations (with 180° period) with the polarization angle, as already observed in previous studies on elongated nano-objects such as nanorods. 2,68 The amplitude of these polarization-induced variations is found here to be strongly particledependent (the minimal extinction cross-section representing 20, 50 and 80% of the maximal measurement for the three investigated nanoparticles), suggesting a morphological dispersion of the three synthesized nanocages.

Strongly contrasting results were obtained for nanocages with twelve windows (Fig. 5). The absorbance of the correspond ding solution shows a single and broad resonance near 600 nm (Fig. 5a). The measured spectra for two individual nanocages (deposited on a thin carbon membrane) present different amplitudes, but their shapes remain similar to that of the solution absorbance spectrum (Fig. 5b), with also a resonance at 600 nm wavelength. Although, the ensemble and the single-particle spectra should be compared with care because the nanoparticle environment is not the same in the two cases (because of the necessity of depositing nanoparticles on a substrate for studying them individually), this indicates that the effect of morphological dispersion (and aggregation) are much weaker for this sample than for the four windows nanocages one. This is further confirmed by the measurement of a weak extinction polarization dependence (<10% variation) for both investigated twelve windows nanocages, which corresponds to the behavior theoretically expected for ideal nanocages (see below).

Optical experiments were complemented with numerical simulations of nanocage optical properties (Fig. 6). These simulations, based on finite-element modeling (FEM), were performed using the mean sizes deduced from a statistical analysis of the TEM images of the synthesized nanocages (Table S2, ESI †), assuming ideal nanocage morphology, and in particular perfectly smooth surfaces and regularly spaced windows. The synthesis intermediates (silica core and PS nodules) were respectively included and omitted when simulating nanocages with twelve and four windows, consistent with the TEM images of the nano-objects optically investigated (Fig. 4 and5).

The inhomogeneous environment of the particles in the SMS experiments resulting from their deposition on a substrate was also taken into account. Direct contact between the nanocages and the substrate was assumed, complementary simulations showing that the obtained results are weakly sensitive to the inclusion of a 1-2 nm separation between them (which could for instance result from an interfacial surfactant layer). Simulations were performed for multiple orientations of the nanocages, showing that this parameter has only a minor influence on their optical properties (Fig. 6).

Fig. 6 Simulated optical properties of nanocages with four (a and b) and twelve (c and d) windows: extinction spectra (a and c) and polarization-dependence (b and d) of extinction at resonance wavelengths (indicated by arrows in the respective panels (a and c)). Simulations were performed in the configuration of SMS experiments (nanocages deposited on a thin membrane), for different nanocage orientations generated by successive 30 degree rotations of the nanocage about an axis orthogonal to light propagation direction (the top views of the considered orientations are shown on top, each one being associated to a specific color). Simulations on nanocages with

four windows were performed with 105 nm core diameter, 160 nm PS satellite diameter, 25 nm gold thickness and 40 nm thick silica substrate. Simulations on nanocages with twelve windows were performed with 125 nm core diameter, 85 nm PS satellite diameter, 20 nm gold thickness and 20 nm thick carbon substrate, including the silica core and PS nodules consistent with the TEM image shown in Fig. 5. An environmental refractive index of 1.1 and 1.3 was used for nanocages with twelve and four windows, respectively. 68 Similarly to those measured via single-particle experiments, the extinction spectra computed for nanocages with four windows display a main, quasi-Lorentzian LSPR in the near-infrared domain. The characteristics of this resonance, extracted from a Lorentzian fit in the energy domain (850 nm central position, 280 meV spectral width and 7 × 10 4 nm 2 eV area) are close to those of the experimental spectra (890-950 nm central position, 210-250 meV and 6-7 × 10 4 nm 2 eV area). Such a good quantitative agreement, with only a slight blue-shift and broadening of the computed resonance as compared to experimental data, is remarkable considering the morphological complexity of the investigated nano-objects.

The extinction spectra computed for nanocages with twelve windows display a strongly asymmetric shape resulting from a partial spectral overlap of a main resonance centered at about 600 nm with an absorption background below 650 nm wavelength due to gold interband transitions. Computed spectra display marked similarities with experimental ones (Fig. 4), with also an extinction maximum near 600 nm. However, the general agreement is not as good as in the case of nanocages with four windows, in particular regarding extinction cross-section amplitudes in the infrared domain, which are larger in the measurements than in the simulations.

This may be the result of uncertainties in the determination of morphological parameters and/or deviation of the actual nanocage morphology from the ideal environment assumed in the modeling. The polarization dependence of nanocage resonance extinction cross-sections was also investigated (Fig. 6b andd) by reproducing the calculations shown in panels a and c for different directions of the polarization of the linearly polarized incident plane wave (wavelength being fixed to its resonance value). These computations yield sinusoidal variations of extinction with polarization angle, similar to the experimental case. Nanocage orientation is predicted to have an impact on the amplitude (and phase) of the polarization dependence. However, the computed polarization-induced P a g e | 8

extinction variations are of less than 5% in all considered cases. This numerical result is consistent with the small polarization dependence observed for nanocages with twelve windows. It also implies that the large polarization dependence measured for the extinction of some nanocages with four windows stems from effects not included in the FEM simulations. It probably originates from imperfections in the window morphology and spacing in the actual nano-objects (while such effects may also occur for nanocages with twelve windows, individual windows imperfections are more likely to average out due to the much higher number of windows).

The synthetic approach presented in this paper can be extended to create hollow nanostructures with a more complex architecture, such as rattle-like nanostructures, consisting of a gold cage containing a guest nano-object.

Hajfathalian et al. recently reported the confinement of a Wulff-shaped core within a metallic nanocage of different shape and nature, 69 but their synthetic approach requires a solid substrate onto which the Wulff-shaped core is first fabricated by thermal dewetting of a thin film, which represents a severe limitation regarding the quantity of nanostructures that can be produced. In our case, all the stages of the synthetic route occur in solution allowing upscaling. We demonstrated that our strategy does actually entrap nano-objects within the gold nanocage by replacing the silica seeds used for the fabrication of the silica/PS multipods by core@shell nanoparticles made of magnetite (γ-Fe3O4) cores surrounded by a thin silica shell. 70 Because their size distribution was larger than that of the conventional silica seeds, the as-obtained maghemite/silica/PS multipod nanoparticles are indeed a more complex mixture of bipods to dodecapods (Table S1, ESI †). Thus in this single experiment, we show that the maghemite/silica core can generate all desired nanocages morphologies. Since the magnetite core can dissolve in hydrofluoric acid, we stopped the fabrication process after the dissolution of the PS nodules. Fig. 7 shows Fe, Si and Au element mapping obtained by STEM-EDX of nanocages with 2, 3, 4, 6 and 12 windows. This approach offers a unique capability to create new hollow nanostructures formed by different components (metal, semiconductor, dielectric), and to combine and modify their individual properties via their interaction (for instance through the strongly enhanced electromagnetic field inside the core of the cage due to plasmonic effect). It opens new ways towards the design of hollow nano-objects.

Conclusions

We demonstrated a new strategy to fabricate gold nanocages, with a control over the number (from 2 to 12) and the relative positioning of the windows, through a multi-step colloidal approach. The frame of the nanocages does exhibit a roughness of a few nanometers that could be smoothed via post-treatment, for example by oxidation etching. The extinction spectra of some of these structures were measured using single-particle spectroscopy. The specific features of the plasmonic hollow nanostructures are well correlated with those computed numerically, P a g e | 9

assuming perfectly symmetrical nanocage geometries. We demonstrated that our synthesis approach allows a guest spherical particle of iron oxide to be included within the nanocages. More broadly, this route offers a promising strategy to design and construct multifunctional architectures based on porous hollow nanostructures.

As it can be adapted to numerous systems (noble and non noble metals, metal oxide or chalcogenide), it may enable a variety of fundamental studies and technological important applications in various fields including biological medicine, sensing and detection, energy conversion and catalysis. [71][72][73] Experimental Synthetic procedures of Au nanocages

Template synthesis.

Silica/polystyrene multipods-like particles. Multipods made of a central silica core and a predetermined number of PS satellite nodules were obtained after a procedure previously published in detail. 63,64 It consists in a seededgrowth emulsion polymerization of styrene using highly monodisperse silica nanoparticles surface-modified with methacryloxyalkyl groups as seeds. We used different batches of multipods dispersed in water (Table S1, ESI †), whose number of PS nodules was controlled by playing with the seed concentration and/or the satellite-to-seed size ratio. Their concentration was determined by the dried extracts method. More details are shown in ESI. †

Regrowth and amination of the silica core. We mixed 1 mL aqueous dispersion of multipods, 45.5 mL absolute ethanol of TEOS (tetraethoxysilane) depending on the expected regrowth extent. We added this solution at the rate of 0.5 mL h -1 and the mixture was stirred at room temperature for 12 h. Then a calculated volume of EDPS, corresponding to 20 functions per nm 2 of free silica surface area, was added to the particle dispersion, which was let to age at 40 °C for 2 h. The as-prepared templates were purified by 3 cycles of centrifugation/redispersion in absolute ethanol. The final concentration of the templates was accurately determined by the dried extracts method and adjusted to 7.3 10 15 particles L -1 . For each batch of silica/PS multipods, the final diameter of the silica core was determined from transmission electron microscopy images (Table S1, ESI †).

Regioselective gold coating. We mixed an aqueous dispersion of gold nanoseeds (500 μL) with the template dispersion (100 μL) and let them to react under stirring at room temperature overnight. The excess of gold nanoseeds was removed by three cycles of centrifugation/redispersion in absolute ethanol. The nanoseedsadsorbed templates were finally redispersed in 400 μL water. Then, we collected 20 μL of this dispersion that we poured into a 500 μL Eppendorf TM tube with 90 μL gold plating solution (GPS), 100 μL polyvinylpyrrolidone (1 wt%) aqueous solution, 200 μL water and 20 μL formaldehyde (9.25 wt%) aqueous solution. The mixture was gently mixed during two days at room temperature. Then, the nanoparticles were washed by three cycles of centrifugation/redispersion in water.

Nanocage development.

The dissolution of the PS nodules was performed in 3 mL tetrahydrofuran at room temperature under stirring for 3 h. After washing by three cycles of centrifugation/redispersion in THF, the particles were finally redispersed in absolute ethanol. The dissolution of the silica core was performed in 3 mL HF (2 vol%) aqueous solution at room temperature under stirring for two hours and washing by three cycles of centrifugation/redispersion in absolute ethanol. Synthetic procedures of filled Au nanocages. The same synthetic protocol was used but we replaced the silica cores used for the fabrication of the templates by Fe3O4@SiO2 particles. The core@shell nanoparticles were synthesized by a previously reported synthesis. 70 More details are shown in ESI. †

Fig. 1

 1 Fig. 1 Schematic representation of the multi-step colloidal strategy for the synthesis of gold nanocages with twelve windows. Details are presented in the main text. Note that the central silica core of the original particle has the same diameter as the PS pods, and is therefore not visible in the drawing until after silica regrowth.

Fig. 2

 2 Fig. 2 Top row: Typical TEM images showing the morphology evolution of the nanoparticles throughout the synthesis stages. Bottom row: TEM, SEM and 3-D reconstruction obtained by electron tomography of the gold nanocages with twelve windows. Scale bars: 100 nm.

Fig. 3

 3 Fig. 3 Top row: Schematic representation of silica/PS templates and intermediate morphologies, leading to the synthesis of nanocages with 2, 3, 4 and 6 windows. Bottom row: STEM-EDX gold maps of the corresponding nanocages (Au in green). Scale bars: 100 nm. TEM or SEM images of the different intermediates and resulting nanocages can be found in Fig. S2-S5 (ESI †), respectively, and the morphological features of the gold nanocages are compiled in Table S2 (ESI †).

Fig. 4

 4 Fig. 4 Optical properties of nanocages with four windows. (a) Absorbance spectrum of the synthesized nanocage solution in ethanol. (b) Quantitative extinction spectra of three individual nanocages deposited on a thin silica membrane, measured using SMS with linearly polarized light. (c) Dependence of their resonance extinction cross-sections on incident light polarization direction, measured by SMS at wavelengths near each LSPR peak (λ = 900-950 nm) (squares), and sinusoidal fits (lines). The TEM image of one of the investigated nanocages (corresponding to the red lines in panels (b and c)) is shown on top (scale bar: 100 nm).

Fig. 7

 7 Fig. 7 STEM-EDX superimposed elemental maps of the Au hollow structures with respectively 2, 3, 4, 6 and 12 windows filled with a spherical Fe3O4 nanoparticle (Fe in red, Si in blue and Au in green). Original STEM images and individual element maps can be seen in Fig. S6-S9 (ESI †), respectively.

Acknowledgements : STEM-EDX analyses were performed at the Plateforme Aquitaine de Caractérisation des Matériaux and Bordeaux Imaging Center of the University of Bordeaux. This work was supported by the Conseil Régional d'Aquitaine, the Agence Nationale de la Recherche (grant ANR-07-BLAN-0271 and ANR-BLAN-SIMI10-LS-100617-15-01), and the LabEx AMADEus (ANR-10-LABX-42) in the framework of IdEx Bordeaux (ANR-10-IDEX-03-02), i.e. the Investissements d'Avenir programme of the French government. The authors acknowledge Dr Véronique Many for some parallel experiments, Dr Laurent Adumeau and Dr Stéphane Mornet for helpful discussion, and Dr Yohan Grand for SEM experiments performed at ITODYS laboratory (UMR 7086, Univ. Paris Diderot).

Conflicts of interest : There are no conflicts to declare.


B François

S Ballandras
email: sylvain.ballandras@femto-st.fr

D Gachon

J.-M Chomeloux

Lesage

T Baron

G Martin

E Lebrasseur

P.-P Lasagne

A Reinhardt

Luc Chomeloux

Jean-Marc Lesage

RF oscillators stabilized by temperature compensated HBARs based on LiNbO 3 /Quartz combination

de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Radio-Frequency (RF) oscillators can be stabilized by various resonating devices. Their stability is mainly conditioned by the spectral quality of the resonator even if the oscillator loop electronics must be optimized to lower the generated noise as much as possible. For mid-term stability, temperature compensation as a key point and allows for notably improving the corresponding figure of merit. In a previous work [START_REF] Baron | Temperature compensated radio-frequency harmonic bulk acoustic resonators[END_REF], we have shown the possibility to build temperature compensated High overtone Bulk Acoustic Resonators (HBARs) combining LiNbO3 and Quartz single crystal plates for the excitation of high order modes exhibiting cubic frequency-temperature behavior. In this work, we develop a radio-frequency oscillator operating near the 434 MHz-centered ISM band to validate the capability of the above-mentioned HBAR for such purposes. The composite substrate have been built using a 3-inch (YXl)/163° LiNbO3 cut wafer bounded and thinned down to 15 µm onto a 350µm thick (YXlt)/34°/90° quartz base.

Single-port resonators operating near 434 MHz (exploiting the third harmonic of the thinned niobate plate as the HBAR transducer) have been then manufactured. Electrical and thermoelectric characterizations have shown quality factor of the resonance in excess of 25000, yielding a QF product in excess of 10 13 and a third order frequency-temperature behavior. A SAW filter was used to select the ISM band and to filter the high spectral density HBAR response. The oscillator then has been measured using a phase noise automatic bench (R&S FSUP26 spectrum and phase noise analyzer). A phase noise better than -160dBc/Hz at 100kHz has been measured as well as a -165dBc/Hz level at 1MHz from the carrier. Mid term stability also have been achieved, first results showing resonator stability better than 10 -9 under room conditions. Further work are expected to lower that level well below 10 -10 .

The first section of the paper discusses the design of the resonator based on thermoelastic computation of the temperature coefficients of frequency (TCF) of each mode of the structure. The basic theory is first recalled and general relations are provided to select the material stack thicknesses for a given mode order/frequency operating condition. A very brief description of the device fabrication is provided and experimental characterization of the resonators is reported. The oscillator architecture is depicted and the stability measurements detailed. As a conclusion, comparison with SAW-based oscillator is achieved and possible improvements of the oscillator is discussed

II. DEVICE ANALYSIS AND DESIGN

A. HBAR Temperature Coefficient of Frequency (TCF)

The celebrated Campbell&Jones method [START_REF] Campbell | A method for estimating crystals cuts and propagation direction for excitation of piezoelectric surface waves[END_REF] is used here for predicting the TCF of any mode of a given HBAR. As it has been reported hundred times in previous papers, only the main basic equation is reported below:

f= V 2 e ƀ df f Ǝ T Ə dV V Ǝ T Ə de e Ǝ T Ə (1)
which means that the frequency changes due to temperature variations are computed as the difference between the development of the velocity and of the stack thickness vs temperature. As discussed in [START_REF] Baron | Temperature compensated radio-frequency harmonic bulk acoustic resonators[END_REF], Lithium Niobate and Quartz have been associated for the fabrication of shear-waver based HBARs. LiNbO3 provides crystal orientations for which very strongly coupled shear waves exist (kt 2 in excess of 45%) whereas AT cut of Quartz allows for compensating second order frequency-temperature effects [START_REF] Ballandras | RESONATEUR HBAR A STABILITE EN TEMPERATURE ELEVEE[END_REF]. Although this idea was already proposed using other material combinations [START_REF] Daniel | [END_REF], no real design process was presented until now and therefore the possibility to actually determine structures allowing for high frequency operation with first order TCF smaller than 1ppm.K -1 was quite hypothetic. Second order compensation was even not imagined in [START_REF] Daniel | [END_REF]. Figure 1 shows the evolution of the TCF1 of almost all the modes of thinned (YXl)/163°L iNbO3 layer atop a (YXlt)/36°/90° Quartz plate for various Niobate/Quartz thickness ratios, always assuming a Niobate layer much thinner than the Quartz substrate (which allows for neglecting the differential thermoelastic stress within the stack). One can see that depending on the harmonic number, the TCF1 changes from +1 to -14ppm.K -1 . Furthermore, depending on the harmonic of the transducer alone, the TCF1 may notably change and thus it cannot be considered as a simple periodic function vs harmonic number. Therefore, it is mandatory to accurately consider all the actual feature of the structure for an accurate design of a resonator, i.e. the operation frequency, the harmonic number and the thickness ratio for a given structure. To complete this, one should also account for the actual thickness of the device as this parameter will control the possibility to select one (frequency/harmonic number) couple. Finally, it clearly appears that the analysis of such HBAR TCF requires a numerical analysis and that if an intuitive approach allows for a first order definition of crystal orientations, the complicated distribution of energy within the stack vs all the structure parameters induces more intrication in the design process. As an illustration of these considerations, the evolution of the TCF of the most strongly coupled mode of the structure (coinciding with the fundamental mode of the niobate plate alone) has been plotted vs niobate thickness (Fig. 2). A TCF1 vs Niobate/Quartz ratio law has been derived from this curve, yielding the following expression:

TCF 1 42. 5* R t Ÿ3.2*log Ǝ 200 R t Ə (2)
where Rt is the Niobate/Quartz ratio, thicknesses and TCF1 being expressed in µm and in ppm.K -1 implicitly.

Considering now the TCF1 evolution vs quartz cut angle ș (rotation around X axis), one can set the following complementary law:

TCF 1 Ǝș35 .Ə*5.10 6 (3)
Using these relations allows one to show that the most coupled mode of the structure is temperature compensated for Rt=1/50 for the (YXlt)/34°/90° quartz cut, whereas the (YXlt)/ 33°/90° and (YXlt)/32°/90° cuts will be selected for Rt respectively equal to 2.5/50 and 5/50.

B. 434 MHz resonator design

The design of the 434 MHz centered resonator has been done considering our capability to thin down LiNbO3 wafers. A typical thickness of 15µm±2µm then must be considered, the Quartz thickness being deduced along the theoretical analysis presented above. Assuming a phase velocity of the shear mode in the LiNbO3 layer near 4000m.s -1 , the third harmonic of the fundamental mode then is easily excited at the expected frequency. To achieve the temperature compensation of the mode, the quartz cut angle and plate thickness then has to be fixed. A Rt value of 2.5/50 is considered as a trade-off between the spectral density of modes and the ease to manufacture the material stack.

Consequently, the quartz thickness is found near 270llm for a cut angle corresponding to (YXlt)/34°/90°. Once again for manufacture simplicity, standard quartz wafer thicknesses are considered, yielding a value of 350llm.The LiNb03 thickness then is adjusted to 141lm according to the above considerations (Rt actual value 2/50). Validation of such a configuration has been achieved theoretically by computing the admittance of the corresponding device and then its theoretical TCF. An equivalent quality coefficient Q of 20,000 has been considered for Quartz whereas a Q of 100,000 was affected to LiNb03• Au electrodes are used at the interface (t = 300nm, Q�1000) and Al ones at the top surface (t = 250nm, Q�2000). An effective Q value slightly larger than 20,000 then is predicted for a k/ of 1,9%0 (Fig. 3).

Temperature compensation is checked by plotting both TCF I and second order TCF2 for each resonance near 434MHz, as shown in FigA. For such a configuration, one can see that TCFI smaller than 1 ppm. K I can be obtained together with TCF2 of less than 5ppb.K 2 .

Figure 3.

Theoretical admittance of a HBAR built on a 14Jlm thick (YXJ)/163° LiNbO, plate onto 350Jlm thick (YXlt)/34°/90° Quartz

III. EXPERIMENTAL IMPLEMENTATION

A. HBARfabrication

As mentioned above, only some information is reported here as most of the process has been already presented in details [START_REF] Baron | Temperature compensated radio-frequency harmonic bulk acoustic resonators[END_REF]. LiNb03 and Quartz are bonded together using thin Cr/ Au films deposited on both wafer sides to be bounded and therefore used both as diffusion layers and interface electrode.

A pre-bonding is first achieved using either an EVG bounder or a Suss Microtech CL200 megasonic cleaner. The bounding then is reinforced by applying a quite strong homogeneous pressure during a few tenth minutes, yielding then an almost perfect acoustic adhesion between both material. Top electrodes are then deposited using standard lithography processes.

B. Resonator characterization

Oscillator stabilization

The implemented oscillator once again is very similar to what has been already presented for SAW devices [START_REF] François | Fabrication and Characterization of a SAW-Oscillatorbased Sensing System Including an Integrated Reciprocal Counter and a Wireless ZigBee Transmission System[END_REF] and therefore will not be detailed here. It is a Colpitts-type negative resistance circuit to which a buffer stage has been added to isolate the oscillating loop and provide sufficient signal for the oscillator characterization. Figure 6 shows the characterization of the oscillator output using a spectrum analyzer, showing the definition of the resonance and also proving the possibility to actually select the ISM band by filtering.

Figure 6.

Spectrum of the oscillator output signal, close to the ISM center (433.7MHz)

Figure 7 shows the phase noise of the oscillator. The noise floor is at -165dBc/Hz and the phase noise at 10kHZ is -140dBc/Hz. .

Phase noise of the Colpitts oscillator.

Figure 8 show Allan variance of the oscillator output signal. The oscillator based on HBAR temperature compensated presents a relative stability of near 2×10 -10 over 2 seconds of integration at room conditions. In this work, we develop a radio-frequency oscillator operating near the 434MHz-centered ISM band to validate the temperature compensated capability of HBAR based on combining LiNbO3 and Quartz single crystal plates for such purposes. Electrical and thermoelectric characterizations have shown QF product in excess of 1013 and a third order frequency-temperature behavior. A phase noise better than -160dBc/Hz at 100kHz has been measured as well as a -165dBc/Hz level at 1MHz from the carrier. First results showing that the resonator stability is better than 10-9 under room conditions over 2 seconds. Further work is expected to lower that level well below 10-10. To achieve this objective, excitation of resonator will be increase, test will be done under controlled temperature condition, and electromechanical coupling coefficient will be increase. Towards ultimate stability allows us competition with SAW based oscillators.

Figure 1 .

 1 Figure 1.Plot of the TCF of a HBAR built on a (YXl)/163°L iNbO3 thinned plate bonded on (YXlt)/36/90 Quartz substrate for various Niobate/Quartz thickness ratio (Quartz thickness arbitrary fixed to 50µm)

Figure 2 .

 2 Figure 2.Plot of the TCF of a HBAR built on a (Yxl)/163°L iNbO3 thinned plate bonded on (Yxlt)/36/90 Quartz substrate for various Niobate/Quartz thickness ratio (Quartz thickness arbitrary fixed to 50µm)

Figure 4 . 1 "

 41 Figure 4.1" and 2nd order TeF of the above LiNbO,/Quartz HBAR, temperature compensation achieved between 430 and 440MHz.

  Conforming to the above design and manufacturing process, several devices have been fabricated and tested to select the most appropriate one for frequency stabilization in the ISM band. Figure 5 shows one example of resonator admittance from which electromechanical coupling k/ and Q factor are deduced, respectively at about 0.2%0 and 25,946, yielding a Q factor of 1.13 x 101 3 , much better than Rayleigh wave based resonators on Quartz considering the same frequency. One can note that Q factor in excess of 30 000 also were measured (QF products in excess of 1.3 x 101 3 ), but for frequencies slightly out of the ISM band. The TCFs have been again tested to demonstrate the predicted compensation effect.

Figure 5 .

 5 Figure 5.

Figure 8 .

 8 Figure 8. Allan Variance of the oscillator output signal, yielding a relative stability near 2×10 -10 over 2 seconds of integration at room conditions IV. CONCLUSION

This work is supported by the FCE -DGCIS under grant #092906659/60/61.