Jean-Baptiste Renard

Michel Chartier

Claude Robert

Gilles Chalumeau

Gwenaël Berthet

Michel Pirre

Jean-Pierre Pommereau

Florence Goutail

Jean-Baptiste Renard

Gwenae ¨ L Berthet

J.-B Renard

M Fr͒

C Chartier

G Robert

M Berthet

Pirre

SALOMON: a new, light balloonborne UV-visible spectrometer for nighttime observations of stratospheric trace-gas species

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Introduction

NO 2 ,N O 3 , and OClO are involved in stratospheric ozone chemistry. These gas-phase species, as well as ozone, possess some absorption bands that are detectable in the near-UV-visible spectral domain. The extinction coefficient of aerosol can also be measured because it presents a strong UV-visible continuum below 25 km. At the present time, several balloonborne UV-visible spectrometers that use the Sun as a light source are in operation, but only one balloonborne UV-visible spectrometer, AMON ͑French acronym for Absorption par les Minoritaires Ozone et NO x ͒, 1,2 takes measurements at night, using a star as the light source. AMON, aboard the stabilized platform of the Geneva Observatory, [START_REF] Huguenin | Design and performance of stratospheric balloon-borne platforms for infrared astrophysical observations[END_REF] which has a pointing system accuracy of 3 arc sec, can achieve a vertical resolution of less than 1 km. Because it has a total weight of 500 kg, AMON must be launched with a balloon whose volume gas capacity is at least 100,000 m [START_REF] Huguenin | Design and performance of stratospheric balloon-borne platforms for infrared astrophysical observations[END_REF] . This requirement implies constraints that reduce the launch opportunities.

Recent studies have shown that, despite good agreement between modeling results and observations of OClO at high latitudes, [START_REF] Pommereau | Observations of the vertical distribution of stratospheric OClO[END_REF][START_REF] Renard | Vertical profile of night-time stratospheric OClO[END_REF] significant discrepancies exist for NO 2 and NO 3 at mid-latitude and high latitude. [START_REF] Renard | Nocturnal vertical distribution of stratospheric O 3 ,N O 2 and NO 3 from balloon measurements[END_REF][START_REF] Lary | Carbon aerosols and atmospheric photochemistry[END_REF][START_REF] Renard | Vertical distribution of nighttime stratospheric NO 2 from balloon measurements-comparison with models[END_REF] Moreover, the possible detection of OBrO by the AMON, [START_REF] Renard | The possible detection of OBrO in the stratosphere[END_REF] although models predict no OBrO at all, [START_REF] Chipperfield | Model calculations of stratospheric OBrO indicating very small abundances[END_REF] as well as the possible observation of BrO at night in the polar vortex [START_REF] Wahner | Near UV atmospheric absorption measurements of column abundance during airborne arctic expedition[END_REF] could imply that bromine chemistry is not well understood. All these facts call for many other nighttime observations at different seasons, latitudes, and local times to improve the database for these species. To perform such measurements, a light instrument with reduced launch constraints is needed.

The light of a star is weak and requires an accurate and heavy pointing system. The Moon, which needs a less accurate pointing system, can be used as a light source to reduce the weight of the instrument. This method of measurement has been already used for ground-based and airborne spectrometers ͑see, e.g., Sanders et al. [START_REF] Sanders | Visible and near-ultraviolet spectroscopy at McMurdo Station, Antarctica. 4. Overview and daily measurements of NO 2 ,O 3 and OClO during 1987[END_REF] and Lal et al. [START_REF] Lal | Atmospheric NO 3 observations over low-latitude northern hemisphere during night[END_REF] ͒ but not by balloonborne instruments.

A new instrument, called SALOMON ͑which is a French acronym for Spectroscopie d'Absorption Lunaire pour l'Observation des Minoritaires Ozone et NO x ͒, funded by the French space agency Centre National d'Etudes Spatiales ͑CNES͒, is now operating.

It was developed based on the experience acquired by the AMON team and by the team that developed the light balloonborne UV-visible spectrometer SAOZ [START_REF] Pommereau | Ozone, nitrogen dioxide and aerosol vertical distribution by UV-visible solar occultation from balloon[END_REF] that uses the Sun as a light source. SALOMON was first flown successfully on 31 October 1998 at midlatitude.

We present below a description of this instrument and the first results, including performance of the pointing system, spectra, and vertical profiles of the species.

Description of the Instrument

A. General Description

SALOMON is made from three boxes ͑Fig. 1͒. The top two boxes, linked by an optical fiber, include a spectrometer and the moon-tracker system. The third, below the two others, contains the CNES telemetry system. The boxes are made from polystyrene, which is well suited for protecting the onboard instruments at landing. The pivot ͑i.e., the primary pointing system͒ is located above the boxes and links the gondola to the flight chain ͑which includes a Global Positioning System, a barometer, and the parachutes͒ and the balloon. The total weight of SALOMON is 80 kg, which permits the use of balloons in the 10,000 -65,000-m 3 range to reach a float altitude of 18 -3 hPa ͑i.e., 27-38 km at mid-latitudes͒. This instrument operates automatically for on-off switching, pointing, data acquisition, and telemetry.

The housekeeping and spectroscopic data are telemetered in real time to the ground.

The measurements are performed by the lunar occultation method. This method consists of recording spectra affected by atmospheric absorption during a moonset or a moonrise. The elevation of the Moon decreases from a few degrees above the gondola's horizon to ϳϪ4°, which implies a duration at float of ϳ40 min. A reference spectrum is recorded when the Moon is as high as possible and the balloon is at float. In fact, observations performed using the Sun or stars have shown that the reference spectrum is only slightly affected by atmospheric species when the light source is more than 2°above the gondola's horizon. In that case, the contribution of the species to the reference spectrum is less than 5% of the maximum contribution of the species to the occultation spectra. We then obtain transmission spectra by dividing the Moon's occultation spectra by the reference spectrum. Finally, we obtain the optical depth spectra that are used to retrieve slant column densities of atmospheric species along the line of sight by taking the opposite of the logarithm of the transmission spectra.

B. The Pivot

The pivot ͑or stabilization unit͒ is designed to stabilize the gondola and to perform rough azimuth control. It is composed of a magnetic damping device to stabilize the flight chain and a mechanical unit with a velocity sensor to perform the gondola rotation. The motor ͑RE35G-071-34, built by Mascon͒ is driven by a numerical control feedback for the gondola's rotation and an analogous control feedback for the gondola's azimuth stabilization. The Moon's location in the sky is determined automatically by two photodiodes mounted 45°from each other. The pivot induces a rotation of the gondola until the fluxes on the two photodiodes are identical. This system is designed to have a precision of less than 0.2°, which is less than half of the Moon's diameter, and to operate when the light flux is greater than 15% of the full Moon's global flux ͑i.e., between its first quarter and last quarter͒.

C. The Moon Tracker

The moon tracker ͑Fig. 2͒ consists of a 25 mm ϫ 76 mm plane mirror that can move on elevation ͑motor BTN 18-D, built by Vernitron͒, mounted upon a turret that turns on azimuth ͑motor 3181V-12I-053, built by Vernitron͒. A portion of the light focused by a lens in the turret is sent to a position sensor, which determines the spot ͑ x, y͒ position. The error in elevation and the error in azimuth are calculated, and the calculated correction is sent directly to the motors by the onboard processor. The system is designed to achieve a precision below 30 arc sec when the gondola is affected by only small perturbations. In the case of strong oscillations of the gondola, as can be encountered during ascent of the balloon, the error on azimuth can be as much as 1 arc min, and the error on elevation can reach a few arc minutes because this axis is not controlled by the pivot.

The flux collected by the mirror is focused onto an optical fiber to send the light to the spectrometer. This device homogenizes the flux and eliminates sensitivity to the variations of the Moon's albedo induced by its nonuniform ground composition.

D. The Spectrometer

The spectrometer, the detector, and the electronics are the same as those used for the SAOZ instrument, [START_REF] Pommereau | Ozone, nitrogen dioxide and aerosol vertical distribution by UV-visible solar occultation from balloon[END_REF] which had performed 80 flights by the end of 1998. The spectrometer is a Jobin-Yvon Model CP200, with a wavelength domain shifted to 350 -700 nm to cover the NO 3 absorption band at 662 nm. The detector is a Hamamatsu photodiode array of 1024 pixels, with a theoretical spectral resolution of 0.34 nm, which is 2.5 times the theoretical resolution of AMON. For the first flight, an entrance slit of 100 m was used to maximize the flux. The exposure time was 52 s, which is suitable for producing a high signal-to-noise ratio without degrading the expected 2-km vertical resolution. A dark current exposure is performed after every ten exposures, and its value is subtracted automatically from the recorded spectra by the onboard processor.

Results of the First Flight

A. Flight Conditions

The first flight of SALOMON occurred during the night of 30 -31 October 1998 from the CNES launching base of Aire sur l'Adour, France ͑43.7 N, 0.3 W͒. This flight was devoted to testing all the technical parameters of the instrument, and a 12,000-m 3 balloon was used. Flight conditions allowed for measurements to be performed during the ascent of the balloon and during the whole moonset. Inasmuch as all the parameters of the pointing system, the moon tracker, and the spectrometer were nominal, scientific results can be extracted from the spectra.

Launch occurred at 00:06 UT, the ascent lasted 75 min, and the float altitude of the balloon was 26.65 km ͑19 hPa͒ with fluctuations of Ϯ150 m. This altitude is suitable for the observation of O 3 but is problematic for observation of NO 2 and NO 3 because the maximum concentrations of these species are above 27 km. Corrections are then needed, as described below in Subsection 3.C. The measurements started at 00:52 UT, just after crossing the tropopause, and stopped at 01:46; 43 spectra were recorded. Because of strong winds and the fast ascent of the balloon, some oscillations of the gondola occurred during the observations and stopped only a few minutes before the end of the measurements at float altitude. Without affecting the spectra, this situation allowed for testing of the pointing system both in perturbed and in quiet conditions.

The phase of the Moon was 68% of full. Inasmuch as the flux was strong enough for both the spectrometer and the pointing system, this flight demonstrated that measurements can be performed less than two days after the first quarter. Thus flights can occur at least 10 days per month, not including the day of the full Moon for which moonrise and moonset occur too close to sunset and sunrise. tracker elevation, respectively. The pivot reached its expected precision, i.e., below 0.2°. The precision of the moon-tracker azimuth was close to 1 arc min during the ascent of the balloon and reached the expected 30 arc sec at float. The precision of the moontracker elevation was of a few arc minutes during the ascent, decreased to 1 arc min at float, but remained above the expected 30 arc sec because of the gondola movements. During the last 10 min at float, these movements stopped, and the expected value was reached.

B. Performance of the Instrument

The analysis of the position of the Moon's Ca, H, and Na absorption lines in all the spectra shows that the wavelength shift from the first spectrum to the last spectrum is less than 0.03 pixel ͑i.e., 0.01 nm͒. This value is close to the theoretical shift induced by the variation of air density and its refractive index. Thus the spectra recorded during the Moon's occultation can be divided by the reference spectrum without shift adjustment, which prevents the appearance of artificial absorption features. The noise is found to be equal to 3.8 digital levels, whereas the signal is close to 10,000 digital levels. Thus a signal-to-noise ratio greater than 2500 per pixel is obtained.

C. Data Reduction and Results

The Moon occultation method requires highly accurate Moon coordinates to enable the inversion be performed and the minimum altitude of each line of sight to be known. The Moon's elevation and azimuth were calculated with the exact location and altitude of the balloon taken into account. The topocentric Moon coordinates were calculated by use of the Bureau des Longitudes on-line data. [START_REF]Bureau des Longitudes[END_REF] Figure 6 presents the raw spectra recorded during the occultation. The cosmic rays, which affect only a few pixels per spectrum, were removed by use of the algorithm previously developed for AMON. [START_REF] Naudet | Balloon measurements of stratospheric trace species using a multichannel UVvisible spectrometer[END_REF] The reference spectrum is the result of averaging five consecutive spectra recorded with a Moon elevation of 2.5°to 1.5°. The algorithms used for data reduction of the transmission spectra are similar to those used for AMON, which are described in detail by Renard et al. [START_REF] Renard | Nocturnal vertical distribution of stratospheric O 3 ,N O 2 and NO 3 from balloon measurements[END_REF][START_REF] Renard | The possible detection of OBrO in the stratosphere[END_REF] The wavelength scale was established from the lunar Ca ͑K͒,Ca͑H͒,H␦,Fe͑d͒,H␤, Na, H␣, and atmospheric O 2 lines and a second-order polynomial fit ͑with a standard deviation smaller than 0.05 nm͒.

Because of the weakness of the Moon's flux in the UV, the spectral domain was reduced to 400 -700 nm. The Rayleigh scattering contribution, calculated by use of the cross sections given by Bucholtz, [START_REF] Bucholtz | Rayleigh-scattering calculations for the terrestrial atmosphere[END_REF] was removed from all spectra. The complete coverage of the visible spectral domain allows O 3 and NO 2 absorption features to be used over large spectral ranges of 455-680 and 400 -550 nm, respectively. Ozone retrieval was performed with the aerosol contribution that follows a second-order polynomial law taken into account. The NO 2 retrieval was performed by use of a differential method with a highpass filter to remove the continuum. With respect to NO 3 , the analysis performed on AMON data has shown that the spectral domain must be restricted to 645-680 nm to avoid inclusion of an oxygen line close to the NO 3 623-nm band. Figure 7 presents an example of the recorded optical depth spectrum, smoothed over 6 pixels, recorded with a Moon elevation of Ϫ1.0°, and the O 3 leastsquares fit with the cross section at 223 K measured at Bremen University. [START_REF] Voigt | information available at their internet site[END_REF] Figures 8 and9 present the NO 2 and the NO 3 retrieval for the same spectrum, smoothed over 5 and 6 pixels, respectively. The NO 2 cross sections used were measured by Harder et al. [START_REF] Harder | Temperature dependent NO 2 cross sections at high spectral resolution[END_REF] at 217 K, and the NO 3 cross sections used were measured at 243 K by Deters and Burrows. [START_REF] Deters | Institute of Environmental Physics and Remote Sensing[END_REF] The error that results from calculations of the standard deviation between the fit and the observed optical depth spectrum is 3 ϫ 10 Ϫ3 ,6ϫ 10 Ϫ4 , and 3 ϫ 10 Ϫ3 for O 3 ,N O 2 , and NO 3 respectively. Finally, we can obtain the aerosol extinction coefficient by subtracting the contribution of all the species described above.

Figure 10 shows the slant column profiles versus Moon elevation for O 3 ,NO 2 ,NO 3 , and aerosols at blue and red wavelengths. Because the measurements were made at the low float altitude of 26.5 km, the contribution of the species to the reference spectrum cannot be neglected; negative values therefore appear for observations performed during the balloon ascent when the Moon elevation was between 6°and 2°. Performing the inversion to obtain concentrations requires that the slant column profile be corrected by the offset contained in the reference spectrum. The first value of the slant column density profile for each species provides an estimation of this offset, or at least of its lower limit. These values will then be added to the slant column profiles.

Comparison with the AMON Instrument

As we discussed in Section 1, only one instrument, AMON, has performed measurements by UV-visible spectrometry at night. The performance of the AMON pointing system is greater than that of the SALOMON pointing system, as stellar observations need greater accuracy ͑the angular diameter of a star is below 1 arc sec, whereas that of the Moon it is 30 arc min͒. Nevertheless, the performance of SALOMON is well suited for Moon observations, as discussed above.

The O 3 and NO 2 retrieval is performed over a larger spectral domain with SALOMON than with AMON: 225 versus 75 nm for O 3 and 150 versus 60 nm for NO 2 . These larger spectral domains ensure that no error exists on the SALOMON wavelength scale, which is essential for the retrieval of small absorption features.

Inasmuch as the Moon flux is greater than the star flux, and because no wavelength shift was detected, one would expect that the SALOMON spectrometer can detect smaller absorption features than the AMON can. This flight showed that NO 2 and NO 3 are detected by the SALOMON with a greater signalto-noise ratio, and therefore sensitivity, than with the AMON ͑sensitivity of 5 ϫ 10 Ϫ4 for the SALOMON compared with 5 ϫ 10 Ϫ3 for the AMON in the case of NO 2 and NO 3 detection͒. This fact allowed us to search for OBrO in the SALOMON spectra. The retrieval of OBrO was performed by the method described by Renard et al. [START_REF] Renard | The possible detection of OBrO in the stratosphere[END_REF] and with the new cross sections measured at 298 K by Fleischmann et al. [START_REF] Fleischmann | Institute of Environmental Physics and Remote Sensing[END_REF] Because the NO 2 cross sections of Harder et al. [START_REF] Harder | Temperature dependent NO 2 cross sections at high spectral resolution[END_REF] stop at 550 nm, and the OBrO cross sections before 470 nm are below our detection limit, the spectral domain for the retrieval is just slightly greater than in the AMON retrieval. A sliding average was performed over three consecutive spectra for lines of sight above 24 km and five consecutive spectra below 24 km to reduce the noise; then the spectra were smoothed over 9 pixels. Figure 11 shows a comparison between a SALOMON spectrum and a fit from the OBrO cross sections. The error, corresponding to the standard deviation between fit and observed spectrum, is equal to 5 ϫ 10 Ϫ5 , which is close to the theoretical limit of the instrument when the smoothing procedures described above are taken into account. This result shows that the SALOMON can detect absorption features of 10 Ϫ4 , which is a slightly better sensitivity than that of ground-based and airborne lunar occultation instruments [START_REF] Sanders | Visible and near-ultraviolet spectroscopy at McMurdo Station, Antarctica. 4. Overview and daily measurements of NO 2 ,O 3 and OClO during 1987[END_REF][START_REF] Lal | Atmospheric NO 3 observations over low-latitude northern hemisphere during night[END_REF] and ten times better than that of AMON, and that the OBrO detection by AMON does not come from instrumental bias.

Figure 12 presents the vertical profiles of these species, after their inversion by an onion-peeling algorithm, and a comparison of them with AMON vertical profiles obtained at the same location on 16 October 1993 ͑the AMON profile retrievals had been improved since the papers of Renard et al. [START_REF] Renard | Nocturnal vertical distribution of stratospheric O 3 ,N O 2 and NO 3 from balloon measurements[END_REF][START_REF] Renard | The possible detection of OBrO in the stratosphere[END_REF] were published͒. As the AMON flight was performed at a float altitude of 39 km, the AMON profiles cover a larger altitude range in the stratosphere than the SALOMON profiles. Nevertheless, the shapes of the two sets of profiles are in good agreement, and the small discrepancies result from different geophysical conditions during the two measurements. Thus these SALOMON observations will be included in the database for further studies of nitrogen species at night by comparison with modeling outputs.

Conclusion

The first flight of SALOMON has shown that the performance of the instrument is as good as expected. Only small modifications ͑the use of optical fiber and lenses with better transmission͒ will be made for the next flights. Thus the global flux on the detector will be increased so the OClO absorption features from 380 to 425 nm can be detected for flights at high latitudes.

As SALOMON can be prepared for launch in only a few days, can be launched in windy conditions ͑as much as 5 m s Ϫ1 at ground͒, and can operate over a wide range of float altitudes, several flights per year can be performed with various scientific objectives. Currently, two flights are planned for the Third European Stratospheric Experiment on Ozone ͑THESEO͒ campaign for the nighttime study of NO 2 and NO 3 , and moonrise measurements will be tried for the second flight. A few flights are planned for years 2000 and 2001, in particular during validation campaigns of instruments onboard such satellite platforms as SAGE III, ILAS͞ADEOS, GOMOS͞EN-VISAT, and SCIAMACHY͞ENVISAT. This instrument may also be involved in the study of the tropics by European groups of scientists that could occur beginning in 2003.

Fig. 1 .

 1 Fig. 1. Global view of the SALOMON. The box that contains the spectrometer is at the left, the box with the moon tracker is at the right, and the box with the CNES telemetry is below them.

Figures 3 , 4 ,Fig. 2 .

 342 Figures 3, 4, and 5 show the pointing precision of the pivot, the moon-tracker azimuth, and the moon-

Fig. 3 .

 3 Fig. 3. Pointing precision of the pivot.

Fig. 4 .

 4 Fig. 4. Pointing precision of the azimuth of the moon tracker.

Fig. 5 .

 5 Fig. 5. Pointing precision of the elevation of the moon tracker.

Fig. 6 .Fig. 7 .

 67 Fig. 6. Raw spectra recorded during the occultation ͑cosmic rays have been removed͒.

Fig. 8 .Fig. 9 .

 89 Fig. 8. Comparison of a NO 2 optical depth spectrum recorded under the same conditions as for Fig. 7 and a least-squares fit.

Fig. 10 .

 10 Fig. 10. Slant column profiles of O 3 ,N O 2 ,N O 3 , and aerosol ͑at two wavelengths͒. The negative values originate from the contributions of these species to the reference spectrum.

Fig. 11 .

 11 Fig. 11. Comparison of an OBrO optical depth spectrum recorded under the same conditions as in Fig. 7 and a least-squares fit.

Fig. 12 .

 12 Fig. 12. Vertical profiles of O 3 ,NO 2 ,NO 3 , and OBrO measured by the SALOMON ͑diamonds͒ compared with the AMON measurements ͑solid curves͒ performed at the same location on 16 October 1993.

We thank all the persons in our laboratories, in particular Pierre Franc ¸ois, who have contributed to the successful performance of this instrument. We also thank the CNES for funding the instrument, and the CNES launching team at Aire sur l'Adour.


Xiaomin Yang

Erika Porcel

Laurent Marichal

Cesar Gonzalez Vargas

Amine Khitous

Daniela Salado-Leza

Xue Li

Jean-Philippe Renault

Serge Pin

Hynd Remita

Frank Wien
email: frank.wien@synchrotron-soleil.fr.

Sandrine Lacombe
email: sandrine.lacombe@u-psud.fr.

Prof Sandrine Lacombe

Prof Frank

Platinum Nanoparticles Stabilize Human Serum Albumin Structure

Keywords: Pt NPs, HSA, synchrotron radiation circular dichroism, thermal stability, thermodynamic properties

Noble metals, especially platinum nanoparticles (Pt NPs) have immense potential in nanomedicine as therapeutic agents on account of their high surface area, intrinsic 2 catalytic properties, etc. However, our understanding of the interaction of nanoparticles with biological entities, especially proteins, is far behind the explosive development of nanotechnology. In this study, we measure the structural and stability changes of proteins at nanomolar concentrations upon interaction with Pt NPs using various techniques. A large increase of 18°C in the thermal unfolding of human serum albumin (HSA) with Pt NPs at the molar ratio 1:1 is observed using synchrotron radiation circular dichroism (SRCD), due to Pt NPs increased thermal stability of HSA through preferential hydration processes. The use of SRCD allows measuring critical parameters on NPs-protein interactions, provides additional information for understanding the influential role of PEG coating in NPs-cell interaction and has implications for nanomedicine and nanotoxicology.

Introduction

After the pioneering work of Rosenberg in 1965, cis-platin have been widely used in cancer therapy [START_REF] Rosenberg | Platinum compounds: a new class of potent antitumour agents[END_REF]. From then on, numerous new Pt compounds have been discovered with better therapeutic performances than cis-platin [START_REF] Dasari | Cisplatin in cancer therapy: molecular mechanisms of action[END_REF]. Despite of the clinical success of the Pt complex, it suffers from a lack of tumor tissue selectivity leading to some severe side effects [START_REF] Medhat | Evaluation of the antitumor activity of platinum nanoparticles in the treatment of hepatocellular carcinoma induced in rats[END_REF].

Advances in nanotechnology have provided new and powerful tools in the development of multifunctional Pt NPs for effective detection and treatment of cancer, overcoming limitations associated with conventional cancer diagnosis and therapy [START_REF] Srinivasan | Multifunctional Nanomaterials and Their Applications in Drug Delivery and Cancer Therapy[END_REF]. Radiolytic synthesis (by gamma rays, X-rays, electron beams or other types of ionizing radiation) provides a major advantage for a large-scale production of monodisperse particles as it generates a uniform distribution of reducing agents in the entire solution during the irradiation process [START_REF] Abidi | One-pot radiolytic synthesis of gold nanorods and their optical properties[END_REF][START_REF] Belloni | Radiation-induced synthesis of mono-and multi-metallic clusters and nanocolloids[END_REF][START_REF] Holade | Advances in electrocatalysis for energy conversion and synthesis of organic molecules[END_REF][START_REF] Uttayarat | Radiolytic synthesis of colloidal silver nanoparticles for antibacterial wound dressings[END_REF]. Another advantage is that these nanoparticles are freshly prepared in water without using potentially toxic solvents. The functionalization of the NPs can be achieved during the radiolytic synthesis by adding an appropriate substance to the reaction (Freitas de [START_REF] Freitas De Freitas | An Overview of the Synthesis of Gold Nanoparticles Using Radiation Technologies[END_REF]. The functionalization with ligands or polymers such as poly(ethylene) glycol PEG enhances the stability and control of their size [START_REF] Wang | One-pot synthesis of AuPt alloyed nanoparticles by intense x-ray irradiation[END_REF].

Pt NPs have received considerable attention in nanomedicine due to their tunable, highly sensitive optical, catalytic, and therapeutic properties. A promising area of research involves the amplification of radiotherapy in the presence of Pt NPs at the tumor site [START_REF] Lacombe | Particle therapy and nanomedicine: state of art and research perspectives[END_REF]. The properties of radioenhancement were demonstrated for Pt NPs at nanoscale [START_REF] Porcel | Platinum nanoparticles: a promising material for future cancer therapy?[END_REF][START_REF] Schlathölter | Improving proton therapy by metal-containing nanoparticles: nanoscale insights[END_REF] and at cellular level [START_REF] Ka | Evaluation of radiosensitization effects by platinum nanodendrites for 6 MV photon beam radiotherapy[END_REF][START_REF] Salado-Leza | Green One-Step Synthesis of Medical Nanoagents for Advanced Radiation Therapy[END_REF]Yang et al., 2020c). For this use, intravenous administration of NPs is preferable than intra tumoral, to avoid pain to the patient and ensure reproducible distribution via enhanced permeability and retention effect (EPR). Thus, further exploration of the interaction of NPs with biological systems and blood proteins in particular is an important issue.

Proteins participate in most of the biological functions of the living matter and play a crucial role in its maintenance. Their biologic activity highly depends on their threedimensional secondary structure [START_REF] Takano | Density functional study of molecular interactions in secondary structures of proteins[END_REF], while undesirable interactions with NPs, may lead to protein aggregation or disfunction [START_REF] Saptarshi | Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle[END_REF]. The present study is focused on the interaction of Pt NPs with human serum albumin (HSA), since serum albumins are major circulatory proteins, which play crucial roles in many important physiological functions like maintaining plasma oncotic pressure, transporting cargo, antioxidant and enzymatic activities [START_REF] Lee | Modifications of human serum albumin and their binding effect[END_REF]. Once NPs enter systemic circulation, particle-protein interaction is the first phenomenon taking place before distribution into various organs [START_REF] Mu | Chemical basis of interactions between engineered nanoparticles and biological systems[END_REF]. The impact of NPs, when they interact with blood proteins, is thus a major question to answer. In particular, a modification of the protein structure is expected to be dramatic, by changing physiological functions [START_REF] Prasanth | Spectroscopic exploration of interaction between PEG-functionalized Ag2S nanoparticles with bovine serum albumin[END_REF].

The analysis of the structure and stability of proteins in protein-Pt NPs is a key to investigate and understand the change of the proteins' properties after their interaction with Pt NPs. The structure analysis can be performed with various techniques. Among them, Circular Dichroism (CD) allows to characterize the secondary structure, and thus folding properties of proteins, and has recently been used to detect structure changes of proteins interacting with NPs [START_REF] Micsonai | Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy[END_REF]. Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy is superior to the conventional CD spectroscopy, thanks to the high intensity of photon emission, and the higher signal-to-noise levels of the CD spectra. This allows to use lower amounts of proteins, and to observe subtle conformational changes as shown by Wallace and Janes [START_REF] Wallace | Synchrotron radiation circular dichroism (SRCD) spectroscopy: an enhanced method for examining protein conformations and protein interactions[END_REF].

Additionally, the spectral range in SRCD extends down to 175 nm (compared to 190 nm in CD). This enables elucidating finer details in the secondary structure of proteins [START_REF] Gobeaux | Reversible assembly of a drug peptide into amyloid fibrils: a dynamic circular dichroism study[END_REF], especially in the presence of nanoparticles [START_REF] Sanchez-Guzman | In situ analysis of weakly bound proteins reveals molecular basis of soft corona formation[END_REF]. SRCD allowed Laera et al. to discover that the thermal unfolding temperature of HSA decreased by 6ºC upon interaction with silver NPs. This indicates that the HSA is significantly destabilized by the NPs, possibly due to a more flexible folded structure formed in the presence of the silver NPs [START_REF] Laera | Measuring protein structure and stability of protein-nanoparticle systems with synchrotron radiation circular dichroism[END_REF]. On the contrary, Robin Capomaccio et al finds a melting temperature (T m ) of 69ºC for free HSA and of 75.4 ℃ for the AuNP-HSA complexes using SRCD, suggesting that Au NPs stabilize HSA, both by increasing its thermal stability, and by reducing its propensity to aggregate [START_REF] Capomaccio | Determination of the structure and morphology of gold nanoparticle-HSA protein complexes[END_REF]. Recently, our group has shown that Gd NPs (AGuIX) can modify the stability of HSA due to an indirect interaction (Yang et al., 2020a).

In this study, Pt NPs were synthesized, characterized and their potential impact on HSA was investigated. Our aim was to understand the interaction between Pt NPs and serum blood proteins by complementary techniques, an area in which the number of available reports is limited. In particular, we measured the secondary structure of proteins in low nanomolar concentrations, and in particular, the structural and thermal stability changes associated with proteins to Pt NPs up to 1:1 particle ratios using the SRCD technique at DISCO, Synchrotron SOLEIL. This extremely sensitive technique has allowed us to show that Pt NPs have significantly increased the thermal stability of HSA, and has also allowed accessing new detailed information about NPs-protein interactions.

Experimental

Materials and chemicals

Tetraammine platinum (II) chloride salt [Pt(NH 3 ) 4 ]Cl 2 ⋅H 2 O (Sigma-Aldrich, USA), poly(ethylene glycol) (H(OCH 2 CH 2 )nOH, M W = 1000 g.mol -1 , Fluka), potassium phosphate monobasic (KH 2 PO 4 ), sodium phosphate dibasic (Na 2 HPO 4 ), HSA was purchased from Sigma-Aldrich, USA. HSA samples were prepared by dissolving HSA powder in 10 mM phosphate buffer at pH 7.0. All chemicals were of analytical grade.

Pure water used in all experiments was filtered (18.4 MΩ cm) by a Milli-Q system (Millipore, Milford, MA, USA).

Synthesis of Pt NPs

Pt NPs were synthesized by facile and ecofriendly one-step radiolysis. The procedure of synthesis and characteristics of these NPs are detailed in the French Patent Application: Nanoparticules et procédé de preparation, FR1900008 [START_REF] Salado-Leza | Green One-Step Synthesis of Medical Nanoagents for Advanced Radiation Therapy[END_REF]Yang et al., 2020b). Briefly, the reduction was achieved in aqueous solution using Pt(NH 3 ) 4 Cl 2 (10 -2 mol/L) as precursor and polyethylene glycol (PEG-OH 1000, 1 mol/L) as radical scavenger, stabilizer and biocompatible capping agent. After thorough deaeration with flushing nitrogen gas, the solutions were irradiated by a γ-source provided by a panoramic 60 Co γ-facility (E= 1.33 MeV, LET= 0.2 keV.μm-1) with dose rate of 68.2 Gy.min -1 . Pt II was completely reduced to Pt 0 at a dose of 1,000 Gy [START_REF] Porcel | Nano-Sensitization under gamma rays and fast ion radiation[END_REF][START_REF] Porcel | Platinum nanoparticles: a promising material for future cancer therapy?[END_REF]. The obtained Pt NPs were lyophilized for storage at 4 ºC for months.

Characterization of Pt NPs

Morphological characterization was conducted by a TEM-STEM JEOL1400 (Ernest) transmission electron microscopy (TEM) with an accelerating voltage of 120 kV. For TEM observations, a drop of the Pt NPs solution was deposited onto copper grids (200 mesh, Agar Scientific Ltd). The grids were dried under a flow of N 2 under ambient conditions before observations. Statistical analysis of Pt NPs diameter was converted to cumulative number-based distributions with 'Image J' program.

Electrokinetic properties (ζ-potential) of Pt NPs were measured with ζ-potential

analyzer model Zeta Potential WALLIS (Cordouan Technologies, France). Pt NPs were diluted in Mili-Q water to reach 0.1 mM, and measured at 25ºC in a disposable folded capillary cell.

The optical absorption measurements were performed on a CARY 300 Scan UV-Visible Spectrophotometer (Int. Agilent) in the frequency range of 200 nm-800 nm at room temperature before and after irradiation. The as-synthesized Pt NPs were diluted with Milli-Q water to a final concentration of Pt [1 mM] for UV-visible absorption measurements.

Preparation of the samples

The HSA mother solution (20 mg/mL) was prepared by dissolving HSA powder in 10 mM phosphate buffer at pH 7.4 [START_REF] Shaw | Spectroscopic studies on the effect of temperature on pHinduced folded states of human serum albumin[END_REF]. The concentration of HSA was determined by absorbance measurements using molar extinction coefficients of 0,58 (g/L) -1 cm -1 at 280 nm [START_REF] Dockal | The three recombinant domains of human serum albumin structural characterization and ligand binding properties[END_REF].

Pt NPs-HSA samples at different Pt NPs/HSA molar ratios (1:30 to 1:1) were prepared by mixing a 15 μM HSA solution with different concentrations of Pt NPs in phosphate buffer at pH 7.0. The samples were incubated at room temperature for 40 minutes before measurements.

Dynamic light scattering

The hydrodynamic diameter of Pt NPs in the absence and presence of proteins (15 μM) (with molar ratios of Pt NPs/HSA from 1:30 to 1:1) was measured by dynamic light scattering (DLS) using a Cordouan VASCO-2 particle size analyzer (Cordouan Technologies, France). The Pt NPs were diluted in Mili-Q water to a final concentration of 0.5 mM, and the measurements were conducted at 25ºC in a semi-micro polystyrene (PS) cuvette.

Isothermal Titration Calorimetry (ITC)

Adsorption isotherms of HSA on Pt NPs were performed by calorimetry using a MicroCal PEAQ-ITC (Malvern). All the solutions were thoroughly degassed prior to the titrations to avoid the formation of bubbles in the calorimeter cell. The reaction cell (280 μL) was loaded with a HSA solution at 1 x 10 -4 mol/L. The syringe (75 μL) was filled with a Pt NPs solution at 2 x 10 -3 mol/L. The proteins and NPs were prepared in the same phosphate buffer (pH 7.4) to prevent any pH effect. The experiments were done in duplicate at 20 °C by adding 2.4 μL of the Pt NPs solution to the HSA solution with an equilibration interval of 200 s. Raw data were obtained as a plot of heat (μcal) against the injection number, and featured a series of peaks for each injection. These raw data peaks were transformed using the instrument software to obtain a plot of the enthalpy change per mole of injectant (∆H 0 , kcal•mol -1 ) against the molar ratio. Control experiments included the titration of the Pt NPs solution into buffer, buffer into HSA, and buffer into buffer, with the same used sample concentration. Corrected data refer to experimental data after subtraction of the control data.

Synchrotron radiation circular dichroism (SRCD) SRCD spectra acquisition

The SRCD spectra of samples were measured on the DISCO beamline, SOLEIL synchrotron radiation facility, Saint-Aubin, France.

Prior to the measurement, the instrument was calibrated using an aqueous solution of (+) camphorsulphonic acid (CSA). To prepare the sample, 4 μL Pt NPs-HSA solution was placed in CaF 2 cuvettes with a 50 μm pathlength [START_REF] Wien | Calcium fluoride micro cells for synchrotron radiation circular dichroism spectroscopy[END_REF].

The HSA spectra were collected between 170 and 261 nm with a step size of 1 nm and an integration time of 1200 ms/nm at 37ºC. All the spectra were recorded in triplicate, averaged, baseline subtracted and zeroed using CDToolX software [START_REF] Lees | CDtool-an integrated software package for circular dichroism spectroscopic data processing, analysis, and archiving[END_REF]. Raw data ellipticity expressed in millidegrees (mdeg) was converted to molar circular dichroism values (Δε). For the molar circular dichroism values (Δε, M -1 .cm -1 = L.mol -1 .cm -1 ), the following Eq. 1 was applied:

(1)

where the MRW (mean residue weight) of HSA is 113.8 Da (g.mol -1 ), P (pathlength) is 0.005 cm (measured by interferometry) and C (protein concentration) was ~1 mg/mL.

Thermal denaturation

Thermal denaturation studies were carried out by collecting SRCD spectra in a temperature range between 26 and 95°C with a step of 3°C. Three spectra were recorded with 5 minutes equilibration time for each temperature.

Ellipticity values were obtained from thermal denaturation curves for each of the three peaks (223 nm, 210 nm and 192 nm) at each temperature. They were normalized by setting the peak values at 26°C to 1.0. The decreasing of the relative intensity of protein with temperature has been fitted to a Boltzmann type equation Eq. 2 with the software OriginLab [START_REF] Laera | Measuring protein structure and stability of protein-nanoparticle systems with synchrotron radiation circular dichroism[END_REF].

(2) (where X is the temperature, X 0 the melting temperature (T m ), and dx the width of the thermal transition) for a simple two-state unfolding process. T m of the protein defined as the midpoint of the unfolding transition.

Structure analysis

The contents of α-helix, β-sheet, turn and other structures of HSA were calculated from SRCD spectra using BeStSel, a web server used for accurate secondary structure components determination [START_REF] Micsonai | BeStSel: A web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra[END_REF][START_REF] Micsonai | Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy[END_REF].

Results and discussions

Synthesis and characterization of Pt NPs

The Pt NPs synthesized in water by radiolysis were precisely characterized using DLS, TEM and the zeta potential technique (Figure 1). The Pt NPs observed by TEM show semi-spherical morphologies (Figure 1a) and a uniform size distribution with an average metallic core diameter of 3.0±0.8 nm (Figure 1b). DLS measurements showed that Pt NPs have an average hydrodynamic diameter of 18±2.6 nm (Figure 1c). Significant differences between TEM analysis and DLS measurement indicate the Pt NPs PEG coating. The zeta potential value of Pt NPs in MiliQ water is -14.2±4.1 mV. This result is in good agreement with experimental results of other works (Ehi-Eromosele, 2016; [START_REF] Ibrahim | SYNTHESIS AND ZETA POTENTIAL OF NOBLE METALS (Pt, Au[END_REF], the PEG coated NPs have an average negative ζ-potential of -14 mV and -17 mV, respectively, in water (pH 6~7), which leads to interparticle repulsion (Ehi-Eromosele, 2016).

[Figure 1]

The reduction of Pt complex and the formation of Pt NPs were monitored using UVvis absorption spectroscopy. Metallic NPs display strong optical absorptions due to their surface plasmon resonance (SPR). Platinum nanoparticles have a plasmon in the UV range [START_REF] Salado-Leza | Radio-enhancing properties of bimetallic Au: Pt nanoparticles: experimental and theoretical evidence[END_REF]Yang et al., 2020b). Figure 2 shows the UV-vis spectra of the aqueous solutions containing [Pt(NH 3 ) 4 ]Cl 2 and PEG-OH before and after irradiation.

The spectra reveal that, before irradiation, the aqueous solutions composed of [Pt(NH 3 ) 4 ]Cl 2 or [Pt(NH 3 ) 4 ]Cl 2 in the presence of PEG-OH, have the absorbance of the main peak close to 240 nm and a small shoulder around 290 nm, which correspond to the ligand-to-metal charge transfer (LMCT) band between (NH 3 ) 4 2+ and Pt II ions [START_REF] Gharibshahi | Influence of dose on particle size and optical properties of colloidal platinum nanoparticles[END_REF]. After irradiation, the two initial absorption peaks disappear due to the complete reduction of Pt II into Pt 0 leading to formation of Pt NPs.

Thus, there was no further charge transfer possible from ligand to metal ions [START_REF] Inwati | In situ free radical growth mechanism of platinum nanoparticles by microwave irradiation and electrocatalytic properties[END_REF]. Moreover, the appearance of a surface plasmon absorption band with a single broad peak centered at around 270 nm indicated the presence of Pt NPs [START_REF] Moores | The plasmon band in noble metal nanoparticles: an introduction to theory and applications[END_REF]Yang et al., 2020b).

The inserted picture in Figure 2 shows the color of the colloidal solution, which changed from colorless (before irradiation) to dark brown (after gamma irradiation).

The colour change is due to the radiolytic reduction of [Pt(NH 3 ) 4 ] 2+ ions to Pt 0 atoms indicating the formation of colloidal Pt NPs [START_REF] Gharibshahi | Influence of dose on particle size and optical properties of colloidal platinum nanoparticles[END_REF][START_REF] Salado-Leza | Green One-Step Synthesis of Medical Nanoagents for Advanced Radiation Therapy[END_REF] as mentioned before.

[Figure 2]

Dynamic light scattering analysis

In the present work, DLS was used to measure the hydrodynamic diameter of different concentrations of Pt NPs in the absence and presence of HSA at pH 7.4 in 10 mmol/L phosphate buffer. No significant change in the NPs size distribution after exposure to HSA at room temperature. It demonstrated there was no complex formation, which is probably due to the grafting of hydrophilic PEG-OH on Pt NPs having a high capacity to prevent the adsorption of proteins [START_REF] Pelaz | Surface functionalization of nanoparticles with polyethylene glycol: effects on protein adsorption and cellular uptake[END_REF][START_REF] Wang | Interaction of gold nanoparticles with proteins and cells[END_REF].

Furthermore, PEGylation might prevents the uptake of Pt NPs by Reticuloendothelial System (RES) and enhance the circulation lifetime of NPs inside the body [START_REF] Suk | PEGylation as a strategy for improving nanoparticle-based drug and gene delivery[END_REF].

[Figure 3]

Isothermal Titration Calorimetry

The binding properties of the Pt NPs with HSA were consecutively studied using ITC.

Figure 4a

and Figure 4b show the plot of enthalpy change (∆H 0 ) against Pt NPs-HSA molar ratio. The amount of heat due to the controls (dilution) is measured and subtracted from the amount of the heat obtained for the Pt NPs-protein system. ITC results did not show any significant binding, and no thermodynamic parameters could be obtained. This finding is consistent with previous studies on telodendrimer-based NPs having PEG in the surface [START_REF] Li | Probing of the assembly structure and dynamics within nanoparticles during interaction with blood proteins[END_REF]. Indeed, the functionalization of NPs by PEG prevents interactions with proteins [START_REF] Gref | Stealth'corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption[END_REF].

[Figure 4]

Synchrotron radiation circular dichroism (SRCD) SRCD spectra acquisition

The adsorption of proteins on the surface of nanomaterials, may induce conformational changes for some proteins, which influence the functionality and biological activity of the proteins [START_REF] Satzer | Protein adsorption onto nanoparticles induces conformational changes: particle size dependency, kinetics, and mechanisms[END_REF][START_REF] Shemetov | Molecular interaction of proteins and peptides with nanoparticles[END_REF]. The SRCD spectroscopy is widely used to study the conformation of proteins in aqueous solution. The far UV SRCD spectra (170~261 nm) provide the variations in the secondary structure of proteins as a result of the molecular interactions with other materials [START_REF] Prasanth | Spectroscopic exploration of interaction between PEG-functionalized Ag2S nanoparticles with bovine serum albumin[END_REF]. Figure 5 shows the SRCD spectra of pure HSA and HSA in the presence of different concentrations of Pt NPs (0.5 mM, 1 mM, 2 mM, and 15 mM), corresponding to Pt NPs-HSA ratios of 1:30, 1:15, 1:7.5, and 1:1. The spectrum exhibits two negative bands at 210 and 223 nm, one positive band at 192 nm, characteristic of the α-helical structure. The negative peak at 223 nm is due to the nπ* transition of the carbonyl group of peptide, while the parallel and perpendicular excitation of the peptide π  π * transition is responsible for the other negative peak at 210 nm and the positive peak at 192 nm [START_REF] Gobeaux | Reversible assembly of a drug peptide into amyloid fibrils: a dynamic circular dichroism study[END_REF].

With the addition of different amounts of Pt NPs to the HSA aqueous solution, the SRCD spectra of the Pt NPs-HSA mixture remained unchanged for three repeated scans at 37°C (Figure 5). This clearly indicates that Pt NPs does not induce a significant conformation change of the protein at 37°C.

[Figure 5]

Thermal denaturation

The thermal stability of proteins without or with Pt NPs was assessed by recording the intensity of SRCD signal at 192 nm as a function of temperature. SRCD spectra of pure HSA and Pt NPs-HSA at different ratios from 1:30 to 1:1 were recorded at temperatures in the range of 26~95°C. The temperature scans of Pt NPs:HSA=1:7.5 are presented in Figure 6. The spectra clearly indicate that the temperature increase causes a gradual decrease of the alpha helical profile.

[Figure 6]

The decreasing of the relative intensity of protein with temperature in Figure 7,

showed that the thermal denaturation curves were shifted with the increasing ratio of Pt NPs-HSA towards higher T m . The resulting T m is reported in Table 1. From the results, we observe the T m increases with increasing the Pt NPs concentration. The free HSA has a T m of 56°C, which is in good agreement with reported results that HSA undergoes morphological changes at 56°C [START_REF] Das | Temperature induced morphological transitions from native to unfolded aggregated states of human serum albumin[END_REF]. The T m of Pt NPs-HSA=1:1 is 74°C, increased 18°C compared to the T m of free HSA. The tremendous increase in T m suggests that Pt NPs increase the thermal stability of HSA. It was shown by other groups that silver NPs decrease the thermal stability of HSA, while gold NPs (using the same preparation method as silver NPs) did not significantly change it [START_REF] Laera | Measuring protein structure and stability of protein-nanoparticle systems with synchrotron radiation circular dichroism[END_REF] even at same ratio of NPs-HSA. However, different experimental conditions must be taken into consideration.

Structure analysis

A more detailed analysis of the two spectra using deconvolution software BeStSel allows estimating the percentage of secondary structure α-helix, β-sheet, turn and random coil elements present in each SRCD spectrum.

Figure 7 shows that the content of α-helix conformation of HSA decreases from 61% to 29%, on contrary β-sheet and random coil increase from 1% to 20% and 30% to 40% respectively, with a subsequent increase of unfolded protein, as a function of the increasing temperature. The turn component of HSA remains practically constant for all the samples ~10% (in the interval 26~95°C). The same trends were found in the presence of Pt NPs, except the structuration in beta sheet was significantly lower at high temperature with the addition of Pt NPs (ratios of Pt NPs-HSA ranging from 1:30 to 1:1).

Protein conformational compositions in pure HSA indicate an increase in β-sheet conformation from 0% to 20% at the expenses of the α-helix content (which diminished from 57% to 28%) when the temperature rises from 38°C to 89°C. As the temperature increases, the cooperative hydrogen bonds that stabilized the helical structure weakened and expose hydrophobic groups to the solvent, partially unfolding the protein structure, which favours the formation of beta-sheet-rich amyloid fibrils [START_REF] Juárez | Existence of different structural intermediates on the fibrillation pathway of human serum albumin[END_REF]. It was shown that the presence of NPs can influence the protein fibrillation process [START_REF] Mahmoudi | Protein fibrillation and nanoparticle interactions: opportunities and challenges[END_REF]. In this case, the alpha helix decreased less in the presence of Pt NPs than in pure HAS, and an increase in random coils substituted the α-helix loss, indicating that Pt NPs prevent inter-protein interactions, and thus increase the conformational stability of proteins.

[Figure 7]

Thermodynamic analysis

The biological responses to NPs are influenced by the forces (such as electrostatic, hydrophobic or Van der Waals forces) present at the bio-nano interface [START_REF] Mariam | Elucidation of structural and functional properties of albumin bound to gold nanoparticles[END_REF]. Hence it is essential to have a complete understanding of the thermodynamics of Pt NPs-HSA interaction. The thermodynamic properties (enthalpy variation, entropy variation) obtained by fitting the temperature changes of local helix content to the Van't Hoff equation Eq. 3 [START_REF] Greenfield | Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions[END_REF]:

(3

)
where K is the binding constant at the corresponding temperature, and R is universal gas constant. ΔH 0 and ΔS 0 are presented in Table 1. We found out a gradual increase of enthalpy variation and entropy variation in the presence of Pt NPs with increasing T m . It can be seen that the enthalpy variation increases significantly from 87 kJ/mol of pure HSA to 116 kJ/mol of Pt NPs-HSA=1:1. The same phenomenon was observed by other groups. It was explained by Senske et al, that nanoparticles can stabilize the native conformation of proteins compared to the unfolded state under external stresses such as temperatures [START_REF] Senske | Protein stabilization by macromolecular crowding through enthalpy rather than entropy[END_REF]. This stabilization is explained by an unfavourable interaction of the protein and the nanoparticles leading to a preferential exclusion of the nanoparticles from the protein surface and a preferential hydration of the protein. Since the solvent accessible surface area is usually increased for the unfolded state, in the presence of Pt NPs, this would increase the thermodynamically unfavourable situation and require more energy for unfolding (unfolding enthalpy variation would increase with the Pt NPs concentration) than in water. As a result, the folding equilibrium should tend to shifted toward the more folded, native state.

Finally, the assumption of the increase of the HSA stability with Pt NPs was supported by good agreement with the thermodynamic data and structure analysis for the thermal unfolding transition determined by far-UV SRCD.

[Figure 8]

Conclusion

In summary, we have successfully synthesized Pt NPs with homogeneous size using radiolytic reduction method by gamma irradiation. The DLS and TEM images indicate that the synthesized Pt NPs are semi-spherical in shape and uniform in size.

UV-Vis spectra suggest the complete reduction of Pt II and the formation of Pt NPs. The coherent results of hydrodynamic diameter and ITC demonstrated that Pt NPs do not bind to HSA even at the high ratio of 1 Pt NP per HSA. SRCD secondary spectra show that even the high concentrations of Pt NPs do not induce significant change of the protein conformation. The thermal stability of protein is assessed by SRCD by monitoring the spectral changes in the spectra with increasing temperature. More importantly, an increase of 18℃ in the thermal unfolding of HSA upon interaction with Pt NPs was found, which support the fact that Pt NPs stabilize the HSA structure. This structure analysis showed that, as the temperature rises, the loss of α-helical content of HSA was decreased whereas a significant reduction in β-sheet was observed. This is due to increased stability of HSA in the presence of Pt NPs. The modification of the protein stability can be attributed to the preferential hydration of the protein. This stabilizing effect renders Pt NPs highly promising and applicable to be used as biocompatible stabilizing agents for proteins.

Author contribution

X.Y. synthesized and characterized Pt NPs, performed and analysed the DLS, UV-Vis spectroscopy, carried out the SRCD spectra analysis, data interpretation and drafted the manuscript. E.P. supervised the experimental protocols. L.M., S.P., J.P.R., A.K. .

  conceived and performed the experiments of ITC. D.S.L, C. G., A. K. and X.L. contributed to the paper modifications. F.W., X. Y., E. P., A.K., C. G. optimized and performed the SRCD experiments. H.R., and D.S.L were engaged in nanoparticles design, synthesis and characterization. S.L. conceived the entire project, reviewing and editing the manuscript. All authors revised the manuscript.

Figure 1

 1 Figure 1 (a) TEM image and (b) HRTEM images of Pt NPs; (c) TEM size distribution

Figure 2

 2 Figure2UV-vis absorption spectra of Pt(NH 3 ) 4 Cl 2 (green line) solution (10 -3 mol.L -1 ), PEG-OH (red line) solution (10 -1 mol.L -1 ), and diluted Pt containing solutions (10 -3 mol.L -1 ) before (blue line) and after (pink line) irradiation (optical path = 2 mm). Inset

Figure 3

 3 Figure 3 Hydrodynamic diameter of Pt NPs and Pt NPs at different Pt NPs-HSA ratios

Figure 4 .

 4 Figure 4. Isothermal titration calorimetry (ITC) plots of differential power throughout

Figure 5

 5 Figure 5 SRCD spectra of HSA alone and Pt NPs-HSA at NPs:HSA molar ratios of

Figure 6

 6 Figure 6 Temperature scans of Pt NPs:HSA=1:7.5 with steps of 3°C (from 26°C to

Figure 7

 7 Figure 7 Thermal denaturation curves of pure HSA and the Pt NPs-HSA complex at

Figure 8

 8 Figure 8 Contribution of the protein conformations: alpha-helix (a), beta sheet (b), turns

Table 1 . Values of the melting temperatures Tm, enthalpy variations ΔH° and entropy variations ΔS° for HSA and Pt NPs-HSA at different concentrations (ratios from 1:30 to 1:1) monitored at 192 nm.

 1

	Sample	Tm,℃	ΔH°, kJmol -1 ΔS°, kJmol -1 K -1
	HSA	56±0.3	87.3±1.9	0.26±0.006
	Pt NPs-HSA 1:30	61±0.3	89.5±1.3	0.27±0.004
	Pt NPs-HSA 1:15	64±0.4	92.8±2.0	0.27±0.006
	Pt NPs-HSA 1:7.5	69±0.4	101.3±4.2	0.28±0.010
	Pt NPs-HSA 1:1	74±0.4	116.4±4.5	0.35±0.014

Acknowledgements X.Y. acknowledges support from China scholarship council (CSC, N° 201607040068). We acknowledge support from the Université Paris Saclay for the "Inititative de Recherche Stratégique" IRS NanoTheRad project. This work also benefited from the RESPORE grant. SRCD measurements on DISCO beamline at SOLEIL synchrotron light source were performed under the proposal 20171494. We thank Ruxandra Gref (Institut des Sciences Moléculaires d'Orsay, Université Paris Saclay) for providing kind help on ITC experiments.


T Widemann

R C Ghail

C F Wilson

D V Titov

W S Kiefer

B Campbell

S Hensley

Alice Le Gall

EnVision: Undestanding why Earth's

Introduction: EnVision [START_REF] Ghail | EnVision M5 proposal[END_REF]2] is a Venus orbiter mission proposal that will determine the nature and current state of geological activity on Venus, and its relationship with the atmosphere, to understand how and why Venus and Earth evolved so differently. EnVision is one of three competing ESA M5 mission concepts currently in their study phase (A1-A2) with a final downselection expected in 2021. The EnVision mission is studied in collaboration with NASA, with the potential sharing of responsibilities currently under science, technical and programmatic assessment.

If selected, the proposed international mission will launch in 2032 on Ariane 62. Following orbit insertion and periapsis walk-down, orbit circularisation will be achieved by aerobraking over a period of several months, followed by a nominal science phase lasting at least 6 Venus days (4 Earth years).

EnVision will use a number of different techniques to search for active geological processes, constrain style and occurrence rate of current-day volcanism on Venus, by searching for thermal, morphological and volatile signatures in repeated observations of the surface and of the atmosphere. It will characterise regional and local geological features, determine crustal support mechanisms and constrain mantle and core properties:

The Synthetic Aperture Radar, VenSAR, will address the key science objectives of the EnVision mission. It will:

-Obtain images at a range of spatial resolutions from regional coverage to images of targeted localities;

-Search for changes in surfa&ce radar imagery; -Measure surface topography regionally by means of stereo radar imaging; and globally by means of nadir altimetry;

-Characterize surface polarimetric reflection and emission properties using both SAR and radiometer measurements.

The Subsurface Sounder, SRS, will be the first instrument to investigate the subsurface structure of Venus and acquire fundamental information on subsurface geology; it will:

-Characterize the vertical structure and stratigraphy of geological units including volcanic flows; and -Determine the depths of weathering and aeolian deposits.

The Venus Spectrometer suite, VenSpec, will: -Obtain global maps of surface emissivity in five wavelength bands in the near-infrared to constrain surface composition and inform evolution scenarios [START_REF] Helbert | The Venus Emissivity Mapper (VEM) -obtaining global mineralogy of Venus from orbit[END_REF]; and -Measure variations of SO2, SO and chemicallyrelated gaseous species in the mesosphere and nightside troposphere, in order to link these variations to atmospheric dynamics, chemistry and volcanism.

The Radio Science experiment will:

-Constrain crustal & lithospheric structure at finer spatial scale than Magellan;

-Measure spin rate and spin axis variations to constrain interior structure; and -Measure spatial and temporal variations of H2SO4 liquid and vapor at 55-35 km.

EnVision will produce a huge dataset of geophysical data of a quality similar to that available for Earth and Mars, and will permit investigation across a large range of disciplines. Lab-based and modelling work will also be required to interpret results from the mission. We therefore invite scientists from across planetary, exoplanetary and Earth science disciplines to participate in the analysis of the data.

References: