Abdallah Amedlous

Charly Hélaine

Rémy Guillet-Nicolas

Oleg Lebedev

Samuel Valable
email: valable@cyceron.fr

Svetlana Mintova
email: svetlana.mintova@ensicaen.fr

Gadolinium-loaded LTL nanosized zeolite for efficient oxygen delivery and magnetic resonance imaging

Keywords: zeolite LTL, injectable nanoparticles, biocompatibility, oxygen delivery, biomedical application

The key challenges in finding efficient gas carriers for biomedical applications are related to the kinetics of gas adsorption/desorption of nontoxic materials over an appropriate time intervals, specifically targeting the affected area. Herein, we report the preparation of nanosized gadolinium (Gd-LTL) zeolite crystals with a diameter of 10-15 nm for oxygen delivery combined with magnetic resonance imaging (MRI) study. The partial ion-exchange of potassium (K) extra framework cations used for the synthesis of nanosized K-LTL zeolite with paramagnetic gadolinium (Gd, 1.53 wt%) changed considerably its adsorption capacity for O2.

The nanosized Gd-LTL zeolite retained high colloidal stability, remarkable crystallinity and no leaching of Gd ions was observed. In addition, the in vitro toxicity study performed in human glioblastoma-derived cell lines revealed that nanosized K-LTL and Gd-LTL nanosized zeolites showed no toxicity even at high concentrations. This study provides promising guidelines regarding the design of smart biocompatible nanosized zeolites coupling gas-delivery and MRI proprieties.

Introduction

Zeolites are microporous, crystalline materials with well-defined compositions and structures with regular pores and cavities of molecular dimensions [START_REF] Gallego | Simple Organic Structure Directing Agents for Synthesizing Nanocrystalline Zeolites[END_REF][START_REF] Moliner | Multipore Zeolites: Synthesis and Catalytic Applications[END_REF] . Zeolite synthesis remains an active field of research and finding new nontraditional applications are highly desired including in biomedicine. The application of zeolites is driven by their physicochemical properties, i.e., chemical composition, pore size, crystalline structure, shape/size of particles as well as chemical/thermal and colloidal stability [START_REF] Yazdi | Zeolites for Theranostic Applications[END_REF][START_REF] Zeng | Microwave Synthesis of Zeolites and Their Related Applications[END_REF][START_REF] Wang | Zeolite Nanosheets for Catalysis[END_REF][START_REF] Mintova | Nanosized Microporous Crystals: Emerging Applications[END_REF] . Currently, there are 255 types zeolite frameworks according to the International Zeolite Association (IZA) database, and most of them are used for catalysis, gas adsorption, and separation [START_REF]Database of Zeolite Structures[END_REF] . In spite of this versatility, several zeolites have been considered for biomedical applications [START_REF] Serati-Nouri | Biomedical Applications of Zeolite-Based Materials: A Review[END_REF] . In recent years, considerable efforts have been devoted to the decrease of zeolite crystal size from micron to nanoscale dimensions resulting in nanozeolites with large surface area, high stability in suspension, short diffusion length, and accessible active sites [START_REF] Awala | Template-Free Nanosized Faujasite-Type Zeolites[END_REF][START_REF] Ng | Capturing Ultrasmall EMT Zeolite from Template-Free Systems[END_REF][START_REF] Grand | Flexible Template-Free RHO Nanosized Zeolite for Selective CO2Adsorption[END_REF][START_REF] Mintova | Advances in Nanosized Zeolites[END_REF] . In addition, the synthesis of nanosized zeolites in the absence of organic templates is highly desired for applications in the biomedical field. The presence of organic template in zeolites required activation at elevated temperatures resulting in irreversible agglomeration and not stabilization of injectable colloidal suspensions [START_REF] Wang | Insights into the Organotemplate-Free Synthesis of Zeolite Catalysts[END_REF][START_REF] Georgieva | Control of Na-EMT Zeolite Synthesis by Organic Additives[END_REF][START_REF] Georgieva | Iron Loaded EMT Nanosized Zeolite with High Affinity towards CO2 and NO[END_REF] . While the organic template-free hydrothermal synthesis allowed the preparation of biocompatible zeolites. The feasibility of using nanosized zeolites for biomedical applications has been demonstrated from several groups already [START_REF] Mintova | Advances in Nanosized Zeolites[END_REF][START_REF] Mintova | Nanosized Zeolites: Quo Vadis?[END_REF] . For instance, the CO2 and O2 sorption properties of faujasite (Na-FAU-X) was controlled by partial ion-exchange of the sodium extra-framework cations by trivalent metals (Gd 3+ , Ce 3+ ) [START_REF] Komaty | Incorporation of Trivalent Cations in NaX Zeolite Nanocrystals for the Adsorption of O2 in the Presence of CO2[END_REF] . The high suspension stability and non-toxicity of nanosized FAU zeolite showed advantages as hyperoxic/hypercapnic gas carriers for glioblastoma treatment [START_REF] Anfray | Nanosized Zeolites as a Gas Delivery Platform in a Glioblastoma Model[END_REF] . More recently, nanosized FAU zeolite with luminescent properties was developed enabling to track the fate of zeolites in vitro experiments on cell culture [START_REF] Komaty | Ruthenium Tris(2,2′-Bipyridyl) Complex Encapsulated in Nanosized Faujasite Zeolite as Intracellular Localization Tracer[END_REF] . Additionally, several studies have examined the efficiency of nanozeolites such as FAU, LTA, and LTL as drug carriers, therapeutic agents, and probes for magnetic resonance imaging [START_REF] Amorim | Zeolite Structures Loading with an Anticancer Compound as Drug Delivery Systems[END_REF][START_REF] Zheng | Ultrasound and Near-Infrared Light Dual-Triggered Upconversion Zeolite-Based Nanocomposite for Hyperthermia-Enhanced Multimodal Melanoma Therapy via a Precise Apoptotic Mechanism[END_REF][START_REF] Zhang | Surface PEG Grafting Density Determines Magnetic Relaxation Properties of Gd-Loaded Porous Nanoparticles for MR Imaging Applications[END_REF][START_REF] Mayer | Nanozeolite-LTL with GdIII Deposited in the Large and Eu III in the Small Cavities as a Magnetic Resonance Optical Imaging Probe[END_REF] . Nevertheless, relatively few studies have explored the use of nanosized zeolites as oxygen delivery systems for hypoxic tumor reoxygenation. Oxygen supply in tumors is highly desired, even transiently because a lack of oxygen is associated with tumor development and resistance to treatment [START_REF] Horsman | Imaging Hypoxia to Improve Radiotherapy Outcome[END_REF][START_REF] Liu | Modulating Hypoxia via Nanomaterials Chemistry for Efficient Treatment of Solid Tumors[END_REF] .

The key challenge of the oxygen delivery system is to release the oxygen molecules over an appropriate time period and specifically target the affected area. Therefore, the development of novel safer nanomaterials with controllable oxygen-release feature for targeted gas therapy applications is urgently needed. Zeolite LTL is an interesting candidate for this purpose since it is composed of uniform onedimensional 12-MR rings running along the c-axis [001] of the crystals with a pore diameter of 0.71 x 0.71 nm, being suitable to host many gas molecules of interest. The LTL zeolite is hexagonal with a space group P6/mmm. The LTL structure is characterized by cancrinite cages linked by double 6-rings (D6R) leading to the formation of columns along the c axis resulting in 12-membered rings (12MR). The classical synthesis of LTL zeolites is performed in potassium-rich aluminosilicate precursors; by varying the amount of potassium cations, the state of the precursor can be varied from dense to clear suspensions. In the LTL zeolite, three extra-framework K + cations sites are present: in the center of the cancrinite cage (site K1), in the center of the 8MR channel midway between adjacent cancrinite cages (site K2), and near the walls of the main 12MR channel (site K3) [START_REF] Price | Determining the Structure of Zeolite Frameworks at High Pressures[END_REF][START_REF] Gigli | Compressibility Behavior and Pressure-Induced over-Hydration of Zeolite KAlSi-L[END_REF] . Cations-exchange in aqueous dispersions of zeolite-LTL always occurs in the K3 sites, while the K2 ones are only involved when K + is substituted by cations with similar ionic sizes, e.g. NH4 +23,28,29 , and the K1 sites are not accessible for exchange. In addition, the ion-exchange process can also generate new cation sites depending on the substituted ions [START_REF] Confalonieri | Ce-Exchange Capacity of Zeolite L in Different Cationic Forms: A Structural Investigation[END_REF] .

In this work, we describe the synthesis of organic template-free nanosized zeolite L (LTL) using only K + as an inorganic structure-directing agent. Loading of Gd 3+ in LTL zeolite was achieved via an ion-exchange procedure. Gd 3+ was selected as a positive contrast agent in order to improve the clarity and definition of the tumor using MRI. The as-prepared K-LTL nanosized zeolite crystals exhibit high colloidal stability and homogenous particle sizes with dimensions of 10-15 nm and Si/Al ratio of 2.59. In addition, in vitro toxicity experiments were performed in human glioblastoma-derived cell lines, and the results confirm that nanocrystals are safe and non-toxic. Furthermore, the O2 adsorption capacity of the LTL samples was investigated by adsorption isotherms and in situ Fourier transform infrared (FTIR) spectroscopy. The high oxygen release in hypoxic conditions and the T1 relaxivity of the Gd-LTL nanocrystals with great potential for MRI are demonstrated.

Experimental section 1.1 Chemicals

Potassium hydroxide (KOH, 90 %), sodium hydroxide pellets (NaOH, 98%), colloidal silica (SiO2, Ludox SM30, 30%), and gadolinium nitrate hexahydrate (Gd(NO3)•6H2O, 99.99%) were purchased from Sigma-Aldrich. Aluminum hydroxide (Al(OH)3, 76.5%) was purchased from Alfa Aesar. All reagents were used as received without further purification. Mili-Q water was used for all synthesis and purification of samples.

Synthesis of LTL nanocrystals

The initial chemical composition used for the synthesis of nanosized LTL zeolite was 5 K2O: 10 SiO2: 0.5 Al2O3: 200 H2O. Typically, in 8 g water, aluminum hydroxide (2.1 g) and potassium hydroxide (0.51 g) were dissolved and stirred until the mixture became transparent.

The silica solution was prepared by mixing 10 g of Ludox ® SM and potassium hydroxide (1.0 g) with 2.86 g of water. The two solutions were mixed by slowly adding the silica solution into the potassium aluminate one under stirring. After 24 hours of stirring, the precursor mixture was transferred to a Teflon-lined stainless-steel autoclave and heated under static conditions in an oven at 170 °C for 18 h. Finally, the colloidal K-LTL nanocrystals (2.5 wt. %) were purified with water using high-speed centrifugation (RPM 20000, G 37565) until neutral pH is reached.

The ion-exchange of LTL zeolite was carried out directly in the purified colloidal suspensions without drying in order to maintain its colloidal stability of the nanocrystals and prevent irreversible aggregation. The ion-exchanged was performed two times with a 0.03 mM of Gd(NO3)•6H2O solution and 5 ml of 2.5 Wt.% of K-LTL zeolite as described elsewhere [START_REF] Komaty | Incorporation of Trivalent Cations in NaX Zeolite Nanocrystals for the Adsorption of O2 in the Presence of CO2[END_REF] . The resulting Gd-exchanged LTL zeolite was purified with water and finally stored in the form of colloidal suspension in aqueous solution.

Characterization

The crystallinity of the samples was characterized by powder X-ray diffraction (PXRD) using a PANalytical X'Pert Pro diffractometer using Cu Kα1 radiation (λ = 1.5406 Å, 45 kV, 40 mA).

The porosity of the samples was determined by N2 adsorption/desorption isotherms measured at -196 °C. The O2 adsorption isotherm on LTL zeolite was measured at 0, 25 and 37 °C using a Micromeritics 3Flex Surface Characterization unit. All samples were degassed at 250 °C under vacuum overnight prior to adsorption analysis. The external surface area and micropore volume were assessed by the t-plot method. The micropore and mesopore size distributions of samples were extracted from the adsorption and desorption branches of the isotherms using the Nonlocal Density Functional Theory (NLDFT) method.

The oxygen adsorption study on nanosized LTL zeolite was performed by in situ Fouriertransform infrared (FTIR) spectroscopy suing a Nicolet 6700 IR spectrometer equipped with a mercury cadmium telluride (MCT) detector. The spectra were recorded in the range of 1300-4000 cm -1 at a resolution of 4 cm -1 after an accumulation of 256 scans. The samples were pressed (~10 7 Pa) into self-supported disks (2 cm 2 area, 20 mg•cm -2 ) and placed in an IR cell equipped with KBr windows. The activation of the zeolite samples was performed in situ at 300 °C for 4 h under vacuum and cooled down to -196 °C. Various amounts of pure oxygen gas were introduced into the cell at 0-100 Torr equilibrium pressure. The spectrum of activated zeolite was used as a background for collecting the difference spectra, which were normalized to the sample mass and plotted as absorbance per gram over wavelength.

The surface morphology and the element composition of samples were studied by Scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX) using a Tescan Mira I LMH under 20 kV. The shape and size of nanoparticles were studied by high-resolution transmission electron microscopy (HRTEM) with an FEI Tecnai G2 30 microscope (Vacc = 300 kV, LaB6).

The thermal stability of samples was evaluated by thermogravimetric analysis (TGA) using a TAG24 SETARAM analyzer under a 40 ml•min -1 airflow. The measurements were carried out between 25 and 800 °C with a heating rate of 10 ºC•min -1 .

Magic-angle spinning nuclear magnetic resonance (MAS-NMR) experiments of samples were

recorded with a single pulse on a Bruker Avance 500 MHz (11.7 T) spectrometer using 4 mm OD zirconia rotors with a spinning frequency of 12 kHz.

The chemical composition of samples was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) using a 7900 ICP-MS from Agilent Technologies. The samples were prepared according to the following procedure: (i) 50 mg of sample was dissolved in 3 mL of hydrofluoric acid (HF) (Sigma-Aldrich 40-45%), then (ii) a 0.5 mL of mixture (HNO3/HCl ≡ 1:3 v/v) was added and heated at 110 °C for 1 h in the polytetrafluoroethylene (PTFE) bottle (100 mL), and (iii) 96.5 mL of double distillated water and 2 g of boric acid (H3BO3) were added. The resulting solution was stirred overnight to facilitate dissolution of the boric acid.

Finally, 10 ml of the solution was diluted 10 times with double distillated water before analysis.

The colloidal stability and average size of particles dispersed in water were determined using dynamic light scattering (DLS) performed with a Malvern Zetasizer Nano instrument. The surface charge of the samples was measured by conducting zeta potential measurements of the same suspensions.

Cell culture and cell viability

The human glioblastoma-derived cell line U251-MG (Cellosaurus_0021) and U87-MG (Cellosaurus_0022) purchased from National Cancer Institute (NCI, Bethesda, MD, USA) and American Type Culture Collection (ATCC, LGC standards, Molsheim, France) respectively, were cultured in DMEM containing 1 g/L of glucose (Dulbecco's Modified Eagle's Medium, Sigma-Aldrich, St Quentin Fallavier, France) supplemented with 10% fetal calf serum (Eurobio, Courtaboeuf, France), 2 mM L-glutamine (Sigma-Aldrich), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich). The cell line was maintained in culture at 37 °C with 5% CO2 and 95 % humidity.

Cell viability analyses were performed under exposure to increased concentration of nanosized zeolites for 24 h and 72 h with a WST-1 assay (Roche, Bale, Switzerland) according to the manufacturer's instruction.

Hemolysis assay

The red blood cells (RBC) were collected from fresh rat blood by centrifugation (1000 rpm, 5 min) and suspended in PBS solution. Then, K-LTL or Gd-LTL zeolite nanoparticles at various concentration were incubated with 500 μL RBC suspension. Saline and 1% Triton X-100 were used as negative and positive controls, respectively. After the incubation at 37 °C for 1 h, the samples were centrifugated (2000 rpm, 10 min) and each supernatant at 540 nm was analysed by a spectrophotometer (Spark®, TECAN, Männedorf, Switzerland). The following formula was used to calculate the percent of hemolysis of RBC.

𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 (%) = 𝑆𝑎𝑚𝑝𝑙𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 -𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 -𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

× 100

Analysis of oxygen release using LTL nanozeolites

The oxygen release capacity of LTL nanozeolite in aqueous solution was studied in a hypoxia workstation (IN VIVO 2 500, Baker Ruskinn, Alliance Bio Expertise, Guipry, France) set at 0.1% O2, 5% CO2 at 37 °C in humidified atmosphere as previously reported [START_REF] Anfray | Nanosized Zeolites as a Gas Delivery Platform in a Glioblastoma Model[END_REF] . A closed reaction tube containing 12 mL of equilibrated PBS at 0.1% O2 and 37 °C was used, and a dissolved oxygen sensor (SevenGo (Duo) pro™/OptiOx™, Mettler Toledo, USA) was immersed in the PBS solution. Prior to experiments, baseline was established by measuring the oxygen saturation in the PBS solution for 30 min. Oxygen-saturated nanosized zeolites (500 µL at 1 wt. %) were then added to the system and oxygen dissolved in the PBS solution was measured for 60 min. The oxygen liberation capacity of as prepared and ion-exchanged nanosized zeolites was compared to oxygen-saturated water (used as control). to the relaxivity (mM -1 •s -1 ). In this study 1 mM Dotarem® and water were used as a control samples.

Results and discussions

Physicochemical characterization of LTL nanozeolites

The crystallinity of as-prepared K-LTL and ion-exchanged Gd-LTL zeolites were investigated by powder X-ray diffraction. As it can be seen in Figure 1a, the PXRD patterns of the pristine K-LTL and Gd-LTL zeolite samples are identical and exhibit similar peaks being assigned to the LTL-type framework topology [START_REF] Treacy | International Zeolite Association. Structure Commission. Collection of Simulated XRD Powder Patterns for Zeolites[END_REF] . The relatively broad peaks are originating from the small size of the crystals. The average crystallite size is approximately 15 nm, according to Debye-Scherrer equation [START_REF] Scherrer | Estimmung Der Grosse Und Der Inneren Struktur von Kolloidteilchen Mittels Rontgenstrahlen[END_REF] . Although the LTL zeolite was subjected to an ion-exchange process, no new crystal phase was detected, and the original crystallinity is maintained. Moreover, no shift was observed in peaks positions of Gd-LTL as compared to the pristine material, indicating that the unit cell parameters, as well as particle size, were not altered after the ion-exchange process.

The K-LTL and Gd-LTL zeolite particle's morphology and size were characterized by SEM and TEM. According to the SEM (Figure 1b), both K-LTL and Gd-LTL consist of welldefined small nanocrystals. After the ion-exchange process, no significant changes were observed. The chemical compositions of the K-LTL and Gd-LTL are obtained by EDX spectrometry (Figure S1 and Figure 1c). The EDX results confirm that potassium was only partially exchanged by gadolinium. Such behavior is not surprising since, potassium ions in K1 and K2 sites are very stable (Figure S2) [START_REF] Confalonieri | Ce-Exchange Capacity of Zeolite L in Different Cationic Forms: A Structural Investigation[END_REF][START_REF] Vezzalini | Ion Exchange Capacity of Synthetic Zeolite L: A Promising Way for Cerium Recovery Giorgia Confalonieri European Synchrotron Radiation Facility[END_REF][START_REF] Mayer | Nanozeolite-LTL with GdIII Deposited in the Large and EuIII in the Small Cavities as a Magnetic Resonance Optical Imaging Probe[END_REF] . The HR-TEM observations show a uniform distribution of well-defined nanocrystals with a high crystallinity. Moreover, HR-TEM showed that LTL particles exhibited elongated cylindrical shape of size of 10 x 20 nm (Figure 2). Since it is of particular importance for the targeted applications, the particle size distribution as well as the colloidal stability of the water suspensions containing K-LTL and Gd-LTL were studied by DLS (Figure 3). The amount of gadolinium loaded in LTL zeolite is about 1.53 wt.%, which corresponds to ∼0.34 Gd 3+ ions per unit cell. The chemical composition of the samples is presented in Table 1. The results show that potassium is only partially exchanged with gadolinium. SiO4 and AlO4 tetrahedra in zeolites [START_REF] Król | Application of IR Spectra in the Studies of Zeolites from D4R and D6R Structural Groups[END_REF] . The bands at 762 and 724 cm -1 correspond to symmetric stretching vibrations of Si-O-Al-O-T (T = Si or Al) and the band centered at 458 cm -1 is attributed to bending vibrations [START_REF] Flanigen | Infrared Structural Studies of Zeolite Frameworks[END_REF] . The Si/Al ratio of the zeolite samples is also calculated using FTIR data according to reference [START_REF] Ma | Facile and Fast Determination of Si/Al Ratio of Zeolites Using FTIR Spectroscopy Technique[END_REF] (Table 1). In addition, the isolated silanols (Si-OH) for K-LTL and Gd-LTL were studied after activation of the samples at 300 °C (Figure 5b). Both samples contain bands in the range of 3765-3505 cm -1 , which are typical of isolated external silanol groups. These bands have a high relative intensity because of the small crystal size, which has already been identified as a spectral characteristic of nanosized zeolites with welldeveloped external surface area [START_REF] Medeiros-Costa | Silanol Defect Engineering and Healing in Zeolites: Opportunities to Fine-Tune Their Properties and Performances[END_REF] .

Figure 5. IR spectra a) using KBr technique in the range 400-4000 cm -1 and b) using selfsupported pallet in the range 3000-3800 cm -1 of K-LTL and Gd-LTL nanosized zeolites The solid-state [START_REF] Vezzalini | Ion Exchange Capacity of Synthetic Zeolite L: A Promising Way for Cerium Recovery Giorgia Confalonieri European Synchrotron Radiation Facility[END_REF] Si and [START_REF] Gigli | Compressibility Behavior and Pressure-Induced over-Hydration of Zeolite KAlSi-L[END_REF] Al NMR spectra of K-LTL and Gd-LTL samples are shown in Figure 6a-d. The spectra of both samples show broad peaks convoluted in five peaks at -84, -90, -96, -100, and -105 attributed to Q0 (4Al), Q1 (3Al), Q2 (2Al), Q3 (1Al), and Q4 (0Al) species, respectively. The Si/Al ratio of the samples is determined by the Engelhardt equation from the deconvolved [START_REF] Vezzalini | Ion Exchange Capacity of Synthetic Zeolite L: A Promising Way for Cerium Recovery Giorgia Confalonieri European Synchrotron Radiation Facility[END_REF] Si MAS NMR spectra. The Si/Al ratio for K-LTL and Gd-LTL is 2.52 and 2.47, respectively. The 27 Al MAS NMR spectra reveal a single resonance peak at 60 ppm, thus confirming that no extra-framework aluminum both K-LTL and Gd-LTL is present.

Figure 6: a) [START_REF] Vezzalini | Ion Exchange Capacity of Synthetic Zeolite L: A Promising Way for Cerium Recovery Giorgia Confalonieri European Synchrotron Radiation Facility[END_REF] Si and b) [START_REF] Gigli | Compressibility Behavior and Pressure-Induced over-Hydration of Zeolite KAlSi-L[END_REF] Al MAS NMR spectra of K-LTL and c) [START_REF] Vezzalini | Ion Exchange Capacity of Synthetic Zeolite L: A Promising Way for Cerium Recovery Giorgia Confalonieri European Synchrotron Radiation Facility[END_REF] Si and d) [START_REF] Gigli | Compressibility Behavior and Pressure-Induced over-Hydration of Zeolite KAlSi-L[END_REF] Al MAS NMR spectra of the Gd-LTL nanosized zeolites.

The porosity of zeolite nanocrystals was further investigated by nitrogen adsorption-desorption measurements. The nitrogen adsorption isotherms and the corresponding pore size distribution are shown in Figure 7a-b; the textural parameters are summarized in Table 2. Both samples display hybrid type I and IV isotherms, according to the IUPAC classification [START_REF] Sing | Reporting Physisorption Data for Gas/Solid Systems with Special Reference to the Determination of Surface Area and Porosity (Recommendations 1984)[END_REF] . Such isotherms are typically observed for monodisperse microporous nanoparticles. In addition, both materials exhibit a H1-type hysteresis loop. The mesoporosity is originating from the interparticle voids. The Brunauer-Emmett-Teller surface area for K-LTL and Gd-LTL samples is 591 and 546 m 2 /g, and the total pore volume is 0.76 and 0.81 cm 3 /g, respectively (Figure 7ab). Both samples exhibit the same micropore volume of 0.14 cm 3 /g. In addition, the pore size distribution for both samples is calculated with a NLDFT model (Figure 7b). The results confirmed that K-LTL and Gd-LTL samples exhibit one family of supermicropore (0.7<w<2 nm) with an average size of 0.75 nm which is in accordance with the mono-dimensional channel system [START_REF] Gigli | Compressibility Behavior and Pressure-Induced over-Hydration of Zeolite KAlSi-L[END_REF] in LTL zeolite and the presence of mesopores with a size of 2-15 nm.

Assessment of potential toxicity of LTL nanozeolites

To assess the biocompatibility of LTL nanozeolites as oxygen-carrier or contrast agent, cytotoxicity measurements were performed on different human cell lines. U251-MG and U87-MG cells were exposed to as-prepared and ion-exchanged nanosized zeolites at concentrations of 10 or 100 μg/mL for 24 h and 72 h, followed by cell viability measurements. As shown in The evaluation of hemocompatibility of nanoparticles is an important point to be considered prior using for in vivo applications. In this study, the hemocompatibility of LTL nanoparticles was evaluated with hemolysis assay using rat blood incubated with variable concentrations of zeolite nanoparticles [10 to 500 µg/mL]. As shown in Figure 8e, the incubation of blood with K-LTL or Gd-LTL zeolite nanocrystals for 1 h resulted in a maximum hemolytic ratio of 0.942 ± 0.771 % and 0.722 ± 0.401 %, respectively for the highest concentration. These results clearly showed an absence of toxicity of nanozeolites to RBC. Indeed, the hemolysis rate of the samples is less than 5%, which is considered as a safe critical value for biomaterials according to the ISO/TR 7406.

Oxygen adsorption capacity of LTL nanozeolites

The equilibrium adsorption isotherms of oxygen on nanosized K-LTL and Gd-LTL zeolites at different temperatures are depicted in Figure 9a-b. The amount of O2 (100 kPa) adsorbed on nanosized K-LTL zeolites at 0, 25 and 37 °C is 5.27 cm 3 g -1 , 3.53 cm 3 g -1, and 3.09 cm 3 g -1 , respectively. As expected, the amount adsorbed oxygen decreases with the temperature. The nanosized K-LTL has a high oxygen adsorption capacity compared to the reported values for micron-sized LTL zeolites of 2.3 cm 3 g -1 (100 KPa, 25 °C) and for commercial faujasite zeolite X of 2.96 cm 3 g -1 (100 KPa, 37 °C) [START_REF] Sebastian | Sorption of Nitrogen, Oxygen, and Argon in Silver-Exchanged Zeolites[END_REF]40 . This can be due to the different chemical composition and diffusion paths in nanosized zeolites It is also proposed that the oxygen molecules interact primarily with the zeolite through its Brønsted acid sites and extra-framework cations.

Therefore, when K + cations are partially ion-exchanged with trivalent Gd 3+ , the O2 adsorption capacities increases. For Gd-LTL, the O2 capacities at 0, 25 and 37 °C is found to be 5.78 cm 3 g -1 , 4.60 cm 3 g -1, and 3.71 cm 3 g -1 respectively. The overall increase in the adsorption capacity is attributed to the removal of K + cations. Indeed, when one trivalent cation is introduced, three K + cations are removed, thus creating additional space and lead to improved accessibility of oxygen molecules. strong absorption band centered at 1552 cm -1 characteristics to the interaction of oxygen molecules with K + sites for K-LTL and K + , Gd 3+ for sample Gd-LTL and consequently, this alteration of its symmetry prevents any IR band from being detected in its gas phase. Since oxygen is a nonpolar molecule, the small shift of the peak located at 1553 cm -1 with increasing oxygen pressure reflects a weak bonding [START_REF] Komaty | A Facile Route toward the Increase of Oxygen Content in Nanosized Zeolite by Insertion of Cerium and Fluorinated Compounds[END_REF] . The total interaction energy of the physical adsorption of these molecules on zeolites includes contributions from dispersion, close-range repulsion, polarization and field-quadrupole 40 . The introduction of the Gd 3+ cations is found to improve the O2 sorption capacity of LTL nanosized zeolite by 27% as shown in Figure 10a.

In addition, the intensity of the oxygen band does not change significantly over several sorption cycles, indicating that the LTL zeolite maintains the O2 capacity after several cycles.

One of the most important property of gas (oxygen) delivery bio-vectors for tumor oxygenation, is the ability to release the oxygen molecules over an appropriate time period. Therefore, the oxygen release from an oxygenated K-LTL and Gd-LTL nanozeolite using a dissolved oxygen probe under hypoxic conditions was studied. A maximum O2 release after 6 min and 8 min using K-LTL (∼1.56 mg•L -1 ) and Gd-LTL (∼2.18 mg•L -1 ), respectively was observed Figure 10b. Afterwards, the oxygen concentration showed a slow decrease over time due to the diffusion of O2 into the air. The above results indicated that our materials have the ability to adsorb and release of oxygen in liquid media. Figure 11a shows greyscale coded T1 maps of Gd-LTL at increasing Gd concentrations (0.001 -0.0625 mM). T1 mapping shows noticeable shortening of the T1 values with increasing the Gd concentration. The resulting r1 (1/T1) value of Gd-LTL calculated at 7 Tesla at room temperature was 79 mM -1 s -1 (Figure 11b). The obtained value of r1 was 20 times higher than those of commercial Gd-chelates (e.g., the relaxivity induced by Dotarem ® Gd-DOTA or Magnevist ® Gd-DTPA is at about 4 mM -1 s -1 at 7T) [START_REF] Rodríguez-Galván | Gadolinium-Containing Carbon Nanomaterials for Magnetic Resonance Imaging: Trends and Challenges[END_REF][START_REF] Hermann | Gadolinium(III) Complexes as MRI Contrast Agents: Ligand Design and Properties of the Complexes[END_REF] .

Conclusions

The synthesis of non-toxic nanosized LTL zeolites containing either K or Gd as extra framework cations for oxygen delivery in hypoxic media and MRI imaging are reported. The ability of LTL nanosized zeolite to adsorb and release O2 under hypoxic conditions is demonstrated. The results show that the Gd-LTL nanosized zeolite exhibits an oxygen capacity of 5.27 cm 3 /g and shows the highest O2 release in the hypoxic medium. Importantly, the incorporation of Gd in the LTL zeolite enabling MRI tracking and preserved structural, textural, colloidal and morphological properties of the nanosized crystals. The toxicity experiments using glioblastoma cell lines demonstrate the non-toxic behavior of the LTL nanosized zeolites.

1. 7

 7 Relaxometry measurementsGd-LTL nanocrystals were dispersed in distilled water at concentration ranging from 0.0625 to 0.001 mM of Gd. Solutions were placed in polypropylene tubes set to a polystyrene support. MRI acquisitions of the phantoms were performed at room temperature on a 7 Tesla BioSpec MRI (Bruker, Ettlingen, Germany). For T1-weighted (T1w) images, a flow sensitive alternating inversion recovery and rapid acquisition with relaxation enhancement (FAIR-RARE) sequence was used (TR: 6000 ms, TE: 77.82 ms, TI: increasing from 25 ms to 5000 ms, RARE factor of 32, image matrix: 128 × 128, 1 slice with a thickness of 3 mm and total acquisition time of 4 min). Image analysis and T1 calculation were performed with Bruker Paravision (version 360.3.3), MATLAB R2021b and ImageJ (version 1.50f). The R1 value (1/T1) was plotted as a function of the Gd concentration and the slope of the line corresponded

Figure 1 .

 1 Figure 1. a) Powder X-ray diffraction patterns b) SEM and c) chemical composition of K-LTL and Gd-LTL nanosized zeolites determined by EDX.

  The mean hydrodynamic diameter of particles obtained by DLS is 15 nm. The hydrodynamic size of the nanoparticles remained stable with no variations with time, making them well-suited for in vivo applications. The zeta potential values of K-LTL and Gd-LTL colloidal suspensions are -46 and -48 mV, respectively, confirming proper colloidal stability and negatively surface charged particles.

Figure 2 .

 2 Figure 2. HAADF-STEM images of (a, b) K-LTL and (c, d) Gd-LTL nanosized zeolites at different magnifications.

Figure 3 .

 3 Figure 3. Dynamic light scattering profile of a) K-LTL and b) Gd-LTL nanosized zeolites.

Figure 4 .

 4 Figure 4. a) TG and b) DTG curves of K-LTL and Gd-LTL nanosized zeolites

Figure 7 .

 7 Figure 7. a) Nitrogen adsorption/desorption isotherms and b) NLDFT pore size distribution and the cumulative pore volume of K-LTL and Gd-LTL nanosized zeolites.

Figure 8 ,

 8 Figure 8, the U251-MG and U87-MG cell viability was not modified by exposure to both

Figure 8 .

 8 Figure 8. Cell viability of human glioblastoma-derived cell lines exposed to K-LTL and Gd-LTL zeolite nanocrystals. Representative phase contrast photographs of a) U251-MG and b)U87-MG cells after 72 h exposure to 100 μg/mL of K-LTL and Gd-LTL zeolite nanocrystals and a control (H2O). Quantification of a) U251-MG and d) U87-MG viability after 24 and 72 h exposed to increasing concentrations of K-LTL and Gd-LTL zeolites. Cell viability was assessed using the WST-1 assay. Mean ± SD, N=5, *p < 0.05 vs control, # p < 0.05 vs 10 µg/mL, Kruskall-Wallis test, e) Hemolysis assay for K-LTL (black), Gd-LTL (blue) nanozeolites incubated with rat RBCs with concentrations 10, 50, 100, 250 and 500 µg/mL at 37 °C for 1 h. Mean ± SD, n=3 independent experiments.

Figure 9 .

 9 Figure 9. Oxygen adsorption isotherms of a) K-LTL and b) Gd-LTL nanosized zeolites at different temperatures

Figure 10 .

 10 Figure 10. a) FTIR band area normalized to the mass of K-LTL and Gd-LTL nanosized zeolites as a function of O2 pressures and b) time-dependent changes of oxygen concentration in PBS solution with addition of oxygen-saturated K-LTL and Gd-LTL nanosized zeolites and the control sample (oxygen-saturated water).

Figure 11 .

 11 Figure 11. a) T1 map MR images as a function of Gd(III) ion concentration and b) Plots of the R1 value (1/T1) as a function of Gd(III) ion concentration. Slope provide r1 value

Table 1 .

 1 Elemental composition of K-LTL and Gd-LTL nanosized zeolites determined by ICP, EDX, NMR and FTIR.

				Chemical composition (wt. %)
	Sample	K	Si	Al	Gd	Si/Al ICP	Si/Al EDX	Si/Al NMR	Si/Al FTIR
	K-LTL	11.81	22.45	8.65	-	2.59	2.22	2.52	3.13
	Gd-LTL	10.76	20.42	7.79	1.53	2.52	2.29	2.47	3.08

Table 2 .

 2 Properties of K-LTL and Gd-LTL nanosized zeolites determined by N2 adsorption characterization

		Surface	Total Pore	NLDFT Micro	NLDFT pore
	Sample	area SBET	volume	Pore volume	size
		(m 2 /g)	(cm 3 /g)	(cm 3 /g)	(nm)
	K-LTL	591	0.76	0.14	0.8
	Gd-LTL	546	0.81	0.14	0.7

Acknowledgments

The financial support from the Conseil Régional de Normandie, FEDER Program Zeoxy, INCA (INCA 11699), CNRS Innovation and the Centre of zeolites and nanoporous materials, Region Normandie, CNRS (CLEAR) is acknowledged.


Al Asier Gómez-Olivencia

Diego López-Onaindia

Nohemi Sala

Antoine Balzeau

Ana Pantoja-Pérez

Ignacio Arganda-Carreras

Mikel Arlegi

Joseba Rios-Garaizar

Aida Gómez-Robles

Asier Gómez-Olivencia

Dept Geología

Ignacio Arganda

Joseba Rios

The human remains found in 1967 in Axlor: still not convincingly Neandertals. A reply to

In 2020, we published a study that described all the human remains found during J. M. de Barandiarán's excavations in Axlor (Dima, Biscay). Our study first presented two deciduous teeth and a parietal fragment found in an undisturbed Mousterian context, all of which show morphological features consistent with a Neandertal classification [START_REF] Gómez-Olivencia | The human remains from Axlor (Dima, Biscay, northern Iberian Peninsula)[END_REF]Supplementary Text S1). Our study also reassessed the human remains previously described by [START_REF] Basabe | Dientes humanos del Musteriense de Axlor (Dima. Vizcaya)[END_REF], likely belonging to a single individual and traditionally classified as a Neandertal (see e.g., [START_REF] Carmona | Estudio comparado de las piezas dentales de Homo neanderthalensis del yacimiento Musteriense de Axlor (Dima, Vizcaya)[END_REF]. However, our metric and morphological assessment suggested stronger affinities with modern humans. Recently, a reply to our article has been published, focusing on the remains previously published by [START_REF] Basabe | Dientes humanos del Musteriense de Axlor (Dima. Vizcaya)[END_REF]. In their reply, González-Urquijo, Bailey, & Lazuen (2021: 553) state that "Axlor's level IV human remains are convincingly Neanderthals", thus concluding that our taxonomic classification "is not supported by the anatomical evidence" and that the "balance of the evidence-morphological and stratigraphic-is most consistent with a Neandertal classification for these teeth" (González-Urquijo et al., 2021: 557).

These authors take issue with two aspects: the taxonomic classification of the human remains found in 1967 and the discussion and reinterpretation of the stratigraphic context of these remains.

Here we address the points raised by [START_REF] González-Urquijo | Axlor's level IV human remains are convincingly Neanderthals: A reply to Gómez-Olivencia et al[END_REF].

1

The taxonomic attribution of the human remains found in 1967

The human remains found in 1967 comprise four teeth (left P 4 -M 3 ) found on September 7 th 1967 in the square 13F at a depth of 265 cm. Two of these teeth (M 1 and M 2 ) were found in a maxillary fragment. Additionally, a left canine was found on September 8 th in the square 13E at a depth of 285 cm (Gómez-Olivencia et al., 2020: Figure S6). Two of these teeth (the C' and the M 2 ) are currently lost, and therefore, our taxonomic assessment was based on the P 4 , M 1 and M 3 . All these remains were originally attributed to the Mousterian level III by [START_REF] Barandiarán | Excavaciones en Axlor[END_REF]; see also Gómez-Olivencia et al., 2020: Figure S6). However, based on the depth of the findings and the depth attributed by Barandiarán to the level IV in 1968 [START_REF] Barandiarán | Excavaciones en Axlor[END_REF], they have been later and repeatedly attributed to level IV [START_REF] Basabe | Restos fósiles humanos de la región Vasco-Cantábrica[END_REF][START_REF] González Urquijo | Aportes de las nuevas excavaciones en Axlor sobre el final del Paleolítico Medio[END_REF]Lazuen Fernández & González-Urquijo, 2019-2020).

González-Urquijo et al. (2021: 553) oversimplified our taxonomic assessment, stating that we concluded that "those remains should instead be assigned to a single Upper Paleolithic Homo sapiens individual". However, our study states that "based on both morphological and size characteristics, this individual shows stronger affinities with modern humans than with

Neandertals" (Gómez-Olivencia et al., 2020: 486), while we also clearly recognized that some of the teeth (e.g., the canine and the P 4 ) showed some features that are present in high percentages in Neandertals, but are not exclusive of this group. In our discussion, we hypothesized that "these human remains could belong to an UPMH [Upper Paleolithic modern human], which should be tested in the near future using direct C14 datings". This claim was based on the results of our morphometric analyses, the absence of Holocene recent prehistory remains from the Axlor sequence and the presence of an early Upper Paleolithic occupation in the site (Gómez-Olivencia et al., 2020: 488). This was summarized in our abstract, which states that the teeth from Axlor "may represent one of the scarce examples of Upper Paleolithic modern human remains in the northern Iberian Peninsula, which should be confirmed by direct dating" (Gómez-Olivencia et al., 2020: 475). Thus, we suggested that these remains may have belonged to an UPMH, the confirmation of which would require further analyses, but we did not discard other possibilities, such as these teeth having a more recent chronology. and UP range of variation. In addition, our size assessment was complemented with shape information (i.e., form analysis). Geometric morphometric analysis of form variation (including size and shape) is a widely used way to assess taxonomic affinity [START_REF] Compton | The morphology of the Late Pleistocene hominin remains from the site of La Cotte de St Brelade, Jersey (Channel Islands)[END_REF][START_REF] Garralda | The Neanderthal teeth from Marillac (Charente, Southwestern France): Morphology, comparisons and paleobiology[END_REF], and it yielded informative results in some teeth from Axlor (M 1 and M 3 ) but not in others (P 4 ), as we clearly stated in our study [START_REF] Gómez-Olivencia | The human remains from Axlor (Dima, Biscay, northern Iberian Peninsula)[END_REF].

González-Urquijo et al. (2021: 553) also consider that the grades of expression of the nonmetric traits for the P 4 , M 1 and M 3 , "rather than being H. sapiens-like […] have trait express that are ambiguous". We struggle to understand where we disagree here, as our study explicitly stated that some of the Axlor teeth have features that are observed in Neandertals in high frequencies (e.g., the bifurcated essential crest of both the lingual and buccal cusps in the P 4 at the EDJ level). González- et al. (2021: 553) also consider that "most of the traits do not clearly distinguish Neanderthals and Upper Paleolithic H. sapiens". For that exact reason, we combined qualitative and quantitative methods for the taxonomic assessment of the Axlor teeth, as opposed to the qualitativeonly evaluation performed by González-Urquijo and colleagues.

Urquijo
González- Urquijo et al. (2021: 553) disagree with one of our assessments of the M 1 characters as they consider that the hypocone is slightly larger than the metacone at the occlusal enamel surface (OES) and the enamel-dentine junction (EDJ). However, they do not provide an image showing how they measured these areas. Indeed, our original study did not include a formal metric comparison of the hypocone and the metacone of the M 1 because the high degree of wear of this molar makes these measurements tentative. Therefore, we simply stated that both the hypocone and the metacone of Axlor show a grade 4 of expression based on the ASUDAS scoring system [START_REF] Turner | Scoring procedures for key morphological traits of the permanent dentition: the Arizona State University dental anthropology system[END_REF]Gómez-Olivencia et al., 2020: Table 4).

González-Urquijo et al. (2021: 553) enumerate a series of methodological issues related to the analysis of the Axlor's M 1 that were explicitly addressed or that indicate their lack of understanding of the methods we used in our study. Firstly, they consider "curious that a quantitative assessment of this tooth was even undertaken since Gómez-Robles et al. explicitly excluded severely worn teeth". Our study explicitly discussed the limitations of identifying cusp tips in worn molars, which is why we performed the geometric morphometric analysis of the M 1 from Axlor twice: once using both the cusp tips and the outline, and once using only the outline.

Discriminant analyses indicated a probability of around 99% for this molar to belong to Homo sapiens in both cases.

González-Urquijo et al. ( 2021) state that we did not indicate whether the outline was corrected. The outline of the M 1 was indeed corrected as it is the standard procedure in geometric morphometric analyses of molar shape [START_REF] Gómez-Robles | A geometric morphometric analysis of hominin upper first molar shape[END_REF]. We clearly stated that we used "the Neandertal and modern human samples used in Gómez-Robles et al. ( 2007), Gómez-Robles, Bermúdez de Castro, Martinón-Torres, Prado-Simón, and Arsuaga (2012), and Gómez-Robles, Martinón-Torres, Bermúdez de Castro, Prado-Simón, and Arsuaga (2011)." (Gómez-Olivencia et al., 2020: 479). While not explicitly stated, this was meant to imply that the same methodology was used: "When mesial and/or distal borders of the teeth were affected by light interproximal wear, original borders were estimated by reference to overall crown shape and the buccolingual extent of the wear facets, following [START_REF] Wood | Analysis of the dental morphology of Plio-Pleistocene hominids. V. Maxillary postcanine tooth morphology[END_REF] and Bailey ( 2004)" (Gómez-Robles et al., 2007: 275-276).

Additionally, [START_REF] González-Urquijo | Axlor's level IV human remains are convincingly Neanderthals: A reply to Gómez-Olivencia et al[END_REF] estimate that between 30% and 37% (between 9 and 11) of the semilandmarks are missing in Axlor's M 1 (see their Figure 1, where they provide a new outline for Axlor's M 1 ). This outline assumes that landmarks 22-28 are affected by wear, and thus the suggested outline enlarges both the metacone and, especially, the hypocone. While we fully agree with González-Urquijo and colleagues on the correction performed on the mesial outline, which is similar to the one we performed to carry out our analysis, their distal correction is clearly

incorrect. An inspection of the Axlor's M 1 3D model (which is freely available in Figshare; see the Data Availability Statement below and in [START_REF] Gómez-Olivencia | The human remains from Axlor (Dima, Biscay, northern Iberian Peninsula)[END_REF] clearly reveals that the surface of the inter-proximal facet is very limited and does not extent to the hypocone semilandmarks (Figure 1). Therefore, the outline correction illustrated by González-Urquijo et al.

on the distal aspect of the molar substantially exceeds the actual interproximal wear of this molar, making it look more Neandertal.

González-Urquijo et al. (2021: 554) state that "the absence of clear divisions between cusps complicates accurately positioning the centroid that is used to place the semilandmarks." Firstly, the location of the centroid depends on the location of the cusp tips, not on the presence of "clear divisions between cusps". As stated above, the degree of wear of this molar does interfere with the accurate location of the four landmarks that the centroid position is based on. However, the specific location of each semilandmark on the outline, which does depend on the calculated centroid, is modified through a sliding algorithm that, again, is a standard step of geometric morphometric analyses [START_REF] Bookstein | Applying landmark methods to biological outline data[END_REF][START_REF] Bookstein | Landmark methods for forms without landmarks: morphometrics of group differences in outline shape[END_REF][START_REF] Bookstein | Midline corpus callosum is a neuroanatomical focus of fetal alcohol damage[END_REF][START_REF] Gunz | Semilandmarks in three dimensions[END_REF]. Hence, any ambiguity in the original positioning of the semilandmarks is ameliorated by the sliding procedure.

[

INSERT FIGURE 1 HERE]

Our geometric morphometric analysis revealed that: a) Axlor's M 1 falls closer to the modern human consensus shape than to the Neandertal consensus shape; and b) the M 1 discriminant analysis provided a very high probability to belong to a modern human, while P 4 and M 3 were not conclusive [START_REF] Gómez-Olivencia | The human remains from Axlor (Dima, Biscay, northern Iberian Peninsula)[END_REF]. However, it is widely accepted that upper first molars are one of the most diagnostic teeth when distinguishing between Neandertals and modern humans clearly present at both the OES and the EDJ". However, we clearly stated that the lingual essential crest is present at the EDJ with a score of 2. This crest was scored as "NO" at the OES level, which means "non observable" (although we failed to spell this out in the legend of our table). Unlike

González-Urquijo and colleagues, we considered that the assessment of this trait at the OES level is too tentative given the substantial degree of wear present in this tooth. In our table, those traits that are absent are clearly indicated by "0 (absent)" (Gómez-Olivencia et al., 2020: Table 3). While we admit our error for not spelling out the abbreviation, this does not make these teeth any more Neandertal.

In the case of the hypocone in the M 3 , we considered that this as an additional cusplet/tubercle, since it is not clearly visible in the original OES (and thus our scoring as 0). As the scoring of other qualitative traits, our assessment (as González-Urquijo and colleagues') bears certain degree of subjectivity, which is why a quantitative assessment is always preferred. In any case, even if the presence of a hypocone in the M 3 were accepted, this trait would still be unable to distinguish between Neandertals and modern humans (see [START_REF] Martinón-Torres | Morphological description and comparison of the dental remains from Atapuerca-Sima de los Huesos site (Spain)[END_REF].

Stratigraphic study

In his field notes and in the published report of Axlor excavations, J. M. de Barandiarán describes quite clearly the context of the human remains: i) The site of Axlor was severely affected during the end of the XIX century and or the beginning of the XX century, resulting in the destruction of a large part of the site, mostly the upper levels, situated in the right part of the site [START_REF] Barandiarán | Excavaciones en Axlor[END_REF]; ii) The human remains under discussion, according to his field notes, were found in the limit of the preserved area, immediately below the mixed sediment that covered the destructed area, and they were the first archaeological remains noted in square 13F; iii) The remains were found in a loose sediment that contrasts with the encrusted sediments excavated beside squares 13E and 13F by J. González Urquijo between 2000and 2008[START_REF] González Urquijo | Axlor. IX Campaña[END_REF][START_REF] González Urquijo | Excavación en el yacimiento arqueológico de Axlor (Dima, Bizkaia). Un estudio arqueológico y antropológico de la transición entre el Paleolítico Medio y el Superior[END_REF].

In their reply, González-Urquijo et al. ( 2021) do not present any new information that contradicts the description made by J. M. de Barandiarán or by González-Urquijo's own team in previous reports and publications [START_REF] Barandiarán | Excavaciones en Axlor[END_REF][START_REF] González Urquijo | Aportes de las nuevas excavaciones en Axlor sobre el final del Paleolítico Medio[END_REF][START_REF] Rios Garaizar | La excavación en Axlor. Las formas de vida de los últimos neandertales[END_REF]. Moreover, some of the interpretations and data presented by González-Urquijo et al. 1).

[INSERT TABLE 1 HERE]

ii) González-Urquijo et al. ( 2021) base their argument on the poor preservation of the Upper Paleolithic (UP) faunal remains found in F8 and F9, but they neglect to mention that a human tooth was found in this level in 2008 [START_REF] González Urquijo | Axlor. IX Campaña[END_REF]. Furthermore, González-Urquijo et al.

(2021) propose a geometry for the UP level omitting that original extension of this level is unknown for two reasons. First, due to the aforementioned partial destruction of the site; and second, due to the excavation of J. M. de Barandiarán who barely recovered, accordingly with the available records, 13 lithic artifacts from level I and 8 lithic artifacts from level II.

iii) The currently available information about the stratigraphic context adjacent to the place where the human remains under discussion were found indicates that the stratigraphic sequence is very difficult to read, that the sediment is encrusted [START_REF] González Urquijo | Aportes de las nuevas excavaciones en Axlor sobre el final del Paleolítico Medio[END_REF], and that no new human remains have been recovered there [START_REF] González Urquijo | Excavación en el yacimiento arqueológico de Axlor (Dima, Bizkaia). Un estudio arqueológico y antropológico de la transición entre el Paleolítico Medio y el Superior[END_REF].

Discussion and conclusions

Here we reiterate that a Neandertal classification for the teeth found in Axlor in 1967 is unlikely. Firstly, the supposedly Neandertal traits discussed by González-Urquijo and colleagues As stated in our original publication, the body of evidence points to a modern human classification for these teeth, with very high probabilities in one of the most informative teeth, the M 1 . Though species-specific morphologies exist, dental size and shape are variable in both species and unable to unquestionably discriminate between them, particularly for specimens that do not show the most extreme species-specific traits. In addition, the Middle to Upper Paleolithic transition entailed a complex scenario that included both cultural changes and population replacement. Recent developments in ancient DNA analyses have complicated the picture, with evidence of admixture between Neandertals and modern humans [START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF]. Fossil remains with ambiguous

Neandertal and modern human traits may represent the anatomical evidence of those admixture events (see [START_REF] Compton | The morphology of the Late Pleistocene hominin remains from the site of La Cotte de St Brelade, Jersey (Channel Islands)[END_REF], although molecular analyses are required to confirm this point. In sum, the teeth found in Axlor in 1967 show a few features which typically appear in Neandertals in high frequencies, but which are also present in H. sapiens. However, most traits, including the quantitative morphometric analysis of the M 1 show H. sapiens affinities. Therefore, we favor this taxonomic classification. Genetic analyses and direct dating, however, are the only way forward to unequivocally clarify their chronology and further inform discussions about their ancestry.

  et al. (2021) disagree with some of our morphological assessments and take issue with the employed methodology. The taxonomic assessment of the Axlor 1967 remains by González-Urquijo et al. (2021) pivots mainly around the morphology of the lost canine, the crown morphology of the P 4 and the morphology of the M 1 .

  (e.g.,[START_REF] Gómez-Robles | A geometric morphometric analysis of hominin upper first molar shape[END_REF]. When considering PC1 and PC2, the Axlor M1 falls closer to both recent and fossil Homo sapiens centroids than to the Neandertal distribution centroid. Overlap is minimal in this plot and only one Neandertal individual from our Neandertal sample (the one from Pech de l'Azé) falls close to Axlor and the recent human centroid (Gómez-Olivencia et al., 2020: Figure6).It is also important to note that the discriminant analyses in[START_REF] Gómez-Olivencia | The human remains from Axlor (Dima, Biscay, northern Iberian Peninsula)[END_REF] were based on the first 10 PCs not just on the two first PCs shown in the figures. Although not reported in our original study, we include here the cross-validated percentages of correct classification: 93.3% overall, with 73.3% of Neandertals and 100% of modern humans correctly classified. Additionally, when the form space is analyzed, the Axlor M 1 falls outside the range of variation of the Neandertal sample[START_REF] Gómez-Olivencia | The human remains from Axlor (Dima, Biscay, northern Iberian Peninsula)[END_REF].With respect to the other teeth, we agree with González-Urquijo et al. (2021) that the welldeveloped mesial ridge and lingual tubercle present in the Axlor canine are traits present in Neandertals in high percentages. Interestingly, they mention the presence of one UP canine (out of 10) with a well-developed mesial ridge, but then question its taxonomic attribution. Our revision of published UP specimens reveals that a tuberculum dentale is also present in other UP specimens (Sunghir 2, Sunghir 3, Mladeč 9;[START_REF] Frayer | Aurignacian female crania and teeth from the Mladeč Caves, Moravia, Czech Republic[END_REF][START_REF] Trinkaus | The People of Sunghir. Burials, Bodies, and Behavior in the Earlier Upper Paleolithic[END_REF]. Moreover, Mladeč 9 also presents a well-developed mesial ridge (although smaller than the distal one), which is attached to the tuberculum dentale as in the canine from Axlor (see[START_REF] Frayer | Aurignacian female crania and teeth from the Mladeč Caves, Moravia, Czech Republic[END_REF]: Figure 19 vs Basabe, 1973: Figure 7).González-Urquijo et al. (2021: 553) disagree with our assessment of two additional traits and state that the "lingual essential crest of the P 4 that Gómez-Olivencia et al. scored as absent is

(

  2021) suffer from important omissions: i) The radiocarbon date table published by González-Urquijo et al. (2021) does not include the spatial information of the samples, nor the information about methods, pretreatment and quality of the samples. Additionally, they omit two published dates from level F made by themselves (Rios-Garaizar, 2017). One of these dates yielded a result of 33,310±360 (sample Beta-225485) which is extremely important for addressing possible disturbances at the site (Rios-Garaizar, 2012). González-Urquijo et al. (2021) reassign one sample (sample Beta-144262) traditionally published as belonging to level D (González Urquijo et al., 2006) to level B without further explanation. Finally, they omit the radiocarbon dates from Axlor published by Marín-Arroyo et al. (2018) (see Table

(

  2021) are ambiguous, unclear or based on an erroneous assessment of the teeth. Second, we show that González-Urquijo et al. (2021) do not present any new information that contradicts the stratigraphic description made by J. M. de Barandiarán or by his team in previous reports and publications, and that they omit important information regarding the archaeological context of the human remains found in 1967.

Figure 1 .

 1 Figure 1. A) Semilandmarks proposed by González-Urquijo et al. (2021) as an accurate correction of

Figure 1 .

 1 Figure 1. A) Semilandmarks proposed by González-Urquijo et al. (2021) as an accurate correction of Axlor's M1 outline; B) Corrected outline used by Gómez-Olivencia et al. (2020) in their original assessment. C and D) Occlusal-distal showing that the extension of the distal facet (marked in red in D) does not affect the occlusal outline. E) Outline of a M1 from Krapina (Krapina 100) showing the bulging hypocone, typical of Neandertal populations (and also of European Middle Pleistocene populations) (Bailey, 2004; Gómez-Robles et al., 2007; Martinón-Torres, Bermúdez de Castro, Gómez-Robles, Prado-Simón, & Arsuaga, 2012).

Table 1

 1 Available radiocarbon dates for Axlor late Middle Paleolithic levels B, D and F.

	Level*	Sample reference	Lab Code	Date (BP)	Method	Material	Taxon, part anatomical	%C	%N	C:N	δ 13 C	δ 13 C VPDB) (‰	δ 15 N AIR) (‰	References
														González Urquijo &
	B**	na	Beta-144262	42,010±1280	AMS	Bone	Non determ.							Ibañez Estévez, 2002; González Urquijo et
														al., 2021
	B	na	Beta-203108	42,720±900	AMS	Bone	Bos/Bison; tibia							González Urquijo et al., 2021
	D	na	Beta-203107	44,920±1950	AMS	Bone	Bos/Bison; axis							González Urquijo et al., 2021
	D	na	Beta-225486	>43,000	AMS	Bone	Cervus; metapodial							Rios-Garaizar, 2012; González Urquijo et al., 2021
	F	na	Beta-225478	>47,500	AMS	Bone	Cervus; metapodial							Rios-Garaizar, 2012
	F	na	Beta-225485	33,310±360	AMS	Bone	Cervus; metapodial							Rios-Garaizar, 2012
	IV	AX.11C.290.149 OxA-32428	>49,300	UF-AMS	Bone	Cervus elaphus; phalanx 2	42.3	3.5	3.3	-19.8	3.5	3.1	Marín-Arroyo et al., 2018
	IV	AX.11C.300. 178	OxA-32429	>49,900	UF-AMS	Bone	Cervus elaphus; carpal	43.0	6.3	3.3	-19.2	6.3	6.0	Marín-Arroyo et al., 2018

John Wiley & Sons, Inc.American Journal of Physical Anthropology

John Wiley & Sons, Inc.

ACKNOWLEDGMENTS

This research has received support from the Spanish Ministerio de Ciencia e Innovación (proyecto PGC2018-093925-B-C33, MCI/AEI/FEDER, UE), Research Group IT1418-19 from the Eusko Jaurlaritza-Gobierno Vasco, and N. Ergal Foundation. AGO was supported by Ramón y Cajal fellowship (RYC-2017-22558). NS was supported by DEATHREVOL project that has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement No. 949330) and by Ramón y Cajal fellowship (RYC2020-029656-I). DLO was supported by an MSC Actions Individual Fellowship (Project N°895713). IA-C acknowledges the support by the University of the Basque Country

UPV/EHU grant GIU19/027. We would like to thank the Editor, the AE, and the reviewers for very useful comments that have helped to improve this paper.

Data Availability Statement

The original micro-ct scans, the derived segmentation files, and 3D volumes are available in figshare at https://doi.org/10.6084/m9.figshare.10308272.


Perlette Totoson

Marilys Pradel

Pierre Thiriet

Contextualized phosphorus recycling: potential diminution of phosphorus criticality at territory scale -Application to agricultural LCA

come

Conclusion

The proposed model will help to provide an effective recycling rate consistent with local context (fertilizer regulation, water framework directive, farmers preferences, agronomy context…). This promotes a better integration of the characteristics of phosphorus recycling into criticality assessment. The model gives a methodology advancement, which would improve or be a complement of the LCA tool to assess territorialized phosphorus recycling scenarii.

  mail : perlette.totoson@inrae.fr Université Clermont Auvergne, INRAE Clermont-Ferrand-Auvergne-Rhône-Alpes, UR TSCF

►P

  content varies according to OR types, treatment processes ►OR diversity is a function of the economic activities present at the spatial scale ►OR reuse in agriculture is presided over by regulations ►Farmers may not use P recycled depending the characteristics of OR (price, agronomic value, location,…) => P recycling depends on the geographical scale studied and its context (regulatory, social, economic, agronomic,...). The context and geographical scale are not taken into account in the evaluation of the recycling rate indicators used in the raw material criticality assessment (as EOL-RR, EOL-RIR,…) There is a need to contextualize P recycling at the local To build the model, four set of parameters are identified ►P-OR supply ►P needs ►Drivers, i.e. any factors that can influence the use of P from OR by farmers ►Preferences of use by farmers

phosphate rock criticality Why phosphorus recycling? Phosphorus recycling refers

  to the reuse in agriculture of collected (i.e. organic effluent) or recovered phosphorus (i.e. struvite) which is contained in organic residues (OR) as digestate, manure, sludge…

		Phosphate rock: main source of phosphorus ► Critical raw material for European Union's (EU) economy ►Non renewable and exhaustible resource ►Long time scale for the exploration of new deposits ►Uneven distribution of global reserves	► Mostly used for phosphate mineral fertilizer manufacturing (86% in EU) ►P as element cannot be substituted in crop fertilizing ►Finland is the only European country producer of phosphate rock (16% of EU supplier) ►High EU dependency on third party countries
	Phosphate rock deposits	Mineral fertilizer	Crop production	Animal foods livestock
						P
		Struvite	Recovery	Reuse	Household Organic residues	Recycling
		Ash	Digestate	Sludge	Compost	Manure

The conceptual model aims to put P recycling back into its context to assess the maximum recoverable and recyclable potential of phosphorus from organic residues deposits in the studied territory.

In the agricultural sector, providing P from recycling sources is currently the only way to mitigate