Marie-Josée Jacobin-Valat

Jeanny Laroche-Traineau

Mélusine Larivière

Stéphane Mornet

Stephane Sanchez

Marc Biran

Caroline Lebaron

PhD Julien Boudon

Sabrina Lacomme

Martine Cérutti

Stéphane Sanchez

Marc Biranphd

Gisèle Clofent-Sanchez
email: gisele.clofent-sanchez@rmsb.u-bordeaux2.fr

Nanoparticles functionalised with an anti

Keywords: MRI contrast agents, Atherosclerosis, Nanoparticles, Platelets, Human antibody

Atherosclerosis is a systemic disorder affecting arterial beds throughout the body, potentially resulting in acute atherothrombotic events such as coronary artery disease (CAD), cerebrovascular disease (CVD), peripheral arterial disease (PAD) or a combination of all (polyvascular or diffuse vascular disease). These cardiovascular diseases cause 19 million deaths per year in the world. They are expected to be the main cause of death globally within the next 10 years owing to their rapidly increasing prevalence in developing countries, due to population aging and other factors, including the increase in unhealthy dietary patterns, physical inactivity, obesity and diabetes mellitus. [START_REF] Psarros | Nanomedicine for the prevention, treatment and imaging of atherosclerosis[END_REF] Thus, the clinical burdent of atherosclerosis is likely to present enormous challenges in the future.

The current opinion is that atherosclerosis is an immune/inflammatory response of the intima to endothelial injury, mainly initiated by the transport of oxidised low-density lipoprotein (Ox-LDL) across the endothelium.2, 3 Several lines of evidence have shown that platelet interactions with modified lipoproteins seem to be quite important in triggering their transfer to the vessel wall. [START_REF] Siegel-Axel | Platelet lipoprotein interplay: trigger of foam cell formation and driver of atherosclerosis[END_REF] Platelets are by themselves inflammatory cells [START_REF] May | Platelets: inflammatory firebugs of vascular walls[END_REF] which can greatly influence monocyte and lymphocyte recruitment through interactions with the dysfunctional endothelium in a well-controlled process involving selectins and integrins. [START_REF] Massberg | A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation[END_REF][START_REF] Gawaz | Platelets in inflammation and atherogenesis[END_REF][START_REF] Badrnya | Platelets mediate oxidized lowdensity lipoprotein-induced monocyte extravasation and foam cell formation[END_REF] P-selectin-dependent formation of platelet-leucocyte aggregates (PLAs) further induces the release of a wealth of adhesive and pro-inflammatory substances. [START_REF] Pitsilos | Platelet factor 4 localization in carotid atherosclerotic plaques: correlation with clinical parameters[END_REF][START_REF] Coppinger | Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions[END_REF] The process continues in a vicious circle-like fashion and blood cells involved in adaptive immunity may play important roles in the self-perpetuating inflammatory process. [START_REF] Yilmaz | Accumulation of immune cells and high expression of chemokines/chemokine receptors in the upstream shoulder of atherosclerotic carotid plaques[END_REF][START_REF] Libby | Transatlantic Network on A Inflammation in atherosclerosis: from pathophysiology to practice[END_REF] Monocytes further differentiate into activated macrophages expressing scavenger receptors which bind different forms of OxLDL, leading to lipid-laden foam cells. [START_REF] Stephen | Scavenger receptors and their potential as therapeutic targets in the treatment of cardiovascular disease[END_REF][START_REF] Stary | A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis[END_REF] Platelets also act on the stability and vulnerability of lipid-rich plaques, through αIIbβ3-mediated platelet-endothelium firm adhesion, CD40L expression and cytokine secretion [START_REF] Tedgui | Cytokines in atherosclerosis: pathogenic and regulatory pathways[END_REF] which coordinate extracellular matrix proteins lysis by matrix metallo-proteases (MMPs), well-known to degrade and fragilize the fibromuscular cap. [START_REF] Sluijter | Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms[END_REF][START_REF] Schulz | Platelets in atherosclerosis and thrombosis[END_REF] Thus, platelets foster an inflammatory environment that influences atherosclerotic plaque development and vulnerability, in addition to their role in acute thrombus formation. [START_REF] Gawaz | Platelets in inflammation and atherogenesis[END_REF] Traditionally, the degree of luminal stenosis has been used as a marker of the stage of atherosclerosis and as an indication for surgical intervention. Coronary angiography is the gold standard technique for lumenography, but unfortunately provides no information about the functional and molecular events leading to plaque rupture. [START_REF] Wasserman | Low-grade carotid stenosis: looking beyond the lumen with MRI[END_REF] Hence, imaging modalities with more pronostic value are highly desirable. MRI approaches have successfully characterised carotid arteries, thanks to its high spatial resolution. [START_REF] Saam | The vulnerable, or high-risk, atherosclerotic plaque: noninvasive MR imaging for characterization and assessment[END_REF] However, up to now, the tortuosity and size of the coronary arteries added to the respiratory and cardiac motion hinder the in vivo imaging of coronary plaque. To overcome these problems and provide information on the molecular and cellular events leading to plaque rupture, we must rely on molecular imaging modalities, capable of reporting on the molecular content of the arterial wall.

In recent years, considerable efforts have been spent in the development of targeted magnetic contrast agents for biomedical imaging in MRI. [START_REF] Klostergaard | Magnetic nanovectors for drug delivery[END_REF] These must be designed to have no toxicity and selective binding to desired epitopes such as cell surface receptors. [START_REF] Choi | Nanoparticles for biomedical imaging: fundamentals of clinical translation[END_REF] With affinities classically in the nanomolar range, antibodies offer binding properties advantages over bio-mimetics and peptides. In order to ensure safety for medical purposes, human antibodies are preferred over murine antibodies (see limitation sections). Moreover, the choice of the targeted biomarker is of fundamental importance because it has to fulfil two criteria: (1) it must sign a pathological state and (2) it should be highly represented.

In light of the above arguments, we believe molecular targeting of platelets is relevant due to their important involvement into every stage of atheroma pathogenesis.

The remaining question concerns their localization and representativity: due to the variety of mecanisms allowing their internalisation -in addition to haemorrhage and thrombi -platelets are certainly retained within the plaque, providing novel means of discriminating atheroma plaques at high risk of rupture.

We thus developed a recombinant human antibody, rIgG4 TEG4, targeting human activated platelets, to be used as a targeting moiety. TEG4 human antibody was obtained through phage-display technology by biopanning on activated platelets. [START_REF] Jacobin | Human IgG monoclonal antialpha(IIb)beta(3)-binding fragments derived from immunized donors using phage display[END_REF] We now produced TEG4 in IgG4 format thanks to the baculovirusinsect cell system, in quantity sufficient to perform biofunctionalisation of nanoparticles. We then designed an original superparamagnetic iron oxide nanoparticle (VUSPIO for Versatile Ultra Small SuperParamagnetic Iron Oxide) [START_REF] Mornet | A method for synthesis and functionalisation of ultrasmall superparamagnetic covalent carriers based on maghemite and dextran[END_REF] (patent FR 2855315 (also published as EP 1627395 and WO 2004107368)) chosen as the contrast agent moiety to covalently couple rIgG4 TEG4 human antibody in order to ensure safety if inoculated in humans.

Methods

Production of TEG4 antibody as a recombinant IgG4 in baculovirus system

The general principle is to replace a non-essential gene with a DNA sequence encoding a foreign protein of interest. This replacement is promoted by homologous recombination between DNA purified from a replication-defective baculovirus [START_REF] Kitts | A method for producing recombinant baculovirus expression vectors at high frequency[END_REF][START_REF] Luckow | Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli[END_REF] and a plasmid called "transfer vector" (pVT). Specific baculovirus cassettes have been designed [START_REF] Juliant | Engineering the baculovirus genome to produce galactosylated antibodies in lepidopteran cells[END_REF] to express the heavy and light chains of an antibody. These cassettes consist of (i) a strong very late viral promoter (P10 or polyedrin (PH)), (ii) a sequence encoding an immunoglobulin signal peptide, (iii) two unique restriction sites (AflII-NheI for heavy chain expression cassette and BssHII-AvrII for light chain expression cassette) to allow the insertion of the VH or VL sequences of the TEG4 anti-αIIbβ3 antibody [START_REF] Jacobin | Human IgG monoclonal antialpha(IIb)beta(3)-binding fragments derived from immunized donors using phage display[END_REF][START_REF] Robert | Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin[END_REF] , in frame with the upstream signal peptide sequence, and (iv) a downstream sequence that encodes the human heavy (γ4) or light chain (λ) constant region. These cassettes are flanked by viral sequences that direct the integration of the foreign genes into a specific P10 or PH locus.

Recombinant IgG4 TEG4 was produced from Sf9 cells infected with the recombinant baculovirus coexpressing the TEG4 heavy and light chains. Recombinant IgG4 TEG4 was further purified on Protein A column (GE HealthCare Life Science,Velizy-Villacoublay, France). Details of cloning into transfer vectors, generation of recombinant viruses and purification of recombinant antibody are provided in online Supplementary Materials.

Synthesis of TEG4-VUSPIO and control-VUSPIO conjugates

The Versatile UltraSmall SuperParamagnetic Iron Oxide (VUSPIO) platform is based on 7.5 nm-sized magnetic cores (maghemite γ-Fe2O3) functionalised by an aminated polysiloxane film grafted on their surface and embedded in a dextran corona. VUSPIO particles differ from USPIO contrast agents by its chemical stability thanks to strong covalent bonds established between magnetic cores and dextran macromolecules. Moreover, their surface is functionalised with long heterobifunctional poly(ethylene oxide) chains serving as cross linkers for derivatisation by fluorochromes and targeting agents. [START_REF] Jacobin-Valat | MRI of inducible Pselectin expression in human activated platelets involved in the early stages of atherosclerosis[END_REF][START_REF] Sonvico | Folate-conjugated iron oxide nanoparticles for solid tumor targeting as potential specific magnetic hyperthermia mediators: synthesis, physicochemical characterization, and in vitro experiments[END_REF] The rIgG4 TEG4 antibody conjugation to VUSPIO contrast agent is achieved by using SM(PEG)24 (Thermo Scientific, Courtaboeuf, France) as coupling agent by converting the remaining primary amine terminal groups into maleimide functions. In parallel, a thiolation of TEG4 is performed with 2-Iminothiolane-HCL (Traut's Reagent), in order to introduce sulfhydryl groups. Detail of TEG4-VUSPIO is provided in online Supplementary Materials. Then the maleimide groups of VUSPIO react with the sulfhydryl groups of the antibody to form stable thioether bonds. A magnetic separation column (MACS, Miltenyi Biotec) was used to separate the unbound rIgG4 TEG4 from the magnetic conjugates.

The same experiments were performed with polyclonal IgG from human serum (Jackson Immunoresearch Laboratories, West Grove, PA, USA) and with human IgG4 isotypic control (kindly provided by M. Cérutti) to obtain IgG cont-VUSPIO and human IgG4 cont-VUSPIO respectively which served as negative controls for unspecific binding.

Flow cytometry and optical microscopy imaging

Aliquots of 10 8 PFA-fixed-activated-platelets (100 μL) [START_REF] Robert | Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin[END_REF] were mixed with 25 μg/mL of rIgG4 TEG4 human antibody, AP-2 murine monoclonal antibody (courtesy of Dr Alan Nurden [START_REF] Pidard | Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets[END_REF] ) and other commercial antibodies (see online Supplementary Materials) targeting the αIIbβ3 integrin or with specified dilutions of TEG4-VUSPIO (48 μg/mL rIgG4 TEG4; 58 mM Fe 3+ ), IgG cont-VUSPIO (50 μg/mL IgG cont; 31 mM Fe 3 + ) and antibody-free VUSPIO (12 mM Fe 3 + ) overnight at 4 °C to obtain in fine 10 μg/mL of antibody and a minimum of 6.2 mM Fe 3 + . See online Supplementary Materials for the quantification of antibody conjugated to VUSPIO. After two washes in PBS, mixtures were re-suspended in appropriate volumes to perform flow cytometry (10 6 platelets) and confocal microscopy studies (10 8 platelets).

In flow cytometry experiments, aliquots of the cell samples were further incubated 30 minutes with secondary Alexa Fluor 488 goat anti-human H + L (Jackson Immuno Research Laboratories) (1/20), goat anti-mouse H + L (Life Technologies, Saint-Aubin, France) (1/20) and fluorescein isothiocyanate (FITC) mouse anti-dextran antibodies (Stem-Cell Technologies, Grenoble, France) (1/5) for detection of respectively, the functionalising primary antibody (rIgG4 TEG4), murine AP-2 antibody and the dextran coating of antibody-conjugated VUSPIO nanoparticles.

For confocal experiments, aliquots of human platelets were re-suspended in 15 μL anti-fading solution (Mowiol® 4-88, Polysciences, Eppelheim, Germany). The digital microscope was a NIKON ECLIPSE 80i. The fluorescence emission was collected between 465 and 555 nm.

Immunohistochemical (IHC) analysis on murine and human atherosclerotic sections

All animal studies were approved under the No. 50120192-A by the Animal Care and Use Committee of Bordeaux, France. ApoE -/-mice were fed a high-cholesterol diet for 21 weeks. Human coronary arteries were harvested from patients with end-stage heart failure having undergone heart transplantation. All of the clinical interventions took place at Haut-Lévèque Hospital (Pessac, France). Human tissue specimens were collected after informed consent. They were immediately processed and embedded in paraffin.

Paraffin-embedded sections of arterial tissue from mouse or human were used in IHC experiments with unconjugated antibodies (rIgG4 TEG4, AP-2, anti-CD41 (αIIb) (Abcam)), PAC-1 antibody [START_REF] Shattil | Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation[END_REF] (a murine antibody that binds to activated platelets, BD Biosciences), anti-CD68 (an antibody targeting macrophages, Dako, Les Ulis, France) or VUSPIO conjugated antibodies (TEG4-VUSPIO, IgG cont-VUSPIO) tested at 10 μg/mL. See Supplementary Materials for details of the IHC experiment.

Transmission electron microscopy

TEG4-VUSPIO bound to activated platelets was visually analysed by using transmission electron microscopy. An electron microscopy study was conducted at BIC (Bordeaux Imaging Center, University Victor Segalen Bordeaux). Aliquots of 10 8 human platelets activated with thrombin were fixed with 0.1% (v/v) glutaraldehyde/1.5% (v/v) paraformaldehyde and mixed with TEG4-VUSPIO and IgG cont-VUSPIO in 0.1 M phosphate buffer. See online Supplementary Materials for details of the experiment.

Magnetic resonance imaging

In vitro MR imaging for relaxivity measurement and for quantification of TEG4-VUSPIO nanoparticles per platelet is described in online Supplementary Materials.

Ex vivo MR imaging was performed using a 9.4 Tesla MRI system (Bruker) (inner coil diameter, 10 mm) on healthy or atherosclerotic aorta, respectively from C57BL/6 wild-type or ApoE -/-mice (n = 3) terminally anaesthesised by inhalation of isoflurane. The chest was opened by thoracotomy, the heart exposed and the right atrium cut. A 30-gauge needle was inserted in the left ventricle. PBS-heparin (50 UI/mL; 2.5 mL (Sanofi Aventis, Vitry-sur-Seine, France)) was inoculated followed by 10 mL PBS. Perfusion was continued with 2 mL of PBS containing a formulation of either TEG4-VUSPIO (20 μL; 58 mM Fe 3 + ) or IgG cont-VUSPIO (38 μL; 31 mM Fe 3+ ) or PBS. After 20 min, mice were again perfused with 10 mL PBS followed by 5 mL of 4% v/v paraformaldehyde (PFA). The aorta was then removed and embedded in a glass MR tube containing 0.8% p/v high-grade, low melting-point agarose. Imaging parameters (FAST low-angle shot three-dimensional sequence) included the following: TE = 8 ms; TR = 27 ms; field of view 10 × 10 × 10 mm 3 ; image resolution 0.039 × 0.067 × 0.067 mm 3 /pixel; acquisition time: overnight; 48 scans.

Results

Production of TEG4 antibody as a recombinant IgG4 in baculovirus system After purification on Protein A, silver-stained SDS-PAGE under reducing and non-reducing conditions revealed proteins corresponding to the expected size of a correctly processed IgG4 and no major additional band (data not shown). Five milligrams of purified recombinant TEG4 (rIgG4 TEG4) was produced and the specificity of the recombinant molecules was controlled by ELISA on the αIIbβ3 integrin (data not shown).

Comparison of rIgG4 TEG4 and AP-2 binding against human platelets and atheroma

The murine monoclonal antibody, designated AP-2, reacts specifically with the complex formed by the human platelet membrane glycoproteins αIIb and β3. The human antibody TEG4 was previously obtained through phage-display biopanning against activated platelets, and characterised as an anti-αIIbβ3 antibody. [START_REF] Jacobin | Human IgG monoclonal antialpha(IIb)beta(3)-binding fragments derived from immunized donors using phage display[END_REF] AP-2 and rIgG4 TEG4 were first tested in flow cytometry and optical microscopy experiments against human thrombin-activated platelets (Figure 1,A). Due to the high number (50,000 copies) of αIIbβ3 integrins on the surface of activated platelets, this specificity results in a significant shift of fluorescence when aliquots of 10 6 PFA-fixed-activated platelets were incubated with AP-2 antibody (Figure 1,A,b; Table 1 (geometric mean: 13,270; median: 12,180)). The same shift of fluorescence was observed with the rIgG4 TEG4 human antibody (Figure 1,A,e; Table 1 (geometric mean: 17,390; median: 25,340)). Activated platelets incubated with either AP-2 or rIgG4 TEG4 showed a fine labelling in optical microscopy (Figure 1,A,c,f).

Figure 1. Flow cytometry and optical fluorescence microscopy assays performed on PFA-activated platelets. Incubations with AP-2 murine monoclonal antibody or rIgG4 TEG4 human monoclonal antibody (A), and TEG4-VUSPIO contrast agent or IgG cont-VUSPIO (B) were followed by a second incubation with species-specific secondary antibodies labelled with Alexa Fluor 488 or anti-dextran antibodies labelled with FITC. The labelling of platelets in flow cytometry (B, i, m) and optical microscopy (B, j) with the TEG4-VUSPIO conjugate is similar to the labelling with rIgG4 TEG4 human antibody (A, e, f).

Bar: 5 μm.

Number of human antibodies per nanoparticle

The absorbance of the VUSPIO samples was measured at 480 nm using a spectrophotometer (U-2800A spectrophotometer, Hitachi). The iron content of samples of conjugated and unconjugated VUSPIO was deduced from the concentration of γ-Fe2O3 calculated as described. [START_REF] Jacobin-Valat | MRI of inducible Pselectin expression in human activated platelets involved in the early stages of atherosclerosis[END_REF] The iron content of the TEG4-VUSPIO conjugate was 3.25 g/L Fe 3+ (Fe 3+ = 58 mM) and the antibody concentration calculated from ELISA curves was 48 μg/mL, leading to a ratio of one antibody per VUSPIO. Analysis of TEG4-VUSPIO reactivity against platelets by fluorescence

The human recombinant monoclonal antibody rIgG4 TEG4 and polyclonal IgG conjugated to VUSPIO nanoparticles (TEG4-VUSPIO and IgG cont-VUSPIO) were incubated with aliquots of PFA-fixed-activated platelets (Figure 1,B). Unconjugated rIgG4 TEG4 antibody at 10 μg/mL and antibody free-VUSPIO at 12 mM Fe 3+ (Figure 1,B,g,k) were used as positive and negative controls respectively. For flow cytometry detection of antibody-VUSPIO, aliquots of the cell samples were further treated with Alexa Fluor 488 goat anti-human H + L or FITC anti-dextran antibodies. The first method indirectly detects targeted contrast agents accumulated all around platelets by staining the targeting ligand (e.g. the functionalising primary antibody) with a secondary antibody (anti-human H + L) recognising the primary human antibody. The second method, using an anti-dextran antibody, is useful to detect the nanoparticle by itself.

Flow cytometry using both secondary antibody systems revealed a distinct fluorescence shift of platelets incubated with TEG4-VUSPIO compared to antibody-free VUSPIO and IgG cont-VUSPIO controls (Figure 1,B). Using anti-human H + L and anti-dextran antibodies, a 7.6 and 5.2 enhancement in targeting was observed respectively for the geometric mean between the contrast agent functionalised with rIgG4 TEG4 and the same agent functionalised with an irrelevant antibody (Figure 1,B,i,m,h,l; Table 1). Moreover, quite the same platelet recognition was shown with free rIgG4 TEG4 at 10 μg/mL and rIgG4 TEG4 grafted on VUSPIO nanoparticles (4040 versus 2090 for the geometric mean and 2260 versus 1970 for the median (Table 1)).

Geometric mean and median values in flow cytometry corroborate the high fluorescence labelling of free rIgG4 TEG4 and TEG4-VUSPIO on activated platelets in optical microscopy.

Analysis of TEG4-VUSPIO reactivity against platelets by TEM and in vitro MRI

Another way to obtain a direct representation of the active targeting of the nanoparticle by itself is to perform TEM analysis on activated platelets. Binding experiments were performed with thrombinactivated platelets using either TEG4-VUSPIO or IgG cont-VUSPIO. TEG4-VUSPIO targeted contrast agent was particularly visible at the platelet periphery, along the plasma membrane (Figure 2, A, blacks arrowheads). Of particular interest was the binding of TEG4-VUSPIO to activated platelets characterised by irregular forms with many irradiating pseudopodes projecting from their surface. In contrast, no labelling was evidenced at the plasma membrane when IgG cont-VUSPIO was used (Figure 2,A,(g,h)). Figure 2, A, i is a TEM analysis of TEG4-VUSPIO nanoparticles.

Figure 2, B describes in vitro MRI platelet experiments assessing the binding of targeted and untargeted VUSPIO to activated platelets. In comparison to the signal obtained with platelets only, T2-weighted MR imaging of platelet phantoms incubated with TEG4-VUSPIO (Figure 2,B,a) revealed a significant signal loss, in the same order of VUSPIO nanoparticles at 250 μM Fe 3+ (Figure 2,B,b). A minor signal loss was observed for platelets incubated with antibody-free VUSPIO particles and IgG4 cont-VUSPIO (Figure 2,B,(e,c)) when compared with platelet alone without any VUSPIO (Figure 2,B,g). Fewer VUSPIO nanoparticles remain trapped after two washes using IgG4 cont-VUSPIO (Figure 2,B,c) compared with antibody-free VUSPIO (Figure 2,B,e). These results suggest that -NH2 functional groups in antibody-free VUSPIOs are involved in residual nonspecific uptake, compared with nanoparticles whose amine groups were shielded by conjugated control antibodies. R2 relaxation rates of cell pellets treated with TEG4-VUSPIO, IgG4 cont-VUSPIO, unconjugated VUSPIO and with PBS only were 47.7 ± 3.5 s -1 , 7.8 ± 2.1 s -1 , 10.9 ± 1.9 s -1 and 4.4 ± 1.2 s -1 respectively.

Figure 2. In vitro validation of platelet targeting by transmission electron microscopy (TEM) and T2-weighted MRI on phantoms. (A) TEM images of TEG4-VUSPIO nanoparticles targeting activated platelets. TEM images clearly show the presence of contrast agents as dots distributed all around the platelets (arrowheads), especially in the pseudopodia developed in activated platelets (a, b, c). b, c and e, f are zoomed frames of a and d respectively, indicated by dashed lines. The same platelets incubated with IgG cont-VUSPIO showed no VUSPIO labelling (g, h). (i) illustrated the TEG4-VUSPIO preparation at 48 mM Fe 3+ . (B) T2 MR images of activated platelets targeted by TEG4-VUSPIO (a) shows the MRI signal obtained with TEG4-VUSPIO compared with the MRI signal of different concentrations of VUSPIO (0 μM Fe 3+ (d), 62.5 μM Fe 3 + (h), 125 μM Fe 3 + (f), 250 μM Fe 3 + (b)) in agarose gel, untargeted VUSPIOs (e), IgG4 cont-VUSPIO (c), platelets only (g). The TEG4-VUSPIO signal loss was in the same range of free VUSPIOs at 250 μM Fe 3 + (b), indicating the efficiency of targeting.

These values and the curve correlating the 1/T2 values measured at 4.7 Tesla with Fe 3+ concentrations were used to estimate the number of TEG4-VUSPIO nanoparticles bound per platelet. We found approximately 170 TEG4-VUSPIO per platelet which corroborates the number of counted targeted nanoparticles on TEM images.

Analysis of TEG4-VUSPIO reactivity against human and murine atheroma by immunohistochemistry

The presence of platelets within the atheroma plaque of ApoE -/-and human coronary specimens was tested using murine (AP-2) and human (rIgG4 TEG4) antibodies targeting the αIIbβ3 integrin and confirmed by commercial antibodies (anti-CD41 and PAC-1, respectively). Arterial sections from ApoE -/- mice showed a strong labelling of the intima in atheroma with both anti-platelet antibodies (Figure 3,D,F). The same staining was obtained with a commercial antibody (Figure 3, B) and controls were performed with appropriate secondary antibodies (Figure 3,A,C,E). The same strong labelling was observed when sections of human coronary specimens were incubated either with AP-2 or rIgG4 TEG4 antibodies (Figure 4, B, C and E, F, respectively). This result was confirmed by the commercial antibody PAC-1 (Figure 4, H, I). Enlarged views (Figure 4, C, F, I) showed the targeting of small neovessels located within the intima, in the desorganized media. The staining of macrophages by anti-CD68 antibody (Figure 4, K, L) matches some of the regions where platelets are found (Figure 4, E, H), suggesting the localisation of platelets in areas of foam cells and platelet-leucocytes-aggregates (PLA) accumulation. No labelling was observed with controls performed with appropriate secondary antibodies (Figure 4,A,D,G,J).

Figure 3. Immunohistochemical detection of integrin αIIbβ3 on aorta sections from ApoE -/-mice. The presence of platelets was shown using two different mouse antibodies: a commercial anti-CD41 (αIIb) antibody (B) and an antibody (AP-2) specific to the complex αIIbβ3 (D). Controls were performed with secondary HRP-labelled anti-rabbit and anti-mouse antibodies (A, C). The human antibody rIgG4 TEG4 (F) recognises platelets at the same level as both murine anti-αIIbβ3 antibodies. The conjugate TEG4-VUSPIO shows a high labelling of platelets within the intima (G) contrary to IgG cont-VUSPIO (H). Controls were performed with a secondary HRP-labelled anti-human H + L antibody (E).

Bar: 100 μm and 50 μm for enlarged views.

Figure 4. Immunohistochemical detection of integrin αIIbβ3 on coronary human sections. AP-2 and rIgG4 TEG4 panels: the presence of platelets was shown using the mouse anti-αIIbβ3 antibody AP-2 (B, C) and the human antibody rIgG4 TEG4 (E, F). PAC-1 panel: the presence of platelets was confirmed with a commercially available mouse antibody specific to human activated αIIbβ3 (H, I). Anti-CD68 panel: foam cells were detected with a commercially available mouse antibody specific to human CD68. (C, F, I, L) are enlerged views of (B, E, H, K). Controls were performed with secondary HRP-labelled anti-mouse IgG antibody (A, J), HRP-labelled anti-human H + L antibody (D) and HRP-labelled anti-mouse IgM antibody (G).

Bars: 100 μm and 50 μm for enlarged views.

The targeted contrast agent TEG4-VUSPIO was tested on sections of ApoE -/-(Figure 3, G) and wild-type C57BL/6J mice (Supplementary data, Figure S3, C), showing a high labelling of platelets within the intima of ApoE -/-mouse (Figure 3, G) contrary to IgG cont-VUSPIO (Figure 3,H). VUSPIO conjugates tested on wild-type C57BL/6J mice (Supplementary data, Figure S3,B,C) and appropriate secondary antibody (Supplementary data, Figure S3, A) showed no labelling.

These in vitro results confirm the maintenance of TEG4 specificity after coupling to VUSPIO nanoparticles.

Ex vivo characterisation of the targeted-contrast agent nanoparticle

Atherosclerotic plaques in ApoE -/-mice thoracic aorta are clearly visible at 9.4 Tesla (Figure 5, B) when compared with healthy aorta of C57BL/6 wild type mice (Figure 5,A). The aorta from atherosclerotic ApoE -/-mice exposed ex vivo to TEG4-VUSPIO preparation shows an intense hypo-signal (black arrows) (Figure 5,C). In contrast, no hypo-signal was observed in the thoracic aorta of ApoE -/-mice that did not receive VUSPIO (Figure 5, B) or did receive IgG cont-VUSPIO (Figure 5, D). Thus, representative ex vivo MRI studies at 9.4 Tesla showed that TEG4-VUSPIO could induce a loss of MRI signal generated by VUSPIO accumulation in the atherosclerotic vessel wall of ApoE -/-mice, due to antibody targeting.

vivo with PBS (A, B), TEG4-VUSPIO (C), IgG-cont VUSPIO (D). No atherosclerotic plaque was visible in healthy aorta (A). Panel C shows an intense hyposignal due to iron deposition within atherosclerotic plaque. In contrast, no loss of signal was observed when IgG cont-VUSPIO was administered (D).

Discussion

This study demonstrates the interest of non invasive imaging of platelets recruited within atherosclerotic lesions by an αIIbβ3-targeted VUSPIO molecular imaging probe. The presence and effects of platelets inside the atherosclerotic plaque are relatively underexplored and this probe could give insight into intraplaque haemorrhage (IPH), neovasculature and vascular wall inflammation which constitute key plaque features underlying atherosclerosis.

The rationale for molecular imaging is to propose an accurate diagnosis method. Prospective clinical trials have shown that measurement of stenosis underestimates carotid plaque burden [START_REF] Wasserman | Low-grade carotid stenosis: looking beyond the lumen with MRI[END_REF] and that the majority of patients with a history of recent transient ischemic attack (TIA) or stroke have mild-to moderate carotid stenosis. Debates still exist on how stenosis severity is related to the unstability of the culprit lesions. [START_REF] Niccoli | Are the culprit lesions severely stenotic?[END_REF] However, data from the PROSPECT (Providing Regional Observations to Study Predictors of Events in the Coronary Tree) study have concluded that a small luminal area is independently associated with the 3year risk of acute coronary events. [START_REF] Stone | A prospective natural-history study of coronary atherosclerosis[END_REF] It is now admitted that looking beyond the lumen with functionalised contrast agents is needed to identify the high-risk plaques across a range of stenoses issuing from both carotid and coronary beds.

Compared to previous approaches using rodent antibodies, the use of a human antibody to functionalise nanoparticles is of particular interest for direct transfer into the clinic, considering the need of repeated injections to follow the progression of the disease. Using human antibodies may reduce immunogenicity by avoiding the development of anti-species antibodies usually described when murine antibodies were injected in humans. [START_REF] Lajus | Thrombocytopenia after abciximab use results from different mechanisms[END_REF] (see Limitations section).

Recombinant IgG4 TEG4 antibody is able to target activated platelets in the same level as the murine AP-2 and anti-CD41 antibodies as demonstrated by fluorescence experiments. Our in vitro experiments conducted with the targeted contrast agent TEG4-VUSPIO confirmed maintenance of specificity against activated platelets using fluorescence and TEM. In vitro MRI results also showed that the activated platelets targeted with TEG4-VUSPIO have the highest signal reduction (R2 relaxation rate of 47.7 ± 3.5 s -1 ) when compared with IgG4-cont VUSPIO, unconjugated VUSPIO and with PBS only (R2 relaxation rates of 7.8 ± 2.1 s -1 , 10.9 ± 1.9 s -1 and 4.4 ± 1.2 s -1 respectively), highlighting the added value of targeting versus passive take up of nanoparticles. All these results demonstrate that TEG4-VUSPIO can selectively accumulate around activated platelets. Recombinant IgG4 TEG4 antibody and TEG4-VUSPIO contrast agent were also shown to highly label atherosclerotic sections of murine and human specimens as demonstrated by IHC studies.

The rationale for targeting platelets within atherosclerotic lesions is that they are highly represented not only in thrombi and intraplaque haemorrhage (IPH) but also in atheroma burden, around necrotic areas and neovessels (see Figure 3, Figure 4). A pioneer study performed by Massberg et al [START_REF] Massberg | A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation[END_REF] showed, by intravital videofluorescence microscopy, that platelets adhering at the carotid bifurcation (lesion-pronesite) in ApoE -/-mice, directly coincided with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Platelet-endothelial interactions linked to increased inflammatory activation and prothrombotic state in atherosclerosis are mediated, in part, by platelet glycoprotein (GP) Ibα. Jonathan Lindner's group [START_REF] Mccarty | Molecular imaging of activated von Willebrand factor to detect high-risk atherosclerotic phenotype[END_REF] used recombinant GPIb as a targeting moiety on microbubble contrast agents to perform contrast-enhanced ultrasound molecular imaging of activated von Willebrand factor (vWF) and thus demonstrated widespread platelet-endothelial interaction in LDLR -/-/ApoBec-1 -/-mice.

Since then, many studies indicate that platelets not only contribute to endothelial dysfunction but also modulate various inflammatory responses by platelet-monocyte complex formation (PMA: plateletmonocyte aggregates) [START_REF] Postea | Contribution of platelet CX(3)CR1 to platelet-monocyte complex formation and vascular recruitment during hyperlipidemia[END_REF][START_REF] Czepluch | Increased proatherogenic monocyteplatelet cross-talk in monocyte subpopulations of patients with stable coronary artery disease[END_REF] or platelet-CD4 + T cell cross-talk [START_REF] Li | CD4 + T cells in atherosclerosis: regulation by platelets[END_REF] , facilitating leucocyte transmigration and thus act on plaque vulnerability. Platelets themselves -like monocytes/macrophages -are able to bind, take up via scavenger receptors (CD36, LOX-1, SR-B1) and transport modified lipoproteins into the intima, thus contributing indirectly and directly to foam cell formation in atherogenesis. [START_REF] Siegel-Axel | Platelet lipoprotein interplay: trigger of foam cell formation and driver of atherosclerosis[END_REF] Inside the plaque they are in close contact with macrophages and phagocytosis of platelets by macrophages was already proposed as an alternative mechanism of foam cell formation 53 years ago by Chandler et al. [START_REF] Chandler | Phagocytized platelets: a source of lipids in human thrombi and atherosclerotic plaques[END_REF] Four decades later, De Meyer et al [START_REF] De Meyer | Platelet phagocytosis and processing of beta-amyloid precursor protein as a mechanism of macrophage activation in atherosclerosis[END_REF] confirmed in vitro that platelet phagocytosis by murine macrophages results in the formation of lipid-laden macrophages. The authors concluded that uptake of modified LDL by platelets and phagocytosis of LDL-laden platelets by macrophages may be critical steps for the development of lipid-rich plaques. Daub et al [START_REF] Daub | Oxidized LDL-activated platelets induce vascular inflammation[END_REF] more recently showed that platelet oxLDL contributes significantly to vascular inflammation and is able to promote atherosclerosis. Platelet-induced lipid accumulation in monocytes thus provides a novel mechanism for how platelets negatively regulate plaque stability. [START_REF] Badrnya | Platelets mediate oxidized lowdensity lipoprotein-induced monocyte extravasation and foam cell formation[END_REF] Furthermore, platelets are contained in large amounts in early-calcified human atherosclerotic plaques as the results of plaque neovascularisation by leaky vessels, blood extravasation and haemorrhage. They have been involved in the release of osteocalcin in atherosclerotic lesions. [START_REF] Foresta | Platelets express and release osteocalcin and co-localize in human calcified atherosclerotic plaques[END_REF] Thus, results from the literature and the high labelling shown in Figure 3, Figure 4 of this study prove that platelets are not only present on plaque surfaces or in IPH. They may play, outside thrombi formation, an important role in inflammation, necrotic core formation, release of factors like osteocalcin and matrix metalloproteinases [START_REF] Seizer | EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation[END_REF] involved in plaque destabilisation.

So far, the proof of entry of platelets into atherosclerotic lesions has only been given in human atheroma and it was correlated with intraplaque microvascular leakage and IPH. [START_REF] Van Lammeren | Platelets enter atherosclerotic plaque via intraplaque microvascular leakage and intraplaque hemorrhage: a histopathological study in carotid plaques[END_REF] Mice atheroma does not typically develop IPH. This means that the different antibodies (TEG4, AP-2, anti-CD41 antibody) used in IHC recognise platelets within ApoE -/-mice atheroma which are not involved in intraplaque thrombi but are rather in interaction with other cells as reported in platelet-leucocyte aggregates (PLA). That is why our study is important from a fundamental point of view because it sheds light into the significant presence of platelets both within mouse and human atheroma as demonstrated in Figure 3, Figure 4 by IHC on sections of ApoE -/-aorta and human coronary arteries.

Moreover, MRI approaches have shown that complex plaque features, such as the necrotic core and intraplaque haemorrhage are prevalent in carotid arteries with minimal stenosis, including those with angiographically normal appearing arteries. [START_REF] Hatsukami | MRI in the early identification and classification of high-risk atherosclerotic carotid plaques[END_REF] Thus, the development of probes that package MRI contrast agents with ligands targeting platelets might show great promise for providing a direct measure of plaque activity and function at the cellular and molecular level. In the current study, the ex vivo results highlighted an accumulation of the targeted contrast agent in the atherosclerotic vessel wall of ApoE -/-mice.

Cheruvu and colleagues showed that plaque composition varies depending on the anatomical site with striking heterogeneity even within the same individual. [START_REF] Cheruvu | Frequency and distribution of thincap fibroatheroma and ruptured plaques in human coronary arteries: a pathologic study[END_REF] Therefore, the finding of a focal distribution of suspected precursor lesions supports the efforts to develop reliable non-invasive imaging tools to identify the molecular and cellular composition of these structures that may occur at sites with and without flowlimiting stenoses.

This manuscript describes the development of a new targeted contrast agent for MRI, from the conception of the targeting moiety, a fully human antibody, to ex vivo MRI images.

Limitations

Today, most therapeutic mAbs in the clinic are humanised or fully human meaning that, theoretically, only a minimal immunogenic potential remains (as compared to murine mAbs). [START_REF] Pavlou | Recombinant protein therapeutics-success rates, market trends and values to 2010[END_REF] However, evolution of recombinant technologies to produce fully human antibodies has only partially solved this historic obstacle of antibodies. Immunogenicity induced by injected proteins is a serious issue because it is directly related to the patient's safety. A risk assessment should be conducted for every biotherapeutic under development to address the occurrence of immunogenicity, as well as the severity of the adverse effects caused. An induced antibody response against human antibodies, called human anti-human antibody (HAHA) response, must always be thoroughly tested in clinical studies. [START_REF] Reichert | Antibodies to watch in 2014[END_REF] In regard of that, the present study has limitations inherent to potential immunogenicity against the injected targeted contrast agent. Even if we have been careful to develop fully human antibodies to serve as the ligand moiety, it is impossible to guarantee, before clinical assays in a cohort of individuals, that anti-idiotypic antibodies will not arise. Moreover, the development of immunogenicity during treatment is highly dependent, not only of the individual status, but also of the pathology treated (immune disorders, chronic illnesses are more at risk to induce auto-antibodies formation) and of the target protein and mechanism of action. [START_REF] Stas | Immunogenicity of therapeutic antibodies[END_REF][START_REF] Chan | Therapeutic antibodies for autoimmunity and inflammation[END_REF]

P a g e | 3

 3

P a g e | 7

 7

Figure 5 .

 5 Figure 5. Ex vivo MRI at 9.4 Tesla. The healthy (A) or atherosclerotic ApoE -/-aorta (B, C, D) were perfused exvivo with PBS (A, B), TEG4-VUSPIO (C), IgG-cont VUSPIO (D). No atherosclerotic plaque was visible in healthy aorta (A). Panel C shows an intense hyposignal due to iron deposition within atherosclerotic plaque. In contrast, no loss of signal was observed whenIgG cont-VUSPIO was administered (D).

This study was supported by two public grants from the French "Agence Nationale de la Recherche" within the context of the Investments for the Future Program, referenced ANR-10-LABX-0057, named IdEx and TRAIL MIMATHUMAB and by public grants from the French "Agence Nationale de la Recherche" within the context of PCV programme, named IMATHABIO and SVSE5 programme, named ATHERANOS.

P a g e | 16

Supplementary material Upper panels: labelling of human activated platelets.

Incubation of human activated platelets with PAC-1 FITC antibody targeting human αIIbβ3 or FITC-labelled isotypic control.

Other panels: labelling of mouse activated platelets.

Incubation of murine activated platelets with commercially available rabbit anti-CD41 monoclonal antibody, AP-2 murine monoclonal antibody or rIgG4 TEG4 human antibody was followed by a second incubation with species-specific secondary antibodies labelled with Alexa Fluor 488. For rIgG4 TEG4, an isotypic control was used.

Figure S3. Immunohistochemical experiment on arterial sections from wild-type C57BL/6J mice. The conjugates TEG4-VUSPIO and IgG cont-VUSPIO were tested on sections of wild-type C57BL/6J mice which did not develop atherosclerosis (C, B). A control was performed with a secondary HRP-labelled anti-human H + L antibody (A). Bar: 100 μm.


Stephen P Thompson
email: stephen.thompson@diamond.ac.uk

Karine Demyk

Sarah J Day

Aneurin Evans

Hugues Leroux

Christophe Depecker

Julia E Parker

Leigh Connor

Heribert Wilhelm

Giannantonio Cibin

Amorphous silicate nanoparticles with controlled Fe-Mg pyroxene compositions

Keywords: Iron and Magnesium silicate, Nanoparticles, Sol-gel, Amorphous material

The production of amorphous pyroxene nanoparticles (∼20 nm) with controlled Fe-Mg content is described. Homogenous particle compositions closely matching required target stoichiometries are obtained by drying a precursor gel under high vacuum conditions. The silicate nature of the particles is characterised using TEM, synchrotron radiation and FTIR. No oxide phase separation occurs, even at high Fe concentration. Structural domains exist within the nanoparticles that are typically ten times smaller than the physical particle size consistent with either a core-shell, or random network with multiple embedded domains, particle structure. Thermal annealing below the crystallisation temperature allows the ordered domain size to be further

Introduction

Nanoparticles are an important class of materials due to the enhancement, over their bulk equivalents, of properties such as optical activity, cation exchange capacity, surface area, chemical reactivity and adsorptivity; and have made significant contributions to a wide range of engineering, technology, biomedical, manufacturing, communications and structural applications as well as being of interest to academic research.

Crystalline nanoparticles have a large fraction of their atoms located on their surface and the presence of structural disorder near the surface results in functional properties significantly different to the bulk crystalline material [1]. Amorphous nanoparticles (ANPs) on the other hand are generally believed [2] to have either a two-part core-shell structure, whereby an amorphous core is surrounded by a more porous surface shell region containing a large amount of structural defects; or to have a layered structure comprising high and low density radial layers. Because of these structural differences ANPs often have very different properties and applications compared to crystalline nanoparticles [2]. Fe and Fe-oxide ANPs, for example, exhibit unique magnetic, catalytic, optical and sorption properties relevant to advanced technological applications [3]. Unfortunately, Fe's preference for forming oxide phases under atmospheric, or similarly oxidising conditions, means that its controlled incorporation into other materials such as silicate is problematic and complicated by its ability to act as both network former and network modifier.

However, Fe-Mg silicate ANPs of pyroxene (and related) silicate compositions are present in the Earth's upper atmosphere [4,5], having formed by the recondensation of Fe, Mg and Si oxide species produced by the ongoing ablation of in-falling meteoroids [6]. These particles are present in sufficient quantities (∼1000's per cm 3 [4]) to play a significant role in a number of atmospheric processes, including cloud nucleation and removal of H 2 SO 4 from the gas phase at high altitude. Laboratory dissolution studies using analogue ANPs provide the physical constraints necessary to assess their environmental impact via modelling [7]. Similarly in astrophysics, Mg-rich pyroxene ANPs condense as dust particles (along with other solid species) in the circumstellar regions surrounding evolved stars and are ubiquitous throughout space. However, the fate of elemental Fe is a significant unsolved problem in astrophysics [8]. Observations show it is highly depleted from the interstellar gas phase and must therefore reside somewhere within the solid phase population. The absence of observable IR features attributable to pure-phase metallic Fe or Fe-oxide phases means that current theories assume Fe is somehow incorporated within the silicate ANP dust, even though fits to observational IR spectra favour Mg-rich, Fe-poor compositions. Since thermodynamics cannot predict the Mg/Fe distribution between amorphous phases, laboratory simulation using analogue ANP materials with variable Fe content represents the only means of investigation. However, the laboratory approach is challenging as the properties of amorphous nanomaterials (and specifically nanosilicates) remain poorly understood and reliable, controlled production methods capable of yielding large volumes suitable for multiple experiments are largely absent [2,9].

Early attempts at synthesising amorphous Fe-bearing silicates met with only limited success and were generally produced as one-offs [10,[START_REF]Krätschmer In Experiments on Cosmic Dust Analogues[END_REF], while more systematic approaches applied glass preparation methods that involved melt quenching [START_REF] Jäger | [END_REF]. However, spectroscopic behaviour and physical homogeneity were strongly influenced by the quenching method rather than composition, as was the Fe oxidation state due to the melting/quenching taking place in air. Furthermore, moderately high Fe content resulted in phase separation and varying crystallisation [13] even in a reducing atmosphere [14]. Attempts to produce silicate ANPs via vapour phase condensation in controlled Mg-Fe-SiO-H 2 -O 2 atmospheres produced only pure Mg or pure Fe silicates whose compositions corresponded to the metastable eutectics in the MgO-SiO 2 and FeO/Fe 2 O 3 -SiO 2 binary phase diagrams [15]. Other researchers found mixed Fe-Mg ANPs could be produced by the laser ablation of mineral targets. However, differences in the optical constants, thermal properties and the melting temperatures of different oxides result in variable evaporation rates, which adversely affect compositional homogeneity [16]. Of more relevance to the present work was an attempt to synthesise amorphous silicates with chondritic composition (i.e. Na 2 O-MgO-Al 2 O 3 -SiO 2 -CaO-FeO-NiO after the composition of C1 meteorites [17]) using a sol-gel approach.

However, the final product consisted of an amorphous matrix containing separated crystallites of magnetite and ferrihydrite [18,8].

In all of the above syntheses, the degree to which Fe is incorporated into the silicate structure could not be controlled. Previously, in producing amorphous Ca-silicate by sol-gel [19] we found that the reaction of Ca with atmospheric CO 2 to form phase separated calcium carbonate could be suppressed by drying the gel under vacuum. In addressing the Fe-Mg silicate ANP synthesis problem we have adapted this procedure to allow the controlled synthesis of Fe-Mg pyroxene silicate ANPs with low size dispersion and tuneable Fe content. In this paper we describe the necessary sol-gel procedure and present the results of laboratory characterisations using electron microscopy, synchrotron radiation and IR spectroscopy.

Experimental

Sol-gel synthesis

Silicate ANPs were produced in the beamline I11 support laboratory at the Diamond Light Source (UK). The sol-gel is formed by rapidly mixing two 0.1 molar solutions (18 MΩ.cm deionised water) of (i) magnesium chloride (MgCl 2 ) and ferrous ammonium sulphate (Fe(II)(NH 4 ) 2 (SO 4 ) 2 ) and (ii) sodium metasilicate (Na 2 SiO 3 ); prepared in stoichiometric ratios necessary to produce Fe x Mg 1-x SiO 3 target compositions with x = 0.1, 0.2, 0.3, 0.4, 0.5, 0.8 and 1.0. All reagents were analytical grade and used as supplied.

Upon mixing, blue-green gelatinous suspensions were formed which were magnetically stirred for 5 minutes to ensure complete mixing/reaction. If left to settle at room temperature, the gels gradually turn brown, resulting in a product with high Fe-oxide content. Thus, immediately after stirring, the suspensions were centrifuged and washed several times in deionised water to remove dissolved reactants. The gels were then transferred to covered alumina boats and dried under vacuum at 50 • C in a Carbolyte vacuum furnace purged with N 2 gas prior to loading. Before the gels could be heated, the sample chamber was pumped overnight to <10 -6 mbar. During heating the pressure inside the vessel increased but by the end of the heating stage (∼6 hours) had returned to below 10 -6 mbar. The chamber was brought to a positive over pressure using N 2 gas and the dry loose powder samples removed and stored under Ar in sealed vials inside a desiccator cabinet. From two 250 ml starting solutions a yield of approximately 2 g of powder was obtained.

The powder colours ranged from very light brown for x = 0.1 to dark brown for x = 1.

Since vacuum drying was at relatively low temperature, the silicate ANPs are likely to be hydrated, either with bonded OH or trapped interstitial H 2 O. Portions of the dried ANPs were therefore also annealed at 200, 300, 400, 450, 500 and 650 • C under vacuum to determine the possible steps required to produce anhydrous product. The furnace chamber was evacuated to ∼2×10 -6 mbar and the ramp rate set to 10 • C min -1 . The set point temperatures were maintained for 12 hours, after which the furnace was switched off and left to cool to room temperature. The annealed powders were again stored under Ar.

Characterisation measurements

FTIR Transmission spectra (6000 -210 cm -1 ) were measured for pressed pellets (0.5-1 mg of sample with 300 mg KBr pressed at 10 T for 4 minutes) using the ESPOIRS instrument at IRAP in Toulouse. This comprises a Bruker VERTEX 70V spectrometer equipped with globar lamp, CsI beamsplitter and DLaTGS detector with CsI window. TEM analysis was performed using a FEI Tecnai G2-20 twin at the University of Lille electron microscopy centre. Small amounts of each composition were crushed in alcohol to form a suspension and a drop deposited on a carbon film supported by a copper TEM grid. Particle sizes were measured by bright field conventional nanoscale imaging, while X-ray energy dispersive spectroscopy (EDS) provided compositional analysis. Synchrotron X-ray powder diffraction (SXPD) measurements were made on beamline I11 [20] at the Diamond Light Source (UK). Diffraction data out to 150 • 2θ were collected in 0.001 • intervals using 15 keV X-rays and the diffractometer's multi-analyser crystal system in Debye-Scherrer geometry.

Total X-ray scattering (TXS) was measured on beamline I15 at Diamond for samples loaded in 1 mm diameter Kapton tubes using 75.99 keV X-rays focused down to 70 μm and a Perkin Elmer 1621 EN area detector located 200 mm from the sample position. Data collection involved summing 200 frames, each comprising ten 1 s exposures. TXS data was transformed to radial pair distribution functions (PDFs) using the PDFgetX2 software2 .

Fe K-edge X-ray absorption spectra were recorded in transmission on Diamond's B18 beamline [21,22]. A few mg of each composition was mixed with 30 mg of cellulose powder, lightly ground and pressed (10 T) into pellets ∼0.5 mm thick. Spectra were recorded between 6.912 and 7.961 keV in 0.3 to 0.4 eV intervals. Fe K-edge Spectra for library samples of hematite (Fe 2 O 3 ), magnetite (Fe 3 O 4 ) and pyrite (FeS 2 ) available at the beamline were also recorded.

Results

Fig. 1a shows mid-IR spectra for the as dried powders. All the spectra contain three main contributions: the characteristic silicate O-Si-O bend and Si-O stretch bands (∼250 -800 cm -1 and ∼800 -1300 cm -1 respectively), plus a broad hydration band (∼3500 cm -1 ) with associated minor bands (1300 -1800 cm -1 and ∼5150 cm -1 ). Although some variation in band widths can be seen, the two silicate bands are devoid of fine structure features, consistent with an absence of crystalline structure. TEM analysis for selected compositions (x = 0.1, 0.5 and 0.8) revealed amorphous nanoparticles with individual sizes ranging from 12 to 33 nm with a distribution peak at ∼20 nm (e.g. Fig. 2) that was found to be independent of composition. EDS for large and small sampling areas (>0.5×0.5 μm 2 and 100×100 nm 2 ) showed good chemical homogeneity with achieved compositions lying close to the target formula (e.g. Fig. 3). The spread in composition was found to be largest for low Fe concentration, suggesting Fe incorporation may be higher in some particles and lower in others. Trace level quantities of Na, Cl, K and Ca left over from the sol-gel were detected; however on a scale up to 10 -3 μm 3 the as dried ANPs were found to be homogeneous with no phase separation to SiO 2 or Mg/Fe-oxide. SXPD (e.g. Fig. 4) showed only the broad features characteristic of a material that at the atomic level is either amorphous or nanostructured. Only minor variations exist between compositions, indicating a relatively consistent particle structure. This is shown in more detail in Fig. 5a which shows PDFs obtained from the TXS measurements. All the PDFs show a very similar structure in terms of feature number, position and profile, although there is a clear change in the relative strengths of the two peaks at ∼1.6 and ∼2.0 Å as the Fe content increases. For the x = 0.8 PDF the structural ordering appears to extend over a slightly longer range which may reflect some minor difference in synthesis conditions. Even so, at short-range the x = 0.8 PDF is still very similar to the other compositions. The Fe K-edge absorption spectra (Fig. 6a) for the as dried ANPs are also all very similar, displaying a pre-edge peak (A) due to 1s → 3p transitions, a weak shoulder at the bottom of the absorption rise due to ligand-to-metal charge transfer (LMCT, [26]) and a sharp absorption rise to a large narrow peak (B) followed by a weaker secondary peak (C), both of which are due to multiple scattering (MS). In transition metal oxides, these post-edge features are due to hybridization of the central atom's 4p orbitals with the 3d octahedral molecular orbitals of the neighbouring O atoms and MS calculations for silicates [27,28,29] show a minimal cluster of a central Fe with six nearest O neighbours is sufficient to account for both the main peak (B) and the broad peak (C); while including larger cluster sizes involving higher shells allows for the appearance of the pre-peak (A) which is therefore an indicator of medium-range structure.

Discussion

Structural environment

The characterisation measurements above confirm the production method yields a set of homogeneous silicate ANPs with pyroxene compositions and atomic-level structures that are very closely related. A semi-quantitative measure of the similarities between the ANP structures can be obtained from the PDF data [25] using the Pearson product-moment correlation, R, calculated pair-wise for all PDF datasets,

R = 1 1 -n n i=0 X i - X σ x Y i - Ȳ σ y (1)
where X and Ȳ are the mean values of datasets X and Y and σ x and σ y their standard deviations. R is insensitive to differences in amplitude scale and for each pair returns a value between 1 (fully correlated) and -1 (fully anticorrelated) with 0 representing uncorrelated. Thus R allows us to gauge how similar/dissimilar the structures are without knowing specific details of atomic coordinates and bond angles etc. [25].

The results of comparing the structures for the as dried ANPs are given in Table 1 (top), calculated using the CORRELATE function in IDL. For compositions up to x = 0.5 the structures are highly correlated irrespective of composition. Although the x = 0.8 PDF shows structural order extending over a longer range, its correlation with the x < 0.5 compositions is still reasonably high and its correlation with x = 1.0 very high, suggesting a structure that is intermediate of x = 0.5 and 1.0. In general, the correlation is high across all the compositions, suggesting that any variation in structure due to increased Fe content is both gradual and proportionate.

The coherent scattering domain size (CSD) is the maximum distance over which structural correlations can be discerned and is represented by the distance at which the amplitude of the PDF oscillations diminish to zero.

From Fig. 5a the CSD for the as dried ANPs is ∼20 Å for all x (∼30 Å for x = 0.8), such that the silicate ANPs are only ordered on a scale that is ∼10× smaller than their average physical dimension determined by TEM. The CSD reduced further by a factor of ∼2 when the ANPs were annealed in vacuum at temperatures in the range of ∼450 • C to ∼500 • C, with temperatures above 500 • C causing the CSD length to increase as the ANPs start to crystallise (Fig. 5b). The reduction in the CSD also brought the x = 0.8 structure into line with the other compositions (Fig. 5c), with all the structures appearing well correlated with each other (Table 1 bottom).

Independent of Fe content, annealing the ANPs below their crystallisation temperature results in amorphous particles that are structurally very similar.

The first two peaks in the PDFs in Fig. 5 2.00 Å) and are closer to octahedral coordination [40,41] than the 1.80 Å typical of tetrahedral bonding, suggesting that few, if any, of the Fe atoms are network formers. Other instances of Fe 2+ and Fe 3+ occupying similar octahedral sites in non-crystalline Fe-silicate environments (e.g. glasses) have also been reported [24].

The absorption edge (B) feature for the as dried ANPs (Fig 6a) appears singular, with its crest at ∼ 7133 eV. However in α-hematite (Fe 2 O 3 ) this feature consists of a main peak and a weaker lower energy shoulder. The Fe in α-hematite are distributed in two parallel sets of alternating layers of equivalent octahedral sites which show different degrees of distortion [30,31].

This manifests as a weak shoulder corresponding to the interaction of Fe with O atoms at 2.115 Å, while the main peak is due to interaction with O atoms at 1.945 Å [31,32]. The energies of these two features are typically ∼7131

eV and ∼7134 eV. For the as dried ANPs, the main peak lies close to ∼7134

eV and, apart from the x ≥0.5 compositions, there is no significant lower energy shoulder. For the high x ANPs the shoulders are very weak and therefore also indicate that for the as dried ANPs the Fe atoms sit mostly within regular octahedral sites. However, for the 450 • C annealed ANPs (Fig. 6b) the shoulder is much stronger, with (B) feature maxima close to 7132 and 7134 eV, showing the formation of two distinct octahedral environments.

Thus, while annealing reduces long-range ordering by reducing the CSD size, it does result in an increase in the short-range ordering.

Fe oxidation state

As well as being an indicator for medium-range structure, the pre-peak (A) in the absorption spectrum (Fig. 6) also carries information regarding the oxidation state of the incorporated Fe, with a 1.4±0.1 eV difference between its centroid position being observed in Fe 2+ and Fe 3+ minerals [33,34,35].

Since hematite, magnetite and pyrite are known to have 100 %, 66.7 % and 0 % Fe 3+ , respectively, their pre-peak positions were taken from the measured library spectra and used to construct a calibration line from which Fe 3+ /ΣFe was calculated for each nanosilicate. This method has been applied to a wide range of minerals and glasses [35,36,37], although it can potentially be subject to error if the difference in site geometry between the reference standards and the unknowns is too large [35,36]. The centroid positions however do allow systematic trends to be identified and Fig. 8 shows Fe 3+ /ΣFe for the as dried and annealed ANPs as a function of composition. For x 0.5, Fe 3+ /ΣFe appears much the same in both, while annealing at 450 % for the ANPs here originates in the hydrolysis reactions that occur during the initial gel formation.

Effect of Fe on IR spectrum

For the as dried ANPs, the Si-O stretch band (Fig 1a) exhibits a broadening with increasing x, while its peak position (1022 cm -1 ) varies only by ∼2 cm -1 . However, for the ANPs annealed at 450 • C (Fig. 1b) it is the band width that shows little variation while the peak shifts from 1023 to 1046 cm -1 for x ≥ 0.8. This is also accompanied by a shift in the bending mode peak to lower wavenumbers as a new peak appears in the form of a shoulder at 458 cm -1 . The presence in the as dried ANP's bending mode of two peaks points to the silicate network not being fully built, with a lower degree of tetrahedral polymerization. However, this needs to be considered in conjunction with the PDF data. In bulk amorphous silicates there are two basic models: the Zachariasen-Warren model [42]- [45], in which Si-O tetrahedra are linked together in a statistically disordered way to form a large-scale continuous random network (CRN) with no long-range periodicity; and the "microcrystal" model attributed to Lebiediev and co-workers [46]- [49], in which the CRN links between the surfaces of ultrafine (∼15-20 Å) crystalline domains [50]. Since the PDF data shows ordering on the 10-20 Å scale, the double peaked bending mode may derive from a structure where the nano sized crystalline domains and interlinking CRN have differing polymerisation. Since the domain structure below 10 Å does not change with annealing, further network building by polymerisation presumably only occurs within the CRN surrounding the nano domain (since this is observed to change when the CSD shrinks during annealing), presumably via the linking together of tetrahedra made possible by the loss of water and/or bonded OH [23,51] and also resulting in a change in the Si-O peak position. However, although our investigations support the domain-structured model, neither the X-ray nor the IR data allow us to distinguish between ANPs with a single core-shell structure or, given the small size of the ordered regions relative to the physical particle size, ANPs containing multiple nano domains linked via a CRN.

ANP Hydration

In Fig. 1a the main hydration band is the strong, broad asymmetric feature peaking at ∼3500 cm -1 . The high x ANPs exhibit a broader asymmetry on the low wavenumber side with a strong side feature at ∼2300 cm -1 ; while the lower x ANPs show a high wavenumber tail at ∼3850 cm -1 . The band itself is attributable to interstitial H 2 O and (Si)O-H groups in differing structural environments [52] and clearly varies with Fe content. The sharp band located at ∼1650 cm -1 is due to the H-O-H bending mode of coordinated and/or adsorbed water [53], while the band at ∼5150 cm -1 is a combination band of molecular water [54,55]. Annealing at 450 • C reduces the 3500 cm -1 band strength by ∼30% with similar behaviour for the 1650 and 5150 cm -1 bands (Fig. 1b). However, doubling the annealing time produced no further change in the shape, position or strength of any of the hydration bands. Annealing at higher temperatures could potentially remove more, or all, H 2 O and bonded OH but as Fig. 5b suggests it will, depending on temperature, also result in increased levels of crystallisation. Some level of persistent hydration is therefore likely to be an inherent feature of sol-gel produced ANPs, but could potentially play an important role in defining their functional or reactive behaviour in future applications.

Conclusions

The tendency for Fe to form oxide phases means that its incorporation during materials syntheses can be problematic and the primary objective of the work reported here was to develop a means of reliably synthesising amorphous pyroxene nanosilicates with controlled Fe/Mg content. In doing so we have developed a robust, well controlled and easy to execute method of producing homogeneous, monodispersed, Fe-bearing nanosilicates in which the Fe atoms are fully incorporated into the amorphous silicate structure.

To verify this we have characterised the structural and spectroscopic prop- As dried at 50

• C x = 0.1 x = 0.2 x = 0.3 x = 0.4 x = 0.5 x = 0.8 x = 1.0 x = 0.

Fe fraction x

  at ∼1.6 Å and ∼2.0 Å correspond to the Si-O and M-O distances (M = Mg or Fe). The Si-O bondlength (Fig. 7a), obtained from the first derivative of the PDF, exhibits no linear compositional dependency as it is also susceptible to other physical factors such as strain and tetrahedral polymerisation [23]. The M-O bond similarly obtained however shows a clearer dependency (Fig. 7b), generally shortening and tending towards a constant value as x increases. The PDF derived M-O distances in Fig. 7b, though indicative of octahedral bondlengths, are of course averaged over both Mg-O and Fe-O. Values for the Fe-O distances (Fig. 7c) obtained from first shell fits to the K-edge EXAFS signal using the Fe-O rocksalt structure show a much smaller range of variation (1.95 to

  erties of the nanosilicates showing that our relatively facile sol-gel based process produces amorphous nanoparticles with selectable Fe content across the Fe x Mg 1-x SiO 3 range. The nanoparticles contain ordered silicate nan-odomains typically 20 Å in size which are reduced further by annealing the particles in vacuum below their crystallisation temperature. Although this reduces correlations in their long-range structure, annealing does increase the ordering of the local short-range environment. The use of a sol-gel as a precursor means that the particles retain a degree of hydration. However, by controlling the annealing temperature it should be possible to selectively produce particles with differing degrees of crystallinity, depending on the requirements of any potential application. Although we have not yet conducted an exhaustive investigation of the optimum necessary conditions required for the transformation of a gel into solid nanoparticles, we believe it is in the application of high vacuum during the drying step that the particle size is determined as this likely interferes with, or disrupts, the post-gelation monomer aggregation process. Tests using a vacuum oven (Thermo Scientific) to dry the gels under medium vacuum (0.01 mbar compared to 10 -6 mbar used in this work) resulted in the formation of large irregular solids several mm in diameter, similar to the material produced from gels dried in air[56]. Thus, by varying either the pumping rate, or the base vacuum pressure, it may prove possible to vary the size distribution. Alternatively, although the present work is aimed at producing Fe-Mg pyroxene silicate ANPs with little, or no, contaminating species, if compositional purity is not a major consideration size control might potentially be achieved by the inclusion of additional steps and/or additives in the production process. For example SiO 2 , TiO 2 and Zn 2 SiO 4 nanoparticles with uniform sizes have been produced via sonochemical methods utilising ultrasonic treatment and Schiff base additives [57]-[60], while "green" Fe 2 O 3 /CuFe 2 O 4 ferromagnetic nanocomposite oxides have been produced via sol-gel-autocombustion in which an onion-derived reductant additive was used [61].

Fig. 1 Fig. 2

 12 Fig. 1 FTIR spectra of amorphous Fe/Mg silicate ANPs in the 6000-210 cm-1 range (a) dried at 50 • C; (b) annealed at 450 • C. All spectra shown normalized to the peak maximum of the Si-O stretching mode at ∼1000 cm -1 .

Fig. 3

 3 Fig. 3 Fe-Mg-Si ternary diagrams showing measured compositions for (a) Fe 0.1 Mg 0.9 SiO 3 and (b) Fe 0.5 Mg 0.5 SiO 3 nanosilicates. The small difference in composition between the large and small sampling sizes is due to a partial volatilization of Mg during the electron beam analysis.

Fig. 4

 4 Fig. 4 Synchrotron X-ray powder diffraction patterns for selected amorphous Fe x Mg 1-x SiO 3 nanoparticles dried at 50 • C.

Fig. 5

 5 Fig. 5 Pair distribution functions (a) for each as dried silicate composition; (b) for the x =0.5 composition as a function of annealing temperature and (c) for each composition after annealing at 450 • C.

Fig. 6

 6 Fig. 6 Fe K-edge spectra for silicate ANPs for x = 0.1 to 1.0 (offset in y-axis direction in order of increasing x) (a) dried at 50 • C and (b) annealed at 450 • C, showing pre-edge feature (A), main absorption edge crest (B) and post edge structure (C) (see text for discussion).

Fig. 7

 7 Fig. 7 (a) Si-O distance (±0.005 Å) as a function of Fe content for as dried ANPs (filled circles) and ANPs annealed at 450 • C (open circles) determined from PDF; (b) Variation of the M-O (Fe/Mg metal to O)

Fig. 8 Fe

 8 Fig. 8 Fe 3+ /ΣFe as a function of composition for ANPs dried at 50 • C (filled circles) and annealed at 450 • C (open circles).

Figure 1 :

 1 Figure 1:

26

 26

Figure 2 :

 2 Figure 2:

Figure 3 :

 3 Figure 3:

Figure 4 :

 4 Figure 4:

Figure 5 :

 5 Figure 5:

Figure 6 :

 6 Figure 6:

Figure 7 :

 7 Figure 7:

Figure 8 :

 8 Figure 8:

  • C significantly reduces the level of Fe 3+ for x 0.5. The variation in Fe 3+ /ΣFe for the lower x compositions may be related to the possible variation of Fe incorporation between particles inferred by the EDS analysis. Recent studies of aqueously altered amorphous Fe-Mg silicate (Fe 1.1 Mg 0.9 SiO 4.15 H 0.3 ), initially produced by pulsed laser deposition and containing only Fe 2+ , found Fe 3+ /ΣFe values of ∼60-75 %[START_REF] Guillou | Proc. Lunar Planet. Sci[END_REF][START_REF] Guillou | [END_REF] suggesting the average Fe 3+ /ΣFe∼50

Table 1 :

 1 Pearson product-momentum correlation, R, calculated out to 30 Å from the Fe x Mg 1-x SiO 3 PDFs.

http://www.totalscattering.lanl.gov

Acknowledgements

This work was supported by Diamond Light Source (beamtime allocations EE8692 and SP8614) and by the ANR: project CIMMES (ANR-11-BS56-029-01). SJD acknowledges financial support by Diamond Light Source and Keele University. HL and CD thank the Chevreul Institute, the European FEDER and the Région Nord-Pas-de-Calais (France).